Agonist anti-IL-2R antibodies and methods of use

Abstract

Anti-IL2R (e.g., anti-IL2RB, anti-IL2RG, anti-IL2RB / G) antibodies are disclosed, along with methods of making such antibodies, compositions, including pharmaceutical compositions, comprising such antibodies, and uses of such antibodies and compositions in the treatment of diseases and disorders mediated by the IL2 / IL2R signaling pathway.

Description

[Technical field]

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 170,383, filed April 2, 2021, and U.S. Provisional Patent Application No. 63 / 239,883, filed September 1, 2021, the contents of each of which are incorporated by reference in their entirety herein.

[0002] This application contains a Sequence Listing that has been submitted electronically in ASCII format, which is incorporated herein by reference in its entirety. Said ASCII copy, created on March 28, 2022, is named 60792_00054WO01_(TNO-0038-WO)_SL.txt and is 90,527 bytes in size.

[0003] The present disclosure relates to antibodies that bind to the interleukin-2 (IL-2) receptor (IL2R) and exhibit agonist activity. The present disclosure further relates to methods of making such antibodies, compositions, including pharmaceutical compositions, that contain such antibodies, and uses of such antibodies and compositions in the treatment of diseases and disorders mediated by the IL2 / IL2R signaling pathway. [Background technology]

[0004] Despite its potent immunoactivating activity and potential to induce durable tumor regression in cancer patients, the success of IL-2 as an immunotherapeutic agent has been limited by severe dose-limiting toxicities. These adverse events are primarily due to preferential uptake of IL-2 by cells expressing the high-affinity, trimeric receptor, IL-2Rαβγ, such as regulatory T (Treg) cells and endothelial cells. The present disclosure relates to antibodies (e.g., multispecific antibodies, multispecific heavy chain-only antibodies, bispecific antibodies, bispecific heavy chain antibodies) that bind to and activate signaling through the dimeric IL-2Rβγ receptor complex expressed on resting T cells and NK cells. By avoiding binding to IL-2Rα, the antibodies described herein abrogate the preferential Treg activation of native IL-2 while maintaining its potent stimulatory effect on other T cell subsets as well as NK cells. Furthermore, the presence of an Fc region in the exemplary antibodies described herein significantly extends the in vivo half-life over that of recombinant IL-2, allowing for more convenient dosing schedules in therapeutic settings. In vivo studies in both mice and cynomolgus monkeys confirmed the in vivo biological activity, broad pharmacokinetics and enhanced safety profile of the exemplary antibodies described herein. Collectively, these results support the use of such antibodies as safe and effective IL-2R agonists and the use of the IL-2 signaling pathway as a therapeutic treatment approach for multiple types of cancer.

[0005] The ability to harness the immune system against tumors is well established, with interleukin-2 (IL-2) being one of the first recombinant proteins successfully used as a treatment for cancer, about 40 years ago. Lotze et al., Journal of Immunology 135(4), 2865-75 (1985); Rosenberg, SAJ Immunol 192, 5451-58 (2014). IL-2 is a master regulator of immune cells, inducing both T cell and natural killer (NK) cell proliferation. However, IL-2 is a pleiotropic cytokine that also induces proliferation of immunosuppressive regulatory T (Treg) cells. Fontenot et al., Nat Immunol 6, 1142-51 (2005). The various functions of IL-2 are determined by the composition of the IL-2 receptor complex subunits expressed on various target cells. Boyman et al., Nat Rev Immunol 12, 180-90 (2012). The high affinity IL-2 receptor complex is composed of IL-2RA (CD25), IL-2RB (CD122) and the common gamma chain receptor IL-2RG (CD132) and is expressed constitutively on CD4+FoxP3+Treg cells and transiently on activated T cells. Waldmann, TA, Nat Rev Immunol 6, 595-601 (2006). The intermediate affinity receptor is composed only of IL-2RB and IL-2RG and is expressed on resting T cells, CD8+memory effector T cells and NK cells. Choudhry, H. et al, Biomed Res Int 2018, 1-7 (2018). Although the IL-2RA subunit is not required for downstream JAK-STAT signaling, its binding with IL-2RB and IL-2RG provides a 100-fold higher affinity for IL-2 compared to the heterodimeric receptor composed only of IL-2RB and IL-2RG. Based on differential binding and cell-specific expression of these receptors, it has been proposed that immunosuppressive Tregs act as a buffer to consume low levels of IL-2, resulting in a threshold effect on IL-2-mediated proliferation of effector lymphocytes. Feinerman, O. et al., Mol Syst Biol 6, 437 (2010).

[0006] Due to its unique signaling properties, low dose IL-2 has been used clinically to stimulate Tregs to treat autoimmunity, while high dose IL-2 has been developed and approved for the treatment of metastatic melanoma and metastatic renal cell carcinoma (Proleukin®), with durable responses observed in 7-12% of patients. McDermott, DF et al., J Clin Oncol 23, 133-141 (2004); Payne, R. et al., J Immunother Cancer 2, 13 (2014); Atkins, MB et al., J Clin Oncol 17, 2105-2105 (1999); Rosenberg, SA et al., Ann Surg 228, 307-319 (1998). However, its short half-life and narrow therapeutic window pose great challenges for the safe and effective use of IL-2 in patients. In particular, Proleukin® has severe side effects including vascular leak syndrome, hypotension and hepatotoxicity that limit its use in cancer immunotherapy. Vascular leak toxicity has been shown to be associated with the expression of high affinity IL-2R on vascular and pulmonary endothelial cells, leading to pulmonary edema. Krieg, C., et al., Proc National Acad Sci 107, 11906-11 (2010). The antitumor effect of Proleukin® is further compromised by its preferential binding to high affinity receptors on Treg cells, blunting its effectiveness as an anticancer treatment. Schwartzentruber, DJ et al., New Engl J Medicine 364, 2119-2127 (2011); Rezvani, K. et al., Blood 108, 1291-1297 (2006). For example, in melanoma patients receiving high-dose IL-2 treatment, costimulatory factor positive (ICOS+) Treg cells were found to be the most proliferating lymphocyte population in the blood after treatment with IL-2, and large numbers of ICOS+ Tregs corresponded to the worst patient outcomes. Sim, GC et al., J Clin Invest 124, 99-110 (2014).

[0007] Due to the pleiotropic nature of native IL-2 and its associated limitations as a therapeutic molecule, there have been considerable efforts to engineer IL-2 variants that reduce dose-limiting toxicity and thereby broaden the therapeutic window. Murer, P. et al., New Biotechnol 52, 42-53 (2019); Arenas-Ramirez, N. et al., Trends Immunol 36, 763-77 (2015). Mutant proteins that avoid preferential activation of high-affinity IL-2R expressing cells such as Tregs and vascular endothelial cells are one approach to achieve this goal. In an effort to generate such molecules, other approaches have involved mutating the IL-2RA binding interface in IL-2, attaching polyethylene glycol to IL-2 proteins, generating synthetic IL-2 proteins, and generating antibodies that block the IL-2RA binding domain. Silva,D.-A.et al.,Nature 565,186-191(2019);Charych,DHet al.,Clin Cancer Res 22,680-690(2016);Arenas-Ramirez,N.et al.,Sci Transl Med 8,367ra166-367ra166(2016);Levin,AMet al.,Nature 484,529-533(2012);Lopes,JEet al.,J Immunother Cancer 8,e000673(2020). As an alternative to IL-2, others have engineered IL-15 variants that bind to the IL-2RB / IL-2RG subunits. The IL-15-specific receptor subunit naturally binds IL-15 in trans from antigen-presenting cells; therefore, active IL-15 recombinant protein requires a single-chain construct expressing both IL-15 and the receptor subunit. Bernett, MJ et al. Abstract 5565:Potency-reduced IL15 / IL15Rα heterodimeric Fc-fusions display enhanced in vivo activity through increased exposure.5565-5565(2018)doi:10.1158 / 1538-7445.am2018-5565.Mutated cytokines have also been fused to antibodies or Fc domains to increase the in vivo half-life of the molecule and localize the cytokine to tumor sites. Murer, P. et al., New Biotechnol 52, 42-53 (2019); Klein, C. et al., Oncoimmunology 6:3 e1277306 (2017); Schliemann, C. et al., Blood 120, 3716-3716 (2012). While some of these engineered proteins have the desired functional activity, many exhibit high levels of in vivo immunogenicity and present challenges with in vivo stability and manufacturing. Brummelen, E. M. J. van et al., Oncotarget 9, 24737-49 (2018); Groot, A. S. Det al., Trends Immunol 28, 482-90 (2007); Schellekens, H., Nephrol Dial Transpl 18, 1257-59 (2003); Verhoef, J. J. F., et al., Drug Discov Today 19, 1945-52 (2014). Thus, generating anti-tumor agonists of the IL-2 pathway with desired biological activity, safety profile and ideal drug-like properties remains a significant challenge in the art. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Lotze et al., Journal of Immunology 135(4), 2865-75(1985) [Non-Patent Document 2] Rosenberg,SAJ Immunol 192,5451-58(2014) [Non-Patent Document 3] Fontenot et al., Nat Immunol 6, 1142-51(2005) [Non-Patent Document 4] Boyman et al., Nat Rev Immunol 12,180-90(2012)

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[0009] The molecules of the present disclosure combine the favorable drug-like properties of antibodies with the functional behavior of molecules that facilitate IL-2RB and IL-2RG binding and downstream signaling. Embodiments of the present disclosure include antibody sequences, such as fully human antibody sequences, such as fully human multispecific (e.g., bispecific) antibodies that simultaneously bind to IL-2RB and IL-2RG subunits, thus mimicking the activity of IL-2 while avoiding binding to IL-2RA. In addition to exhibiting the desired activation and proliferation of immune effector cells, the bispecific IL-2RB / G agonist antibodies described herein also avoid the preferential proliferation of inhibitory Tregs both in vitro and in vivo.

[0010] Non-limiting example embodiments (set 1) Some example embodiments / features of the present disclosure include, but are not limited to, the following.

[0011] 1. (a) a CDR1 sequence having no more than two substitutions (e.g., 0, 1 or 2) in any one of SEQ ID NOs: 1-3; and / or (b) a CDR2 sequence having two or less substitutions (e.g., 0, 1 or 2) in any one of SEQ ID NOs: 4 to 6; and / or (c) a CDR3 sequence having two or less substitutions (e.g., 0, 1, or 2) in any one of SEQ ID NOs: 7 to 10. An antibody that binds to IL2RB, comprising a heavy chain variable region comprising:

[0012] 2. (a) a CDR1 sequence comprising any one of SEQ ID NOs: 1 to 3; and / or (b) a CDR2 sequence comprising any one of SEQ ID NOs: 4 to 6; and / or (c) a CDR3 sequence comprising any one of SEQ ID NOs: 7 to 10; 2. The antibody of feature 1, comprising:

[0013] 3. (a) a CDR1 sequence comprising any one of SEQ ID NOs: 1 to 3; and (b) a CDR2 sequence comprising any one of SEQ ID NOs: 4 to 6; and (c) a CDR3 sequence comprising any one of SEQ ID NOs: 7 to 10; 3. The antibody according to feature 1 or 2, comprising:

[0014] 4. (a) the CDR1 sequence of SEQ ID NO:1, the CDR2 sequence of SEQ ID NO:4, and the CDR3 sequence of SEQ ID NO:7; or (b) the CDR1 sequence of SEQ ID NO: 1, the CDR2 sequence of SEQ ID NO: 4 and the CDR3 sequence of SEQ ID NO: 8; or (c) the CDR1 sequence of SEQ ID NO: 2, the CDR2 sequence of SEQ ID NO: 5, and the CDR3 sequence of SEQ ID NO: 9; or (d) the CDR1 sequence of SEQ ID NO: 3, the CDR2 sequence of SEQ ID NO: 6, and the CDR3 sequence of SEQ ID NO: 10 The antibody according to any one of Features 1 to 3, comprising:

[0015] 5. The antibody according to any one of Features 1 to 4, comprising a heavy chain variable region having at least 95% sequence identity to any one of SEQ ID NOs: 11 to 14.

[0016] 6. The antibody according to any one of Features 1 to 5, comprising a heavy chain variable region sequence selected from SEQ ID NOs: 11 to 14.

[0017] 7.Formula (a): GGSISSS X1 W (SEQ ID NO: 26) (wherein X1 is D or N); and Formula (b): I X2 HSGST (SEQ ID NO:27) wherein X2 is D or S; and Formula (c): X3 RG X4 WEL X5 DAFDI (SEQ ID NO: 28) (Wherein, X3 is G or A; X4 is S or Q; and X5 is S or T; and An antibody that binds to IL2RB, comprising a heavy chain variable region comprising:

[0018] 8.Formula (a): GFTFS X1 YG (SEQ ID NO: 29) (wherein X1 is S or T); and Formula (b): ISYDGSN X2 (SEQ ID NO: 30) (wherein X2 is K or R); and Formula (c): ARDLDYD X3 LTGDPVGGFDI (SEQ ID NO: 31) (wherein X3 is V or I); and An antibody that binds to IL2RB, comprising a heavy chain variable region comprising:

[0019] 9. (a) a CDR1 sequence having two or less substitutions (e.g., 0, 1 or 2) in any one of SEQ ID NOs: 15-16; and / or (b) a CDR2 sequence having two or less substitutions (e.g., 0, 1 or 2) in any one of SEQ ID NOs: 17 to 19; and / or (c) a CDR3 sequence having two or less substitutions (e.g., 0, 1, or 2) in any one of SEQ ID NOs: 20 to 21. An antibody that binds to IL2RG, comprising a heavy chain variable region comprising:

[0020] 10. (a) a CDR1 sequence comprising any one of SEQ ID NOs: 15 to 16; and / or (b) a CDR2 sequence comprising any one of SEQ ID NOs: 17 to 19; and / or (c) a CDR3 sequence comprising any one of SEQ ID NOs: 20 to 21 10. The antibody of feature 9, comprising:

[0021] 11. (a) a CDR1 sequence comprising any one of SEQ ID NOs: 15 to 16; and (b) a CDR2 sequence comprising any one of SEQ ID NOs: 17 to 19; and (c) a CDR3 sequence comprising any one of SEQ ID NOs: 20 to 21 11. The antibody of feature 9 or 10, comprising:

[0022] 12. (a) the CDR1 sequence of SEQ ID NO: 15, the CDR2 sequence of SEQ ID NO: 17, and the CDR3 sequence of SEQ ID NO: 20; or (b) the CDR1 sequence of SEQ ID NO: 15, the CDR2 sequence of SEQ ID NO: 18, and the CDR3 sequence of SEQ ID NO: 20; or (c) the CDR1 sequence of SEQ ID NO: 16, the CDR2 sequence of SEQ ID NO: 18, and the CDR3 sequence of SEQ ID NO: 20; or (d) the CDR1 sequence of SEQ ID NO: 15, the CDR2 sequence of SEQ ID NO: 19, and the CDR3 sequence of SEQ ID NO: 21 12. The antibody according to any one of Features 9 to 11, comprising:

[0023] 13. The antibody according to any one of Features 9 to 12, comprising a heavy chain variable region having at least 95% sequence identity to any one of SEQ ID NOs: 22 to 25.

[0024] 14. The antibody according to any one of Features 9 to 13, comprising a heavy chain variable region sequence selected from SEQ ID NOs: 22 to 25.

[0025] 15.Formula (a): GF X1 X2 X3 X4 YY (SEQ ID NO: 32) (Wherein, X1 is T or I; X2 is F or V; X3 is S, N or G; and X4 is D or N; Formula (b): IS X5 SG X6 X7 I (Sequence number 33) (Wherein, X5 is S or N; X6 is D, S, G or N; and X7 is T or I; (c) a CDR3 sequence comprising the sequence ARGDAVSITGDY (SEQ ID NO: 20); An antibody that binds to IL2RG, comprising a heavy chain variable region comprising:

[0026] 16. The antibody according to any one of features 1 to 15, wherein the CDR1, CDR2 and CDR3 sequences are present in a human VH framework.

[0027] 17. The antibody according to any one of features 1 to 16, which is multispecific.

[0028] 18. The antibody according to any one of features 1 to 17, which is bispecific.

[0029] 19. An antibody according to any one of features 1 to 18, which binds to IL2RB and IL2RG.

[0030] 20. CDR1 sequence of SEQ ID NO:1; CDR2 sequence of SEQ ID NO:4; and CDR3 sequence of SEQ ID NO:7 a first heavy chain variable region that binds to IL2RB, comprising: CDR1 sequence of SEQ ID NO: 15; CDR2 sequence of SEQ ID NO: 17; and CDR3 sequence of SEQ ID NO: 20 and a second heavy chain variable region that binds to IL2RG, comprising: An antibody comprising:

[0031] 21. The antibody of feature 20, wherein the first heavy chain variable region has at least 95% sequence identity to SEQ ID NO:11, and the second heavy chain variable region has at least 95% sequence identity to SEQ ID NO:22.

[0032] 22. The antibody of feature 20 or 21, wherein the first heavy chain variable region comprises SEQ ID NO:11, and the second heavy chain variable region comprises SEQ ID NO:22.

[0033] 23. The antibody according to any one of features 20 to 22, comprising a first polypeptide comprising SEQ ID NO:53 and a second polypeptide comprising SEQ ID NO:61.

[0034] 24. CDR1 sequence of SEQ ID NO:1; CDR2 sequence of SEQ ID NO:4; and CDR3 sequence of SEQ ID NO:8 a first heavy chain variable region that binds to IL2RB, comprising: CDR1 sequence of SEQ ID NO: 15; CDR2 sequence of SEQ ID NO: 18; and CDR3 sequence of SEQ ID NO: 20 and a second heavy chain variable region that binds to IL2RG, comprising: An antibody comprising:

[0035] 25. The antibody of feature 24, wherein the first heavy chain variable region has at least 95% sequence identity to SEQ ID NO: 12, and the second heavy chain variable region has at least 95% sequence identity to SEQ ID NO: 23.

[0036] 26. The antibody of feature 24 or 25, wherein the first heavy chain variable region comprises SEQ ID NO: 12, and the second heavy chain variable region comprises SEQ ID NO: 23.

[0037] 27. The antibody according to any one of features 24 to 26, comprising a first polypeptide comprising SEQ ID NO: 62 and a second polypeptide comprising SEQ ID NO: 63.

[0038] 28. CDR1 sequence of SEQ ID NO:2; CDR2 sequence of SEQ ID NO:5; and CDR3 sequence of SEQ ID NO:9 a first heavy chain variable region that binds to IL2RB; CDR1 sequence of SEQ ID NO: 15; CDR2 sequence of SEQ ID NO: 18; and CDR3 sequence of SEQ ID NO: 20 and a second heavy chain variable region that binds to IL2RG, comprising: An antibody comprising:

[0039] 29. The antibody of feature 28, wherein the first heavy chain variable region has at least 95% sequence identity to SEQ ID NO: 13, and the second heavy chain variable region has at least 95% sequence identity to SEQ ID NO: 23.

[0040] 30. The antibody of feature 28 or 29, wherein the first heavy chain variable region comprises SEQ ID NO: 13, and the second heavy chain variable region comprises SEQ ID NO: 23.

[0041] 31. The antibody according to any one of features 28 to 30, comprising a first polypeptide comprising SEQ ID NO: 64 and a second polypeptide comprising SEQ ID NO: 65.

[0042] 32. CDR1 sequence of SEQ ID NO:3; CDR2 sequence of SEQ ID NO:6; and CDR3 sequence of SEQ ID NO: 10 a first heavy chain variable region that binds to IL2RB, comprising: CDR1 sequence of SEQ ID NO: 15; CDR2 sequence of SEQ ID NO: 17; and CDR3 sequence of SEQ ID NO: 20 and a second heavy chain variable region that binds to IL2RG, comprising: An antibody comprising:

[0043] 33. The antibody of feature 32, wherein the first heavy chain variable region has at least 95% sequence identity to SEQ ID NO: 14, and the second heavy chain variable region has at least 95% sequence identity to SEQ ID NO: 22.

[0044] 34. The antibody of feature 32 or 33, wherein the first heavy chain variable region comprises SEQ ID NO: 14, and the second heavy chain variable region comprises SEQ ID NO: 22.

[0045] 35. The antibody according to any one of features 32 to 34, comprising a first polypeptide comprising SEQ ID NO: 66 and a second polypeptide comprising SEQ ID NO: 67.

[0046] 36. CDR1 sequence of SEQ ID NO:1; CDR2 sequence of SEQ ID NO:4; and CDR3 sequence of SEQ ID NO:8 a first heavy chain variable region that binds to IL2RB, comprising: CDR1 sequence of SEQ ID NO: 16; CDR2 sequence of SEQ ID NO: 18; and CDR3 sequence of SEQ ID NO: 20 and a second heavy chain variable region that binds to IL2RG, comprising: An antibody comprising:

[0047] 37. The antibody of feature 36, wherein the first heavy chain variable region has at least 95% sequence identity to SEQ ID NO: 12, and the second heavy chain variable region has at least 95% sequence identity to SEQ ID NO: 24.

[0048] 38. The antibody of feature 36 or 37, wherein the first heavy chain variable region comprises SEQ ID NO: 12, and the second heavy chain variable region comprises SEQ ID NO: 24.

[0049] 39. The antibody according to any one of features 36 to 38, comprising a first polypeptide comprising SEQ ID NO: 34 and a second polypeptide comprising SEQ ID NO: 35.

[0050] 40. CDR1 sequence of SEQ ID NO:1; CDR2 sequence of SEQ ID NO:4; and CDR3 sequence of SEQ ID NO:8 a first heavy chain variable region that binds to IL2RB, comprising: CDR1 sequence of SEQ ID NO: 15; CDR2 sequence of SEQ ID NO: 19; and CDR3 sequence of SEQ ID NO:21 and a second heavy chain variable region that binds to IL2RG, comprising: An antibody comprising:

[0051] 41. The antibody of feature 40, wherein the first heavy chain variable region has at least 95% sequence identity to SEQ ID NO: 12, and the second heavy chain variable region has at least 95% sequence identity to SEQ ID NO: 25.

[0052] 42. The antibody of feature 40 or 41, wherein the first heavy chain variable region comprises SEQ ID NO: 12, and the second heavy chain variable region comprises SEQ ID NO: 25.

[0053] 43. The antibody according to any one of features 40 to 42, comprising a first polypeptide comprising SEQ ID NO: 36 and a second polypeptide comprising SEQ ID NO: 37.

[0054] 44. The antibody of any one of features 20, 24, 28, 32, 36, or 40, wherein the CDR1, CDR2 and CDR3 sequences in the first heavy chain variable region are present in a VH human framework.

[0055] 45. The antibody of any one of features 20, 24, 28, 32, 36, 40, or 44, wherein the CDR1, CDR2 and CDR3 sequences in the second heavy chain variable region are present in a VH human framework.

[0056] 46. ​​An antibody according to any one of features 1 to 22, 24 to 26, 28 to 30, 32 to 34, 36 to 38, 40 to 42, 44, or 45, which comprises an Fc region.

[0057] 47. The antibody of feature 46, wherein the Fc region is a variant Fc region.

[0058] 48. The antibody of feature 47, wherein the variant Fc region has at least about 80% (e.g., at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%) homology to the native sequence Fc region.

[0059] 49. The antibody of feature 47 or 48, wherein the variant Fc region comprises a heterodimerization modification.

[0060] 50. Heterodimerization modifications include knob-and-hole substitutions (e.g., in a variant IgG1 Fc region, 1) Y407T in one chain and T366Y in the other chain; 2) Y407A in one chain and T366W in the other chain; 3) F405A in one chain and T394W in the other chain; 4) F405W in one chain and T394S in the other chain; 5) Y407T in one chain and T366Y in the other chain; 6) T366Y and F405A in one chain. and T394W and Y407T in the other chain; 7) T366W and F405W in one chain and T394S and Y407A in the other chain; 8) F405W and Y407A in one chain and T366W and T394S in the other chain; or 9) T366W in one polypeptide of Fc and T366S, L368A, and Y407V in the other chain.

[0061] 51. The antibody of feature 49 or 50, wherein the heterodimerization modification comprises substitutions that create new disulfide bridges (e.g., in a variant IgG1 Fc region: 1) Y349C in one Fc polypeptide chain and S354C in the other chain; 2) Y349C in one Fc polypeptide chain and E356C in the other chain; 3) Y349C in one Fc polypeptide chain and E357C in the other chain; 4) L351C in one Fc polypeptide chain and S354C in the other chain; 5) T394C in one Fc polypeptide chain and E397C in the other chain; or 6) D399C in one Fc polypeptide chain and K392C in the other chain).

[0062] 52. Heterodimerization modifications include charge pair substitutions (e.g., 1) K409E in one chain plus D399K in the other chain; 2) K409E in one chain plus D399R in the other chain; 3) K409D in one chain plus D399K in the other chain; 4) K409D in one chain plus D399R in the other chain; 5) K392E in one chain plus D392R in the other chain. 99R; 6) K392E in one chain plus D399K in the other chain; 7) K392D in one chain plus D399R in the other chain; 8) K392D in one chain plus D399K in the other chain; 9) K409D and K360D in one chain plus D399K and E356K in the other chain; 10) K409D and K370D in one chain plus D399K and E356K in the other chain. D399K and E357K; 11) K409D and K392D in one chain plus D399K, E356K, and E357K in the other chain; 12) K409D and K392D in one chain and D399K in the other chain; 13) K409D and K392D in one chain plus D399K and E356K in the other chain; 14) K409D and K392D in one chain plus etc. 52) D399K and D357K in one chain; 15) K409D and K370D in one chain plus D399K and D357K in the other chain; 16) D399K in one chain plus K409D and K360D in the other chain; or 17) K409D and K439D in one chain plus D399K and E356K in the other chain.

[0063] 53. The antibody of any one of features 47 to 52, wherein the variant Fc region is a silenced Fc region.

[0064] 54. The antibody of feature 53, wherein the silenced Fc region comprises a substitution of one or more (e.g., two or more) of Fc region residues 238, 265, 269, 270, 297, 327, and 329 according to EU numbering.

[0065] 55. The antibody of feature 53 or 54, wherein the silenced Fc region comprises substitutions that alter glycosylation.

[0066] 56. The antibody of any one of features 53 to 55, wherein the silenced Fc region comprises an effectorless mutation (e.g., N297A, N297G, a DANA mutation (D265A+N297A) or a DANG mutation (D265A+N297G) in the CH2 region) and / or a K322A and L234A / L235A mutation.

[0067] 57. The antibody according to any one of features 1 to 22, 24 to 26, 28 to 30, 32 to 34, 36 to 38, 40 to 42, or 44 to 56, comprising a heavy chain constant region sequence in the absence of a CH1 sequence.

[0068] 58. The antibody according to any one of features 1 to 22, 24 to 26, 28 to 30, 32 to 34, 36 to 38, 40 to 42, or 44 to 57, comprising a heavy chain constant region including a hinge region, a CH2 domain, and a CH3 domain.

[0069] 59. The antibody of feature 58, wherein the hinge region comprises a wild-type human IgG4 hinge region sequence (sequence number 54).

[0070] 60. The antibody of feature 58, wherein the hinge region comprises a variant human IgG4 hinge region sequence (sequence number 55) comprising a S228P mutation.

[0071] 61. The antibody according to any one of Features 58 to 60, wherein the CH2 domain comprises a wild-type human IgG4 CH2 domain sequence (SEQ ID NO: 56).

[0072] 62. The antibody according to any one of features 58 to 61, wherein the CH2 domain comprises a variant human IgG4 CH2 domain comprising a F234A mutation, a L235A mutation or both a F234A mutation and a L235A mutation.

[0073] 63. The antibody according to any one of Features 58 to 62, wherein the CH3 domain comprises a wild-type human IgG4 CH3 domain sequence (SEQ ID NO: 58).

[0074] 64. The antibody according to any one of features 58 to 62, wherein the CH3 domain comprises a variant human IgG4 CH3 domain sequence comprising a T366W mutation.

[0075] 65. The antibody according to any one of features 58 to 62, wherein the CH3 domain comprises a variant human IgG4 CH3 domain sequence comprising a T366S, a L368A mutation and a Y407V mutation.

[0076] 66. The antibody according to any one of features 1 to 65, which is a human antibody.

[0077] 67. The antibody according to any one of features 1 to 66, which is an isolated antibody.

[0078] 68. The antibody according to any one of features 1 to 67, which is an intact IgG molecule.

[0079] 69. The antibody according to any one of features 1 to 68, which is an intact IgG1 molecule.

[0080] 70. The antibody according to any one of features 1 to 68, which is an intact IgG2 molecule.

[0081] 71. The antibody according to any one of features 1 to 68, which is an intact IgG4 molecule.

[0082] 72. The antibody according to any one of features 1 to 67, which is an immunologically active portion of an intact IgG molecule.

[0083] 73. The antibody according to any one of features 1 to 67, which is an immunologically active portion of an intact IgG1 molecule.

[0084] 74. The antibody according to any one of features 1 to 67, which is an immunologically active portion of an intact IgG2 molecule.

[0085] 75. The antibody according to any one of features 1 to 67, which is an immunologically active portion of an intact IgG4 molecule.

[0086] 76. The antibody according to any one of Features 1 to 67, which is a triple-chain antibody-like molecule.

[0087] 77. The antibody according to any one of features 1 to 67, which is a heavy chain antibody.

[0088] 78. The antibody according to any one of Features 1 to 77, having a Tm of about 55°C to about 65°C.

[0089] 79. The antibody according to any one of Features 1 to 78, having a Tag of about 55°C to about 65°C.

[0090] 80.about 10 -11 M~about 10 -6 M (for example, about 10 -10 M~about 10 -6 Around M; about 10 -9 M ~ about 10 -6 Around M; about 10 -8 M~about 10 -6 Around M; about 10 -11 M~about 10 -8 Around M; about 10 -10 M~about 10 -8 Around M; about 10 -9 M~about 10 -8 Around M; about 10 -11 M~about 10 -9 Around M; about 10 -10 M ~ about 10 -9 80. The antibody according to any one of Features 1 to 79, having affinity for IL2R with a Kd of approximately 1 M.

[0091] 81.About 10 -8 M ~ approx. 2.5×10 -7 81. The antibody according to any one of Features 1 to 80, having affinity for IL2RB with a Kd of around M.

[0092] 82.About 10 -9 M ~ approx. 2.5×10 -8 82. The antibody according to any one of Features 1 to 81, having affinity for IL2RG with a Kd of around M.

[0093] 83. The antibody of any one of features 80 to 82, wherein the Kd is measured using a ForteBio Octet Qk384 instrument in kinetic mode.

[0094] 84. The antibody according to any one of features 80 to 83, wherein Kd is measured using a ForteBio Octet Qk384 instrument containing an anti-human Fc capture (AHC, 18-5005) sensor in kinetic mode.

[0095] 85. The antibody according to any one of features 80 to 82, wherein Kd is measured according to the method described in the Examples herein.

[0096] 86. An antibody according to any one of features 1 to 85, which functions as an IL2 receptor beta / gamma agonist.

[0097] 87. An antibody according to any one of features 1 to 86; and Pharmaceutically acceptable excipients 23. A pharmaceutical composition comprising:

[0098] 88. The pharmaceutical composition according to feature 87, adapted for intravenous delivery.

[0099] 89. The pharmaceutical composition according to feature 87, adapted for subcutaneous delivery.

[0100] 90. A polynucleotide encoding the antibody according to any one of features 1 to 86.

[0101] 91. A vector comprising the polynucleotide according to feature 90.

[0102] 92. A cell (e.g., a CHO cell) comprising the vector according to feature 91.

[0103] 93. A method for producing an antibody according to any one of features 1 to 86, comprising: Growing a cell (e.g., a CHO cell) according to feature 92 under conditions permissive for expression of the antibody; isolating the antibody from the cells and / or the cell culture medium in which the cells are grown; The method includes:

[0104] 94. A method for making an antibody according to any one of features 1-86, comprising immunizing a transgenic animal (e.g., a transgenic rat, a UniRat™ animal) with an IL2R and identifying an IL2R-binding heavy chain sequence.

[0105] 95. A kit for treating a disease or disorder in an individual in need thereof, comprising: An antibody according to any one of features 1 to 86 or a pharmaceutical composition according to any one of features 87 to 89; and User manual Kit including:

[0106] 96. The kit of feature 95, further comprising at least one additional reagent.

[0107] 97. The kit of feature 96, wherein the at least one additional reagent comprises a chemotherapeutic agent.

[0108] 98. A method for treating a disease or disorder, comprising administering to an individual in need thereof an effective dose of an antibody described in any one of Features 1-86 or a pharmaceutical composition described in any one of Features 87-89.

[0109] 99. The method of feature 98, wherein the antibody or pharmaceutical composition is administered in conjunction with another course of treatment.

[0110] 100. The method according to feature 98 or 99, wherein the antibody or pharmaceutical composition is administered in conjunction with a chemotherapy regimen.

[0111] 101. Use of an antibody according to any one of features 1 to 86 in the preparation of a medicament for treating a disease or disorder in an individual in need thereof.

[0112] 102. The use according to feature 101, wherein the drug is intended for administration with another course of treatment.

[0113] 103. The use according to feature 101 or 102, wherein the medicament is intended for administration in conjunction with a chemotherapy regimen.

[0114] 104. An antibody according to any one of features 1-86 or a pharmaceutical composition according to any one of features 87-89 for use in treating a disease or disorder in an individual in need thereof.

[0115] 105. The antibody for use or the pharmaceutical composition for use according to feature 104, wherein the antibody or pharmaceutical composition is intended for use in conjunction with another course of treatment.

[0116] 106. The antibody for use or the pharmaceutical composition for use according to feature 104, wherein the antibody or pharmaceutical composition is intended for use in conjunction with a chemotherapy regimen.

[0117] 107. The kit, method, use, antibody for use, or pharmaceutical composition for use according to any one of Features 95 to 106, wherein the disease or disorder is cancer.

[0118] 108. The kit, method, use, antibody for use, or pharmaceutical composition for use according to feature 107, wherein the cancer is advanced or metastatic cancer.

[0119] 109. The kit, method, use, antibody for use, or pharmaceutical composition for use according to feature 107 or 108, wherein the cancer is a solid tumor cancer.

[0120] 110. The kit, method, use, antibody for use, or pharmaceutical composition for use according to feature 109, wherein the solid tumor cancer is selected from renal cell carcinoma, melanoma, urothelial carcinoma, triple-negative breast cancer, non-small cell lung cancer (NSCLC), colorectal cancer, sarcoma, squamous cell carcinoma of the head and neck, and metastatic castration-resistant prostate cancer.

[0121] 111. The kit, method, use, antibody for use, or pharmaceutical composition for use according to feature 107 or 108, wherein the cancer is a liquid cancer.

[0122] 112. The kit, method, use, antibody for use, or pharmaceutical composition for use according to feature 111, wherein the liquid cancer is multiple myeloma or acute myeloid leukemia.

[0123] 113. A method for stimulating IL2R signaling in an immune cell, comprising contacting the immune cell with an antibody according to any one of features 1-86 or a pharmaceutical composition according to any one of features 87-89.

[0124] 114. A method for stimulating the IL2RB / IL2RG dimeric receptor complex on an immune cell, comprising contacting the immune cell with an antibody according to any one of features 1-86 or a pharmaceutical composition according to any one of features 87-89.

[0125] 115. The method according to feature 113 or 114, wherein the immune cells are selected from CD4+ T cells, CD8+ T cells and natural killer (NK) cells.

[0126] 116. Use of an antibody according to any one of features 1 to 86 in the preparation of a medicament for stimulating IL2R signalling in immune cells in an individual in need thereof.

[0127] 117. Use of an antibody according to any one of features 1 to 86 in the preparation of a medicament for the stimulation of an IL2RB / IL2RG dimeric receptor complex on an immune cell in an individual in need thereof.

[0128] 118. The use according to feature 116 or 117, wherein the immune cells are selected from CD4+ T cells, CD8+ T cells and natural killer (NK) cells.

[0129] 119. An antibody according to any one of features 1-86 or a pharmaceutical composition according to any one of features 87-89 for use in a method for stimulating IL2R signaling in immune cells.

[0130] 120. An antibody according to any one of features 1 to 86 or a pharmaceutical composition according to any one of features 87 to 89 for use in a method for stimulating the IL2RB / IL2RG dimeric receptor complex on an immune cell.

[0131] 121. The antibody for use or the pharmaceutical composition for use according to feature 119 or 120, wherein the immune cells are selected from CD4+ T cells, CD8+ T cells and natural killer (NK) cells.

[0132] Non-limiting example embodiments (set 2) Without being limited thereto, some example embodiments / sections of the present disclosure include the following.

[0133] 1. (a) a CDR1 sequence having no more than two substitutions in any one of SEQ ID NOs: 1 to 3; and / or (b) a CDR2 sequence having no more than two substitutions in any one of SEQ ID NOs: 4 to 6; and / or (c) a CDR3 sequence having two or less substitutions in any one of SEQ ID NOs: 7 to 10; A heavy chain antibody that binds to IL2RB, comprising a heavy chain variable region comprising:

[0134] 2. The heavy chain antibody of clause 1, wherein the CDR1, CDR2 and CDR3 sequences are present in a human VH framework.

[0135] 3. The heavy chain antibody of paragraph 1 or 2, further comprising a heavy chain constant region sequence in the absence of a CH1 sequence.

[0136] 4. (a) a CDR1 sequence comprising any one of SEQ ID NOs: 1 to 3; and / or (b) a CDR2 sequence comprising any one of SEQ ID NOs: 4 to 6; and / or (c) a CDR3 sequence comprising any one of SEQ ID NOs: 7 to 10; The heavy chain antibody according to any one of Items 1 to 3, comprising:

[0137] 5. (a) a CDR1 sequence comprising any one of SEQ ID NOs: 1 to 3; and (b) a CDR2 sequence comprising any one of SEQ ID NOs: 4 to 6; and (c) a CDR3 sequence comprising any one of SEQ ID NOs: 7 to 10; The heavy chain antibody according to item 4, comprising:

[0138] 6. (a) the CDR1 sequence of SEQ ID NO:1, the CDR2 sequence of SEQ ID NO:4, and the CDR3 sequence of SEQ ID NO:7; or (b) the CDR1 sequence of SEQ ID NO: 1, the CDR2 sequence of SEQ ID NO: 4 and the CDR3 sequence of SEQ ID NO: 8; or (c) the CDR1 sequence of SEQ ID NO: 2, the CDR2 sequence of SEQ ID NO: 5, and the CDR3 sequence of SEQ ID NO: 9; or (d) the CDR1 sequence of SEQ ID NO: 3, the CDR2 sequence of SEQ ID NO: 6, and the CDR3 sequence of SEQ ID NO: 10 The heavy chain antibody of item 5, comprising:

[0139] 7. The heavy chain antibody according to any one of items 1 to 5, comprising a heavy chain variable region having at least 95% sequence identity to any one of SEQ ID NOs: 11 to 14.

[0140] 8. The heavy chain antibody according to item 7, comprising a heavy chain variable region sequence selected from the group consisting of SEQ ID NOs: 11 to 14.

[0141] 9.Formula (a): GGSISSS X1 W (SEQ ID NO: 26) (wherein X1 is D or N); and Formula (b): I X2 HSGST (SEQ ID NO:27) wherein X2 is D or S; and Formula (c): X3 RG X4 WEL X5 DAFDI (SEQ ID NO: 28) (Wherein, X3 is G or A; X4 is S or Q; and X5 is S or T; and A heavy chain antibody that binds to IL2RB, comprising a heavy chain variable region comprising:

[0142] 10.Formula (a): GFTFS X1 YG (SEQ ID NO: 29) (wherein X1 is S or T); and Formula (b): ISYDGSN X2 (SEQ ID NO: 30) (wherein X2 is K or R); and Formula (c): ARDLDYD X3 LTGDPVGGFDI (SEQ ID NO: 31) (wherein X3 is V or I); and A heavy chain antibody that binds to IL2RB, comprising a heavy chain variable region comprising:

[0143] 11. The heavy chain antibody according to any one of items 9 to 10, wherein the CDR1, CDR2 and CDR3 sequences are present in a VH human framework.

[0144] 12. (a) a CDR1 sequence having no more than two substitutions in any one of SEQ ID NOs: 15-16; and / or (b) a CDR2 sequence having no more than two substitutions in any one of SEQ ID NOs: 17 to 19; and / or (c) a CDR3 sequence having two or less substitutions in any one of SEQ ID NOs: 20 to 21 A heavy chain antibody that binds to IL2RG, comprising a heavy chain variable region comprising:

[0145] 13. The heavy chain antibody of clause 12, wherein the CDR1, CDR2 and CDR3 sequences are in a human VH framework.

[0146] 14. The heavy chain antibody of clause 12 or 13, further comprising a heavy chain constant region sequence in the absence of a CH1 sequence.

[0147] 15. (a) a CDR1 sequence comprising any one of SEQ ID NOs: 15 to 16; and / or (b) a CDR2 sequence comprising any one of SEQ ID NOs: 17 to 19; and / or (c) a CDR3 sequence comprising any one of SEQ ID NOs: 20 to 21 15. The heavy chain antibody according to any one of Items 12 to 14, comprising:

[0148] 16. (a) a CDR1 sequence comprising any one of SEQ ID NOs: 15 to 16; and (b) a CDR2 sequence comprising any one of SEQ ID NOs: 17 to 19; and (c) a CDR3 sequence comprising any one of SEQ ID NOs: 20 to 21 The heavy chain antibody of claim 15, comprising:

[0149] 17. (a) the CDR1 sequence of SEQ ID NO: 15, the CDR2 sequence of SEQ ID NO: 17, and the CDR3 sequence of SEQ ID NO: 20; or (b) the CDR1 sequence of SEQ ID NO: 15, the CDR2 sequence of SEQ ID NO: 18, and the CDR3 sequence of SEQ ID NO: 20; or (c) the CDR1 sequence of SEQ ID NO: 16, the CDR2 sequence of SEQ ID NO: 18, and the CDR3 sequence of SEQ ID NO: 20; or (d) the CDR1 sequence of SEQ ID NO: 15, the CDR2 sequence of SEQ ID NO: 19, and the CDR3 sequence of SEQ ID NO: 21 The heavy chain antibody of claim 16, comprising:

[0150] 18. The heavy chain antibody according to any one of items 12 to 16, comprising a heavy chain variable region having at least 95% sequence identity to any one of SEQ ID NOs: 22 to 25.

[0151] 19. The heavy chain antibody according to item 18, comprising a heavy chain variable region sequence selected from the group consisting of SEQ ID NOs: 22 to 25.

[0152] 20.Formula (a): GF X1 X2 X3 X4 YY (SEQ ID NO: 32) (Wherein, X1 is T or I; X2 is F or V; X3 is S, N or G; and X4 is D or N; Formula (b): IS X5 SG X6 X7 I (Sequence number 33) (Wherein, X5 is S or N; X6 is D, S, G or N; and X7 is T or I; (c) a CDR3 sequence comprising the sequence ARGDAVSITGDY (SEQ ID NO: 20); A heavy chain antibody that binds to IL2RG, comprising a heavy chain variable region comprising:

[0153] 21. The heavy chain antibody of clause 20, wherein the CDR1, CDR2 and CDR3 sequences are present in a VH human framework.

[0154] 22. The heavy chain antibody according to any one of items 1 to 21, which is polyspecific.

[0155] 23. The heavy chain antibody of clause 22, which is bispecific.

[0156] 24. The heavy chain antibody of item 22 or 23, which binds to IL2RB and IL2RG.

[0157] 25. The heavy chain antibody according to any one of items 1 to 24, further comprising a heavy chain constant region comprising a hinge region, a CH2 domain, and a CH3 domain.

[0158] 26. The heavy chain antibody of clause 25, wherein the hinge region comprises a wild-type human IgG4 hinge region sequence (SEQ ID NO:54).

[0159] 27. The heavy chain antibody of clause 25, wherein the hinge region comprises a variant human IgG4 hinge region sequence (SEQ ID NO:55) containing a S228P mutation.

[0160] 28. The heavy chain antibody according to any one of items 25 to 27, wherein the CH2 domain comprises a wild-type human IgG4 CH2 domain sequence (SEQ ID NO: 56).

[0161] 29. The heavy chain antibody according to any one of clauses 25 to 27, wherein the CH2 domain comprises a variant human IgG4 CH2 domain comprising a F234A mutation, a L235A mutation or both a F234A mutation and a L235A mutation.

[0162] 30. The heavy chain antibody according to any one of items 25 to 29, wherein the CH3 domain comprises a wild-type human IgG4 CH3 domain sequence (SEQ ID NO: 58).

[0163] 31. The heavy chain antibody of any one of clauses 25 to 29, wherein the CH3 domain comprises a variant human IgG4 CH3 domain sequence comprising a T366W mutation.

[0164] 32. The heavy chain antibody of any one of clauses 25 to 29, wherein the CH3 domain comprises a variant human IgG4 CH3 domain sequence comprising a T366S, an L368A and a Y407V mutation.

[0165] 33. The heavy chain antibody according to any one of items 22 to 32, which functions as an IL2 receptor beta / gamma agonist.

[0166] 34. CDR1 sequence of SEQ ID NO:1; CDR2 sequence of SEQ ID NO:4; and CDR3 sequence of SEQ ID NO:7 a first heavy chain variable region that binds to IL2RB; CDR1 sequence of SEQ ID NO: 15; CDR2 sequence of SEQ ID NO: 17; and CDR3 sequence of SEQ ID NO: 20 a second heavy chain variable region that binds to IL2RG, 4. A bispecific agonist anti-IL2R heavy chain antibody comprising:

[0167] 35. The antibody of clause 34, wherein the first heavy chain variable region has at least 95% sequence identity to SEQ ID NO:11, and the second heavy chain variable region has at least 95% sequence identity to SEQ ID NO:22.

[0168] 36. The antibody of clause 35, wherein the first heavy chain variable region comprises SEQ ID NO:11 and the second heavy chain variable region comprises SEQ ID NO:22.

[0169] 37. The antibody of clause 36, comprising a first polypeptide comprising SEQ ID NO:53 and a second polypeptide comprising SEQ ID NO:61.

[0170] 38. CDR1 sequence of SEQ ID NO:1; CDR2 sequence of SEQ ID NO:4; and CDR3 sequence of SEQ ID NO:8 a first heavy chain variable region that binds to IL2RB, comprising: CDR1 sequence of SEQ ID NO: 15; CDR2 sequence of SEQ ID NO: 18; and CDR3 sequence of SEQ ID NO: 20 and a second heavy chain variable region that binds to IL2RG, comprising: 4. A bispecific agonist anti-IL2R heavy chain antibody comprising:

[0171] 39. The antibody of clause 38, wherein the first heavy chain variable region has at least 95% sequence identity to SEQ ID NO: 12, and the second heavy chain variable region has at least 95% sequence identity to SEQ ID NO: 23.

[0172] 40. The antibody of clause 39, wherein the first heavy chain variable region comprises SEQ ID NO: 12 and the second heavy chain variable region comprises SEQ ID NO: 23.

[0173] 41. The antibody of clause 40, comprising a first polypeptide comprising SEQ ID NO:62 and a second polypeptide comprising SEQ ID NO:63.

[0174] 42. CDR1 sequence of SEQ ID NO:2; CDR2 sequence of SEQ ID NO:5; and CDR3 sequence of SEQ ID NO:9 a first heavy chain variable region that binds to IL2RB, comprising: CDR1 sequence of SEQ ID NO: 15; CDR2 sequence of SEQ ID NO: 18; and CDR3 sequence of SEQ ID NO: 20 and a second heavy chain variable region that binds to IL2RG, comprising: 4. A bispecific agonist anti-IL2R heavy chain antibody comprising:

[0175] 43. The antibody of clause 42, wherein the first heavy chain variable region has at least 95% sequence identity to SEQ ID NO: 13, and the second heavy chain variable region has at least 95% sequence identity to SEQ ID NO: 23.

[0176] 44. The antibody of clause 43, wherein the first heavy chain variable region comprises SEQ ID NO: 13 and the second heavy chain variable region comprises SEQ ID NO: 23.

[0177] 45. The antibody of paragraph 44, comprising a first polypeptide comprising SEQ ID NO:64 and a second polypeptide comprising SEQ ID NO:65.

[0178] 46. ​​CDR1 sequence of SEQ ID NO:3; CDR2 sequence of SEQ ID NO:6; and CDR3 sequence of SEQ ID NO: 10 a first heavy chain variable region that binds to IL2RB, comprising: CDR1 sequence of SEQ ID NO: 15; CDR2 sequence of SEQ ID NO: 17; and CDR3 sequence of SEQ ID NO: 20 and a second heavy chain variable region that binds to IL2RG, comprising: 4. A bispecific agonist anti-IL2R heavy chain antibody comprising:

[0179] 47. The antibody of clause 46, wherein the first heavy chain variable region has at least 95% sequence identity to SEQ ID NO: 14, and the second heavy chain variable region has at least 95% sequence identity to SEQ ID NO: 22.

[0180] 48. The antibody of clause 47, wherein the first heavy chain variable region comprises SEQ ID NO: 14 and the second heavy chain variable region comprises SEQ ID NO: 22.

[0181] 49. The antibody of clause 48, comprising a first polypeptide comprising SEQ ID NO:66 and a second polypeptide comprising SEQ ID NO:67.

[0182] 50. CDR1 sequence of SEQ ID NO:1; CDR2 sequence of SEQ ID NO:4; and CDR3 sequence of SEQ ID NO:8 a first heavy chain variable region that binds to IL2RB, comprising: CDR1 sequence of SEQ ID NO: 16; CDR2 sequence of SEQ ID NO: 18; and CDR3 sequence of SEQ ID NO: 20 and a second heavy chain variable region that binds to IL2RG, comprising: 4. A bispecific agonist anti-IL2R heavy chain antibody comprising:

[0183] 51. The antibody of clause 50, wherein the first heavy chain variable region has at least 95% sequence identity to SEQ ID NO:12, and the second heavy chain variable region has at least 95% sequence identity to SEQ ID NO:24.

[0184] 52. The antibody of clause 51, wherein the first heavy chain variable region comprises SEQ ID NO: 12 and the second heavy chain variable region comprises SEQ ID NO: 24.

[0185] 53. The antibody of clause 52, comprising a first polypeptide comprising SEQ ID NO:34 and a second polypeptide comprising SEQ ID NO:35.

[0186] 54. CDR1 sequence of SEQ ID NO:1; CDR2 sequence of SEQ ID NO:4; and CDR3 sequence of SEQ ID NO:8 a first heavy chain variable region that binds to IL2RB, comprising: CDR1 sequence of SEQ ID NO: 15; CDR2 sequence of SEQ ID NO: 19; and CDR3 sequence of SEQ ID NO:21 and a second heavy chain variable region that binds to IL2RG, comprising: 4. A bispecific agonist anti-IL2R heavy chain antibody comprising:

[0187] 55. The antibody of clause 54, wherein the first heavy chain variable region has at least 95% sequence identity to SEQ ID NO:12, and the second heavy chain variable region has at least 95% sequence identity to SEQ ID NO:25.

[0188] 56. The antibody of clause 55, wherein the first heavy chain variable region comprises SEQ ID NO: 12 and the second heavy chain variable region comprises SEQ ID NO: 25.

[0189] 57. The antibody of paragraph 56, comprising a first polypeptide comprising SEQ ID NO:36 and a second polypeptide comprising SEQ ID NO:37.

[0190] 58. A pharmaceutical composition comprising the antibody according to any one of items 1 to 57.

[0191] 59. A polynucleotide encoding the antibody according to any one of items 1 to 57.

[0192] 60. A vector comprising the polynucleotide according to paragraph 59.

[0193] 61. A cell comprising the vector according to paragraph 60.

[0194] 62. A method for producing an antibody according to any one of claims 1 to 57, comprising growing a cell according to claim 61 under conditions permissive for expression of the antibody, and isolating the antibody from the cell and / or the cell culture medium in which the cell is grown.

[0195] 63. A method for producing an antibody according to any one of paragraphs 1 to 57, comprising immunizing a UniRat animal with IL2R and identifying an IL2R-binding heavy chain sequence.

[0196] 64. A kit for treating a disease or disorder in an individual in need thereof, comprising the antibody according to any one of items 1 to 57 or the pharmaceutical composition according to item 58, and instructions for use.

[0197] 65. The kit of paragraph 64, further comprising at least one additional reagent.

[0198] 66. The kit of paragraph 65, wherein the at least one additional reagent comprises a chemotherapeutic agent.

[0199] 67. A method of treatment comprising administering to an individual in need thereof an effective dose of the antibody according to any one of paragraphs 1 to 57 or the pharmaceutical composition according to paragraph 58.

[0200] 68. Use of an antibody according to any one of paragraphs 1 to 57 in the preparation of a medicament for the treatment of a disease or disorder in an individual in need thereof.

[0201] 69. The antibody according to any one of clauses 1 to 57 or the pharmaceutical composition according to clause 58 for use in therapy in an individual in need thereof.

[0202] 70. A method for treating cancer, comprising administering the antibody according to any one of items 1 to 57 or the pharmaceutical composition according to item 58 to a subject having said cancer.

[0203] 71. The method or use according to any one of items 67 to 70, wherein the cancer is advanced or metastatic cancer.

[0204] 72. The method or use according to any one of items 67 to 71, wherein the cancer is a solid tumor cancer.

[0205] 73. The method or use according to paragraph 72, wherein the solid tumor cancer is selected from the group consisting of renal cell carcinoma, melanoma, urothelial carcinoma, triple-negative breast cancer, non-small cell lung cancer (NSCLC), colorectal cancer, sarcoma, squamous cell carcinoma of the head and neck, and metastatic castration-resistant prostate cancer.

[0206] 74. A method for stimulating IL2R signaling in immune cells, the method comprising contacting immune cells with the antibody according to any one of items 1 to 57 or the pharmaceutical composition according to item 58.

[0207] 75. A method for stimulating the IL2RB / IL2RG dimeric receptor complex on an immune cell, the method comprising contacting the immune cell with the antibody according to any one of items 1 to 57 or the pharmaceutical composition according to item 58.

[0208] 76. The method of paragraph 74 or 75, wherein the immune cells are selected from the group consisting of CD4+ T cells, CD8+ T cells and natural killer (NK) cells.

[0209] IL-2R, also known as the interleukin-2 receptor, is a heterodimeric protein expressed on the surface of various immune cells that serves as the cognate ligand for interleukin-2 (IL-2). The IL-2R complex is composed of various combinations of IL-2Rα (ILR2A), IL-2Rβ (IL2RB) and IL-2Rγ (IL2RG) protein chains. IL-2RA is also known as CD25, and the human IL2RA sequence (UniProtKB No. P01589) is provided herein as SEQ ID NO: 38. IL-2RB is also known as CD122, and the human IL2RB sequence (UniProtKB No. P14784) is provided herein as SEQ ID NO: 39. IL-2RG is also known as CD132, and the human IL2RG sequence (UniProtKB No. P31785) is provided herein as SEQ ID NO: 40. The human IL-2 sequence (UniProtKB No. P60568) is provided herein as SEQ ID NO:41.

[0210] An embodiment of the present disclosure relates to an antibody that binds to IL2RB, comprising a heavy chain variable region including: (a) a CDR1 sequence having two or less (e.g., 0, 1 or 2) substitutions in any one of SEQ ID NOs: 1 to 3; and / or (b) a CDR2 sequence having two or less (e.g., 0, 1 or 2) substitutions in any one of SEQ ID NOs: 4 to 6; and / or (c) a CDR3 sequence having two or less (e.g., 0, 1 or 2) substitutions in any one of SEQ ID NOs: 7 to 10.

[0211] In some embodiments, the antibody comprises: (a) a CDR1 sequence comprising any one of SEQ ID NOs: 1-3; and / or (b) a CDR2 sequence comprising any one of SEQ ID NOs: 4-6; and / or (c) a CDR3 sequence comprising any one of SEQ ID NOs: 7-10.

[0212] In some embodiments, the antibody comprises: (a) a CDR1 sequence comprising any one of SEQ ID NOs: 1-3; and (b) a CDR2 sequence comprising any one of SEQ ID NOs: 4-6; and (c) a CDR3 sequence comprising any one of SEQ ID NOs: 7-10.

[0213] In some embodiments, the antibody comprises: (a) the CDR1 sequence of SEQ ID NO: 1, the CDR2 sequence of SEQ ID NO: 4 and the CDR3 sequence of SEQ ID NO: 7; or (b) the CDR1 sequence of SEQ ID NO: 1, the CDR2 sequence of SEQ ID NO: 4 and the CDR3 sequence of SEQ ID NO: 8; or (c) the CDR1 sequence of SEQ ID NO: 2, the CDR2 sequence of SEQ ID NO: 5, and the CDR3 sequence of SEQ ID NO: 9; or (d) the CDR1 sequence of SEQ ID NO: 3, the CDR2 sequence of SEQ ID NO: 6, and the CDR3 sequence of SEQ ID NO: 10 Includes.

[0214] In some embodiments, the CDR1, CDR2 and CDR3 sequences are present in a human VH framework.

[0215] In some embodiments, the antibody comprises a heavy chain variable region comprising a sequence having six or fewer (e.g., five or fewer, four or fewer, three or fewer, two or fewer; 6, 5, 4, 3, 2, 1, 0) substitutions in any one of SEQ ID NOs: 11-14. In some embodiments, the antibody comprises a heavy chain variable region having at least 95% (e.g., at least 96%, at least 97%, at least 98%, at least 99%) sequence identity to any one of SEQ ID NOs: 11-14. In some embodiments, the antibody comprises a heavy chain variable region sequence selected from the group consisting of SEQ ID NOs: 11-14.

[0216] In some embodiments, the antibody is an isolated antibody. In some embodiments, the antibody is a human antibody. In some embodiments, the antibody is an isolated human antibody.

[0217] In some embodiments, the antibody is an intact IgG molecule. In some embodiments, the antibody is an intact IgG1 molecule. In some embodiments, the antibody is an intact IgG2 molecule. In some embodiments, the antibody is an intact IgG4 molecule. In some embodiments, the antibody is an immunologically active portion of an intact IgG molecule. In some embodiments, the antibody is an immunologically active portion of an intact IgG1 molecule. In some embodiments, the antibody is an immunologically active portion of an intact IgG2 molecule. In some embodiments, the antibody is an immunologically active portion of an intact IgG4 molecule. In some embodiments, the antibody is a triple chain antibody-like molecule. In some embodiments, the antibody is a heavy chain antibody.

[0218] In some embodiments, the antibody comprises an Fc region. In some embodiments, the antibody comprises a variant Fc region. In some embodiments, the variant Fc region has at least about 80% homology (e.g., at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%) with a native sequence Fc region.

[0219] In some embodiments, the variant Fc region comprises a heterodimerization modification. In some embodiments, the heterodimerization modification is a knob-and-hole substitution (e.g., in a variant IgG1 Fc region, 1) Y407T in one chain and T366Y in the other chain; 2) Y407A in one chain and T366W in the other chain; 3) F405A in one chain and T394W in the other chain; 4) F405W in one chain and T394S in the other chain; 5) Y407T in one chain and T366Y in the other chain; 6) T366Y in one chain and T394S in the other chain). and F405A in one chain and T394W and Y407T in the other chain; 7) T366W and F405W in one chain and T394S and Y407A in the other chain; 8) F405W and Y407A in one chain and T366W and T394S in the other chain; or 9) T366W in one polypeptide of Fc and T366S, L368A, and Y407V in the other chain. In some embodiments, heterodimerization modifications include substitutions that create new disulfide bridges (e.g., in a variant IgG1 Fc region, 1) Y349C in one Fc polypeptide chain and S354C in the other chain; 2) Y349C in one Fc polypeptide chain and E356C in the other chain; 3) Y349C in one Fc polypeptide chain and E357C in the other chain; 4) L351C in one Fc polypeptide chain and S354C in the other chain; 5) T394C in one Fc polypeptide chain and E397C in the other chain; or 6) D399C in one Fc polypeptide chain and K392C in the other chain).In some embodiments, the heterodimerization modification is a charge pair substitution (e.g., 1) K409E in one chain plus D399K in the other chain; 2) K409E in one chain plus D399R in the other chain; 3) K409D in one chain plus D399K in the other chain; 4) K409D in one chain plus D399R in the other chain; 5) K392E in one chain plus D399R in the other chain; 6) K392E in one chain plus D399K in the other chain; 7) K392D in one chain plus D399R in the other chain; 8) K392D in one chain plus D399K in the other chain; 9) K409D and K360D in one chain plus D399K and E356K in the other chain; 10) K409D and K 370D plus D399K and E357K in the other chain; 11) K409D and K392D in one chain plus D399K, E356K, and E357K in the other chain; 12) K409D and K392D in one chain and D399K in the other chain; 13) K409D and K392D in one chain plus D399K and E356K in the other chain; 14) K409D and K392D in one chain plus D399K and E356K in the other chain; 14) K409D and K392D in one chain plus D399K and D357K in the other chain; 15) K409D and K370D in one chain plus D399K and D357K in the other chain; 16) D399K in one chain plus K409D and K360D in the other chain; or 17) K409D and K439D in one chain plus D399K and E356K in the other chain.

[0220] In some embodiments, the Fc region is a silenced Fc region. In some embodiments, the silenced Fc region comprises one or more (e.g., two or more) substitutions of Fc region residues 238, 265, 269, 270, 297, 327, and 329 according to EU numbering. In some embodiments, the silenced Fc region comprises a substitution that alters glycosylation. In some embodiments, the silenced Fc region comprises an effectorless mutation (e.g., N297A, N297G, DANA mutation (D265A+N297A) or DANG mutation (D265A+N297G) in the CH2 region). In some embodiments, the silenced Fc region comprises K322A and L234A / L235A mutations.

[0221] In some embodiments, the antibody further comprises a heavy chain constant region sequence in the absence of a CH1 sequence. In some embodiments, the antibody comprises a heavy chain constant region comprising a hinge region, a CH2 domain, and a CH3 domain. In some embodiments, the hinge region comprises a wild-type human IgG4 hinge region sequence (SEQ ID NO: 54). In some embodiments, the hinge region comprises a variant human IgG4 hinge region sequence (SEQ ID NO: 55) comprising a S228P mutation. In some embodiments, the CH2 domain comprises a wild-type human IgG4 CH2 domain sequence (SEQ ID NO: 56). In some embodiments, the CH2 domain comprises a variant human IgG4 CH2 domain comprising a F234A mutation, a L235A mutation, or both a F234A mutation and a L235A mutation. In some embodiments, the CH3 domain comprises a wild-type human IgG4 CH3 domain sequence (SEQ ID NO: 58). In some embodiments, the CH3 domain comprises a variant human IgG4 CH3 domain sequence comprising a T366W mutation. In some embodiments, the CH3 domain comprises a variant human IgG4 CH3 domain sequence comprising a T366S, an L368A mutation, and a Y407V mutation.

[0222] In some embodiments, the antibody has a Tm of about 55° C. to about 65° C. In some embodiments, the antibody has a Tag of about 55° C. to about 65° C. In some embodiments, the antibody has a Tm of about 55° C. to about 65° C. and a Tag of about 55° C. to about 65° C.

[0223] In some embodiments, the antibody is multispecific. In some embodiments, the antibody is bispecific. In some embodiments, the antibody binds to IL2RB and IL2RG. In some embodiments, the antibody functions as an IL2 receptor beta / gamma agonist.

[0224] In some embodiments, the antibody is -11 M ~ about 10 -6 M (for example, about 10 -10 M ~ about 10 -6 Around M; about 10 -9 M ~ about 10 -6 Around M; about 10 -8 M ~ about 10 -6 Around M; about 10 -11 M ~ about 10 -8 Around M; about 10 -10 M ~ about 10 -8 Around M; about 10 -9 M ~ about 10 -8 Around M; about 10 -11 M ~ about 10 -9 Around M; about 10 -10 M ~ about 10 -9 It has affinity for IL2R with a Kd of approximately 1 M.

[0225] In some embodiments, the antibody is -8 M ~ approx. 2.5×10 -7 It has affinity for IL2RB with a Kd of around M.

[0226] In some embodiments, the antibody is -9 M ~ approx. 2.5×10 -8 It has affinity for IL2RG with a Kd of around M.

[0227] In some embodiments, the antibody is-8 M ~ approx. 2.5×10 -7 The affinity for IL2RB is approximately 10 -9 M ~ approx. 2.5×10 -8 It has affinity for IL2RG with a Kd of around M.

[0228] In some embodiments, Kd is measured using a ForteBio Octet Qk384 instrument in kinetic mode. In some embodiments, Kd is measured using a ForteBio Octet Qk384 instrument with an anti-human Fc capture (AHC, 18-5005) sensor in kinetic mode. In some embodiments, Kd is measured according to the method described in the Examples herein.

[0229] An aspect of the present disclosure is (a) Formula: GGSISSS X1 W (SEQ ID NO: 26) (wherein X1 is D or N); and Formula (b): I X2 HSGST (SEQ ID NO:27) wherein X2 is D or S; and Formula (c): X3 RG X4 WEL X5 DAFDI (SEQ ID NO: 28) wherein X3 is G or A; X4 is S or Q; and X5 is S or T; The present invention includes an antibody that binds to IL2RB, comprising a heavy chain variable region comprising:

[0230] In some embodiments, the CDR1, CDR2 and CDR3 sequences are present in a VH human framework.

[0231] In some embodiments, the antibody is an isolated antibody. In some embodiments, the antibody is a human antibody. In some embodiments, the antibody is an isolated human antibody.

[0232] In some embodiments, the antibody is an intact IgG molecule. In some embodiments, the antibody is an intact IgG1 molecule. In some embodiments, the antibody is an intact IgG2 molecule. In some embodiments, the antibody is an intact IgG4 molecule. In some embodiments, the antibody is an immunologically active portion of an intact IgG molecule. In some embodiments, the antibody is an immunologically active portion of an intact IgG1 molecule. In some embodiments, the antibody is an immunologically active portion of an intact IgG2 molecule. In some embodiments, the antibody is an immunologically active portion of an intact IgG4 molecule. In some embodiments, the antibody is a triple chain antibody-like molecule. In some embodiments, the antibody is a heavy chain antibody.

[0233] In some embodiments, the antibody comprises an Fc region. In some embodiments, the antibody comprises a variant Fc region. In some embodiments, the variant Fc region has at least about 80% (e.g., at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%) homology to a native sequence Fc region.

[0234] In some embodiments, the variant Fc region comprises a heterodimerization modification. In some embodiments, the heterodimerization modification is a knob-and-hole substitution (e.g., in a variant IgG1 Fc region, 1) Y407T in one chain and T366Y in the other chain; 2) Y407A in one chain and T366W in the other chain; 3) F405A in one chain and T394W in the other chain; 4) F405W in one chain and T394S in the other chain; 5) Y407T in one chain and T366Y in the other chain; 6) T366Y in one chain and T394S in the other chain). and F405A in one chain and T394W and Y407T in the other chain; 7) T366W and F405W in one chain and T394S and Y407A in the other chain; 8) F405W and Y407A in one chain and T366W and T394S in the other chain; or 9) T366W in one polypeptide of Fc and T366S, L368A, and Y407V in the other chain. In some embodiments, heterodimerization modifications include substitutions that create new disulfide bridges (e.g., in a variant IgG1 Fc region, 1) Y349C in one Fc polypeptide chain and S354C in the other chain; 2) Y349C in one Fc polypeptide chain and E356C in the other chain; 3) Y349C in one Fc polypeptide chain and E357C in the other chain; 4) L351C in one Fc polypeptide chain and S354C in the other chain; 5) T394C in one Fc polypeptide chain and E397C in the other chain; or 6) D399C in one Fc polypeptide chain and K392C in the other chain).In some embodiments, the heterodimerization modification is a charge pair substitution (e.g., 1) K409E in one chain plus D399K in the other chain; 2) K409E in one chain plus D399R in the other chain; 3) K409D in one chain plus D399K in the other chain; 4) K409D in one chain plus D399R in the other chain; 5) K392E in one chain plus D399R in the other chain; 6) K392E in one chain plus D399K in the other chain; 7) K392D in one chain plus D399R in the other chain; 8) K392D in one chain plus D399K in the other chain; 9) K409D and K360D in one chain plus D399K and E356K in the other chain; 10) K409D and K 370D plus D399K and E357K in the other chain; 11) K409D and K392D in one chain plus D399K, E356K, and E357K in the other chain; 12) K409D and K392D in one chain and D399K in the other chain; 13) K409D and K392D in one chain plus D399K and E356K in the other chain; 14) K409D and K392D in one chain plus D399K and E356K in the other chain; 14) K409D and K392D in one chain plus D399K and D357K in the other chain; 15) K409D and K370D in one chain plus D399K and D357K in the other chain; 16) D399K in one chain plus K409D and K360D in the other chain; or 17) K409D and K439D in one chain plus D399K and E356K in the other chain.

[0235] In some embodiments, the Fc region is a silenced Fc region. In some embodiments, the silenced Fc region comprises one or more (e.g., two or more) substitutions of Fc region residues 238, 265, 269, 270, 297, 327, and 329 according to EU numbering. In some embodiments, the silenced Fc region comprises a substitution that alters glycosylation. In some embodiments, the silenced Fc region comprises an effectorless mutation (e.g., N297A, N297G, DANA mutation (D265A+N297A) or DANG mutation (D265A+N297G) in the CH2 region). In some embodiments, the silenced Fc region comprises K322A and L234A / L235A mutations.

[0236] In some embodiments, the antibody further comprises a heavy chain constant region sequence in the absence of a CH1 sequence. In some embodiments, the antibody comprises a heavy chain constant region comprising a hinge region, a CH2 domain, and a CH3 domain. In some embodiments, the hinge region comprises a wild-type human IgG4 hinge region sequence (SEQ ID NO: 54). In some embodiments, the hinge region comprises a variant human IgG4 hinge region sequence (SEQ ID NO: 55) comprising a S228P mutation. In some embodiments, the CH2 domain comprises a wild-type human IgG4 CH2 domain sequence (SEQ ID NO: 56). In some embodiments, the CH2 domain comprises a variant human IgG4 CH2 domain comprising a F234A mutation, a L235A mutation, or both a F234A mutation and a L235A mutation. In some embodiments, the CH3 domain comprises a wild-type human IgG4 CH3 domain sequence (SEQ ID NO: 58). In some embodiments, the CH3 domain comprises a variant human IgG4 CH3 domain sequence comprising a T366W mutation. In some embodiments, the CH3 domain comprises a variant human IgG4 CH3 domain sequence comprising a T366S, an L368A mutation, and a Y407V mutation.

[0237] In some embodiments, the antibody has a Tm of about 55° C. to about 65° C. In some embodiments, the antibody has a Tag of about 55° C. to about 65° C. In some embodiments, the antibody has a Tm of about 55° C. to about 65° C. and a Tag of about 55° C. to about 65° C.

[0238] In some embodiments, the antibody is multispecific. In some embodiments, the antibody is bispecific. In some embodiments, the antibody binds to IL2RB and IL2RG. In some embodiments, the antibody functions as an IL2 receptor beta / gamma agonist.

[0239] In some embodiments, the antibody is -11 M ~ about 10 -6 M (for example, about 10 -10 M ~ about 10 -6 Around M; about 10 -9 M ~ about 10 -6 Around M; about 10 -8 M ~ about 10 -6 Around M; about 10 -11 M ~ about 10 -8 Around M; about 10 -10 M ~ about 10 -8 Around M; about 10 -9 M ~ about 10 -8 Around M; about 10 -11 M ~ about 10 -9 Around M; about 10 -10 M ~ about 10 -9 It has affinity for IL2R with a Kd of approximately 1 M.

[0240] In some embodiments, the antibody is -8 M ~ approx. 2.5×10 -7 It has affinity for IL2RB with a Kd of around M.

[0241] In some embodiments, the antibody is -9 M ~ approx. 2.5×10 -8 It has affinity for IL2RG with a Kd of around M.

[0242] In some embodiments, the antibody is-8 M ~ approx. 2.5×10 -7 The affinity for IL2RB is approximately 10 -9 M ~ approx. 2.5×10 -8 It has affinity for IL2RG with a Kd of around M.

[0243] In some embodiments, Kd is measured using a ForteBio Octet Qk384 instrument in kinetic mode. In some embodiments, Kd is measured using a ForteBio Octet Qk384 instrument with an anti-human Fc capture (AHC, 18-5005) sensor in kinetic mode. In some embodiments, Kd is measured according to the method described in the Examples herein.

[0244] An aspect of the present disclosure is (a) Formula: GFTFS X1 YG (SEQ ID NO: 29) (wherein X1 is S or T); and Formula (b): ISYDGSN X2 (SEQ ID NO: 30) (wherein X2 is K or R); and Formula (c): ARDLDYD X3 LTGDPVGGFDI (SEQ ID NO: 31) (wherein X3 is V or I); and The present invention includes an antibody that binds to IL2RB, comprising a heavy chain variable region comprising:

[0245] In some embodiments, the CDR1, CDR2 and CDR3 sequences are present in a VH human framework.

[0246] In some embodiments, the antibody is an isolated antibody. In some embodiments, the antibody is a human antibody. In some embodiments, the antibody is an isolated human antibody.

[0247] In some embodiments, the antibody is an intact IgG molecule. In some embodiments, the antibody is an intact IgG1 molecule. In some embodiments, the antibody is an intact IgG2 molecule. In some embodiments, the antibody is an intact IgG4 molecule. In some embodiments, the antibody is an immunologically active portion of an intact IgG molecule. In some embodiments, the antibody is an immunologically active portion of an intact IgG1 molecule. In some embodiments, the antibody is an immunologically active portion of an intact IgG2 molecule. In some embodiments, the antibody is an immunologically active portion of an intact IgG4 molecule. In some embodiments, the antibody is a triple chain antibody-like molecule. In some embodiments, the antibody is a heavy chain antibody.

[0248] In some embodiments, the antibody comprises an Fc region. In some embodiments, the antibody comprises a variant Fc region. In some embodiments, the variant Fc region has at least about 80% (e.g., at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%) homology to a native sequence Fc region.

[0249] In some embodiments, the variant Fc region comprises a heterodimerization modification. In some embodiments, the heterodimerization modification is a knob-and-hole substitution (e.g., in a variant IgG1 Fc region, 1) Y407T in one chain and T366Y in the other chain; 2) Y407A in one chain and T366W in the other chain; 3) F405A in one chain and T394W in the other chain; 4) F405W in one chain and T394S in the other chain; 5) Y407T in one chain and T366Y in the other chain; 6) T366Y in one chain and T394S in the other chain). and F405A in one chain and T394W and Y407T in the other chain; 7) T366W and F405W in one chain and T394S and Y407A in the other chain; 8) F405W and Y407A in one chain and T366W and T394S in the other chain; or 9) T366W in one polypeptide of Fc and T366S, L368A, and Y407V in the other chain. In some embodiments, heterodimerization modifications include substitutions that create new disulfide bridges (e.g., in a variant IgG1 Fc region, 1) Y349C in one Fc polypeptide chain and S354C in the other chain; 2) Y349C in one Fc polypeptide chain and E356C in the other chain; 3) Y349C in one Fc polypeptide chain and E357C in the other chain; 4) L351C in one Fc polypeptide chain and S354C in the other chain; 5) T394C in one Fc polypeptide chain and E397C in the other chain; or 6) D399C in one Fc polypeptide chain and K392C in the other chain).In some embodiments, the heterodimerization modification is a charge pair substitution (e.g., 1) K409E in one chain plus D399K in the other chain; 2) K409E in one chain plus D399R in the other chain; 3) K409D in one chain plus D399K in the other chain; 4) K409D in one chain plus D399R in the other chain; 5) K392E in one chain plus D399R in the other chain; 6) K392E in one chain plus D399K in the other chain; 7) K392D in one chain plus D399R in the other chain; 8) K392D in one chain plus D399K in the other chain; 9) K409D and K360D in one chain plus D399K and E356K in the other chain; 10) K409D and K 370D plus D399K and E357K in the other chain; 11) K409D and K392D in one chain plus D399K, E356K, and E357K in the other chain; 12) K409D and K392D in one chain and D399K in the other chain; 13) K409D and K392D in one chain plus D399K and E356K in the other chain; 14) K409D and K392D in one chain plus D399K and E356K in the other chain; 14) K409D and K392D in one chain plus D399K and D357K in the other chain; 15) K409D and K370D in one chain plus D399K and D357K in the other chain; 16) D399K in one chain plus K409D and K360D in the other chain; or 17) K409D and K439D in one chain plus D399K and E356K in the other chain.

[0250] In some embodiments, the Fc region is a silenced Fc region. In some embodiments, the silenced Fc region comprises one or more (e.g., two or more) substitutions of Fc region residues 238, 265, 269, 270, 297, 327, and 329 according to EU numbering. In some embodiments, the silenced Fc region comprises a substitution that alters glycosylation. In some embodiments, the silenced Fc region comprises an effectorless mutation (e.g., N297A, N297G, DANA mutation (D265A+N297A) or DANG mutation (D265A+N297G) in the CH2 region). In some embodiments, the silenced Fc region comprises K322A and L234A / L235A mutations.

[0251] In some embodiments, the antibody further comprises a heavy chain constant region sequence in the absence of a CH1 sequence. In some embodiments, the antibody comprises a heavy chain constant region comprising a hinge region, a CH2 domain, and a CH3 domain. In some embodiments, the hinge region comprises a wild-type human IgG4 hinge region sequence (SEQ ID NO: 54). In some embodiments, the hinge region comprises a variant human IgG4 hinge region sequence (SEQ ID NO: 55) comprising a S228P mutation. In some embodiments, the CH2 domain comprises a wild-type human IgG4 CH2 domain sequence (SEQ ID NO: 56). In some embodiments, the CH2 domain comprises a variant human IgG4 CH2 domain comprising a F234A mutation, a L235A mutation, or both a F234A mutation and a L235A mutation. In some embodiments, the CH3 domain comprises a wild-type human IgG4 CH3 domain sequence (SEQ ID NO: 58). In some embodiments, the CH3 domain comprises a variant human IgG4 CH3 domain sequence comprising a T366W mutation. In some embodiments, the CH3 domain comprises a variant human IgG4 CH3 domain sequence comprising a T366S, an L368A mutation, and a Y407V mutation.

[0252] In some embodiments, the antibody has a Tm of about 55° C. to about 65° C. In some embodiments, the antibody has a Tag of about 55° C. to about 65° C. In some embodiments, the antibody has a Tm of about 55° C. to about 65° C. and a Tag of about 55° C. to about 65° C.

[0253] In some embodiments, the antibody is multispecific. In some embodiments, the antibody is bispecific. In some embodiments, the antibody binds to IL2RB and IL2RG. In some embodiments, the antibody functions as an IL2 receptor beta / gamma agonist.

[0254] In some embodiments, the antibody is -11 M ~ about 10 -6 M (for example, about 10 -10 M ~ about 10 -6 Around M; about 10 -9 M ~ about 10 -6 Around M; about 10 -8 M ~ about 10 -6 Around M; about 10 -11 M ~ about 10 -8 Around M; about 10 -10 M ~ about 10 -8 Around M; about 10 -9 M ~ about 10 -8 Around M; about 10 -11 M ~ about 10 -9 Around M; about 10 -10 M ~ about 10 -9 It has affinity for IL2R with a Kd of approximately 1 M.

[0255] In some embodiments, the antibody is -8 M ~ approx. 2.5×10 -7 It has affinity for IL2RB with a Kd of around M.

[0256] In some embodiments, the antibody is -9 M ~ approx. 2.5×10 -8 It has affinity for IL2RG with a Kd of around M.

[0257] In some embodiments, the antibody is-8 M ~ approx. 2.5×10 -7 The affinity for IL2RB is approximately 10 -9 M ~ approx. 2.5×10 -8 It has affinity for IL2RG with a Kd of around M.

[0258] In some embodiments, Kd is measured using a ForteBio Octet Qk384 instrument in kinetic mode. In some embodiments, Kd is measured using a ForteBio Octet Qk384 instrument with an anti-human Fc capture (AHC, 18-5005) sensor in kinetic mode. In some embodiments, Kd is measured according to the method described in the Examples herein.

[0259] Embodiments of the present disclosure include antibodies that bind to IL2RG, comprising a heavy chain variable region comprising: (a) a CDR1 sequence having two or less (e.g., 0, 1 or 2) substitutions in any one of SEQ ID NOs: 15 to 16; and / or (b) a CDR2 sequence having two or less (e.g., 0, 1 or 2) substitutions in any one of SEQ ID NOs: 17 to 19; and / or (c) a CDR3 sequence having two or less (e.g., 0, 1 or 2) substitutions in any one of SEQ ID NOs: 20 to 21.

[0260] In some embodiments, the antibody comprises: (a) a CDR1 sequence comprising any one of SEQ ID NOs: 15-16; and / or (b) a CDR2 sequence comprising any one of SEQ ID NOs: 17-19; and / or (c) a CDR3 sequence comprising any one of SEQ ID NOs: 20-21.

[0261] In some embodiments, the antibody comprises: (a) a CDR1 sequence comprising any one of SEQ ID NOs: 15-16; and (b) a CDR2 sequence comprising any one of SEQ ID NOs: 17-19; and (c) a CDR3 sequence comprising any one of SEQ ID NOs: 20-21.

[0262] In some embodiments, the antibody comprises: (a) the CDR1 sequence of SEQ ID NO: 15, the CDR2 sequence of SEQ ID NO: 17, and the CDR3 sequence of SEQ ID NO: 20; or (b) the CDR1 sequence of SEQ ID NO: 15, the CDR2 sequence of SEQ ID NO: 18, and the CDR3 sequence of SEQ ID NO: 20; or (c) the CDR1 sequence of SEQ ID NO: 16, the CDR2 sequence of SEQ ID NO: 18, and the CDR3 sequence of SEQ ID NO: 20; or (d) the CDR1 sequence of SEQ ID NO: 15, the CDR2 sequence of SEQ ID NO: 19, and the CDR3 sequence of SEQ ID NO: 21 Includes.

[0263] In some embodiments, the CDR1, CDR2 and CDR3 sequences are present in a VH human framework.

[0264] In some embodiments, the antibody comprises a heavy chain variable region comprising a sequence having six or fewer (e.g., five or fewer, four or fewer, three or fewer, two or fewer; 6, 5, 4, 3, 2, 1, 0) substitutions in any one of SEQ ID NOs: 22-25. In some embodiments, the antibody comprises a heavy chain variable region having at least 95% (e.g., at least 96%, at least 97%, at least 98%, at least 99%) sequence identity to any one of SEQ ID NOs: 22-25. In some embodiments, the antibody comprises a heavy chain variable region sequence selected from the group consisting of SEQ ID NOs: 22-25.

[0265] In some embodiments, the antibody is an isolated antibody. In some embodiments, the antibody is a human antibody. In some embodiments, the antibody is an isolated human antibody.

[0266] In some embodiments, the antibody is an intact IgG molecule. In some embodiments, the antibody is an intact IgG1 molecule. In some embodiments, the antibody is an intact IgG2 molecule. In some embodiments, the antibody is an intact IgG4 molecule. In some embodiments, the antibody is an immunologically active portion of an intact IgG molecule. In some embodiments, the antibody is an immunologically active portion of an intact IgG1 molecule. In some embodiments, the antibody is an immunologically active portion of an intact IgG2 molecule. In some embodiments, the antibody is an immunologically active portion of an intact IgG4 molecule. In some embodiments, the antibody is a triple chain antibody-like molecule. In some embodiments, the antibody is a heavy chain antibody.

[0267] In some embodiments, the antibody comprises an Fc region. In some embodiments, the antibody comprises a variant Fc region. In some embodiments, the variant Fc region has at least about 80% (e.g., at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%) homology to a native sequence Fc region.

[0268] In some embodiments, the variant Fc region comprises a heterodimerization modification. In some embodiments, the heterodimerization modification is a knob-and-hole substitution (e.g., in a variant IgG1 Fc region, 1) Y407T in one chain and T366Y in the other chain; 2) Y407A in one chain and T366W in the other chain; 3) F405A in one chain and T394W in the other chain; 4) F405W in one chain and T394S in the other chain; 5) Y407T in one chain and T366Y in the other chain; 6) T366Y in one chain and T394S in the other chain). and F405A in one chain and T394W and Y407T in the other chain; 7) T366W and F405W in one chain and T394S and Y407A in the other chain; 8) F405W and Y407A in one chain and T366W and T394S in the other chain; or 9) T366W in one polypeptide of Fc and T366S, L368A, and Y407V in the other chain. In some embodiments, heterodimerization modifications include substitutions that create new disulfide bridges (e.g., in a variant IgG1 Fc region, 1) Y349C in one Fc polypeptide chain and S354C in the other chain; 2) Y349C in one Fc polypeptide chain and E356C in the other chain; 3) Y349C in one Fc polypeptide chain and E357C in the other chain; 4) L351C in one Fc polypeptide chain and S354C in the other chain; 5) T394C in one Fc polypeptide chain and E397C in the other chain; or 6) D399C in one Fc polypeptide chain and K392C in the other chain).In some embodiments, the heterodimerization modification is a charge pair substitution (e.g., 1) K409E in one chain plus D399K in the other chain; 2) K409E in one chain plus D399R in the other chain; 3) K409D in one chain plus D399K in the other chain; 4) K409D in one chain plus D399R in the other chain; 5) K392E in one chain plus D399R in the other chain; 6) K392E in one chain plus D399K in the other chain; 7) K392D in one chain plus D399R in the other chain; 8) K392D in one chain plus D399K in the other chain; 9) K409D and K360D in one chain plus D399K and E356K in the other chain; 10) K409D and K 370D plus D399K and E357K in the other chain; 11) K409D and K392D in one chain plus D399K, E356K, and E357K in the other chain; 12) K409D and K392D in one chain and D399K in the other chain; 13) K409D and K392D in one chain plus D399K and E356K in the other chain; 14) K409D and K392D in one chain plus D399K and E356K in the other chain; 14) K409D and K392D in one chain plus D399K and D357K in the other chain; 15) K409D and K370D in one chain plus D399K and D357K in the other chain; 16) D399K in one chain plus K409D and K360D in the other chain; or 17) K409D and K439D in one chain plus D399K and E356K in the other chain.

[0269] In some embodiments, the Fc region is a silenced Fc region. In some embodiments, the silenced Fc region comprises one or more (e.g., two or more) substitutions of Fc region residues 238, 265, 269, 270, 297, 327, and 329 according to EU numbering. In some embodiments, the silenced Fc region comprises a substitution that alters glycosylation. In some embodiments, the silenced Fc region comprises an effectorless mutation (e.g., N297A, N297G, DANA mutation (D265A+N297A) or DANG mutation (D265A+N297G) in the CH2 region). In some embodiments, the silenced Fc region comprises K322A and L234A / L235A mutations.

[0270] In some embodiments, the antibody further comprises a heavy chain constant region sequence in the absence of a CH1 sequence. In some embodiments, the antibody comprises a heavy chain constant region comprising a hinge region, a CH2 domain, and a CH3 domain. In some embodiments, the hinge region comprises a wild-type human IgG4 hinge region sequence (SEQ ID NO: 54). In some embodiments, the hinge region comprises a variant human IgG4 hinge region sequence (SEQ ID NO: 55) comprising a S228P mutation. In some embodiments, the CH2 domain comprises a wild-type human IgG4 CH2 domain sequence (SEQ ID NO: 56). In some embodiments, the CH2 domain comprises a variant human IgG4 CH2 domain comprising a F234A mutation, a L235A mutation, or both a F234A mutation and a L235A mutation. In some embodiments, the CH3 domain comprises a wild-type human IgG4 CH3 domain sequence (SEQ ID NO: 58). In some embodiments, the CH3 domain comprises a variant human IgG4 CH3 domain sequence comprising a T366W mutation. In some embodiments, the CH3 domain comprises a variant human IgG4 CH3 domain sequence comprising a T366S, an L368A mutation, and a Y407V mutation.

[0271] In some embodiments, the antibody has a Tm of about 55° C. to about 65° C. In some embodiments, the antibody has a Tag of about 55° C. to about 65° C. In some embodiments, the antibody has a Tm of about 55° C. to about 65° C. and a Tag of about 55° C. to about 65° C.

[0272] In some embodiments, the antibody is multispecific. In some embodiments, the antibody is bispecific. In some embodiments, the antibody binds to IL2RB and IL2RG. In some embodiments, the antibody functions as an IL2 receptor beta / gamma agonist.

[0273] In some embodiments, the antibody is -11 M ~ about 10 -6 M (for example, about 10 -10 M ~ about 10 -6 Around M; about 10 -9 M ~ about 10 -6 Around M; about 10 -8 M ~ about 10 -6 Around M; about 10 -11 M ~ about 10 -8 Around M; about 10 -10 M ~ about 10 -8 Around M; about 10 -9 M ~ about 10 -8 Around M; about 10 -11 M ~ about 10 -9 Around M; about 10 -10 M ~ about 10 -9 It has affinity for IL2R with a Kd of approximately 1 M.

[0274] In some embodiments, the antibody is -8 M ~ approx. 2.5×10 -7 It has affinity for IL2RB with a Kd of around M.

[0275] In some embodiments, the antibody is -9 M ~ approx. 2.5×10 -8 It has affinity for IL2RG with a Kd of around M.

[0276] In some embodiments, the antibody is-8 M ~ approx. 2.5×10 -7 The affinity for IL2RB is approximately 10 -9 M ~ approx. 2.5×10 -8 It has affinity for IL2RG with a Kd of around M.

[0277] In some embodiments, Kd is measured using a ForteBio Octet Qk384 instrument in kinetic mode. In some embodiments, Kd is measured using a ForteBio Octet Qk384 instrument with an anti-human Fc capture (AHC, 18-5005) sensor in kinetic mode. In some embodiments, Kd is measured according to the method described in the Examples herein.

[0278] An aspect of the present disclosure is (a) Formula: GF X1 X2 X3 X4 YY (SEQ ID NO: 32) wherein X1 is T or I; X2 is F or V; X3 is S, N or G; and X4 is D or N; Formula (b): IS X5 SG X6 X7 I (Sequence number 33) (wherein X5 is S or N; X6 is D, S, G or N; and X7 is T or I); (c) a CDR3 sequence comprising the sequence ARGDAVSITGDY (SEQ ID NO: 20); The present invention includes an antibody that binds to IL2RG, comprising a heavy chain variable region comprising:

[0279] In some embodiments, the CDR1, CDR2 and CDR3 sequences are present in a VH human framework.

[0280] In some embodiments, the antibody is multispecific. In some embodiments, the antibody is bispecific. In some embodiments, the antibody binds to IL2RB and IL2RG.

[0281] In some embodiments, the antibody is an isolated antibody. In some embodiments, the antibody is a human antibody. In some embodiments, the antibody is an isolated human antibody.

[0282] In some embodiments, the antibody is an intact IgG molecule. In some embodiments, the antibody is an intact IgG1 molecule. In some embodiments, the antibody is an intact IgG2 molecule. In some embodiments, the antibody is an intact IgG4 molecule. In some embodiments, the antibody is an immunologically active portion of an intact IgG molecule. In some embodiments, the antibody is an immunologically active portion of an intact IgG1 molecule. In some embodiments, the antibody is an immunologically active portion of an intact IgG2 molecule. In some embodiments, the antibody is an immunologically active portion of an intact IgG4 molecule. In some embodiments, the antibody is a triple chain antibody-like molecule. In some embodiments, the antibody is a heavy chain antibody.

[0283] In some embodiments, the antibody is an Fc region-containing antibody. In some embodiments, the antibody further comprises a heavy chain constant region comprising a hinge region, a CH2 domain, and a CH3 domain. In some embodiments, the heavy chain constant region does not contain a CH1 sequence. In some embodiments, the hinge region comprises a wild-type human IgG4 hinge region sequence (SEQ ID NO: 54). In some embodiments, the hinge region comprises a variant human IgG4 hinge region sequence comprising a S228P mutation (SEQ ID NO: 55). In some embodiments, the CH2 domain comprises a wild-type human IgG4 CH2 domain sequence (SEQ ID NO: 56). In some embodiments, the CH2 domain comprises a variant human IgG4 CH2 domain comprising a F234A mutation, a L235A mutation, or both a F234A mutation and a L235A mutation. In some embodiments, the CH3 domain comprises a wild-type human IgG4 CH3 domain sequence (SEQ ID NO: 58). In some embodiments, the CH3 domain comprises a variant human IgG4 CH3 domain sequence comprising a T366W mutation. In some embodiments, the CH3 domain comprises a variant human IgG4 CH3 domain sequence comprising a T366S, an L368A mutation, and a Y407V mutation.

[0284] In some embodiments, the antibody has a Tm of about 55° C. to about 65° C. In some embodiments, the antibody has a Tag of about 55° C. to about 65° C. In some embodiments, the antibody has a Tm of about 55° C. to about 65° C. and a Tag of about 55° C. to about 65° C.

[0285] In some embodiments, the antibody functions as an IL2 receptor beta / gamma agonist.

[0286] In some embodiments, the antibody is -11 M ~ about 10 -6 M (for example, about 10 -10 M ~ about 10 -6 Around M; about 10 -9 M ~ about 10 -6 Around M; about 10 -8 M ~ about 10 -6 Around M; about 10-11 M ~ about 10 -8 Around M; about 10 -10 M ~ about 10 -8 Around M; about 10 -9 M ~ about 10 -8 Around M; about 10 -11 M ~ about 10 -9 Around M; about 10 -10 M ~ about 10 -9 It has affinity for IL2R with a Kd of approximately 1 M.

[0287] In some embodiments, the antibody is -8 M ~ approx. 2.5×10 -7 It has affinity for IL2RB with a Kd of around M.

[0288] In some embodiments, the antibody is -9 M ~ approx. 2.5×10 -8 It has affinity for IL2RG with a Kd of around M.

[0289] In some embodiments, the antibody is -8 M ~ approx. 2.5×10 -7 The affinity for IL2RB is approximately 10 -9 M ~ approx. 2.5×10 -8 It has affinity for IL2RG with a Kd of around M.

[0290] In some embodiments, Kd is measured using a ForteBio Octet Qk384 instrument in kinetic mode. In some embodiments, Kd is measured using a ForteBio Octet Qk384 instrument with an anti-human Fc capture (AHC, 18-5005) sensor in kinetic mode. In some embodiments, Kd is measured according to the method described in the Examples herein.

[0291] An aspect of the present disclosure is a first heavy chain variable region that binds to IL2RB, comprising a CDR1 sequence of SEQ ID NO:1; a CDR2 sequence of SEQ ID NO:4; and a CDR3 sequence of SEQ ID NO:7; a second heavy chain variable region that binds to IL2RG, comprising: a CDR1 sequence of SEQ ID NO: 15; a CDR2 sequence of SEQ ID NO: 17; and a CDR3 sequence of SEQ ID NO: 20; The antibody comprises

[0292] In some embodiments, the CDR1, CDR2 and CDR3 sequences in the first heavy chain variable region are present in a VH human framework. In some embodiments, the CDR1, CDR2 and CDR3 sequences in the second heavy chain variable region are present in a VH human framework. In some embodiments, the CDR1, CDR2 and CDR3 sequences in the first and second heavy chain variable regions are present in a VH human framework.

[0293] In some embodiments, the first heavy chain variable region has at least 95% (e.g., at least 96%, at least 97%, at least 98%, at least 99%) sequence identity to SEQ ID NO:11, and the second heavy chain variable region has at least 95% (e.g., at least 96%, at least 97%, at least 98%, at least 99%) sequence identity to SEQ ID NO:22. In some embodiments, the first heavy chain variable region comprises SEQ ID NO:11, and the second heavy chain variable region comprises SEQ ID NO:22.

[0294] In some embodiments, the antibody is an isolated antibody. In some embodiments, the antibody is a human antibody. In some embodiments, the antibody is an isolated human antibody.

[0295] In some embodiments, the antibody is an intact IgG molecule. In some embodiments, the antibody is an intact IgG1 molecule. In some embodiments, the antibody is an intact IgG2 molecule. In some embodiments, the antibody is an intact IgG4 molecule. In some embodiments, the antibody is an immunologically active portion of an intact IgG molecule. In some embodiments, the antibody is an immunologically active portion of an intact IgG1 molecule. In some embodiments, the antibody is an immunologically active portion of an intact IgG2 molecule. In some embodiments, the antibody is an immunologically active portion of an intact IgG4 molecule. In some embodiments, the antibody is a triple chain antibody-like molecule. In some embodiments, the antibody is a heavy chain antibody.

[0296] In some embodiments, the antibody comprises an Fc region. In some embodiments, the antibody comprises a variant Fc region. In some embodiments, the variant Fc region has at least about 80% (e.g., at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%) homology to a native sequence Fc region.

[0297] In some embodiments, the variant Fc region comprises a heterodimerization modification. In some embodiments, the heterodimerization modification is a knob-and-hole substitution (e.g., in a variant IgG1 Fc region, 1) Y407T in one chain and T366Y in the other chain; 2) Y407A in one chain and T366W in the other chain; 3) F405A in one chain and T394W in the other chain; 4) F405W in one chain and T394S in the other chain; 5) Y407T in one chain and T366Y in the other chain; 6) T366Y in one chain and T394S in the other chain). and F405A in one chain and T394W and Y407T in the other chain; 7) T366W and F405W in one chain and T394S and Y407A in the other chain; 8) F405W and Y407A in one chain and T366W and T394S in the other chain; or 9) T366W in one polypeptide of Fc and T366S, L368A, and Y407V in the other chain. In some embodiments, heterodimerization modifications include substitutions that create new disulfide bridges (e.g., in a variant IgG1 Fc region, 1) Y349C in one Fc polypeptide chain and S354C in the other chain; 2) Y349C in one Fc polypeptide chain and E356C in the other chain; 3) Y349C in one Fc polypeptide chain and E357C in the other chain; 4) L351C in one Fc polypeptide chain and S354C in the other chain; 5) T394C in one Fc polypeptide chain and E397C in the other chain; or 6) D399C in one Fc polypeptide chain and K392C in the other chain).In some embodiments, the heterodimerization modification is a charge pair substitution (e.g., 1) K409E in one chain plus D399K in the other chain; 2) K409E in one chain plus D399R in the other chain; 3) K409D in one chain plus D399K in the other chain; 4) K409D in one chain plus D399R in the other chain; 5) K392E in one chain plus D399R in the other chain; 6) K392E in one chain plus D399K in the other chain; 7) K392D in one chain plus D399R in the other chain; 8) K392D in one chain plus D399K in the other chain; 9) K409D and K360D in one chain plus D399K and E356K in the other chain; 10) K409D and K 370D plus D399K and E357K in the other chain; 11) K409D and K392D in one chain plus D399K, E356K, and E357K in the other chain; 12) K409D and K392D in one chain and D399K in the other chain; 13) K409D and K392D in one chain plus D399K and E356K in the other chain; 14) K409D and K392D in one chain plus D399K and E356K in the other chain; 14) K409D and K392D in one chain plus D399K and D357K in the other chain; 15) K409D and K370D in one chain plus D399K and D357K in the other chain; 16) D399K in one chain plus K409D and K360D in the other chain; or 17) K409D and K439D in one chain plus D399K and E356K in the other chain.

[0298] In some embodiments, the Fc region is a silenced Fc region. In some embodiments, the silenced Fc region comprises one or more (e.g., two or more) substitutions of Fc region residues 238, 265, 269, 270, 297, 327, and 329 according to EU numbering. In some embodiments, the silenced Fc region comprises a substitution that alters glycosylation. In some embodiments, the silenced Fc region comprises an effectorless mutation (e.g., N297A, N297G, DANA mutation (D265A+N297A) or DANG mutation (D265A+N297G) in the CH2 region). In some embodiments, the silenced Fc region comprises K322A and L234A / L235A mutations.

[0299] In some embodiments, the antibody further comprises a heavy chain constant region sequence in the absence of a CH1 sequence. In some embodiments, the antibody comprises a heavy chain constant region comprising a hinge region, a CH2 domain, and a CH3 domain. In some embodiments, the hinge region comprises a wild-type human IgG4 hinge region sequence (SEQ ID NO: 54). In some embodiments, the hinge region comprises a variant human IgG4 hinge region sequence (SEQ ID NO: 55) comprising a S228P mutation. In some embodiments, the CH2 domain comprises a wild-type human IgG4 CH2 domain sequence (SEQ ID NO: 56). In some embodiments, the CH2 domain comprises a variant human IgG4 CH2 domain comprising a F234A mutation, a L235A mutation, or both a F234A mutation and a L235A mutation. In some embodiments, the CH3 domain comprises a wild-type human IgG4 CH3 domain sequence (SEQ ID NO: 58). In some embodiments, the CH3 domain comprises a variant human IgG4 CH3 domain sequence comprising a T366W mutation. In some embodiments, the CH3 domain comprises a variant human IgG4 CH3 domain sequence comprising a T366S, an L368A mutation, and a Y407V mutation.

[0300] In some embodiments, the antibody comprises a first polypeptide comprising SEQ ID NO:53 and a second polypeptide comprising SEQ ID NO:61.

[0301] In some embodiments, the antibody has a Tm of about 55° C. to about 65° C. In some embodiments, the antibody has a Tag of about 55° C. to about 65° C. In some embodiments, the antibody has a Tm of about 55° C. to about 65° C. and a Tag of about 55° C. to about 65° C.

[0302] In some embodiments, the antibody is -11 M ~ about 10 -6 M (for example, about 10 -10 M ~ about 10 -6 Around M; about 10 -9 M ~ about 10 -6 Around M; about 10 -8 M ~ about 10 -6 Around M; about 10 -11 M ~ about 10 -8 Around M; about 10 -10 M ~ about 10 -8 Around M; about 10 -9 M ~ about 10 -8 Around M; about 10 -11 M ~ about 10 -9 Around M; about 10 -10 M ~ about 10 -9 It has affinity for IL2R with a Kd of approximately 1 M.

[0303] In some embodiments, the antibody is -8 M ~ approx. 2.5×10 -7 It has affinity for IL2RB with a Kd of around M.

[0304] In some embodiments, the antibody is -9 M ~ approx. 2.5×10 -8 It has affinity for IL2RG with a Kd of around M.

[0305] In some embodiments, the antibody is -8 M ~ approx. 2.5×10 -7 The affinity for IL2RB is approximately 10 -9M ~ approx. 2.5×10 -8 It has affinity for IL2RG with a Kd of around M.

[0306] In some embodiments, Kd is measured using a ForteBio Octet Qk384 instrument in kinetic mode. In some embodiments, Kd is measured using a ForteBio Octet Qk384 instrument with an anti-human Fc capture (AHC, 18-5005) sensor in kinetic mode. In some embodiments, Kd is measured according to the method described in the Examples herein.

[0307] An aspect of the present disclosure is a first heavy chain variable region that binds to IL2RB, comprising a CDR1 sequence of SEQ ID NO:1; a CDR2 sequence of SEQ ID NO:4; and a CDR3 sequence of SEQ ID NO:8; a second heavy chain variable region that binds to IL2RG, comprising: a CDR1 sequence of SEQ ID NO: 15; a CDR2 sequence of SEQ ID NO: 18; and a CDR3 sequence of SEQ ID NO: 20; The antibody comprises

[0308] In some embodiments, the CDR1, CDR2 and CDR3 sequences in the first heavy chain variable region are present in a VH human framework. In some embodiments, the CDR1, CDR2 and CDR3 sequences in the second heavy chain variable region are present in a VH human framework. In some embodiments, the CDR1, CDR2 and CDR3 sequences in the first and second heavy chain variable regions are present in a VH human framework.

[0309] In some embodiments, the first heavy chain variable region has at least 95% (e.g., at least 96%, at least 97%, at least 98%, at least 99%) sequence identity to SEQ ID NO: 12, and the second heavy chain variable region has at least 95% (e.g., at least 96%, at least 97%, at least 98%, at least 99%) sequence identity to SEQ ID NO: 23. In some embodiments, the first heavy chain variable region comprises SEQ ID NO: 12, and the second heavy chain variable region comprises SEQ ID NO:23.

[0310] In some embodiments, the antibody is an isolated antibody. In some embodiments, the antibody is a human antibody. In some embodiments, the antibody is an isolated human antibody.

[0311] In some embodiments, the antibody is an intact IgG molecule. In some embodiments, the antibody is an intact IgG1 molecule. In some embodiments, the antibody is an intact IgG2 molecule. In some embodiments, the antibody is an intact IgG4 molecule. In some embodiments, the antibody is an immunologically active portion of an intact IgG molecule. In some embodiments, the antibody is an immunologically active portion of an intact IgG1 molecule. In some embodiments, the antibody is an immunologically active portion of an intact IgG2 molecule. In some embodiments, the antibody is an immunologically active portion of an intact IgG4 molecule. In some embodiments, the antibody is a triple chain antibody-like molecule. In some embodiments, the antibody is a heavy chain antibody.

[0312] In some embodiments, the antibody comprises an Fc region. In some embodiments, the antibody comprises a variant Fc region. In some embodiments, the variant Fc region has at least about 80% (e.g., at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%) homology to a native sequence Fc region.

[0313] In some embodiments, the variant Fc region comprises a heterodimerization modification. In some embodiments, the heterodimerization modification is a knob-and-hole substitution (e.g., in a variant IgG1 Fc region, 1) Y407T in one chain and T366Y in the other chain; 2) Y407A in one chain and T366W in the other chain; 3) F405A in one chain and T394W in the other chain; 4) F405W in one chain and T394S in the other chain; 5) Y407T in one chain and T366Y in the other chain; 6) T366Y in one chain and T394S in the other chain). and F405A in one chain and T394W and Y407T in the other chain; 7) T366W and F405W in one chain and T394S and Y407A in the other chain; 8) F405W and Y407A in one chain and T366W and T394S in the other chain; or 9) T366W in one polypeptide of Fc and T366S, L368A, and Y407V in the other chain. In some embodiments, heterodimerization modifications include substitutions that create new disulfide bridges (e.g., in a variant IgG1 Fc region, 1) Y349C in one Fc polypeptide chain and S354C in the other chain; 2) Y349C in one Fc polypeptide chain and E356C in the other chain; 3) Y349C in one Fc polypeptide chain and E357C in the other chain; 4) L351C in one Fc polypeptide chain and S354C in the other chain; 5) T394C in one Fc polypeptide chain and E397C in the other chain; or 6) D399C in one Fc polypeptide chain and K392C in the other chain).In some embodiments, the heterodimerization modification is a charge pair substitution (e.g., 1) K409E in one chain plus D399K in the other chain; 2) K409E in one chain plus D399R in the other chain; 3) K409D in one chain plus D399K in the other chain; 4) K409D in one chain plus D399R in the other chain; 5) K392E in one chain plus D399R in the other chain; 6) K392E in one chain plus D399K in the other chain; 7) K392D in one chain plus D399R in the other chain; 8) K392D in one chain plus D399K in the other chain; 9) K409D and K360D in one chain plus D399K and E356K in the other chain; 10) K409D and K 370D plus D399K and E357K in the other chain; 11) K409D and K392D in one chain plus D399K, E356K, and E357K in the other chain; 12) K409D and K392D in one chain and D399K in the other chain; 13) K409D and K392D in one chain plus D399K and E356K in the other chain; 14) K409D and K392D in one chain plus D399K and E356K in the other chain; 14) K409D and K392D in one chain plus D399K and D357K in the other chain; 15) K409D and K370D in one chain plus D399K and D357K in the other chain; 16) D399K in one chain plus K409D and K360D in the other chain; or 17) K409D and K439D in one chain plus D399K and E356K in the other chain.

[0314] In some embodiments, the Fc region is a silenced Fc region. In some embodiments, the silenced Fc region comprises one or more (e.g., two or more) substitutions of Fc region residues 238, 265, 269, 270, 297, 327, and 329 according to EU numbering. In some embodiments, the silenced Fc region comprises a substitution that alters glycosylation. In some embodiments, the silenced Fc region comprises an effectorless mutation (e.g., N297A, N297G, DANA mutation (D265A+N297A) or DANG mutation (D265A+N297G) in the CH2 region). In some embodiments, the silenced Fc region comprises K322A and L234A / L235A mutations.

[0315] In some embodiments, the antibody further comprises a heavy chain constant region sequence in the absence of a CH1 sequence. In some embodiments, the antibody comprises a heavy chain constant region comprising a hinge region, a CH2 domain, and a CH3 domain. In some embodiments, the hinge region comprises a wild-type human IgG4 hinge region sequence (SEQ ID NO: 54). In some embodiments, the hinge region comprises a variant human IgG4 hinge region sequence (SEQ ID NO: 55) comprising a S228P mutation. In some embodiments, the CH2 domain comprises a wild-type human IgG4 CH2 domain sequence (SEQ ID NO: 56). In some embodiments, the CH2 domain comprises a variant human IgG4 CH2 domain comprising a F234A mutation, a L235A mutation, or both a F234A mutation and a L235A mutation. In some embodiments, the CH3 domain comprises a wild-type human IgG4 CH3 domain sequence (SEQ ID NO: 58). In some embodiments, the CH3 domain comprises a variant human IgG4 CH3 domain sequence comprising a T366W mutation. In some embodiments, the CH3 domain comprises a variant human IgG4 CH3 domain sequence comprising a T366S, an L368A mutation, and a Y407V mutation.

[0316] In some embodiments, the antibody comprises a first polypeptide comprising SEQ ID NO:62 and a second polypeptide comprising SEQ ID NO:63.

[0317] In some embodiments, the antibody has a Tm of about 55° C. to about 65° C. In some embodiments, the antibody has a Tag of about 55° C. to about 65° C. In some embodiments, the antibody has a Tm of about 55° C. to about 65° C. and a Tag of about 55° C. to about 65° C.

[0318] In some embodiments, the antibody is -11 M ~ about 10 -6 M (for example, about 10 -10 M ~ about 10 -6 Around M; about 10 -9 M ~ about 10 -6 Around M; about 10 -8 M ~ about 10 -6 Around M; about 10 -11 M ~ about 10 -8 Around M; about 10 -10 M ~ about 10 -8 Around M; about 10 -9 M ~ about 10 -8 Around M; about 10 -11 M ~ about 10 -9 Around M; about 10 -10 M ~ about 10 -9 It has affinity for IL2R with a Kd of approximately 1 M.

[0319] In some embodiments, the antibody is -8 M ~ approx. 2.5×10 -7 It has affinity for IL2RB with a Kd of around M.

[0320] In some embodiments, the antibody is -9 M ~ approx. 2.5×10 -8 It has affinity for IL2RG with a Kd of around M.

[0321] In some embodiments, the antibody is -8 M ~ approx. 2.5×10 -7 The affinity for IL2RB is approximately 10 -9M ~ approx. 2.5×10 -8 It has affinity for IL2RG with a Kd of around M.

[0322] In some embodiments, Kd is measured using a ForteBio Octet Qk384 instrument in kinetic mode. In some embodiments, Kd is measured using a ForteBio Octet Qk384 instrument with an anti-human Fc capture (AHC, 18-5005) sensor in kinetic mode. In some embodiments, Kd is measured according to the method described in the Examples herein.

[0323] An aspect of the present disclosure is a first heavy chain variable region that binds to IL2RB, comprising a CDR1 sequence of SEQ ID NO:2; a CDR2 sequence of SEQ ID NO:5; and a CDR3 sequence of SEQ ID NO:9; a second heavy chain variable region that binds to IL2RG, comprising: a CDR1 sequence of SEQ ID NO: 15; a CDR2 sequence of SEQ ID NO: 18; and a CDR3 sequence of SEQ ID NO: 20; The antibody comprises

[0324] In some embodiments, the CDR1, CDR2 and CDR3 sequences in the first heavy chain variable region are present in a VH human framework. In some embodiments, the CDR1, CDR2 and CDR3 sequences in the second heavy chain variable region are present in a VH human framework. In some embodiments, the CDR1, CDR2 and CDR3 sequences in the first and second heavy chain variable regions are present in a VH human framework.

[0325] In some embodiments, the first heavy chain variable region has at least 95% (e.g., at least 96%, at least 97%, at least 98%, at least 99%) sequence identity to SEQ ID NO: 13, and the second heavy chain variable region has at least 95% (e.g., at least 96%, at least 97%, at least 98%, at least 99%) sequence identity to SEQ ID NO: 23. In some embodiments, the first heavy chain variable region comprises SEQ ID NO: 13, and the second heavy chain variable region comprises SEQ ID NO:23.

[0326] In some embodiments, the antibody is an isolated antibody. In some embodiments, the antibody is a human antibody. In some embodiments, the antibody is an isolated human antibody.

[0327] In some embodiments, the antibody is an intact IgG molecule. In some embodiments, the antibody is an intact IgG1 molecule. In some embodiments, the antibody is an intact IgG2 molecule. In some embodiments, the antibody is an intact IgG4 molecule. In some embodiments, the antibody is an immunologically active portion of an intact IgG molecule. In some embodiments, the antibody is an immunologically active portion of an intact IgG1 molecule. In some embodiments, the antibody is an immunologically active portion of an intact IgG2 molecule. In some embodiments, the antibody is an immunologically active portion of an intact IgG4 molecule. In some embodiments, the antibody is a triple chain antibody-like molecule. In some embodiments, the antibody is a heavy chain antibody.

[0328] In some embodiments, the antibody comprises an Fc region. In some embodiments, the antibody comprises a variant Fc region. In some embodiments, the variant Fc region has at least about 80% (e.g., at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%) homology to a native sequence Fc region.

[0329] In some embodiments, the variant Fc region comprises a heterodimerization modification. In some embodiments, the heterodimerization modification is a knob-and-hole substitution (e.g., in a variant IgG1 Fc region, 1) Y407T in one chain and T366Y in the other chain; 2) Y407A in one chain and T366W in the other chain; 3) F405A in one chain and T394W in the other chain; 4) F405W in one chain and T394S in the other chain; 5) Y407T in one chain and T366Y in the other chain; 6) T366Y in one chain and T394S in the other chain). and F405A in one chain and T394W and Y407T in the other chain; 7) T366W and F405W in one chain and T394S and Y407A in the other chain; 8) F405W and Y407A in one chain and T366W and T394S in the other chain; or 9) T366W in one polypeptide of Fc and T366S, L368A, and Y407V in the other chain. In some embodiments, heterodimerization modifications include substitutions that create new disulfide bridges (e.g., in a variant IgG1 Fc region, 1) Y349C in one Fc polypeptide chain and S354C in the other chain; 2) Y349C in one Fc polypeptide chain and E356C in the other chain; 3) Y349C in one Fc polypeptide chain and E357C in the other chain; 4) L351C in one Fc polypeptide chain and S354C in the other chain; 5) T394C in one Fc polypeptide chain and E397C in the other chain; or 6) D399C in one Fc polypeptide chain and K392C in the other chain).In some embodiments, the heterodimerization modification is a charge pair substitution (e.g., 1) K409E in one chain plus D399K in the other chain; 2) K409E in one chain plus D399R in the other chain; 3) K409D in one chain plus D399K in the other chain; 4) K409D in one chain plus D399R in the other chain; 5) K392E in one chain plus D399R in the other chain; 6) K392E in one chain plus D399K in the other chain; 7) K392D in one chain plus D399R in the other chain; 8) K392D in one chain plus D399K in the other chain; 9) K409D and K360D in one chain plus D399K and E356K in the other chain; 10) K409D and K 370D plus D399K and E357K in the other chain; 11) K409D and K392D in one chain plus D399K, E356K, and E357K in the other chain; 12) K409D and K392D in one chain and D399K in the other chain; 13) K409D and K392D in one chain plus D399K and E356K in the other chain; 14) K409D and K392D in one chain plus D399K and E356K in the other chain; 14) K409D and K392D in one chain plus D399K and D357K in the other chain; 15) K409D and K370D in one chain plus D399K and D357K in the other chain; 16) D399K in one chain plus K409D and K360D in the other chain; or 17) K409D and K439D in one chain plus D399K and E356K in the other chain.

[0330] In some embodiments, the Fc region is a silenced Fc region. In some embodiments, the silenced Fc region comprises one or more (e.g., two or more) substitutions of Fc region residues 238, 265, 269, 270, 297, 327, and 329 according to EU numbering. In some embodiments, the silenced Fc region comprises a substitution that alters glycosylation. In some embodiments, the silenced Fc region comprises an effectorless mutation (e.g., N297A, N297G, DANA mutation (D265A+N297A) or DANG mutation (D265A+N297G) in the CH2 region). In some embodiments, the silenced Fc region comprises K322A and L234A / L235A mutations.

[0331] In some embodiments, the antibody further comprises a heavy chain constant region sequence in the absence of a CH1 sequence. In some embodiments, the antibody comprises a heavy chain constant region comprising a hinge region, a CH2 domain, and a CH3 domain. In some embodiments, the hinge region comprises a wild-type human IgG4 hinge region sequence (SEQ ID NO: 54). In some embodiments, the hinge region comprises a variant human IgG4 hinge region sequence (SEQ ID NO: 55) comprising a S228P mutation. In some embodiments, the CH2 domain comprises a wild-type human IgG4 CH2 domain sequence (SEQ ID NO: 56). In some embodiments, the CH2 domain comprises a variant human IgG4 CH2 domain comprising a F234A mutation, a L235A mutation, or both a F234A mutation and a L235A mutation. In some embodiments, the CH3 domain comprises a wild-type human IgG4 CH3 domain sequence (SEQ ID NO: 58). In some embodiments, the CH3 domain comprises a variant human IgG4 CH3 domain sequence comprising a T366W mutation. In some embodiments, the CH3 domain comprises a variant human IgG4 CH3 domain sequence comprising a T366S, an L368A mutation, and a Y407V mutation.

[0332] In some embodiments, the antibody comprises a first polypeptide comprising SEQ ID NO:64 and a second polypeptide comprising SEQ ID NO:65.

[0333] In some embodiments, the antibody has a Tm of about 55° C. to about 65° C. In some embodiments, the antibody has a Tag of about 55° C. to about 65° C. In some embodiments, the antibody has a Tm of about 55° C. to about 65° C. and a Tag of about 55° C. to about 65° C.

[0334] In some embodiments, the antibody is -11 M ~ about 10 -6 M (for example, about 10 -10 M ~ about 10 -6 Around M; about 10 -9 M ~ about 10 -6 Around M; about 10 -8 M ~ about 10 -6 Around M; about 10 -11 M ~ about 10 -8 Around M; about 10 -10 M ~ about 10 -8 Around M; about 10 -9 M ~ about 10 -8 Around M; about 10 -11 M ~ about 10 -9 Around M; about 10 -10 M ~ about 10 -9 It has affinity for IL2R with a Kd of approximately 1 M.

[0335] In some embodiments, the antibody is -8 M ~ approx. 2.5×10 -7 It has affinity for IL2RB with a Kd of around M.

[0336] In some embodiments, the antibody is -9 M ~ approx. 2.5×10 -8 It has affinity for IL2RG with a Kd of around M.

[0337] In some embodiments, the antibody is -8 M ~ approx. 2.5×10 -7 The affinity for IL2RB is approximately 10 -9M ~ approx. 2.5×10 -8 It has affinity for IL2RG with a Kd of around M.

[0338] In some embodiments, Kd is measured using a ForteBio Octet Qk384 instrument in kinetic mode. In some embodiments, Kd is measured using a ForteBio Octet Qk384 instrument with an anti-human Fc capture (AHC, 18-5005) sensor in kinetic mode. In some embodiments, Kd is measured according to the method described in the Examples herein.

[0339] An aspect of the present disclosure is a first heavy chain variable region that binds to IL2RB, comprising a CDR1 sequence of SEQ ID NO:3; a CDR2 sequence of SEQ ID NO:6; and a CDR3 sequence of SEQ ID NO:10; a second heavy chain variable region that binds to IL2RG, comprising: a CDR1 sequence of SEQ ID NO: 15; a CDR2 sequence of SEQ ID NO: 17; and a CDR3 sequence of SEQ ID NO: 20; The antibody comprises

[0340] In some embodiments, the CDR1, CDR2 and CDR3 sequences in the first heavy chain variable region are present in a VH human framework. In some embodiments, the CDR1, CDR2 and CDR3 sequences in the second heavy chain variable region are present in a VH human framework. In some embodiments, the CDR1, CDR2 and CDR3 sequences in the first and second heavy chain variable regions are present in a VH human framework.

[0341] In some embodiments, the first heavy chain variable region has at least 95% (e.g., at least 96%, at least 97%, at least 98%, at least 99%) sequence identity to SEQ ID NO: 14, and the second heavy chain variable region has at least 95% (e.g., at least 96%, at least 97%, at least 98%, at least 99%) sequence identity to SEQ ID NO: 22. In some embodiments, the first heavy chain variable region comprises SEQ ID NO: 14, and the second heavy chain variable region comprises SEQ ID NO:22.

[0342] In some embodiments, the antibody is an isolated antibody. In some embodiments, the antibody is a human antibody. In some embodiments, the antibody is an isolated human antibody.

[0343] In some embodiments, the antibody is an intact IgG molecule. In some embodiments, the antibody is an intact IgG1 molecule. In some embodiments, the antibody is an intact IgG2 molecule. In some embodiments, the antibody is an intact IgG4 molecule. In some embodiments, the antibody is an immunologically active portion of an intact IgG molecule. In some embodiments, the antibody is an immunologically active portion of an intact IgG1 molecule. In some embodiments, the antibody is an immunologically active portion of an intact IgG2 molecule. In some embodiments, the antibody is an immunologically active portion of an intact IgG4 molecule. In some embodiments, the antibody is a triple chain antibody-like molecule. In some embodiments, the antibody is a heavy chain antibody.

[0344] In some embodiments, the antibody comprises an Fc region. In some embodiments, the antibody comprises a variant Fc region. In some embodiments, the variant Fc region has at least about 80% (e.g., at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%) homology to a native sequence Fc region.

[0345] In some embodiments, the variant Fc region comprises a heterodimerization modification. In some embodiments, the heterodimerization modification is a knob-and-hole substitution (e.g., in a variant IgG1 Fc region, 1) Y407T in one chain and T366Y in the other chain; 2) Y407A in one chain and T366W in the other chain; 3) F405A in one chain and T394W in the other chain; 4) F405W in one chain and T394S in the other chain; 5) Y407T in one chain and T366Y in the other chain; 6) T366Y in one chain and T394S in the other chain). and F405A in one chain and T394W and Y407T in the other chain; 7) T366W and F405W in one chain and T394S and Y407A in the other chain; 8) F405W and Y407A in one chain and T366W and T394S in the other chain; or 9) T366W in one polypeptide of Fc and T366S, L368A, and Y407V in the other chain. In some embodiments, heterodimerization modifications include substitutions that create new disulfide bridges (e.g., in a variant IgG1 Fc region, 1) Y349C in one Fc polypeptide chain and S354C in the other chain; 2) Y349C in one Fc polypeptide chain and E356C in the other chain; 3) Y349C in one Fc polypeptide chain and E357C in the other chain; 4) L351C in one Fc polypeptide chain and S354C in the other chain; 5) T394C in one Fc polypeptide chain and E397C in the other chain; or 6) D399C in one Fc polypeptide chain and K392C in the other chain).In some embodiments, the heterodimerization modification is a charge pair substitution (e.g., 1) K409E in one chain plus D399K in the other chain; 2) K409E in one chain plus D399R in the other chain; 3) K409D in one chain plus D399K in the other chain; 4) K409D in one chain plus D399R in the other chain; 5) K392E in one chain plus D399R in the other chain; 6) K392E in one chain plus D399K in the other chain; 7) K392D in one chain plus D399R in the other chain; 8) K392D in one chain plus D399K in the other chain; 9) K409D and K360D in one chain plus D399K and E356K in the other chain; 10) K409D and K 370D plus D399K and E357K in the other chain; 11) K409D and K392D in one chain plus D399K, E356K, and E357K in the other chain; 12) K409D and K392D in one chain and D399K in the other chain; 13) K409D and K392D in one chain plus D399K and E356K in the other chain; 14) K409D and K392D in one chain plus D399K and E356K in the other chain; 14) K409D and K392D in one chain plus D399K and D357K in the other chain; 15) K409D and K370D in one chain plus D399K and D357K in the other chain; 16) D399K in one chain plus K409D and K360D in the other chain; or 17) K409D and K439D in one chain plus D399K and E356K in the other chain.

[0346] In some embodiments, the Fc region is a silenced Fc region. In some embodiments, the silenced Fc region comprises one or more (e.g., two or more) substitutions of Fc region residues 238, 265, 269, 270, 297, 327, and 329 according to EU numbering. In some embodiments, the silenced Fc region comprises a substitution that alters glycosylation. In some embodiments, the silenced Fc region comprises an effectorless mutation (e.g., N297A, N297G, DANA mutation (D265A+N297A) or DANG mutation (D265A+N297G) in the CH2 region). In some embodiments, the silenced Fc region comprises K322A and L234A / L235A mutations.

[0347] In some embodiments, the antibody further comprises a heavy chain constant region sequence in the absence of a CH1 sequence. In some embodiments, the antibody comprises a heavy chain constant region comprising a hinge region, a CH2 domain, and a CH3 domain. In some embodiments, the hinge region comprises a wild-type human IgG4 hinge region sequence (SEQ ID NO: 54). In some embodiments, the hinge region comprises a variant human IgG4 hinge region sequence (SEQ ID NO: 55) comprising a S228P mutation. In some embodiments, the CH2 domain comprises a wild-type human IgG4 CH2 domain sequence (SEQ ID NO: 56). In some embodiments, the CH2 domain comprises a variant human IgG4 CH2 domain comprising a F234A mutation, a L235A mutation, or both a F234A mutation and a L235A mutation. In some embodiments, the CH3 domain comprises a wild-type human IgG4 CH3 domain sequence (SEQ ID NO: 58). In some embodiments, the CH3 domain comprises a variant human IgG4 CH3 domain sequence comprising a T366W mutation. In some embodiments, the CH3 domain comprises a variant human IgG4 CH3 domain sequence comprising a T366S, an L368A mutation, and a Y407V mutation.

[0348] In some embodiments, the antibody comprises a first polypeptide comprising SEQ ID NO:66 and a second polypeptide comprising SEQ ID NO:67.

[0349] In some embodiments, the antibody has a Tm of about 55° C. to about 65° C. In some embodiments, the antibody has a Tag of about 55° C. to about 65° C. In some embodiments, the antibody has a Tm of about 55° C. to about 65° C. and a Tag of about 55° C. to about 65° C.

[0350] In some embodiments, the antibody is -11 M ~ about 10 -6 M (for example, about 10 -10 M ~ about 10 -6 Around M; about 10 -9 M ~ about 10 -6 Around M; about 10 -8 M ~ about 10 -6 Around M; about 10 -11 M ~ about 10 -8 Around M; about 10 -10 M ~ about 10 -8 Around M; about 10 -9 M ~ about 10 -8 Around M; about 10 -11 M ~ about 10 -9 Around M; about 10 -10 M ~ about 10 -9 It has affinity for IL2R with a Kd of approximately 1 M.

[0351] In some embodiments, the antibody is -8 M ~ approx. 2.5×10 -7 It has affinity for IL2RB with a Kd of around M.

[0352] In some embodiments, the antibody is -9 M ~ approx. 2.5×10 -8 It has affinity for IL2RG with a Kd of around M.

[0353] In some embodiments, the antibody is -8 M ~ approx. 2.5×10 -7 The affinity for IL2RB is approximately 10 -9M ~ approx. 2.5×10 -8 It has affinity for IL2RG with a Kd of around M.

[0354] In some embodiments, Kd is measured using a ForteBio Octet Qk384 instrument in kinetic mode. In some embodiments, Kd is measured using a ForteBio Octet Qk384 instrument with an anti-human Fc capture (AHC, 18-5005) sensor in kinetic mode. In some embodiments, Kd is measured according to the method described in the Examples herein.

[0355] An aspect of the present disclosure is a first heavy chain variable region that binds to IL2RB, comprising a CDR1 sequence of SEQ ID NO:1; a CDR2 sequence of SEQ ID NO:4; and a CDR3 sequence of SEQ ID NO:8; a second heavy chain variable region that binds to IL2RG, comprising: a CDR1 sequence of SEQ ID NO: 16; a CDR2 sequence of SEQ ID NO: 18; and a CDR3 sequence of SEQ ID NO: 20; The antibody comprises

[0356] In some embodiments, the CDR1, CDR2 and CDR3 sequences in the first heavy chain variable region are present in a VH human framework. In some embodiments, the CDR1, CDR2 and CDR3 sequences in the second heavy chain variable region are present in a VH human framework. In some embodiments, the CDR1, CDR2 and CDR3 sequences in the first and second heavy chain variable regions are present in a VH human framework.

[0357] In some embodiments, the first heavy chain variable region has at least 95% (e.g., at least 96%, at least 97%, at least 98%, at least 99%) sequence identity to SEQ ID NO: 12, and the second heavy chain variable region has at least 95% (e.g., at least 96%, at least 97%, at least 98%, at least 99%) sequence identity to SEQ ID NO: 24. In some embodiments, the first heavy chain variable region comprises SEQ ID NO: 12, and the second heavy chain variable region comprises SEQ ID NO:24.

[0358] In some embodiments, the antibody is an isolated antibody. In some embodiments, the antibody is a human antibody. In some embodiments, the antibody is an isolated human antibody.

[0359] In some embodiments, the antibody is an intact IgG molecule. In some embodiments, the antibody is an intact IgG1 molecule. In some embodiments, the antibody is an intact IgG2 molecule. In some embodiments, the antibody is an intact IgG4 molecule. In some embodiments, the antibody is an immunologically active portion of an intact IgG molecule. In some embodiments, the antibody is an immunologically active portion of an intact IgG1 molecule. In some embodiments, the antibody is an immunologically active portion of an intact IgG2 molecule. In some embodiments, the antibody is an immunologically active portion of an intact IgG4 molecule. In some embodiments, the antibody is a triple chain antibody-like molecule. In some embodiments, the antibody is a heavy chain antibody.

[0360] In some embodiments, the antibody comprises an Fc region. In some embodiments, the antibody comprises a variant Fc region. In some embodiments, the variant Fc region has at least about 80% (e.g., at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%) homology to a native sequence Fc region.

[0361] In some embodiments, the variant Fc region comprises a heterodimerization modification. In some embodiments, the heterodimerization modification is a knob-and-hole substitution (e.g., in a variant IgG1 Fc region, 1) Y407T in one chain and T366Y in the other chain; 2) Y407A in one chain and T366W in the other chain; 3) F405A in one chain and T394W in the other chain; 4) F405W in one chain and T394S in the other chain; 5) Y407T in one chain and T366Y in the other chain; 6) T366Y in one chain and T394S in the other chain). and F405A in one chain and T394W and Y407T in the other chain; 7) T366W and F405W in one chain and T394S and Y407A in the other chain; 8) F405W and Y407A in one chain and T366W and T394S in the other chain; or 9) T366W in one polypeptide of Fc and T366S, L368A, and Y407V in the other chain. In some embodiments, heterodimerization modifications include substitutions that create new disulfide bridges (e.g., in a variant IgG1 Fc region, 1) Y349C in one Fc polypeptide chain and S354C in the other chain; 2) Y349C in one Fc polypeptide chain and E356C in the other chain; 3) Y349C in one Fc polypeptide chain and E357C in the other chain; 4) L351C in one Fc polypeptide chain and S354C in the other chain; 5) T394C in one Fc polypeptide chain and E397C in the other chain; or 6) D399C in one Fc polypeptide chain and K392C in the other chain).In some embodiments, the heterodimerization modification is a charge pair substitution (e.g., 1) K409E in one chain plus D399K in the other chain; 2) K409E in one chain plus D399R in the other chain; 3) K409D in one chain plus D399K in the other chain; 4) K409D in one chain plus D399R in the other chain; 5) K392E in one chain plus D399R in the other chain; 6) K392E in one chain plus D399K in the other chain; 7) K392D in one chain plus D399R in the other chain; 8) K392D in one chain plus D399K in the other chain; 9) K409D and K360D in one chain plus D399K and E356K in the other chain; 10) K409D and K 370D plus D399K and E357K in the other chain; 11) K409D and K392D in one chain plus D399K, E356K, and E357K in the other chain; 12) K409D and K392D in one chain and D399K in the other chain; 13) K409D and K392D in one chain plus D399K and E356K in the other chain; 14) K409D and K392D in one chain plus D399K and E356K in the other chain; 14) K409D and K392D in one chain plus D399K and D357K in the other chain; 15) K409D and K370D in one chain plus D399K and D357K in the other chain; 16) D399K in one chain plus K409D and K360D in the other chain; or 17) K409D and K439D in one chain plus D399K and E356K in the other chain.

[0362] In some embodiments, the Fc region is a silenced Fc region. In some embodiments, the silenced Fc region comprises one or more (e.g., two or more) substitutions of Fc region residues 238, 265, 269, 270, 297, 327, and 329 according to EU numbering. In some embodiments, the silenced Fc region comprises a substitution that alters glycosylation. In some embodiments, the silenced Fc region comprises an effectorless mutation (e.g., N297A, N297G, DANA mutation (D265A+N297A) or DANG mutation (D265A+N297G) in the CH2 region). In some embodiments, the silenced Fc region comprises K322A and L234A / L235A mutations.

[0363] In some embodiments, the antibody further comprises a heavy chain constant region sequence in the absence of a CH1 sequence. In some embodiments, the antibody comprises a heavy chain constant region comprising a hinge region, a CH2 domain, and a CH3 domain. In some embodiments, the hinge region comprises a wild-type human IgG4 hinge region sequence (SEQ ID NO: 54). In some embodiments, the hinge region comprises a variant human IgG4 hinge region sequence (SEQ ID NO: 55) comprising a S228P mutation. In some embodiments, the CH2 domain comprises a wild-type human IgG4 CH2 domain sequence (SEQ ID NO: 56). In some embodiments, the CH2 domain comprises a variant human IgG4 CH2 domain comprising a F234A mutation, a L235A mutation, or both a F234A mutation and a L235A mutation. In some embodiments, the CH3 domain comprises a wild-type human IgG4 CH3 domain sequence (SEQ ID NO: 58). In some embodiments, the CH3 domain comprises a variant human IgG4 CH3 domain sequence comprising a T366W mutation. In some embodiments, the CH3 domain comprises a variant human IgG4 CH3 domain sequence comprising a T366S, an L368A mutation, and a Y407V mutation.

[0364] In some embodiments, the antibody comprises a first polypeptide comprising SEQ ID NO:34 and a second polypeptide comprising SEQ ID NO:35.

[0365] In some embodiments, the antibody has a Tm of about 55° C. to about 65° C. In some embodiments, the antibody has a Tag of about 55° C. to about 65° C. In some embodiments, the antibody has a Tm of about 55° C. to about 65° C. and a Tag of about 55° C. to about 65° C.

[0366] In some embodiments, the antibody is -11 M ~ about 10 -6 M (for example, about 10 -10 M ~ about 10 -6 Around M; about 10 -9 M ~ about 10 -6 Around M; about 10 -8 M ~ about 10 -6 Around M; about 10 -11 M ~ about 10 -8 Around M; about 10 -10 M ~ about 10 -8 Around M; about 10 -9 M ~ about 10 -8 Around M; about 10 -11 M ~ about 10 -9 Around M; about 10 -10 M ~ about 10 -9 It has affinity for IL2R with a Kd of approximately 1 M.

[0367] In some embodiments, the antibody is -8 M ~ approx. 2.5×10 -7 It has affinity for IL2RB with a Kd of around M.

[0368] In some embodiments, the antibody is -9 M ~ approx. 2.5×10 -8 It has affinity for IL2RG with a Kd of around M.

[0369] In some embodiments, the antibody is -8 M ~ approx. 2.5×10 -7 The affinity for IL2RB is approximately 10 -9M ~ approx. 2.5×10 -8 It has affinity for IL2RG with a Kd of around M.

[0370] In some embodiments, Kd is measured using a ForteBio Octet Qk384 instrument in kinetic mode. In some embodiments, Kd is measured using a ForteBio Octet Qk384 instrument with an anti-human Fc capture (AHC, 18-5005) sensor in kinetic mode. In some embodiments, Kd is measured according to the method described in the Examples herein.

[0371] An aspect of the present disclosure is a first heavy chain variable region that binds to IL2RB, comprising a CDR1 sequence of SEQ ID NO:1; a CDR2 sequence of SEQ ID NO:4; and a CDR3 sequence of SEQ ID NO:8; a second heavy chain variable region that binds to IL2RG, comprising: a CDR1 sequence of SEQ ID NO: 15; a CDR2 sequence of SEQ ID NO: 19; and a CDR3 sequence of SEQ ID NO: 21; The antibody comprises

[0372] In some embodiments, the CDR1, CDR2 and CDR3 sequences in the first heavy chain variable region are present in a VH human framework. In some embodiments, the CDR1, CDR2 and CDR3 sequences in the second heavy chain variable region are present in a VH human framework. In some embodiments, the CDR1, CDR2 and CDR3 sequences in the first and second heavy chain variable regions are present in a VH human framework.

[0373] In some embodiments, the first heavy chain variable region has at least 95% (e.g., at least 96%, at least 97%, at least 98%, at least 99%) sequence identity to SEQ ID NO: 12, and the second heavy chain variable region has at least 95% (e.g., at least 96%, at least 97%, at least 98%, at least 99%) sequence identity to SEQ ID NO: 25. In some embodiments, the first heavy chain variable region comprises SEQ ID NO: 12, and the second heavy chain variable region comprises SEQ ID NO:25.

[0374] In some embodiments, the antibody is an isolated antibody. In some embodiments, the antibody is a human antibody. In some embodiments, the antibody is an isolated human antibody.

[0375] In some embodiments, the antibody is an intact IgG molecule. In some embodiments, the antibody is an intact IgG1 molecule. In some embodiments, the antibody is an intact IgG2 molecule. In some embodiments, the antibody is an intact IgG4 molecule. In some embodiments, the antibody is an immunologically active portion of an intact IgG molecule. In some embodiments, the antibody is an immunologically active portion of an intact IgG1 molecule. In some embodiments, the antibody is an immunologically active portion of an intact IgG2 molecule. In some embodiments, the antibody is an immunologically active portion of an intact IgG4 molecule. In some embodiments, the antibody is a triple chain antibody-like molecule. In some embodiments, the antibody is a heavy chain antibody.

[0376] In some embodiments, the antibody comprises an Fc region. In some embodiments, the antibody comprises a variant Fc region. In some embodiments, the variant Fc region has at least about 80% (e.g., at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%) homology to a native sequence Fc region.

[0377] In some embodiments, the variant Fc region comprises a heterodimerization modification. In some embodiments, the heterodimerization modification is a knob-and-hole substitution (e.g., in a variant IgG1 Fc region, 1) Y407T in one chain and T366Y in the other chain; 2) Y407A in one chain and T366W in the other chain; 3) F405A in one chain and T394W in the other chain; 4) F405W in one chain and T394S in the other chain; 5) Y407T in one chain and T366Y in the other chain; 6) T366Y in one chain and T394S in the other chain). and F405A in one chain and T394W and Y407T in the other chain; 7) T366W and F405W in one chain and T394S and Y407A in the other chain; 8) F405W and Y407A in one chain and T366W and T394S in the other chain; or 9) T366W in one polypeptide of Fc and T366S, L368A, and Y407V in the other chain. In some embodiments, heterodimerization modifications include substitutions that create new disulfide bridges (e.g., in a variant IgG1 Fc region, 1) Y349C in one Fc polypeptide chain and S354C in the other chain; 2) Y349C in one Fc polypeptide chain and E356C in the other chain; 3) Y349C in one Fc polypeptide chain and E357C in the other chain; 4) L351C in one Fc polypeptide chain and S354C in the other chain; 5) T394C in one Fc polypeptide chain and E397C in the other chain; or 6) D399C in one Fc polypeptide chain and K392C in the other chain).In some embodiments, the heterodimerization modification is a charge pair substitution (e.g., 1) K409E in one chain plus D399K in the other chain; 2) K409E in one chain plus D399R in the other chain; 3) K409D in one chain plus D399K in the other chain; 4) K409D in one chain plus D399R in the other chain; 5) K392E in one chain plus D399R in the other chain; 6) K392E in one chain plus D399K in the other chain; 7) K392D in one chain plus D399R in the other chain; 8) K392D in one chain plus D399K in the other chain; 9) K409D and K360D in one chain plus D399K and E356K in the other chain; 10) K409D and K 370D plus D399K and E357K in the other chain; 11) K409D and K392D in one chain plus D399K, E356K, and E357K in the other chain; 12) K409D and K392D in one chain and D399K in the other chain; 13) K409D and K392D in one chain plus D399K and E356K in the other chain; 14) K409D and K392D in one chain plus D399K and E356K in the other chain; 14) K409D and K392D in one chain plus D399K and D357K in the other chain; 15) K409D and K370D in one chain plus D399K and D357K in the other chain; 16) D399K in one chain plus K409D and K360D in the other chain; or 17) K409D and K439D in one chain plus D399K and E356K in the other chain.

[0378] In some embodiments, the Fc region is a silenced Fc region. In some embodiments, the silenced Fc region comprises one or more (e.g., two or more) substitutions of Fc region residues 238, 265, 269, 270, 297, 327, and 329 according to EU numbering. In some embodiments, the silenced Fc region comprises a substitution that alters glycosylation. In some embodiments, the silenced Fc region comprises an effectorless mutation (e.g., N297A, N297G, DANA mutation (D265A+N297A) or DANG mutation (D265A+N297G) in the CH2 region). In some embodiments, the silenced Fc region comprises K322A and L234A / L235A mutations.

[0379] In some embodiments, the antibody further comprises a heavy chain constant region sequence in the absence of a CH1 sequence. In some embodiments, the antibody comprises a heavy chain constant region comprising a hinge region, a CH2 domain, and a CH3 domain. In some embodiments, the hinge region comprises a wild-type human IgG4 hinge region sequence (SEQ ID NO: 54). In some embodiments, the hinge region comprises a variant human IgG4 hinge region sequence (SEQ ID NO: 55) comprising a S228P mutation. In some embodiments, the CH2 domain comprises a wild-type human IgG4 CH2 domain sequence (SEQ ID NO: 56). In some embodiments, the CH2 domain comprises a variant human IgG4 CH2 domain comprising a F234A mutation, a L235A mutation, or both a F234A mutation and a L235A mutation. In some embodiments, the CH3 domain comprises a wild-type human IgG4 CH3 domain sequence (SEQ ID NO: 58). In some embodiments, the CH3 domain comprises a variant human IgG4 CH3 domain sequence comprising a T366W mutation. In some embodiments, the CH3 domain comprises a variant human IgG4 CH3 domain sequence comprising a T366S, an L368A mutation, and a Y407V mutation.

[0380] In some embodiments, the antibody comprises a first polypeptide comprising SEQ ID NO:36 and a second polypeptide comprising SEQ ID NO:37.

[0381] In some embodiments, the antibody has a Tm of about 55° C. to about 65° C. In some embodiments, the antibody has a Tag of about 55° C. to about 65° C. In some embodiments, the antibody has a Tm of about 55° C. to about 65° C. and a Tag of about 55° C. to about 65° C.

[0382] In some embodiments, the antibody is -11 M ~ about 10 -6 M (for example, about 10 -10 M ~ about 10 -6 Around M; about 10 -9 M ~ about 10 -6 Around M; about 10 -8 M ~ about 10 -6 Around M; about 10 -11 M ~ about 10 -8 Around M; about 10 -10 M ~ about 10 -8 Around M; about 10 -9 M ~ about 10 -8 Around M; about 10 -11 M ~ about 10 -9 Around M; about 10 -10 M ~ about 10 -9 It has affinity for IL2R with a Kd of approximately 1 M.

[0383] In some embodiments, the antibody is -8 M ~ approx. 2.5×10 -7 It has affinity for IL2RB with a Kd of around M.

[0384] In some embodiments, the antibody is -9 M ~ approx. 2.5×10 -8 It has affinity for IL2RG with a Kd of around M.

[0385] In some embodiments, the antibody is -8 M ~ approx. 2.5×10 -7 The affinity for IL2RB is approximately 10 -9M ~ approx. 2.5×10 -8 It has affinity for IL2RG with a Kd of around M.

[0386] In some embodiments, Kd is measured using a ForteBio Octet Qk384 instrument in kinetic mode. In some embodiments, Kd is measured using a ForteBio Octet Qk384 instrument with an anti-human Fc capture (AHC, 18-5005) sensor in kinetic mode. In some embodiments, Kd is measured according to the method described in the Examples herein.

[0387] In conventional IgG antibodies, the association of the heavy and light chains is due in part to hydrophobic interactions between the light chain constant region and the CH1 constant domain of the heavy chain. There are additional residues in the framework 2 (FR2) and framework 4 (FR4) regions of the heavy chain that also contribute to this hydrophobic interaction between the heavy and light chains.

[0388] However, it is known that the sera of camelids (the suborder Tylopoda, which includes camels, dromedaries and llamas) contain a major type of antibody composed only of paired H chains (heavy chain antibodies or HCAbs).The heavy chain antibodies of Camelidae (Camelus dromedarius, Camelus bactrianus, Lama glama, Lama guanaco, Lama alpaca and Lama vicugna) have a unique structure consisting of a single variable domain (VHH), a hinge region and two constant domains (CH2 and CH3) that are highly homologous to the CH2 and CH3 domains of classical antibodies. These heavy chain antibodies lack the first domain (CH1) of the constant region, which is present in the genome but is spliced ​​out during mRNA processing. The absence of the CH1 domain explains the absence of light chains in heavy chain antibodies, since this domain is the anchor position for the constant domain of the light chain. Such heavy chain antibodies have naturally evolved to confer antigen binding specificity and high affinity by three CDRs from a conventional antibody or a fragment thereof. Muyldermans, 2001; J Biotechnol 74:277-302; Revets et al., 2005; Expert Opin Biol Ther 5:111-124. Cartilaginous fishes such as sharks have also evolved a unique type of immunoglobulin, called IgNAR, which lacks light chain polypeptides and is entirely composed of heavy chains. IgNAR molecules can be engineered by molecular engineering to create variable domains of a single heavy chain polypeptide (vNAR). Nuttall et al.Eur.J.Biochem.270,3543-3554(2003);Nuttall et al.Function and Bioinformatics 55,187-197(2004);Dooley et al.,Molecular Immunology 40,25-33(2003).

[0389] The ability of heavy chain antibodies devoid of light chains to bind antigen was established in the 1960s (Jaton et al. (1968) Biochemistry, 7, 4185-4195). Heavy chain immunoglobulins physically separated from light chains retained 80% of the antigen-binding activity of tetrameric antibodies. Sitia et al. (1990) Cell, 60, 781-790 showed that removal of the CH1 domain from a rearranged mouse μ gene resulted in the production of heavy chain antibodies devoid of light chains in mammalian cell culture. The antibodies produced retained VH binding specificity and effector functions.

[0390] Heavy chain antibodies with high specificity and affinity can be generated against various antigens through immunization (van der Linden, RH, et al. Biochim. Biophys. Acta. 1431, 37-46 (1999)) and VHH moieties can be easily cloned and expressed in yeast (Frenken, LG J, et al. J. Biotechnol. 78, 11-21 (2000)). Their expression, solubility and stability levels are significantly higher than classical F(ab) or Fv fragments. Ghahroudi, M A. et al. FEBS Lett. 414, 521-526 (1997).

[0391] Mice functionally silenced at the lambda (lambda) light chain (L) locus and / or at the lambda and kappa (kappa) light chain loci and antibodies produced by such mice are described in U.S. Patent Nos. 7,541,513 and 8,367,888. Recombinant production of heavy chain antibodies in mice and rats has been reported, for example, in WO 2006008548, U.S. Patent Publication No. 20100122358, Nguyen et al., 2003, Immunology; 109(1), 93-101; Bruggemann et al., Crit. Rev. Immunol.; 2006, 26(5):377-90; and Zou et al., 2007, J Exp Med; 204(13):3271-3283. The generation of knockout rats by embryonic microinjection of zinc finger nucleases is described in Geurts et al., 2009, Science, 325(5939):433. Soluble heavy chain antibodies and transgenic rodents containing heterologous heavy chain loci producing such antibodies are described in U.S. Patents 8,883,150 and 9,365,655. CAR-T structures containing single domain antibodies as binding (targeting) domains are described, for example, in Iri-Sofla et al., 2011, Experimental Cell Research 317:2630-2641 and Jamnani et al., 2014, Biochim Biophys Acta, 1840:378-386.

[0392] Embodiments of the present disclosure include heavy chain antibodies that bind to IL2RB, comprising a heavy chain variable region comprising: (a) a CDR1 sequence having two or less (e.g., 0, 1 or 2) substitutions in any one of SEQ ID NOs: 1 to 3; and / or (b) a CDR2 sequence having two or less (e.g., 0, 1 or 2) substitutions in any one of SEQ ID NOs: 4 to 6; and / or (c) a CDR3 sequence having two or less (e.g., 0, 1 or 2) substitutions in any one of SEQ ID NOs: 7 to 10.

[0393] In some embodiments, the heavy chain antibody comprises: (a) a CDR1 sequence comprising any one of SEQ ID NOs: 1-3; and / or (b) a CDR2 sequence comprising any one of SEQ ID NOs: 4-6; and / or (c) a CDR3 sequence comprising any one of SEQ ID NOs: 7-10.

[0394] In some embodiments, the heavy chain antibody comprises: (a) a CDR1 sequence comprising any one of SEQ ID NOs: 1-3; and (b) a CDR2 sequence comprising any one of SEQ ID NOs: 4-6; and (c) a CDR3 sequence comprising any one of SEQ ID NOs: 7-10.

[0395] In some embodiments, the heavy chain antibody comprises: (a) the CDR1 sequence of SEQ ID NO: 1, the CDR2 sequence of SEQ ID NO: 4 and the CDR3 sequence of SEQ ID NO: 7; or (b) the CDR1 sequence of SEQ ID NO: 1, the CDR2 sequence of SEQ ID NO: 4 and the CDR3 sequence of SEQ ID NO: 8; or (c) the CDR1 sequence of SEQ ID NO: 2, the CDR2 sequence of SEQ ID NO: 5, and the CDR3 sequence of SEQ ID NO: 9; or (d) the CDR1 sequence of SEQ ID NO: 3, the CDR2 sequence of SEQ ID NO: 6, and the CDR3 sequence of SEQ ID NO: 10 Includes.

[0396] In some embodiments, the CDR1, CDR2 and CDR3 sequences are present in a human VH framework.

[0397] In some embodiments, the heavy chain antibody comprises a heavy chain variable region comprising a sequence having six or fewer (e.g., five or fewer, four or fewer, three or fewer, two or fewer; 6, 5, 4, 3, 2, 1, 0) substitutions in any one of SEQ ID NOs: 11-14. In some embodiments, the heavy chain antibody comprises a heavy chain variable region having at least 95% (e.g., at least 96%, at least 97%, at least 98%, at least 99%) sequence identity to any one of SEQ ID NOs: 11-14. In some embodiments, the heavy chain antibody comprises a heavy chain variable region sequence selected from the group consisting of SEQ ID NOs: 11-14.

[0398] In some embodiments, the heavy chain antibody comprises an Fc region. In some embodiments, the heavy chain antibody comprises a variant Fc region. In some embodiments, the variant Fc region has at least about 80% homology (e.g., at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%) with a native sequence Fc region.

[0399] In some embodiments, the variant Fc region comprises a heterodimerization modification. In some embodiments, the heterodimerization modification is a knob-and-hole substitution (e.g., in a variant IgG1 Fc region, 1) Y407T in one chain and T366Y in the other chain; 2) Y407A in one chain and T366W in the other chain; 3) F405A in one chain and T394W in the other chain; 4) F405W in one chain and T394S in the other chain; 5) Y407T in one chain and T366Y in the other chain; 6) T366Y in one chain and T394S in the other chain). and F405A in one chain and T394W and Y407T in the other chain; 7) T366W and F405W in one chain and T394S and Y407A in the other chain; 8) F405W and Y407A in one chain and T366W and T394S in the other chain; or 9) T366W in one polypeptide of Fc and T366S, L368A, and Y407V in the other chain. In some embodiments, heterodimerization modifications include substitutions that create new disulfide bridges (e.g., in a variant IgG1 Fc region, 1) Y349C in one Fc polypeptide chain and S354C in the other chain; 2) Y349C in one Fc polypeptide chain and E356C in the other chain; 3) Y349C in one Fc polypeptide chain and E357C in the other chain; 4) L351C in one Fc polypeptide chain and S354C in the other chain; 5) T394C in one Fc polypeptide chain and E397C in the other chain; or 6) D399C in one Fc polypeptide chain and K392C in the other chain).In some embodiments, the heterodimerization modification is a charge pair substitution (e.g., 1) K409E in one chain plus D399K in the other chain; 2) K409E in one chain plus D399R in the other chain; 3) K409D in one chain plus D399K in the other chain; 4) K409D in one chain plus D399R in the other chain; 5) K392E in one chain plus D399R in the other chain; 6) K392E in one chain plus D399K in the other chain; 7) K392D in one chain plus D399R in the other chain; 8) K392D in one chain plus D399K in the other chain; 9) K409D and K360D in one chain plus D399K and E356K in the other chain; 10) K409D and K 370D plus D399K and E357K in the other chain; 11) K409D and K392D in one chain plus D399K, E356K, and E357K in the other chain; 12) K409D and K392D in one chain and D399K in the other chain; 13) K409D and K392D in one chain plus D399K and E356K in the other chain; 14) K409D and K392D in one chain plus D399K and E356K in the other chain; 14) K409D and K392D in one chain plus D399K and D357K in the other chain; 15) K409D and K370D in one chain plus D399K and D357K in the other chain; 16) D399K in one chain plus K409D and K360D in the other chain; or 17) K409D and K439D in one chain plus D399K and E356K in the other chain.

[0400] In some embodiments, the Fc region is a silenced Fc region. In some embodiments, the silenced Fc region comprises one or more (e.g., two or more) substitutions of Fc region residues 238, 265, 269, 270, 297, 327, and 329 according to EU numbering. In some embodiments, the silenced Fc region comprises a substitution that alters glycosylation. In some embodiments, the silenced Fc region comprises an effectorless mutation (e.g., N297A, N297G, DANA mutation (D265A+N297A) or DANG mutation (D265A+N297G) in the CH2 region). In some embodiments, the silenced Fc region comprises K322A and L234A / L235A mutations.

[0401] In some embodiments, the heavy chain antibody further comprises a heavy chain constant region sequence in the absence of a CH1 sequence. In some embodiments, the heavy chain antibody comprises a heavy chain constant region comprising a hinge region, a CH2 domain, and a CH3 domain. In some embodiments, the hinge region comprises a wild-type human IgG4 hinge region sequence (SEQ ID NO: 54). In some embodiments, the hinge region comprises a variant human IgG4 hinge region sequence (SEQ ID NO: 55) comprising a S228P mutation. In some embodiments, the CH2 domain comprises a wild-type human IgG4 CH2 domain sequence (SEQ ID NO: 56). In some embodiments, the CH2 domain comprises a variant human IgG4 CH2 domain comprising a F234A mutation, a L235A mutation, or both a F234A mutation and a L235A mutation. In some embodiments, the CH3 domain comprises a wild-type human IgG4 CH3 domain sequence (SEQ ID NO: 58). In some embodiments, the CH3 domain comprises a variant human IgG4 CH3 domain sequence comprising a T366W mutation. In some embodiments, the CH3 domain comprises a variant human IgG4 CH3 domain sequence comprising a T366S, an L368A mutation, and a Y407V mutation.

[0402] In some embodiments, the heavy chain antibody has a Tm of about 55° C. to about 65° C. In some embodiments, the heavy chain antibody has a Tag of about 55° C. to about 65° C. In some embodiments, the heavy chain antibody has a Tm of about 55° C. to about 65° C. and a Tag of about 55° C. to about 65° C.

[0403] In some embodiments, the heavy chain antibody is multispecific. In some embodiments, the heavy chain antibody is bispecific. In some embodiments, the heavy chain antibody binds to IL2RB and IL2RG. In some embodiments, the heavy chain antibody functions as an IL2 receptor beta / gamma agonist.

[0404] In some embodiments, the heavy chain antibody has a length of about 10 -11 M ~ about 10 -6 M (for example, about 10 -10 M ~ about 10 -6 Around M; about 10 -9 M ~ about 10 -6 Around M; about 10 -8 M ~ about 10 -6 Around M; about 10 -11 M ~ about 10 -8 Around M; about 10 -10 M ~ about 10 -8 Around M; about 10 -9 M ~ about 10 -8 Around M; about 10 -11 M ~ about 10 -9 Around M; about 10 -10 M ~ about 10 -9 It has affinity for IL2R with a Kd of approximately 1 M.

[0405] In some embodiments, the heavy chain antibody has a length of about 10 -8 M ~ approx. 2.5×10 -7 It has affinity for IL2RB with a Kd of around M.

[0406] In some embodiments, the heavy chain antibody has a length of about 10 -9 M ~ approx. 2.5×10 -8 It has affinity for IL2RG with a Kd of around M.

[0407] In some embodiments, the heavy chain antibody has a length of about 10 -8 M ~ approx. 2.5×10 -7 The affinity for IL2RB is approximately 10 -9 M ~ approx. 2.5×10 -8 It has affinity for IL2RG with a Kd of around M.

[0408] In some embodiments, Kd is measured using a ForteBio Octet Qk384 instrument in kinetic mode. In some embodiments, Kd is measured using a ForteBio Octet Qk384 instrument with an anti-human Fc capture (AHC, 18-5005) sensor in kinetic mode. In some embodiments, Kd is measured according to the method described in the Examples herein.

[0409] An aspect of the present disclosure is (a) Formula: GGSISSS X1 W (SEQ ID NO: 26) (wherein X1 is D or N); and Formula (b): I X2 HSGST (SEQ ID NO:27) wherein X2 is D or S; and Formula (c): X3 RG X4 WEL X5 DAFDI (SEQ ID NO: 28) wherein X3 is G or A; X4 is S or Q; and X5 is S or T; The present invention also includes a heavy chain antibody that binds to IL2RB, comprising a heavy chain variable region comprising:

[0410] In some embodiments, the CDR1, CDR2 and CDR3 sequences are present in a VH human framework.

[0411] In some embodiments, the heavy chain antibody comprises an Fc region. In some embodiments, the heavy chain antibody comprises a variant Fc region. In some embodiments, the variant Fc region has at least about 80% (e.g., at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%) homology to a native sequence Fc region.

[0412] In some embodiments, the variant Fc region comprises a heterodimerization modification. In some embodiments, the heterodimerization modification is a knob-and-hole substitution (e.g., in a variant IgG1 Fc region, 1) Y407T in one chain and T366Y in the other chain; 2) Y407A in one chain and T366W in the other chain; 3) F405A in one chain and T394W in the other chain; 4) F405W in one chain and T394S in the other chain; 5) Y407T in one chain and T366Y in the other chain; 6) T366Y in one chain and T394S in the other chain). and F405A in one chain and T394W and Y407T in the other chain; 7) T366W and F405W in one chain and T394S and Y407A in the other chain; 8) F405W and Y407A in one chain and T366W and T394S in the other chain; or 9) T366W in one polypeptide of Fc and T366S, L368A, and Y407V in the other chain. In some embodiments, heterodimerization modifications include substitutions that create new disulfide bridges (e.g., in a variant IgG1 Fc region, 1) Y349C in one Fc polypeptide chain and S354C in the other chain; 2) Y349C in one Fc polypeptide chain and E356C in the other chain; 3) Y349C in one Fc polypeptide chain and E357C in the other chain; 4) L351C in one Fc polypeptide chain and S354C in the other chain; 5) T394C in one Fc polypeptide chain and E397C in the other chain; or 6) D399C in one Fc polypeptide chain and K392C in the other chain).In some embodiments, the heterodimerization modification is a charge pair substitution (e.g., 1) K409E in one chain plus D399K in the other chain; 2) K409E in one chain plus D399R in the other chain; 3) K409D in one chain plus D399K in the other chain; 4) K409D in one chain plus D399R in the other chain; 5) K392E in one chain plus D399R in the other chain; 6) K392E in one chain plus D399K in the other chain; 7) K392D in one chain plus D399R in the other chain; 8) K392D in one chain plus D399K in the other chain; 9) K409D and K360D in one chain plus D399K and E356K in the other chain; 10) K409D and K 370D plus D399K and E357K in the other chain; 11) K409D and K392D in one chain plus D399K, E356K, and E357K in the other chain; 12) K409D and K392D in one chain and D399K in the other chain; 13) K409D and K392D in one chain plus D399K and E356K in the other chain; 14) K409D and K392D in one chain plus D399K and E356K in the other chain; 14) K409D and K392D in one chain plus D399K and D357K in the other chain; 15) K409D and K370D in one chain plus D399K and D357K in the other chain; 16) D399K in one chain plus K409D and K360D in the other chain; or 17) K409D and K439D in one chain plus D399K and E356K in the other chain.

[0413] In some embodiments, the Fc region is a silenced Fc region. In some embodiments, the silenced Fc region comprises one or more (e.g., two or more) substitutions of Fc region residues 238, 265, 269, 270, 297, 327, and 329 according to EU numbering. In some embodiments, the silenced Fc region comprises a substitution that alters glycosylation. In some embodiments, the silenced Fc region comprises an effectorless mutation (e.g., N297A, N297G, DANA mutation (D265A+N297A) or DANG mutation (D265A+N297G) in the CH2 region). In some embodiments, the silenced Fc region comprises K322A and L234A / L235A mutations.

[0414] In some embodiments, the heavy chain antibody further comprises a heavy chain constant region sequence in the absence of a CH1 sequence. In some embodiments, the heavy chain antibody comprises a heavy chain constant region comprising a hinge region, a CH2 domain, and a CH3 domain. In some embodiments, the hinge region comprises a wild-type human IgG4 hinge region sequence (SEQ ID NO: 54). In some embodiments, the hinge region comprises a variant human IgG4 hinge region sequence (SEQ ID NO: 55) comprising a S228P mutation. In some embodiments, the CH2 domain comprises a wild-type human IgG4 CH2 domain sequence (SEQ ID NO: 56). In some embodiments, the CH2 domain comprises a variant human IgG4 CH2 domain comprising a F234A mutation, a L235A mutation, or both a F234A mutation and a L235A mutation. In some embodiments, the CH3 domain comprises a wild-type human IgG4 CH3 domain sequence (SEQ ID NO: 58). In some embodiments, the CH3 domain comprises a variant human IgG4 CH3 domain sequence comprising a T366W mutation. In some embodiments, the CH3 domain comprises a variant human IgG4 CH3 domain sequence comprising a T366S, an L368A mutation, and a Y407V mutation.

[0415] In some embodiments, the heavy chain antibody has a Tm of about 55° C. to about 65° C. In some embodiments, the heavy chain antibody has a Tag of about 55° C. to about 65° C. In some embodiments, the heavy chain antibody has a Tm of about 55° C. to about 65° C. and a Tag of about 55° C. to about 65° C.

[0416] In some embodiments, the heavy chain antibody is multispecific. In some embodiments, the heavy chain antibody is bispecific. In some embodiments, the heavy chain antibody binds to IL2RB and IL2RG. In some embodiments, the heavy chain antibody functions as an IL2 receptor beta / gamma agonist.

[0417] In some embodiments, the heavy chain antibody has a length of about 10 -11 M ~ about 10 -6 M (for example, about 10 -10 M ~ about 10 -6 Around M; about 10 -9 M ~ about 10 -6 Around M; about 10 -8 M ~ about 10 -6 Around M; about 10 -11 M ~ about 10 -8 Around M; about 10 -10 M ~ about 10 -8 Around M; about 10 -9 M ~ about 10 -8 Around M; about 10 -11 M ~ about 10 -9 Around M; about 10 -10 M ~ about 10 -9 It has affinity for IL2R with a Kd of approximately 1 M.

[0418] In some embodiments, the heavy chain antibody has a length of about 10 -8 M ~ approx. 2.5×10 -7 It has affinity for IL2RB with a Kd of around M.

[0419] In some embodiments, the heavy chain antibody has a length of about 10 -9 M ~ approx. 2.5×10 -8 It has affinity for IL2RG with a Kd of around M.

[0420] In some embodiments, the heavy chain antibody has a length of about 10 -8 M ~ approx. 2.5×10 -7 The affinity for IL2RB is approximately 10 -9 M ~ approx. 2.5×10 -8 It has affinity for IL2RG with a Kd of around M.

[0421] In some embodiments, Kd is measured using a ForteBio Octet Qk384 instrument in kinetic mode. In some embodiments, Kd is measured using a ForteBio Octet Qk384 instrument with an anti-human Fc capture (AHC, 18-5005) sensor in kinetic mode. In some embodiments, Kd is measured according to the method described in the Examples herein.

[0422] An aspect of the present disclosure is (a) Formula: GFTFS X1 YG (SEQ ID NO: 29) (wherein X1 is S or T); and Formula (b): ISYDGSN X2 (SEQ ID NO: 30) (wherein X2 is K or R); and Formula (c): ARDLDYD X3 LTGDPVGGFDI (SEQ ID NO: 31) (wherein X3 is V or I); and The present invention also includes a heavy chain antibody that binds to IL2RB, comprising a heavy chain variable region comprising:

[0423] In some embodiments, the CDR1, CDR2 and CDR3 sequences are present in a VH human framework.

[0424] In some embodiments, the heavy chain antibody comprises an Fc region. In some embodiments, the heavy chain antibody comprises a variant Fc region. In some embodiments, the variant Fc region has at least about 80% (e.g., at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%) homology to a native sequence Fc region.

[0425] In some embodiments, the variant Fc region comprises a heterodimerization modification. In some embodiments, the heterodimerization modification is a knob-and-hole substitution (e.g., in a variant IgG1 Fc region, 1) Y407T in one chain and T366Y in the other chain; 2) Y407A in one chain and T366W in the other chain; 3) F405A in one chain and T394W in the other chain; 4) F405W in one chain and T394S in the other chain; 5) Y407T in one chain and T366Y in the other chain; 6) T366Y in one chain and T394S in the other chain). and F405A in one chain and T394W and Y407T in the other chain; 7) T366W and F405W in one chain and T394S and Y407A in the other chain; 8) F405W and Y407A in one chain and T366W and T394S in the other chain; or 9) T366W in one polypeptide of Fc and T366S, L368A, and Y407V in the other chain. In some embodiments, heterodimerization modifications include substitutions that create new disulfide bridges (e.g., in a variant IgG1 Fc region, 1) Y349C in one Fc polypeptide chain and S354C in the other chain; 2) Y349C in one Fc polypeptide chain and E356C in the other chain; 3) Y349C in one Fc polypeptide chain and E357C in the other chain; 4) L351C in one Fc polypeptide chain and S354C in the other chain; 5) T394C in one Fc polypeptide chain and E397C in the other chain; or 6) D399C in one Fc polypeptide chain and K392C in the other chain).In some embodiments, the heterodimerization modification is a charge pair substitution (e.g., 1) K409E in one chain plus D399K in the other chain; 2) K409E in one chain plus D399R in the other chain; 3) K409D in one chain plus D399K in the other chain; 4) K409D in one chain plus D399R in the other chain; 5) K392E in one chain plus D399R in the other chain; 6) K392E in one chain plus D399K in the other chain; 7) K392D in one chain plus D399R in the other chain; 8) K392D in one chain plus D399K in the other chain; 9) K409D and K360D in one chain plus D399K and E356K in the other chain; 10) K409D and K 370D plus D399K and E357K in the other chain; 11) K409D and K392D in one chain plus D399K, E356K, and E357K in the other chain; 12) K409D and K392D in one chain and D399K in the other chain; 13) K409D and K392D in one chain plus D399K and E356K in the other chain; 14) K409D and K392D in one chain plus D399K and E356K in the other chain; 14) K409D and K392D in one chain plus D399K and D357K in the other chain; 15) K409D and K370D in one chain plus D399K and D357K in the other chain; 16) D399K in one chain plus K409D and K360D in the other chain; or 17) K409D and K439D in one chain plus D399K and E356K in the other chain.

[0426] In some embodiments, the Fc region is a silenced Fc region. In some embodiments, the silenced Fc region comprises a substitution at one or more (e.g., two or more) of Fc region residues 238, 265, 269, 270, 297, 327, and 329 according to EU numbering. In some embodiments, the silenced Fc region comprises a substitution that alters glycosylation. In some embodiments, the silenced Fc region comprises an effectorless mutation (e.g., N297A, N297G, a DANA mutation (D265A+N297A) or a DANG mutation (D265A+N297G) in the CH2 region. In some embodiments, the silenced Fc region comprises K322A and L234A / L235A mutations.

[0427] In some embodiments, the heavy chain antibody further comprises a heavy chain constant region sequence in the absence of a CH1 sequence. In some embodiments, the heavy chain antibody comprises a heavy chain constant region comprising a hinge region, a CH2 domain, and a CH3 domain. In some embodiments, the hinge region comprises a wild-type human IgG4 hinge region sequence (SEQ ID NO: 54). In some embodiments, the hinge region comprises a variant human IgG4 hinge region sequence (SEQ ID NO: 55) comprising a S228P mutation. In some embodiments, the CH2 domain comprises a wild-type human IgG4 CH2 domain sequence (SEQ ID NO: 56). In some embodiments, the CH2 domain comprises a variant human IgG4 CH2 domain comprising a F234A mutation, a L235A mutation, or both a F234A mutation and a L235A mutation. In some embodiments, the CH3 domain comprises a wild-type human IgG4 CH3 domain sequence (SEQ ID NO: 58). In some embodiments, the CH3 domain comprises a variant human IgG4 CH3 domain comprising a T366W mutation. In some embodiments, the CH3 domain comprises a variant human IgG4 CH3 domain sequence comprising a T366S, an L368A mutation, and a Y407V mutation.

[0428] In some embodiments, the heavy chain antibody has a Tm of about 55° C. to about 65° C. In some embodiments, the heavy chain antibody has a Tag of about 55° C. to about 65° C. In some embodiments, the heavy chain antibody has a Tm of about 55° C. to about 65° C. and a Tag of about 55° C. to about 65° C.

[0429] In some embodiments, a heavy chain antibody is multispecific. In some embodiments, a heavy chain antibody is bispecific. In some embodiments, a heavy chain antibody binds to IL2RB and IL2RG. In some embodiments, a heavy chain antibody functions as an IL2 receptor beta / gamma agonist.

[0430] In some embodiments, the heavy chain antibody has a length of about 10 -11 M~10 -6 M (for example, about 10 -10 M~10 -6 Around M; about 10 -9 M~10 -6 Around M; about 10 -8 M~10 -6 Around M; about 10 -11 M~10 -8 Around M; about 10 -10 M~10 -8 Around M; about 10 -9 M~10 -8 Around M; about 10 -11 M~10 -9 Around M; about 10 -10 M~10 -9 It has affinity for IL2R with a Kd of approximately 1 M.

[0431] In some embodiments, the heavy chain antibody has a length of about 10 -8 M~2.5×10 -7 It has affinity for IL2RB with a Kd of around M.

[0432] In some embodiments, the heavy chain antibody has a length of about 10 -9 M~2.5×10 -8 It has affinity for IL2RG with a Kd of around M.

[0433] In some embodiments, the heavy chain antibody has a length of about 10 -8 M~2.5×10 -7 It has an affinity for IL2RB with a Kd of about 10 -9 M~2.5×10 -8 It has affinity for IL2RG with a Kd of around M.

[0434] In some embodiments, Kd is measured in kinetic mode using a ForteBio Octet Qk384 instrument. In some embodiments, Kd is measured in kinetic mode using a ForteBio Octet Qk384 instrument equipped with an anti-human Fc capture (AHC, 18-5005) sensor. In some embodiments, Kd is measured according to the method described in the Examples herein.

[0435] Embodiments of the present disclosure include heavy chain antibodies that bind to IL2RG, comprising a heavy chain variable region comprising: (a) a CDR1 sequence having two or less (e.g., 0, 1 or 2) substitutions in any one of SEQ ID NOs: 15 to 16; and / or (b) a CDR2 sequence having two or less (e.g., 0, 1 or 2) substitutions in any one of SEQ ID NOs: 17 to 19; and / or (c) a CDR3 sequence having two or less (e.g., 0, 1 or 2) substitutions in any one of SEQ ID NOs: 20 to 21.

[0436] In some embodiments, the heavy chain antibody comprises: (a) a CDR1 sequence comprising any one of SEQ ID NOs: 15-16; and / or (b) a CDR2 sequence comprising any one of SEQ ID NOs: 17-19; and / or (c) a CDR3 sequence comprising any one of SEQ ID NOs: 20-21.

[0437] In some embodiments, the heavy chain antibody comprises: (a) a CDR1 sequence comprising any one of SEQ ID NOs: 15-16; and (b) a CDR2 sequence comprising any one of SEQ ID NOs: 17-19; and (c) a CDR3 sequence comprising any one of SEQ ID NOs: 20-21.

[0438] In some embodiments, the heavy chain antibody comprises: (a) the CDR1 sequence of SEQ ID NO: 15, the CDR2 sequence of SEQ ID NO: 17, and the CDR3 sequence of SEQ ID NO: 20; or (b) the CDR1 sequence of SEQ ID NO: 15, the CDR2 sequence of SEQ ID NO: 18, and the CDR3 sequence of SEQ ID NO: 20; or (c) the CDR1 sequence of SEQ ID NO: 16, the CDR2 sequence of SEQ ID NO: 18, and the CDR3 sequence of SEQ ID NO: 20; or (d) the CDR1 sequence of SEQ ID NO: 15, the CDR2 sequence of SEQ ID NO: 19, and the CDR3 sequence of SEQ ID NO: 21 Includes.

[0439] In some embodiments, the CDR1, CDR2 and CDR3 sequences are present in a VH human framework.

[0440] In some embodiments, the heavy chain antibody comprises a heavy chain variable region comprising a sequence having six or fewer (e.g., five or fewer, four or fewer, three or fewer, two or fewer, six, five, four, three, two, one, zero) substitutions in any one of SEQ ID NOs: 22-25. In some embodiments, the heavy chain antibody comprises a heavy chain variable region having at least 95% (e.g., at least 96%, at least 97%, at least 98%, at least 99%) sequence identity to any one of SEQ ID NOs: 22-25. In some embodiments, the heavy chain antibody comprises a heavy chain variable region selected from the group consisting of SEQ ID NOs: 22-25.

[0441] In some embodiments, the heavy chain antibody comprises an Fc region. In some embodiments, the heavy chain antibody comprises a variant Fc region. In some embodiments, the variant Fc region retains at least about 80% (e.g., at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%) homology to a native sequence Fc region.

[0442] In some embodiments, the variant Fc region comprises heterodimerization modifications. In some embodiments, the heterodimerization modifications comprise knob-and-hole substitutions (e.g., in a variant IgG1 Fc region: 1) Y407T in one chain and T366Y in the other chain; 2) Y407A in one chain and T366W in the other chain; 3) F405A in one chain and T394W in the other chain; 4) F405W in one chain and T394S in the other chain; 5) Y407T in one chain and T366Y in the other chain; 6) F405S in one chain and T394S in the other chain; 7) T366W and F405W in one chain and T394S and Y407A in the other chain; 8) Y405W and Y407A in one chain and T366W and T394S in the other chain; or 9) T366W and T366S, L368A in one polypeptide of Fc and Y407V in the other. In some embodiments, heterodimerization modifications comprise substitutions that create new disulfide bridges (e.g., in a variant IgG1 Fc region: 1) Y349C in one Fc polypeptide chain and S354C in the other polypeptide chain; 2) Y349C in one Fc polypeptide chain and E356C in the other polypeptide chain; 3) Y349C in one Fc polypeptide chain and E357C in the other polypeptide chain; 4) L351C in one Fc polypeptide chain and S354C in the other polypeptide chain; 5) T394C in one Fc polypeptide chain and E397C in the other polypeptide chain; or 6) D399C in one Fc polypeptide chain and K392C in the other polypeptide chain).In some embodiments, heterodimerization modifications include charge pair substitutions (e.g., 1) K409E in one chain plus D399K in the other chain; 2) K409E in one chain plus D399R in the other chain; 3) K409D in one chain plus D399K in the other chain; 4) K409D in one chain plus D399R in the other chain; 5) K392E in one chain plus D399R in the other chain; 6) K392E in one chain plus D399K in the other chain; 7) K392D in one chain plus D399R in the other chain; 8) K392D in one chain plus D399K in the other chain; 9) K409D and K360D in one chain plus D399K and E356 in the other chain. K; 10) K409D and K370D in one chain plus D399K and E357K in the other chain; 11) K409D and K392D in one chain plus D399K, E356K, and E357K in the other chain; 12) K409D and K392D in one chain and D399K in the other chain; 13) K409D and K392D in one chain plus D399K and E356K; 14) K409D and K392D on one chain plus D399K and D357K on the other chain; 15) K409D and K370D on one chain plus D399K and D357K on the other chain; 16) D399K on one chain plus K409D and K360D on the other chain; or 17) K409D and K439D on one chain plus D399K and E356K on the other chain.

[0443] In some embodiments, the Fc region is a silenced Fc region. In some embodiments, the silenced Fc region comprises a substitution at one or more (e.g., two or more) of Fc region residues 238, 265, 269, 270, 297, 327, and 329 according to EU numbering. In some embodiments, the silenced Fc region comprises a substitution that alters glycosylation. In some embodiments, the silenced Fc region comprises an effectorless mutation (e.g., N297A, N297G, DANA mutation (D265A+N297A) or DANG mutation (D265A+N297G) in the CH2 region). In some embodiments, the silenced Fc region comprises K322A and L234A / L235A mutations.

[0444] In some embodiments, the heavy chain antibody further comprises a heavy chain constant region sequence in the absence of a CH1 sequence. In some embodiments, the heavy chain antibody comprises a heavy chain constant region comprising a hinge region, a CH2 domain, and a CH3 domain. In some embodiments, the hinge region comprises a wild-type human IgG4 hinge region sequence (SEQ ID NO: 54). In some embodiments, the hinge region comprises a variant human IgG4 hinge region sequence (SEQ ID NO: 55) comprising a S228P mutation. In some embodiments, the CH2 domain comprises a wild-type human IgG4 CH2 domain sequence (SEQ ID NO: 56). In some embodiments, the CH2 domain comprises a variant human IgG4 CH2 domain comprising a F234A mutation, a L235A mutation, or both a F234A mutation and a L235A mutation. In some embodiments, the CH3 domain comprises a wild-type human IgG4 CH3 domain sequence (SEQ ID NO: 58). In some embodiments, the CH3 domain comprises a variant human IgG4 CH3 domain comprising a T366W mutation. In some embodiments, the CH3 domain comprises a variant human IgG4 CH3 domain sequence comprising a T366S, an L368A mutation, and a Y407V mutation.

[0445] In some embodiments, the heavy chain antibody has a Tm of about 55° C. to about 65° C. In some embodiments, the heavy chain antibody has a Tag of about 55° C. to about 65° C. In some embodiments, the heavy chain antibody has a Tm of about 55° C. to about 65° C. and a Tag of about 55° C. to about 65° C.

[0446] In some embodiments, a heavy chain antibody is multispecific. In some embodiments, a heavy chain antibody is bispecific. In some embodiments, a heavy chain antibody binds to IL2RB and IL2RG. In some embodiments, a heavy chain antibody functions as an IL2 receptor beta / gamma agonist.

[0447] In some embodiments, the heavy chain antibody has a length of about 10 -11 M~10 -6 M (for example, about 10 -10 M~10 -6 Around M; about 10 -9 M~10 -6 Around M; about 10 -8 M~10 -6 Around M; about 10 -11 M~10 -8 Around M; about 10 -10 M~10 -8 Around M; about 10 -9 M~10 -8 Around M; about 10 -11 M~10 -9 Around M; about 10 -10 M~10 -9 It has affinity for IL2R with a Kd of approximately 1 M.

[0448] In some embodiments, the heavy chain antibody has a length of about 10 -8 M~2.5×10 -7 It has affinity for IL2RB with a Kd of around M.

[0449] In some embodiments, the heavy chain antibody has a length of about 10 -9 M~2.5×10 -8 It has affinity for IL2RG with a Kd of around M.

[0450] In some embodiments, the heavy chain antibody has a length of about 10 -8 M~2.5×10 -7 It has an affinity for IL2RB with a Kd of about 10 -9 M~2.5×10 -8 It has affinity for IL2RG with a Kd of around M.

[0451] In some embodiments, Kd is measured in kinetic mode using a ForteBio Octet Qk384 instrument. In some embodiments, Kd is measured in kinetic mode using a ForteBio Octet Qk384 instrument equipped with an anti-human Fc capture (AHC, 18-5005) sensor. In some embodiments, Kd is measured according to the method described in the Examples herein.

[0452] An embodiment of the disclosure is a heavy chain antibody that binds to IL2RG, comprising: (a) Formula: GF X1 X2 X3 X4 YY (SEQ ID NO: 32) wherein X1 is T or I; X2 is F or V; X3 is S, N or G; and X4 is D or N; Formula (b): IS X5 SG X6 X7 I (Sequence number 33) wherein X5 is S or N; X6 is D, S, G or N; and X7 is T or I; (c) a CDR3 sequence comprising the sequence ARGDAVSITGDY (SEQ ID NO: 20); The present invention also includes heavy chain antibodies, which include a heavy chain variable region comprising:

[0453] In some embodiments, the CDR1, CDR2 and CDR3 sequences are present in a VH human framework.

[0454] In some embodiments, the heavy chain antibody is multispecific. In some embodiments, the heavy chain antibody is bispecific. In some embodiments, the heavy chain antibody binds to IL2RB and IL2RG.

[0455] In some embodiments, the heavy chain antibody comprises an Fc region. In some embodiments, the heavy chain antibody further comprises a heavy chain constant region comprising a hinge region, a CH2 domain, and a CH3 domain. In some embodiments, the heavy chain constant region does not contain a CH1 sequence. In some embodiments, the hinge region comprises a wild-type human IgG4 hinge region sequence (SEQ ID NO: 54). In some embodiments, the hinge region comprises a variant human IgG4 hinge region sequence (SEQ ID NO: 55) comprising a S228P mutation. In some embodiments, the CH2 domain comprises a wild-type human IgG4 CH2 domain sequence (SEQ ID NO: 56). In some embodiments, the CH2 domain comprises a variant human IgG4 CH2 domain comprising a F234A mutation, a L235A mutation, or both a F234A mutation and a L235A mutation. In some embodiments, the CH3 domain comprises a wild-type human IgG4 CH3 domain sequence (SEQ ID NO: 58). In some embodiments, the CH3 domain comprises a variant human IgG4 CH3 domain comprising a T366W mutation. In some embodiments, the CH3 domain comprises a variant human IgG4 CH3 domain sequence comprising a T366S, an L368A mutation, and a Y407V mutation.

[0456] In some embodiments, the heavy chain antibody has a Tm of about 55° C. to about 65° C. In some embodiments, the heavy chain antibody has a Tag of about 55° C. to about 65° C. In some embodiments, the heavy chain antibody has a Tm of about 55° C. to about 65° C. and a Tag of about 55° C. to about 65° C.

[0457] In some embodiments, the heavy chain antibodies function as IL2 receptor beta / gamma agonists.

[0458] In some embodiments, the heavy chain antibody has a length of about 10 -11 M~10 -6 M (for example, about 10 -10 M~10 -6 Around M; about 10 -9 M~10 -6Around M; about 10 -8 M~10 -6 Around M; about 10 -11 M~10 -8 Around M; about 10 -10 M~10 -8 Around M; about 10 -9 M~10 -8 Around M; about 10 -11 M~10 -9 Around M; about 10 -10 M~10 -9 It has affinity for IL2R with a Kd of approximately 1 M.

[0459] In some embodiments, the heavy chain antibody has a length of about 10 -8 M~2.5×10 -7 It has affinity for IL2RB with a Kd of around M.

[0460] In some embodiments, the heavy chain antibody has a length of about 10 -9 M~2.5×10 -8 It has affinity for IL2RG with a Kd of around M.

[0461] In some embodiments, the heavy chain antibody has a length of about 10 -8 M~2.5×10 -7 It has an affinity for IL2RB with a Kd of about 10 -9 M~2.5×10 -8 It has affinity for IL2RG with a Kd of around M.

[0462] In some embodiments, Kd is measured in kinetic mode using a ForteBio Octet Qk384 instrument. In some embodiments, Kd is measured in kinetic mode using a ForteBio Octet Qk384 instrument equipped with an anti-human Fc capture (AHC, 18-5005) sensor. In some embodiments, Kd is measured according to the method described in the Examples herein.

[0463] An embodiment of the disclosure is a bispecific agonist anti-IL2R heavy chain antibody, comprising: A first heavy chain variable region that binds to IL2RB, comprising a CDR1 sequence of SEQ ID NO: 1, a CDR2 sequence of SEQ ID NO: 4, and a CDR3 sequence of SEQ ID NO: 7; a second heavy chain variable region that binds to IL2RG, comprising a CDR1 sequence of SEQ ID NO: 15, a CDR2 sequence of SEQ ID NO: 17, and a CDR3 sequence of SEQ ID NO: 20; The present invention also includes a bispecific agonist anti-IL2R heavy chain antibody comprising:

[0464] In some embodiments, the CDR1, CDR2 and CDR3 sequences in the first heavy chain variable region are present in a VH human framework. In some embodiments, the CDR1, CDR2 and CDR3 sequences in the second heavy chain variable region are present in a VH human framework. In some embodiments, the CDR1, CDR2 and CDR3 sequences in the first and second heavy chain variable regions are present in a VH human framework.

[0465] In some embodiments, the first heavy chain variable region has at least 95% (e.g., at least 96%, at least 97%, at least 98%, at least 99%) sequence identity to SEQ ID NO:11, and the second heavy chain variable region has at least 95% (e.g., at least 96%, at least 97%, at least 98%, at least 99%) sequence identity to SEQ ID NO:22. In some embodiments, the first heavy chain variable region comprises SEQ ID NO:11, and the second heavy chain variable region comprises SEQ ID NO:22.

[0466] In some embodiments, the bispecific agonist anti-IL2R heavy chain antibody comprises an Fc region. In some embodiments, the bispecific agonist anti-IL2R heavy chain antibody comprises a variant Fc region. In some embodiments, the variant Fc region retains at least about 80% (e.g., at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%) homology to a native sequence Fc region.

[0467] In some embodiments, the variant Fc region comprises heterodimerization modifications. In some embodiments, the heterodimerization modifications comprise knob-and-hole substitutions (e.g., in a variant IgG1 Fc region: 1) Y407T in one chain and T366Y in the other chain; 2) Y407A in one chain and T366W in the other chain; 3) F405A in one chain and T394W in the other chain; 4) F405W in one chain and T394S in the other chain; 5) Y407T in one chain and T366Y in the other chain; 6) F405S in one chain and T394S in the other chain; 7) T366W and F405W in one chain and T394S and Y407A in the other chain; 8) F405W and Y407A in one chain and T366W and T394S in the other chain; or 9) T366W and T366S, L368A in one polypeptide of Fc and Y407V in the other. In some embodiments, heterodimerization modifications comprise substitutions that create new disulfide bridges (e.g., in a variant IgG1 Fc region: 1) Y349C in one Fc polypeptide chain and S354C in the other polypeptide chain; 2) Y349C in one Fc polypeptide chain and E356C in the other polypeptide chain; 3) Y349C in one Fc polypeptide chain and E357C in the other polypeptide chain; 4) L351C in one Fc polypeptide chain and S354C in the other polypeptide chain; 5) T394C in one Fc polypeptide chain and E397C in the other polypeptide chain; or 6) D399C in one Fc polypeptide chain and K392C in the other polypeptide chain).In some embodiments, heterodimerization modifications include charge pair substitutions (e.g., 1) K409E in one chain plus D399K in the other chain; 2) K409E in one chain plus D399R in the other chain; 3) K409D in one chain plus D399K in the other chain; 4) K409D in one chain plus D399R in the other chain; 5) K392E in one chain plus D399R in the other chain; 6) K392E in one chain plus D399K in the other chain; 7) K392D in one chain plus D399R in the other chain; 8) K392D in one chain plus D399K in the other chain; 9) K409D and K360D in one chain plus D399K and E356 in the other chain. K; 10) K409D and K370D in one chain plus D399K and E357K in the other chain; 11) K409D and K392D in one chain plus D399K, E356K, and E357K in the other chain; 12) K409D and K392D in one chain and D399K in the other chain; 13) K409D and K392D in one chain plus D399K and E356K; 14) K409D and K392D on one chain plus D399K and D357K on the other chain; 15) K409D and K370D on one chain plus D399K and D357K on the other chain; 16) D399K on one chain plus K409D and K360D on the other chain; or 17) K409D and K439D on one chain plus D399K and E356K on the other chain.

[0468] In some embodiments, the Fc region is a silenced Fc region. In some embodiments, the silenced Fc region comprises a substitution at one or more (e.g., two or more) of Fc region residues 238, 265, 269, 270, 297, 327, and 329 according to EU numbering. In some embodiments, the silenced Fc region comprises a substitution that alters glycosylation. In some embodiments, the silenced Fc region comprises an effectorless mutation (e.g., N297A, N297G, DANA mutation (D265A+N297A) or DANG mutation (D265A+N297G) in the CH2 region). In some embodiments, the silenced Fc region comprises K322A and L234A / L235A mutations.

[0469] In some embodiments, the bispecific agonist anti-IL2R heavy chain antibody further comprises a heavy chain constant region sequence in the absence of a CH1 sequence. In some embodiments, the bispecific agonist anti-IL2R heavy chain antibody comprises a heavy chain constant region comprising a hinge region, a CH2 domain and a CH3 domain. In some embodiments, the hinge region comprises a wild-type human IgG4 hinge region sequence (SEQ ID NO: 54). In some embodiments, the hinge region comprises a variant human IgG4 hinge region sequence (SEQ ID NO: 55) comprising a S228P mutation. In some embodiments, the CH2 domain comprises a wild-type human IgG4 CH2 domain sequence (SEQ ID NO: 56). In some embodiments, the CH2 domain comprises a variant human IgG4 CH2 domain comprising a F234A mutation, a L235A mutation or both a F234A mutation and a L235A mutation. In some embodiments, the CH3 domain comprises a wild-type human IgG4 CH3 domain sequence (SEQ ID NO: 58). In some embodiments, the CH3 domain comprises a variant human IgG4 CH3 domain comprising a T366W mutation.In some embodiments, the CH3 domain comprises a variant human IgG4 CH3 domain sequence comprising a T366S, a L368A mutation, and a Y407V mutation.

[0470] In some embodiments, the bispecific agonist anti-IL2R heavy chain antibody comprises a first polypeptide comprising SEQ ID NO:53 and a second polypeptide comprising SEQ ID NO:61.

[0471] In some embodiments, the bispecific agonist anti-IL2R heavy chain antibody has a Tm of about 55° C. to about 65° C. In some embodiments, the bispecific agonist anti-IL2R heavy chain antibody has a Tag of about 55° C. to about 65° C. In some embodiments, the bispecific agonist anti-IL2R heavy chain antibody has a Tm of about 55° C. to about 65° C. and a Tag of about 55° C. to about 65° C.

[0472] In some embodiments, the bispecific agonist anti-IL2R heavy chain antibody is about 10 -11 M~10 -6 M (for example, about 10 -10 M~10 -6 Around M; about 10 -9 M~10 -6 Around M; about 10 -8 M~10 -6 Around M; about 10 -11 M~10 -8 Around M; about 10 -10 M~10 -8 Around M; about 10 -9 M~10 -8 Around M; about 10 -11 M~10 -9 Around M; about 10 -10 M~10 -9 It has affinity for IL2R with a Kd of approximately 1 M.

[0473] In some embodiments, the bispecific agonist anti-IL2R heavy chain antibody is about 10 -8 M~2.5×10 -7 It has affinity for IL2RB with a Kd of around M.

[0474] In some embodiments, the bispecific agonist anti-IL2R heavy chain antibody is about 10 -9 M~2.5×10 -8 It has affinity for IL2RG with a Kd of around M.

[0475] In some embodiments, the bispecific agonist anti-IL2R heavy chain antibody is about 10 -8 M~2.5×10 -7 It has an affinity for IL2RB with a Kd of about 10 -9 M~2.5×10 -8 It has affinity for IL2RG with a Kd of around M.

[0476] In some embodiments, Kd is measured in kinetic mode using a ForteBio Octet Qk384 instrument. In some embodiments, Kd is measured in kinetic mode using a ForteBio Octet Qk384 instrument equipped with an anti-human Fc capture (AHC, 18-5005) sensor. In some embodiments, Kd is measured according to the method described in the Examples herein.

[0477] An embodiment of the disclosure is a bispecific agonist anti-IL2R heavy chain antibody, comprising: A first heavy chain variable region that binds to IL2RB, comprising a CDR1 sequence of SEQ ID NO: 1, a CDR2 sequence of SEQ ID NO: 4, and a CDR3 sequence of SEQ ID NO: 8; a second heavy chain variable region that binds to IL2RG, comprising a CDR1 sequence of SEQ ID NO: 15, a CDR2 sequence of SEQ ID NO: 18, and a CDR3 sequence of SEQ ID NO: 20; The present invention also includes a bispecific agonist anti-IL2R heavy chain antibody comprising:

[0478] In some embodiments, the CDR1, CDR2 and CDR3 sequences in the first heavy chain variable region are present in a VH human framework. In some embodiments, the CDR1, CDR2 and CDR3 sequences in the second heavy chain variable region are present in a VH human framework. In some embodiments, the CDR1, CDR2 and CDR3 sequences in the first and second heavy chain variable regions are present in a VH human framework.

[0479] In some embodiments, the first heavy chain variable region has at least 95% (e.g., at least 96%, at least 97%, at least 98%, at least 99%) sequence identity to SEQ ID NO: 12, and the second heavy chain variable region has at least 95% (e.g., at least 96%, at least 97%, at least 98%, at least 99%) sequence identity to SEQ ID NO: 23. In some embodiments, the first heavy chain variable region comprises SEQ ID NO: 12, and the second heavy chain variable region comprises SEQ ID NO:23.

[0480] In some embodiments, the bispecific agonist anti-IL2R heavy chain antibody comprises an Fc region. In some embodiments, the bispecific agonist anti-IL2R heavy chain antibody comprises a variant Fc region. In some embodiments, the variant Fc region retains at least about 80% (e.g., at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%) homology to a native sequence Fc region.

[0481] In some embodiments, the variant Fc region comprises heterodimerization modifications. In some embodiments, the heterodimerization modifications comprise knob-and-hole substitutions (e.g., in a variant IgG1 Fc region: 1) Y407T in one chain and T366Y in the other chain; 2) Y407A in one chain and T366W in the other chain; 3) F405A in one chain and T394W in the other chain; 4) F405W in one chain and T394S in the other chain; 5) Y407T in one chain and T366Y in the other chain; 6) F405S in one chain and T394S in the other chain; 7) T366W and F405W in one chain and T394S and Y407A in the other chain; 8) F405W and Y407A in one chain and T366W and T394S in the other chain; or 9) T366W and T366S, L368A in one polypeptide of Fc and Y407V in the other. In some embodiments, heterodimerization modifications comprise substitutions that create new disulfide bridges (e.g., in a variant IgG1 Fc region: 1) Y349C in one Fc polypeptide chain and S354C in the other polypeptide chain; 2) Y349C in one Fc polypeptide chain and E356C in the other polypeptide chain; 3) Y349C in one Fc polypeptide chain and E357C in the other polypeptide chain; 4) L351C in one Fc polypeptide chain and S354C in the other polypeptide chain; 5) T394C in one Fc polypeptide chain and E397C in the other polypeptide chain; or 6) D399C in one Fc polypeptide chain and K392C in the other polypeptide chain).In some embodiments, heterodimerization modifications include charge pair substitutions (e.g., 1) K409E in one chain plus D399K in the other chain; 2) K409E in one chain plus D399R in the other chain; 3) K409D in one chain plus D399K in the other chain; 4) K409D in one chain plus D399R in the other chain; 5) K392E in one chain plus D399R in the other chain; 6) K392E in one chain plus D399K in the other chain; 7) K392D in one chain plus D399R in the other chain; 8) K392D in one chain plus D399K in the other chain; 9) K409D and K360D in one chain plus D399K and E356 in the other chain. K; 10) K409D and K370D in one chain plus D399K and E357K in the other chain; 11) K409D and K392D in one chain plus D399K, E356K, and E357K in the other chain; 12) K409D and K392D in one chain and D399K in the other chain; 13) K409D and K392D in one chain plus D399K and E356K; 14) K409D and K392D on one chain plus D399K and D357K on the other chain; 15) K409D and K370D on one chain plus D399K and D357K on the other chain; 16) D399K on one chain plus K409D and K360D on the other chain; or 17) K409D and K439D on one chain plus D399K and E356K on the other chain.

[0482] In some embodiments, the Fc region is a silenced Fc region. In some embodiments, the silenced Fc region comprises a substitution at one or more (e.g., two or more) of Fc region residues 238, 265, 269, 270, 297, 327, and 329 according to EU numbering. In some embodiments, the silenced Fc region comprises a substitution that alters glycosylation. In some embodiments, the silenced Fc region comprises an effectorless mutation (e.g., N297A, N297G, DANA mutation (D265A+N297A) or DANG mutation (D265A+N297G) in the CH2 region). In some embodiments, the silenced Fc region comprises K322A and L234A / L235A mutations.

[0483] In some embodiments, the bispecific agonist anti-IL2R heavy chain antibody further comprises a heavy chain constant region sequence in the absence of a CH1 sequence. In some embodiments, the bispecific agonist anti-IL2R heavy chain antibody comprises a heavy chain constant region comprising a hinge region, a CH2 domain and a CH3 domain. In some embodiments, the hinge region comprises a wild-type human IgG4 hinge region sequence (SEQ ID NO: 54). In some embodiments, the hinge region comprises a variant human IgG4 hinge region sequence (SEQ ID NO: 55) comprising a S228P mutation. In some embodiments, the CH2 domain comprises a wild-type human IgG4 CH2 domain sequence (SEQ ID NO: 56). In some embodiments, the CH2 domain comprises a variant human IgG4 CH2 domain comprising a F234A mutation, a L235A mutation or both a F234A mutation and a L235A mutation. In some embodiments, the CH3 domain comprises a wild-type human IgG4 CH3 domain sequence (SEQ ID NO: 58). In some embodiments, the CH3 domain comprises a variant human IgG4 CH3 domain comprising a T366W mutation.In some embodiments, the CH3 domain comprises a variant human IgG4 CH3 domain sequence comprising a T366S, a L368A mutation, and a Y407V mutation.

[0484] In some embodiments, the bispecific agonist anti-IL2R heavy chain antibody comprises a first polypeptide comprising SEQ ID NO:62 and a second polypeptide comprising SEQ ID NO:63.

[0485] In some embodiments, the bispecific agonist anti-IL2R heavy chain antibody has a Tm of about 55° C. to about 65° C. In some embodiments, the bispecific agonist anti-IL2R heavy chain antibody has a Tag of about 55° C. to about 65° C. In some embodiments, the bispecific agonist anti-IL2R heavy chain antibody has a Tm of about 55° C. to about 65° C. and a Tag of about 55° C. to about 65° C.

[0486] In some embodiments, the bispecific agonist anti-IL2R heavy chain antibody is about 10 -11 M~10 -6 M (for example, about 10 -10 M~10 -6 Around M; about 10 -9 M~10 -6 Around M; about 10 -8 M~10 -6 Around M; about 10 -11 M~10 -8 Around M; about 10 -10 M~10 -8 Around M; about 10 -9 M~10 -8 Around M; about 10 -11 M~10 -9 Around M; about 10 -10 M~10 -9 It has affinity for IL2R with a Kd of approximately 1 M.

[0487] In some embodiments, the bispecific agonist anti-IL2R heavy chain antibody is about 10 -8 M~2.5×10 -7 It has affinity for IL2RB with a Kd of around M.

[0488] In some embodiments, the bispecific agonist anti-IL2R heavy chain antibody is about 10 -9 M~2.5×10 -8 It has affinity for IL2RG with a Kd of around M.

[0489] In some embodiments, the bispecific agonist anti-IL2R heavy chain antibody is about 10 -8 M~2.5×10 -7 It has an affinity for IL2RB with a Kd of about 10 -9 M~2.5×10 -8 It has affinity for IL2RG with a Kd of around M.

[0490] In some embodiments, Kd is measured in kinetic mode using a ForteBio Octet Qk384 instrument. In some embodiments, Kd is measured in kinetic mode using a ForteBio Octet Qk384 instrument equipped with an anti-human Fc capture (AHC, 18-5005) sensor. In some embodiments, Kd is measured according to the method described in the Examples herein.

[0491] An embodiment of the disclosure is a bispecific agonist anti-IL2R heavy chain antibody, comprising: A first heavy chain variable region that binds to IL2RB, comprising a CDR1 sequence of SEQ ID NO: 2, a CDR2 sequence of SEQ ID NO: 5, and a CDR3 sequence of SEQ ID NO: 9; a second heavy chain variable region that binds to IL2RG, comprising a CDR1 sequence of SEQ ID NO: 15, a CDR2 sequence of SEQ ID NO: 18, and a CDR3 sequence of SEQ ID NO: 20; The present invention also includes a bispecific agonist anti-IL2R heavy chain antibody comprising:

[0492] In some embodiments, the CDR1, CDR2 and CDR3 sequences in the first heavy chain variable region are present in a VH human framework. In some embodiments, the CDR1, CDR2 and CDR3 sequences in the second heavy chain variable region are present in a VH human framework. In some embodiments, the CDR1, CDR2 and CDR3 sequences in the first and second heavy chain variable regions are present in a VH human framework.

[0493] In some embodiments, the first heavy chain variable region has at least 95% (e.g., at least 96%, at least 97%, at least 98%, at least 99%) sequence identity to SEQ ID NO: 13, and the second heavy chain variable region has at least 95% (e.g., at least 96%, at least 97%, at least 98%, at least 99%) sequence identity to SEQ ID NO: 23. In some embodiments, the first heavy chain variable region comprises SEQ ID NO: 13, and the second heavy chain variable region comprises SEQ ID NO:23.

[0494] In some embodiments, the bispecific agonist anti-IL2R heavy chain antibody comprises an Fc region. In some embodiments, the bispecific agonist anti-IL2R heavy chain antibody comprises a variant Fc region. In some embodiments, the variant Fc region retains at least about 80% (e.g., at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%) homology to a native sequence Fc region.

[0495] In some embodiments, the variant Fc region comprises heterodimerization modifications. In some embodiments, the heterodimerization modifications comprise knob-and-hole substitutions (e.g., in a variant IgG1 Fc region: 1) Y407T in one chain and T366Y in the other chain; 2) Y407A in one chain and T366W in the other chain; 3) F405A in one chain and T394W in the other chain; 4) F405W in one chain and T394S in the other chain; 5) Y407T in one chain and T366Y in the other chain; 6) F405S in one chain and T394S in the other chain; 7) T366W and F405W in one chain and T394S and Y407A in the other chain; 8) F405W and Y407A in one chain and T366W and T394S in the other chain; or 9) T366W and T366S, L368A in one polypeptide of Fc and Y407V in the other. In some embodiments, heterodimerization modifications comprise substitutions that create new disulfide bridges (e.g., in a variant IgG1 Fc region: 1) Y349C in one Fc polypeptide chain and S354C in the other polypeptide chain; 2) Y349C in one Fc polypeptide chain and E356C in the other polypeptide chain; 3) Y349C in one Fc polypeptide chain and E357C in the other polypeptide chain; 4) L351C in one Fc polypeptide chain and S354C in the other polypeptide chain; 5) T394C in one Fc polypeptide chain and E397C in the other polypeptide chain; or 6) D399C in one Fc polypeptide chain and K392C in the other polypeptide chain).In some embodiments, heterodimerization modifications include charge pair substitutions (e.g., 1) K409E in one chain plus D399K in the other chain; 2) K409E in one chain plus D399R in the other chain; 3) K409D in one chain plus D399K in the other chain; 4) K409D in one chain plus D399R in the other chain; 5) K392E in one chain plus D399R in the other chain; 6) K392E in one chain plus D399K in the other chain; 7) K392D in one chain plus D399R in the other chain; 8) K392D in one chain plus D399K in the other chain; 9) K409D and K360D in one chain plus D399K and E356 in the other chain. K; 10) K409D and K370D in one chain plus D399K and E357K in the other chain; 11) K409D and K392D in one chain plus D399K, E356K, and E357K in the other chain; 12) K409D and K392D in one chain and D399K in the other chain; 13) K409D and K392D in one chain plus D399K and E356K; 14) K409D and K392D on one chain plus D399K and D357K on the other chain; 15) K409D and K370D on one chain plus D399K and D357K on the other chain; 16) D399K on one chain plus K409D and K360D on the other chain; or 17) K409D and K439D on one chain plus D399K and E356K on the other chain.

[0496] In some embodiments, the Fc region is a silenced Fc region. In some embodiments, the silenced Fc region comprises a substitution at one or more (e.g., two or more) of Fc region residues 238, 265, 269, 270, 297, 327, and 329 according to EU numbering. In some embodiments, the silenced Fc region comprises a substitution that alters glycosylation. In some embodiments, the silenced Fc region comprises an effectorless mutation (e.g., N297A, N297G, DANA mutation (D265A+N297A) or DANG mutation (D265A+N297G) in the CH2 region). In some embodiments, the silenced Fc region comprises K322A and L234A / L235A mutations.

[0497] In some embodiments, the bispecific agonist anti-IL2R heavy chain antibody further comprises a heavy chain constant region sequence in the absence of a CH1 sequence. In some embodiments, the bispecific agonist anti-IL2R heavy chain antibody comprises a heavy chain constant region comprising a hinge region, a CH2 domain and a CH3 domain. In some embodiments, the hinge region comprises a wild-type human IgG4 hinge region sequence (SEQ ID NO: 54). In some embodiments, the hinge region comprises a variant human IgG4 hinge region sequence (SEQ ID NO: 55) comprising a S228P mutation. In some embodiments, the CH2 domain comprises a wild-type human IgG4 CH2 domain sequence (SEQ ID NO: 56). In some embodiments, the CH2 domain comprises a variant human IgG4 CH2 domain comprising a F234A mutation, a L235A mutation or both a F234A mutation and a L235A mutation. In some embodiments, the CH3 domain comprises a wild-type human IgG4 CH3 domain sequence (SEQ ID NO: 58). In some embodiments, the CH3 domain comprises a variant human IgG4 CH3 domain comprising a T366W mutation.In some embodiments, the CH3 domain comprises a variant human IgG4 CH3 domain sequence comprising a T366S, a L368A mutation, and a Y407V mutation.

[0498] In some embodiments, the bispecific agonist anti-IL2R heavy chain antibody comprises a first polypeptide comprising SEQ ID NO:64 and a second polypeptide comprising SEQ ID NO:65.

[0499] In some embodiments, the bispecific agonist anti-IL2R heavy chain antibody has a Tm of about 55° C. to about 65° C. In some embodiments, the bispecific agonist anti-IL2R heavy chain antibody has a Tag of about 55° C. to about 65° C. In some embodiments, the bispecific agonist anti-IL2R heavy chain antibody has a Tm of about 55° C. to about 65° C. and a Tag of about 55° C. to about 65° C.

[0500] In some embodiments, the bispecific agonist anti-IL2R heavy chain antibody is about 10 -11 M~10 -6 M (for example, about 10 -10 M~10 -6 Around M; about 10 -9 M~10 -6 Around M; about 10 -8 M~10 -6 Around M; about 10 -11 M~10 -8 Around M; about 10 -10 M~10 -8 Around M; about 10 -9 M~10 -8 Around M; about 10 -11 M~10 -9 Around M; about 10 -10 M~10 -9 It has affinity for IL2R with a Kd of approximately 1 M.

[0501] In some embodiments, the bispecific agonist anti-IL2R heavy chain antibody is about 10 -8 M~2.5×10 -7 It has affinity for IL2RB with a Kd of around M.

[0502] In some embodiments, the bispecific agonist anti-IL2R heavy chain antibody is about 10 -9 M~2.5×10 -8 It has affinity for IL2RG with a Kd of around M.

[0503] In some embodiments, the bispecific agonist anti-IL2R heavy chain antibody is about 10 -8 M~2.5×10 -7 It has an affinity for IL2RB with a Kd of about 10 -9 M~2.5×10 -8 It has affinity for IL2RG with a Kd of around M.

[0504] In some embodiments, Kd is measured in kinetic mode using a ForteBio Octet Qk384 instrument. In some embodiments, Kd is measured in kinetic mode using a ForteBio Octet Qk384 instrument equipped with an anti-human Fc capture (AHC, 18-5005) sensor. In some embodiments, Kd is measured according to the method described in the Examples herein.

[0505] An embodiment of the disclosure is a bispecific agonist anti-IL2R heavy chain antibody, comprising: A first heavy chain variable region that binds to IL2RB, comprising a CDR1 sequence of SEQ ID NO: 3, a CDR2 sequence of SEQ ID NO: 6, and a CDR3 sequence of SEQ ID NO: 10; a second heavy chain variable region that binds to IL2RG, comprising a CDR1 sequence of SEQ ID NO: 15, a CDR2 sequence of SEQ ID NO: 17, and a CDR3 sequence of SEQ ID NO: 20; The present invention also includes a bispecific agonist anti-IL2R heavy chain antibody comprising:

[0506] In some embodiments, the CDR1, CDR2 and CDR3 sequences in the first heavy chain variable region are present in a VH human framework. In some embodiments, the CDR1, CDR2 and CDR3 sequences in the second heavy chain variable region are present in a VH human framework. In some embodiments, the CDR1, CDR2 and CDR3 sequences in the first and second heavy chain variable regions are present in a VH human framework.

[0507] In some embodiments, the first heavy chain variable region has at least 95% (e.g., at least 96%, at least 97%, at least 98%, at least 99%) sequence identity to SEQ ID NO: 14, and the second heavy chain variable region has at least 95% (e.g., at least 96%, at least 97%, at least 98%, at least 99%) sequence identity to SEQ ID NO: 22. In some embodiments, the first heavy chain variable region comprises SEQ ID NO: 14, and the second heavy chain variable region comprises SEQ ID NO:22.

[0508] In some embodiments, the bispecific agonist anti-IL2R heavy chain antibody comprises an Fc region. In some embodiments, the bispecific agonist anti-IL2R heavy chain antibody comprises a variant Fc region. In some embodiments, the variant Fc region retains at least about 80% (e.g., at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%) homology to a native sequence Fc region.

[0509] In some embodiments, the variant Fc region comprises heterodimerization modifications. In some embodiments, the heterodimerization modifications comprise knob-and-hole substitutions (e.g., in a variant IgG1 Fc region: 1) Y407T in one chain and T366Y in the other chain; 2) Y407A in one chain and T366W in the other chain; 3) F405A in one chain and T394W in the other chain; 4) F405W in one chain and T394S in the other chain; 5) Y407T in one chain and T366Y in the other chain; 6) F405S in one chain and T394S in the other chain; 7) T366W and F405W in one chain and T394S and Y407A in the other chain; 8) F405W and Y407A in one chain and T366W and T394S in the other chain; or 9) T366W and T366S, L368A in one polypeptide of Fc and Y407V in the other. In some embodiments, heterodimerization modifications comprise substitutions that create new disulfide bridges (e.g., in a variant IgG1 Fc region: 1) Y349C in one Fc polypeptide chain and S354C in the other polypeptide chain; 2) Y349C in one Fc polypeptide chain and E356C in the other polypeptide chain; 3) Y349C in one Fc polypeptide chain and E357C in the other polypeptide chain; 4) L351C in one Fc polypeptide chain and S354C in the other polypeptide chain; 5) T394C in one Fc polypeptide chain and E397C in the other polypeptide chain; or 6) D399C in one Fc polypeptide chain and K392C in the other polypeptide chain).In some embodiments, heterodimerization modifications include charge pair substitutions (e.g., 1) K409E in one chain plus D399K in the other chain; 2) K409E in one chain plus D399R in the other chain; 3) K409D in one chain plus D399K in the other chain; 4) K409D in one chain plus D399R in the other chain; 5) K392E in one chain plus D399R in the other chain; 6) K392E in one chain plus D399K in the other chain; 7) K392D in one chain plus D399R in the other chain; 8) K392D in one chain plus D399K in the other chain; 9) K409D and K360D in one chain plus D399K and E356 in the other chain. K; 10) K409D and K370D in one chain plus D399K and E357K in the other chain; 11) K409D and K392D in one chain plus D399K, E356K, and E357K in the other chain; 12) K409D and K392D in one chain and D399K in the other chain; 13) K409D and K392D in one chain plus D399K and E356K; 14) K409D and K392D on one chain plus D399K and D357K on the other chain; 15) K409D and K370D on one chain plus D399K and D357K on the other chain; 16) D399K on one chain plus K409D and K360D on the other chain; or 17) K409D and K439D on one chain plus D399K and E356K on the other chain.

[0510] In some embodiments, the Fc region is a silenced Fc region. In some embodiments, the silenced Fc region comprises a substitution at one or more (e.g., two or more) of Fc region residues 238, 265, 269, 270, 297, 327, and 329 according to EU numbering. In some embodiments, the silenced Fc region comprises a substitution that alters glycosylation. In some embodiments, the silenced Fc region comprises an effectorless mutation (e.g., N297A, N297G, DANA mutation (D265A+N297A) or DANG mutation (D265A+N297G) in the CH2 region). In some embodiments, the silenced Fc region comprises K322A and L234A / L235A mutations.

[0511] In some embodiments, the bispecific agonist anti-IL2R heavy chain antibody further comprises a heavy chain constant region sequence in the absence of a CH1 sequence. In some embodiments, the bispecific agonist anti-IL2R heavy chain antibody comprises a heavy chain constant region comprising a hinge region, a CH2 domain and a CH3 domain. In some embodiments, the hinge region comprises a wild-type human IgG4 hinge region sequence (SEQ ID NO: 54). In some embodiments, the hinge region comprises a variant human IgG4 hinge region sequence (SEQ ID NO: 55) comprising a S228P mutation. In some embodiments, the CH2 domain comprises a wild-type human IgG4 CH2 domain sequence (SEQ ID NO: 56). In some embodiments, the CH2 domain comprises a variant human IgG4 CH2 domain comprising a F234A mutation, a L235A mutation or both a F234A mutation and a L235A mutation. In some embodiments, the CH3 domain comprises a wild-type human IgG4 CH3 domain sequence (SEQ ID NO: 58). In some embodiments, the CH3 domain comprises a variant human IgG4 CH3 domain comprising a T366W mutation.In some embodiments, the CH3 domain comprises a variant human IgG4 CH3 domain sequence comprising a T366S, a L368A mutation, and a Y407V mutation.

[0512] In some embodiments, the bispecific agonist anti-IL2R heavy chain antibody comprises a first polypeptide comprising SEQ ID NO:66 and a second polypeptide comprising SEQ ID NO:67.

[0513] In some embodiments, the bispecific agonist anti-IL2R heavy chain antibody has a Tm of about 55° C. to about 65° C. In some embodiments, the bispecific agonist anti-IL2R heavy chain antibody has a Tag of about 55° C. to about 65° C. In some embodiments, the bispecific agonist anti-IL2R heavy chain antibody has a Tm of about 55° C. to about 65° C. and a Tag of about 55° C. to about 65° C.

[0514] In some embodiments, the bispecific agonist anti-IL2R heavy chain antibody is about 10 -11 M~10 -6 M (for example, about 10 -10 M~10 -6 Around M; about 10 -9 M~10 -6 Around M; about 10 -8 M~10 -6 Around M; about 10 -11 M~10 -8 Around M; about 10 -10 M~10 -8 Around M; about 10 -9 M~10 -8 Around M; about 10 -11 M~10 -9 Around M; about 10 -10 M~10 -9 It has affinity for IL2R with a Kd of approximately 1 M.

[0515] In some embodiments, the bispecific agonist anti-IL2R heavy chain antibody is about 10 -8 M~2.5×10 -7 It has affinity for IL2RB with a Kd of around M.

[0516] In some embodiments, the bispecific agonist anti-IL2R heavy chain antibody is about 10 -9 M~2.5×10 -8 It has affinity for IL2RG with a Kd of around M.

[0517] In some embodiments, the bispecific agonist anti-IL2R heavy chain antibody is about 10 -8 M~2.5×10 -7 It has an affinity for IL2RB with a Kd of about 10 -9 M~2.5×10 -8 It has affinity for IL2RG with a Kd of around M.

[0518] In some embodiments, Kd is measured in kinetic mode using a ForteBio Octet Qk384 instrument. In some embodiments, Kd is measured in kinetic mode using a ForteBio Octet Qk384 instrument equipped with an anti-human Fc capture (AHC, 18-5005) sensor. In some embodiments, Kd is measured according to the method described in the Examples herein.

[0519] An embodiment of the disclosure is a bispecific agonist anti-IL2R heavy chain antibody, comprising: A first heavy chain variable region that binds to IL2RB, comprising a CDR1 sequence of SEQ ID NO: 1, a CDR2 sequence of SEQ ID NO: 4, and a CDR3 sequence of SEQ ID NO: 8; a second heavy chain variable region that binds to IL2RG, comprising a CDR1 sequence of SEQ ID NO: 16, a CDR2 sequence of SEQ ID NO: 18, and a CDR3 sequence of SEQ ID NO: 20; The present invention also includes a bispecific agonist anti-IL2R heavy chain antibody comprising:

[0520] In some embodiments, the CDR1, CDR2 and CDR3 sequences in the first heavy chain variable region are present in a VH human framework. In some embodiments, the CDR1, CDR2 and CDR3 sequences in the second heavy chain variable region are present in a VH human framework. In some embodiments, the CDR1, CDR2 and CDR3 sequences in the first and second heavy chain variable regions are present in a VH human framework.

[0521] In some embodiments, the first heavy chain variable region has at least 95% (e.g., at least 96%, at least 97%, at least 98%, at least 99%) sequence identity to SEQ ID NO: 12, and the second heavy chain variable region has at least 95% (e.g., at least 96%, at least 97%, at least 98%, at least 99%) sequence identity to SEQ ID NO: 24. In some embodiments, the first heavy chain variable region comprises SEQ ID NO: 12, and the second heavy chain variable region comprises SEQ ID NO:24.

[0522] In some embodiments, the bispecific agonist anti-IL2R heavy chain antibody comprises an Fc region. In some embodiments, the bispecific agonist anti-IL2R heavy chain antibody comprises a variant Fc region. In some embodiments, the variant Fc region retains at least about 80% (e.g., at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%) homology to a native sequence Fc region.

[0523] In some embodiments, the variant Fc region comprises heterodimerization modifications. In some embodiments, the heterodimerization modifications comprise knob-and-hole substitutions (e.g., in a variant IgG1 Fc region: 1) Y407T in one chain and T366Y in the other chain; 2) Y407A in one chain and T366W in the other chain; 3) F405A in one chain and T394W in the other chain; 4) F405W in one chain and T394S in the other chain; 5) Y407T in one chain and T366Y in the other chain; 6) F405S in one chain and T394S in the other chain; 7) T366W and F405W in one chain and T394S and Y407A in the other chain; 8) F405W and Y407A in one chain and T366W and T394S in the other chain; or 9) T366W and T366S, L368A in one polypeptide of Fc and Y407V in the other. In some embodiments, heterodimerization modifications comprise substitutions that create new disulfide bridges (e.g., in a variant IgG1 Fc region: 1) Y349C in one Fc polypeptide chain and S354C in the other polypeptide chain; 2) Y349C in one Fc polypeptide chain and E356C in the other polypeptide chain; 3) Y349C in one Fc polypeptide chain and E357C in the other polypeptide chain; 4) L351C in one Fc polypeptide chain and S354C in the other polypeptide chain; 5) T394C in one Fc polypeptide chain and E397C in the other polypeptide chain; or 6) D399C in one Fc polypeptide chain and K392C in the other polypeptide chain).In some embodiments, heterodimerization modifications include charge pair substitutions (e.g., 1) K409E in one chain plus D399K in the other chain; 2) K409E in one chain plus D399R in the other chain; 3) K409D in one chain plus D399K in the other chain; 4) K409D in one chain plus D399R in the other chain; 5) K392E in one chain plus D399R in the other chain; 6) K392E in one chain plus D399K in the other chain; 7) K392D in one chain plus D399R in the other chain; 8) K392D in one chain plus D399K in the other chain; 9) K409D and K360D in one chain plus D399K and E356 in the other chain. K; 10) K409D and K370D in one chain plus D399K and E357K in the other chain; 11) K409D and K392D in one chain plus D399K, E356K, and E357K in the other chain; 12) K409D and K392D in one chain and D399K in the other chain; 13) K409D and K392D in one chain plus D399K and E356K; 14) K409D and K392D on one chain plus D399K and D357K on the other chain; 15) K409D and K370D on one chain plus D399K and D357K on the other chain; 16) D399K on one chain plus K409D and K360D on the other chain; or 17) K409D and K439D on one chain plus D399K and E356K on the other chain.

[0524] In some embodiments, the Fc region is a silenced Fc region. In some embodiments, the silenced Fc region comprises a substitution at one or more (e.g., two or more) of Fc region residues 238, 265, 269, 270, 297, 327, and 329 according to EU numbering. In some embodiments, the silenced Fc region comprises a substitution that alters glycosylation. In some embodiments, the silenced Fc region comprises an effectorless mutation (e.g., N297A, N297G, DANA mutation (D265A+N297A) or DANG mutation (D265A+N297G) in the CH2 region). In some embodiments, the silenced Fc region comprises K322A and L234A / L235A mutations.

[0525] In some embodiments, the bispecific agonist anti-IL2R heavy chain antibody further comprises a heavy chain constant region sequence in the absence of a CH1 sequence. In some embodiments, the bispecific agonist anti-IL2R heavy chain antibody comprises a heavy chain constant region comprising a hinge region, a CH2 domain and a CH3 domain. In some embodiments, the hinge region comprises a wild-type human IgG4 hinge region sequence (SEQ ID NO: 54). In some embodiments, the hinge region comprises a variant human IgG4 hinge region sequence (SEQ ID NO: 55) comprising a S228P mutation. In some embodiments, the CH2 domain comprises a wild-type human IgG4 CH2 domain sequence (SEQ ID NO: 56). In some embodiments, the CH2 domain comprises a variant human IgG4 CH2 domain comprising a F234A mutation, a L235A mutation or both a F234A mutation and a L235A mutation. In some embodiments, the CH3 domain comprises a wild-type human IgG4 CH3 domain sequence (SEQ ID NO: 58). In some embodiments, the CH3 domain comprises a variant human IgG4 CH3 domain comprising a T366W mutation.In some embodiments, the CH3 domain comprises a variant human IgG4 CH3 domain sequence comprising a T366S, a L368A mutation, and a Y407V mutation.

[0526] In some embodiments, the bispecific agonist anti-IL2R heavy chain antibody comprises a first polypeptide comprising SEQ ID NO:34 and a second polypeptide comprising SEQ ID NO:35.

[0527] In some embodiments, the bispecific agonist anti-IL2R heavy chain antibody has a Tm of about 55° C. to about 65° C. In some embodiments, the bispecific agonist anti-IL2R heavy chain antibody has a Tag of about 55° C. to about 65° C. In some embodiments, the bispecific agonist anti-IL2R heavy chain antibody has a Tm of about 55° C. to about 65° C. and a Tag of about 55° C. to about 65° C.

[0528] In some embodiments, the bispecific agonist anti-IL2R heavy chain antibody is about 10 -11 M~10 -6 M (for example, about 10 -10 M~10 -6 Around M; about 10 -9 M~10 -6 Around M; about 10 -8 M~10 -6 Around M; about 10 -11 M~10 -8 Around M; about 10 -10 M~10 -8 Around M; about 10 -9 M~10 -8 Around M; about 10 -11 M~10 -9 Around M; about 10 -10 M~10 -9 It has affinity for IL2R with a Kd of approximately 1 M.

[0529] In some embodiments, the bispecific agonist anti-IL2R heavy chain antibody is about 10 -8 M~2.5×10 -7 It has affinity for IL2RB with a Kd of around M.

[0530] In some embodiments, the bispecific agonist anti-IL2R heavy chain antibody is about 10 -9 M~2.5×10 -8 It has affinity for IL2RG with a Kd of around M.

[0531] In some embodiments, the bispecific agonist anti-IL2R heavy chain antibody is about 10 -8 M~2.5×10 -7 It has an affinity for IL2RB with a Kd of about 10 -9 M~2.5×10 -8 It has affinity for IL2RG with a Kd of around M.

[0532] In some embodiments, Kd is measured in kinetic mode using a ForteBio Octet Qk384 instrument. In some embodiments, Kd is measured in kinetic mode using a ForteBio Octet Qk384 instrument equipped with an anti-human Fc capture (AHC, 18-5005) sensor. In some embodiments, Kd is measured according to the method described in the Examples herein.

[0533] An embodiment of the disclosure is a bispecific agonist anti-IL2R heavy chain antibody, comprising: A first heavy chain variable region that binds to IL2RB, comprising a CDR1 sequence of SEQ ID NO: 1, a CDR2 sequence of SEQ ID NO: 4, and a CDR3 sequence of SEQ ID NO: 8; a second heavy chain variable region that binds to IL2RG, comprising a CDR1 sequence of SEQ ID NO: 15, a CDR2 sequence of SEQ ID NO: 19, and a CDR3 sequence of SEQ ID NO: 21; The present invention also includes a bispecific agonist anti-IL2R heavy chain antibody comprising:

[0534] In some embodiments, the CDR1, CDR2 and CDR3 sequences in the first heavy chain variable region are present in a VH human framework. In some embodiments, the CDR1, CDR2 and CDR3 sequences in the second heavy chain variable region are present in a VH human framework. In some embodiments, the CDR1, CDR2 and CDR3 sequences in the first and second heavy chain variable regions are present in a VH human framework.

[0535] In some embodiments, the first heavy chain variable region has at least 95% (e.g., at least 96%, at least 97%, at least 98%, at least 99%) sequence identity to SEQ ID NO: 12, and the second heavy chain variable region has at least 95% (e.g., at least 96%, at least 97%, at least 98%, at least 99%) sequence identity to SEQ ID NO: 25. In some embodiments, the first heavy chain variable region comprises SEQ ID NO: 12, and the second heavy chain variable region comprises SEQ ID NO:25.

[0536] In some embodiments, the bispecific agonist anti-IL2R heavy chain antibody comprises an Fc region. In some embodiments, the bispecific agonist anti-IL2R heavy chain antibody comprises a variant Fc region. In some embodiments, the variant Fc region retains at least about 80% (e.g., at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%) homology to a native sequence Fc region.

[0537] In some embodiments, the variant Fc region comprises heterodimerization modifications. In some embodiments, the heterodimerization modifications comprise knob-and-hole substitutions (e.g., in a variant IgG1 Fc region: 1) Y407T in one chain and T366Y in the other chain; 2) Y407A in one chain and T366W in the other chain; 3) F405A in one chain and T394W in the other chain; 4) F405W in one chain and T394S in the other chain; 5) Y407T in one chain and T366Y in the other chain; 6) F405S in one chain and T394S in the other chain; 7) T366W and F405W in one chain and T394S and Y407A in the other chain; 8) F405W and Y407A in one chain and T366W and T394S in the other chain; or 9) T366W and T366S, L368A in one polypeptide of Fc and Y407V in the other. In some embodiments, heterodimerization modifications comprise substitutions that create new disulfide bridges (e.g., in a variant IgG1 Fc region: 1) Y349C in one Fc polypeptide chain and S354C in the other polypeptide chain; 2) Y349C in one Fc polypeptide chain and E356C in the other polypeptide chain; 3) Y349C in one Fc polypeptide chain and E357C in the other polypeptide chain; 4) L351C in one Fc polypeptide chain and S354C in the other polypeptide chain; 5) T394C in one Fc polypeptide chain and E397C in the other polypeptide chain; or 6) D399C in one Fc polypeptide chain and K392C in the other polypeptide chain).In some embodiments, heterodimerization modifications include charge pair substitutions (e.g., 1) K409E in one chain plus D399K in the other chain; 2) K409E in one chain plus D399R in the other chain; 3) K409D in one chain plus D399K in the other chain; 4) K409D in one chain plus D399R in the other chain; 5) K392E in one chain plus D399R in the other chain; 6) K392E in one chain plus D399K in the other chain; 7) K392D in one chain plus D399R in the other chain; 8) K392D in one chain plus D399K in the other chain; 9) K409D and K360D in one chain plus D399K and E356 in the other chain. K; 10) K409D and K370D in one chain plus D399K and E357K in the other chain; 11) K409D and K392D in one chain plus D399K, E356K, and E357K in the other chain; 12) K409D and K392D in one chain and D399K in the other chain; 13) K409D and K392D in one chain plus D399K and E356K; 14) K409D and K392D on one chain plus D399K and D357K on the other chain; 15) K409D and K370D on one chain plus D399K and D357K on the other chain; 16) D399K on one chain plus K409D and K360D on the other chain; or 17) K409D and K439D on one chain plus D399K and E356K on the other chain.

[0538] In some embodiments, the Fc region is a silenced Fc region. In some embodiments, the silenced Fc region comprises a substitution at one or more (e.g., two or more) of Fc region residues 238, 265, 269, 270, 297, 327, and 329 according to EU numbering. In some embodiments, the silenced Fc region comprises a substitution that alters glycosylation. In some embodiments, the silenced Fc region comprises an effectorless mutation (e.g., N297A, N297G, DANA mutation (D265A+N297A) or DANG mutation (D265A+N297G) in the CH2 region). In some embodiments, the silenced Fc region comprises K322A and L234A / L235A mutations.

[0539] In some embodiments, the bispecific agonist anti-IL2R heavy chain antibody further comprises a heavy chain constant region sequence in the absence of a CH1 sequence. In some embodiments, the bispecific agonist anti-IL2R heavy chain antibody comprises a heavy chain constant region comprising a hinge region, a CH2 domain and a CH3 domain. In some embodiments, the hinge region comprises a wild-type human IgG4 hinge region sequence (SEQ ID NO: 54). In some embodiments, the hinge region comprises a variant human IgG4 hinge region sequence (SEQ ID NO: 55) comprising a S228P mutation. In some embodiments, the CH2 domain comprises a wild-type human IgG4 CH2 domain sequence (SEQ ID NO: 56). In some embodiments, the CH2 domain comprises a variant human IgG4 CH2 domain comprising a F234A mutation, a L235A mutation or both a F234A mutation and a L235A mutation. In some embodiments, the CH3 domain comprises a wild-type human IgG4 CH3 domain sequence (SEQ ID NO: 58). In some embodiments, the CH3 domain comprises a variant human IgG4 CH3 domain comprising a T366W mutation.In some embodiments, the CH3 domain comprises a variant human IgG4 CH3 domain sequence comprising a T366S, a L368A mutation, and a Y407V mutation.

[0540] In some embodiments, the bispecific agonist anti-IL2R heavy chain antibody comprises a first polypeptide comprising SEQ ID NO:36 and a second polypeptide comprising SEQ ID NO:37.

[0541] In some embodiments, the bispecific agonist anti-IL2R heavy chain antibody has a Tm of about 55° C. to about 65° C. In some embodiments, the bispecific agonist anti-IL2R heavy chain antibody has a Tag of about 55° C. to about 65° C. In some embodiments, the bispecific agonist anti-IL2R heavy chain antibody has a Tm of about 55° C. to about 65° C. and a Tag of about 55° C. to about 65° C.

[0542] In some embodiments, the bispecific agonist anti-IL2R heavy chain antibody is about 10 -11 M~10 -6 M (for example, about 10 -10 M~10 -6 Around M; about 10 -9 M~10 -6 Around M; about 10 -8 M~10 -6 Around M; about 10 -11 M~10 -8 Around M; about 10 -10 M~10 -8 Around M; about 10 -9 M~10 -8 Around M; about 10 -11 M~10 -9 Around M; about 10 -10 M~10 -9 It has affinity for IL2R with a Kd of approximately 1 M.

[0543] In some embodiments, the bispecific agonist anti-IL2R heavy chain antibody is about 10 -8 M~2.5×10 -7 It has affinity for IL2RB with a Kd of around M.

[0544] In some embodiments, the bispecific agonist anti-IL2R heavy chain antibody is about 10 -9 M~2.5×10 -8 It has affinity for IL2RG with a Kd of around M.

[0545] In some embodiments, the bispecific agonist anti-IL2R heavy chain antibody is about 10 -8 M~2.5×10 -7 It has an affinity for IL2RB with a Kd of about 10 -9 M~2.5×10 -8 It has affinity for IL2RG with a Kd of around M.

[0546] In some embodiments, Kd is measured in kinetic mode using a ForteBio Octet Qk384 instrument. In some embodiments, Kd is measured in kinetic mode using a ForteBio Octet Qk384 instrument equipped with an anti-human Fc capture (AHC, 18-5005) sensor. In some embodiments, Kd is measured according to the method described in the Examples herein.

[0547] Aspects of the disclosure include pharmaceutical compositions comprising the antibodies (e.g., heavy chain antibodies; bispecific agonist anti-IL2R heavy chain antibodies) described herein.

[0548] Aspects of the disclosure include polynucleotides encoding the antibodies (eg, heavy chain antibodies; bispecific agonist anti-IL2R heavy chain antibodies) described herein.

[0549] Aspects of the present disclosure include vectors that include the polynucleotides described herein.

[0550] Aspects of the present disclosure include cells comprising the vectors described herein.

[0551] Aspects of the disclosure include methods of producing an antibody described herein (e.g., a heavy chain antibody; a bispecific agonist anti-IL2R heavy chain antibody), the method comprising growing a cell described herein under conditions that allow for expression of the antibody, and isolating the antibody from the cell and / or the cell culture medium in which the cell is grown.

[0552] Aspects of the disclosure include a method of making the antibodies described herein, the method comprising immunizing a UniRat™ animal with IL2R and identifying an IL2R-binding heavy chain sequence.

[0553] Aspects of the disclosure include kits for treating a disease or disorder in an individual in need thereof, the kit comprising an antibody described herein (e.g., a heavy chain antibody; a bispecific agonist anti-IL2R heavy chain antibody) or a pharmaceutical composition described herein and instructions for use.

[0554] In some embodiments, the kit further comprises at least one additional reagent. In some embodiments, the at least one additional reagent comprises a chemotherapeutic agent.

[0555] Aspects of the disclosure include methods of treatment comprising administering to an individual in need thereof an effective amount of an antibody (e.g., a heavy chain antibody; a bispecific agonist anti-IL2R heavy chain antibody) described herein, or a pharmaceutical composition described herein.

[0556] Aspects of the disclosure include the use of an antibody (e.g., a heavy chain antibody; a bispecific agonist anti-IL2R heavy chain antibody) described herein in the preparation of a medicament for treating a disease or disorder in an individual in need thereof. In some embodiments, the disease or disorder is cancer. In some embodiments, the cancer is an advanced or metastatic cancer. In some embodiments, the cancer is a liquid cancer, such as, for example, multiple myeloma or acute myeloid leukemia. In some embodiments, the cancer is a solid tumor cancer. In some embodiments, the solid tumor cancer is selected from the group consisting of renal cell carcinoma, melanoma, urothelial cancer, triple-negative breast cancer, non-small cell lung cancer (NSCLC), colorectal cancer, sarcoma, squamous cell carcinoma of the head and neck, and metastatic castration-resistant prostate cancer.

[0557] Aspects of the disclosure include an antibody (e.g., a heavy chain antibody; a bispecific agonist anti-IL2R heavy chain antibody) described herein, or a pharmaceutical composition described herein, for use in therapy in an individual in need thereof.

[0558] Aspects of the disclosure include an antibody (e.g., a heavy chain antibody; a bispecific agonist anti-IL2R heavy chain antibody) described herein or a pharmaceutical composition described herein for use in treating a disease or disorder in an individual in need thereof. In some embodiments, the disease or disorder is cancer. In some embodiments, the cancer is an advanced or metastatic cancer. In some embodiments, the cancer is a liquid cancer, such as, for example, multiple myeloma or acute myeloid leukemia. In some embodiments, the cancer is a solid tumor cancer. In some embodiments, the solid tumor cancer is selected from the group consisting of renal cell carcinoma, melanoma, urothelial cancer, triple-negative breast cancer, non-small cell lung cancer (NSCLC), colorectal cancer, sarcoma, squamous cell carcinoma of the head and neck, and metastatic castration-resistant prostate cancer.

[0559] Aspects of the disclosure include a method of treating cancer comprising administering to a subject an antibody described herein (e.g., a heavy chain antibody; a bispecific agonist anti-IL2R heavy chain antibody) or a pharmaceutical composition described herein. In some embodiments, the cancer is an advanced or metastatic cancer. In some embodiments, the cancer is a liquid cancer, such as, for example, multiple myeloma or acute myeloid leukemia. In some embodiments, the cancer is a solid tumor cancer. In some embodiments, the solid tumor cancer is selected from the group consisting of renal cell carcinoma, melanoma, urothelial carcinoma, triple-negative breast cancer, non-small cell lung cancer (NSCLC), colorectal cancer, sarcoma, squamous cell carcinoma of the head and neck, and metastatic castration-resistant prostate cancer.

[0560] Aspects of the disclosure include a method of stimulating IL2R signaling in an immune cell, the method comprising contacting an immune cell with an antibody described herein (e.g., a heavy chain antibody; a bispecific agonist anti-IL2R heavy chain antibody) or a pharmaceutical composition described herein.

[0561] Aspects of the disclosure include a method of stimulating the IL2RB / IL2RG dimeric receptor complex on an immune cell, the method comprising contacting the immune cell with an antibody described herein (e.g., a heavy chain antibody; a bispecific agonist anti-IL2R heavy chain antibody) or a pharmaceutical composition described herein.

[0562] In some embodiments, the immune cells are selected from the group consisting of CD4+ T cells, CD8+ T cells, and natural killer (NK) cells.

[0563] These and additional aspects will be further described in the remainder of the disclosure, including the Examples. [Brief description of the drawings]

[0564] [Figure 1] FIG. 1 is a table summarizing the binding kinetics of the listed bispecific antibody constructs for human and cynomolgus IL2RB and IL2RG. [Figure 2A] Panels AC are a series of heat map tables displaying fold induction of phosphorylated STAT5 (pSTAT5) in CD8+ T cells from human PBMCs treated for 1 hour with 50 nM of anti-IL2Rβ / γ bispecific UniAbs™ (Panel A); a 1:1 mixture of anti-IL2Rβ and anti-IL2Rγ monospecific UniAbs™ or single agents (Panel B); or IL-2 as a control (Panel C). pSTAT5 levels were determined by flow cytometry and reported as geometric mean fluorescence intensity (gMFI) relative to the gMFI of unstimulated cells. [Figure 2B]Panels AC are a series of heat map tables displaying fold induction of phosphorylated STAT5 (pSTAT5) in CD8+ T cells from human PBMCs treated for 1 hour with 50 nM of anti-IL2Rβ / γ bispecific UniAbs™ (Panel A); a 1:1 mixture of anti-IL2Rβ and anti-IL2Rγ monospecific UniAbs™ or single agents (Panel B); or IL-2 as a control (Panel C). pSTAT5 levels were determined by flow cytometry and reported as geometric mean fluorescence intensity (gMFI) relative to the gMFI of unstimulated cells. [Figure 2C] Panels AC are a series of heat map tables displaying fold induction of phosphorylated STAT5 (pSTAT5) in CD8+ T cells from human PBMCs treated for 1 hour with 50 nM of anti-IL2Rβ / γ bispecific UniAbs™ (Panel A); a 1:1 mixture of anti-IL2Rβ and anti-IL2Rγ monospecific UniAbs™ or single agents (Panel B); or IL-2 as a control (Panel C). pSTAT5 levels were determined by flow cytometry and reported as geometric mean fluorescence intensity (gMFI) relative to the gMFI of unstimulated cells. [Figure 3A] Panels A-C are a series of graphs showing cell binding of the indicated cell types as a function of concentration for the indicated bispecific antibody constructs. Cell binding was determined by flow cytometry and reported as geometric mean fluorescence intensity (gMFI) relative to the gMFI of cells stained only with the secondary detection antibody. [Figure 3B] Panels A-C are a series of graphs showing cell binding of the indicated cell types as a function of concentration for the indicated bispecific antibody constructs. Cell binding was determined by flow cytometry and reported as geometric mean fluorescence intensity (gMFI) relative to the gMFI of cells stained only with the secondary detection antibody. [Figure 3C] Panels A-C are a series of graphs showing cell binding of the indicated cell types as a function of concentration for the indicated bispecific antibody constructs. Cell binding was determined by flow cytometry and reported as geometric mean fluorescence intensity (gMFI) relative to the gMFI of cells stained only with the secondary detection antibody. [Figure 4A] Panels A-E are a series of graphs showing STAT5 phosphorylation dose curves in human and cynomolgus monkey PBMCs as a function of concentration for the indicated bispecific antibody constructs and control molecules (IL-2 and IL-2 variants). pSTAT5 levels were determined by flow cytometry and reported as percentages of the indicated cell types. [Figure 4B] Panels A-E are a series of graphs showing STAT5 phosphorylation dose curves in human and cynomolgus monkey PBMCs as a function of concentration for the indicated bispecific antibody constructs and control molecules (IL-2 and IL-2 variants). pSTAT5 levels were determined by flow cytometry and reported as percentages of the indicated cell types. [Figure 4C] Panels A-E are a series of graphs showing STAT5 phosphorylation dose curves in human and cynomolgus monkey PBMCs as a function of concentration for the indicated bispecific antibody constructs and control molecules (IL-2 and IL-2 variants). pSTAT5 levels were determined by flow cytometry and reported as percentages of the indicated cell types. [Figure 4D] Panels A-E are a series of graphs showing STAT5 phosphorylation dose curves in human and cynomolgus monkey PBMCs as a function of concentration for the indicated bispecific antibody constructs and control molecules (IL-2 and IL-2 variants). pSTAT5 levels were determined by flow cytometry and reported as percentages of the indicated cell types. [Figure 4E] Panels A-E are a series of graphs showing STAT5 phosphorylation dose curves in human and cynomolgus monkey PBMCs as a function of concentration for the indicated bispecific antibody constructs and control molecules (IL-2 and IL-2 variants). pSTAT5 levels were determined by flow cytometry and reported as percentages of the indicated cell types. [Figure 5A]Panels A-D are a series of graphs showing proliferation in the indicated human cells as a function of concentration (Ki67 dose curves) for the indicated bispecific antibody constructs and control molecules (IL-2 and IL-2 variants). Ki67 levels were determined by flow cytometry and reported as a percentage of the indicated cell types. [Figure 5B] Panels A-D are a series of graphs showing proliferation in the indicated human cells as a function of concentration (Ki67 dose curves) for the indicated bispecific antibody constructs and control molecules (IL-2 and IL-2 variants). Ki67 levels were determined by flow cytometry and reported as a percentage of the indicated cell types. [Figure 5C] Panels A-D are a series of graphs showing proliferation in the indicated human cells as a function of concentration (Ki67 dose curves) for the indicated bispecific antibody constructs and control molecules (IL-2 and IL-2 variants). Ki67 levels were determined by flow cytometry and reported as a percentage of the indicated cell types. [Figure 5D] Panels A-D are a series of graphs showing proliferation in the indicated human cells as a function of concentration (Ki67 dose curves) for the indicated bispecific antibody constructs and control molecules (IL-2 and IL-2 variants). Ki67 levels were determined by flow cytometry and reported as a percentage of the indicated cell types. [Figure 6A] Panels AD are a series of graphs showing cytokine secretion in human whole blood as a function of concentration for the indicated bispecific antibody constructs and a control molecule (IL-2). [Figure 6B] Panels AD are a series of graphs showing cytokine secretion in human whole blood as a function of concentration for the indicated bispecific antibody constructs and a control molecule (IL-2). [Figure 6C] Panels AD are a series of graphs showing cytokine secretion in human whole blood as a function of concentration for the indicated bispecific antibody constructs and a control molecule (IL-2). [Figure 6D]Panels AD are a series of graphs showing cytokine secretion in human whole blood as a function of concentration for the indicated bispecific antibody constructs and a control molecule (IL-2). [Figure 7A] Panels A-B provide cellular internalization data for the indicated bispecific antibody constructs. Panel A displays the internalization of the indicated anti-IL2Rβ / γ UniAbs by CD8+ T cells from human PBMC as a function of time. Panel B provides a tabular representation of this data. Surface levels of UniAbs were detected by flow cytometry and reported relative to cells that were not internalized. The observed half-lives ranged from 0.27 h to 0.81 h. As observed here, internalization may be partially dependent on the specific anti-IL2RG arm of the bispecific antibody, since molecules containing the IL2RG_F16B binding sequence were internalized to a greater extent and faster than molecules containing a different anti-IL2RG binding sequence. [Figure 7B] Panels A-B provide cellular internalization data for the indicated bispecific antibody constructs. Panel A displays the internalization of the indicated anti-IL2Rβ / γ UniAbs by CD8+ T cells from human PBMC as a function of time. Panel B provides a tabular representation of this data. Surface levels of UniAbs were detected by flow cytometry and reported relative to cells that were not internalized. The observed half-lives ranged from 0.27 h to 0.81 h. As observed here, internalization may be partially dependent on the specific anti-IL2RG arm of the bispecific antibody, since molecules containing the IL2RG_F16B binding sequence were internalized to a greater extent and faster than molecules containing a different anti-IL2RG binding sequence. [Figure 8A] Panels A-B provide mouse model PK data in graphical (Panel A) and tabular (Panel B) formats. BALB / c mice (n=3 per group per time point) were administered 1 mg / kg of the indicated anti-IL2Rβ / γ UniAbs by tail vein injection. Serum was collected at six time points over a two week period and tested together by ELISA against human IgG4. Results are shown as a function of time (Panel A) or in tabular format (Panel B). [Figure 8B] Panels A-B provide mouse model PK data in graphical (Panel A) and tabular (Panel B) formats. BALB / c mice (n=3 per group per time point) were administered 1 mg / kg of the indicated anti-IL2Rβ / γ UniAbs by tail vein injection. Serum was collected at six time points over a two week period and tested together by ELISA against human IgG4. Results are shown as a function of time (Panel A) or in tabular format (Panel B). [Figure 9] 1 is a table summarizing some properties of the bispecific antibody constructs shown. All constructs were expressed in the ExpiCHO expression system and purified in two steps after heat stress and stability was determined based on percent aggregation by SE-HPLC. Tm and Tagg were measured using the UNcle platform. For the SE-HPLC experiments, 20 μg of protein was run on a TSK gel G3000 5 μm column. [Figure 10A]Panels A-C provide summary data from the mouse model of GVHD. Irradiated NSG mice (5 per treatment group) were each transplanted with 20 million human PBMCs. Animals were then treated twice weekly with either vehicle alone (100 μL), 22 μg rhIL-2 per day or the two indicated bispecific antibody constructs at 1 mg / kg in 100 μL until euthanasia (20% weight loss). Panel A provides an overview of the mouse model of GVHD and the subsequent dosing scheme. Panel B shows the animal weights as a function of time for the indicated experimental groups. Panel C displays the analysis of cells from the spleens of mice in this study harvested 5 days after treatment. CD8+ and CD4+ T cell proliferation was compared between the four treatment groups by measuring CSFE staining in different lymphocyte populations. Both of the two tested bispecific antibody constructs (IL2RB_F09C**IL2RG_F16A (BsAb-1) and IL2RB_F09G**IL2RG_F16B (BsAb-2)) showed significantly more proliferating CD8+ T cells compared to rhIL-2 and vehicle controls. CD4+ T cells proliferated to a lesser extent, but a significant increase in proliferating CD4+ T cells was seen in IL2RB_F09G**IL2RG_F16B (BsAb-2) treated mice compared to vehicle controls (Panel C). This data demonstrates that cytokine receptor agonists promote immune effector activation and proliferation in vivo and accelerate GVHD in huPBMC engrafted NSG mice at a rate similar to cytokine controls. [Figure 10B]Panels A-C provide summary data from the mouse model of GVHD. Irradiated NSG mice (5 per treatment group) were each transplanted with 20 million human PBMCs. Animals were then treated twice weekly with either vehicle alone (100 μL), 22 μg rhIL-2 per day or the two indicated bispecific antibody constructs at 1 mg / kg in 100 μL until euthanasia (20% weight loss). Panel A provides an overview of the mouse model of GVHD and the subsequent dosing scheme. Panel B shows the animal weights as a function of time for the indicated experimental groups. Panel C displays the analysis of cells from the spleens of mice in this study harvested 5 days after treatment. CD8+ and CD4+ T cell proliferation was compared between the four treatment groups by measuring CSFE staining in different lymphocyte populations. Both of the two tested bispecific antibody constructs (IL2RB_F09C**IL2RG_F16A (BsAb-1) and IL2RB_F09G**IL2RG_F16B (BsAb-2)) showed significantly more proliferating CD8+ T cells compared to rhIL-2 and vehicle controls. CD4+ T cells proliferated to a lesser extent, but a significant increase in proliferating CD4+ T cells was seen in IL2RB_F09G**IL2RG_F16B (BsAb-2) treated mice compared to vehicle controls (Panel C). This data demonstrates that cytokine receptor agonists promote immune effector activation and proliferation in vivo and accelerate GVHD in huPBMC engrafted NSG mice at a rate similar to cytokine controls. [Figure 10C]Panels A-C provide summary data from the mouse model of GVHD. Irradiated NSG mice (5 per treatment group) were each transplanted with 20 million human PBMCs. Animals were then treated twice weekly with either vehicle alone (100 μL), 22 μg rhIL-2 per day or the two indicated bispecific antibody constructs at 1 mg / kg in 100 μL until euthanasia (20% weight loss). Panel A provides an overview of the mouse model of GVHD and the subsequent dosing scheme. Panel B shows the animal weights as a function of time for the indicated experimental groups. Panel C displays the analysis of cells from the spleens of mice in this study harvested 5 days after treatment. CD8+ and CD4+ T cell proliferation was compared between the four treatment groups by measuring CSFE staining in different lymphocyte populations. Both of the two tested bispecific antibody constructs (IL2RB_F09C**IL2RG_F16A (BsAb-1) and IL2RB_F09G**IL2RG_F16B (BsAb-2)) showed significantly more proliferating CD8+ T cells compared to rhIL-2 and vehicle controls. CD4+ T cells proliferated to a lesser extent, but a significant increase in proliferating CD4+ T cells was seen in IL2RB_F09G**IL2RG_F16B (BsAb-2) treated mice compared to vehicle controls (Panel C). This data demonstrates that cytokine receptor agonists promote immune effector activation and proliferation in vivo and accelerate GVHD in huPBMC engrafted NSG mice at a rate similar to cytokine controls. [Figure 11A] Panels A-J are a series of graphs summarizing in vivo pharmacodynamic (PD) data from non-GLP cynomolgus monkey studies. Panels A-E display the percentage of the indicated cell type as a function of time post-dose. Panels F-J display the concentration (x105) of the indicated cell type per μL of blood as a function of time post-dose. Panel K displays the ratio of CD8+ T cells to CD4+ T cells as a function of time post-dose. [Figure 11B]Panels A-J are a series of graphs summarizing in vivo pharmacodynamic (PD) data from non-GLP cynomolgus monkey studies. Panels A-E display the percentage of the indicated cell type as a function of time post-dose. Panels F-J display the concentration (x105) of the indicated cell type per μL of blood as a function of time post-dose. Panel K displays the ratio of CD8+ T cells to CD4+ T cells as a function of time post-dose. [Figure 11C] Panels A-J are a series of graphs summarizing in vivo pharmacodynamic (PD) data from non-GLP cynomolgus monkey studies. Panels A-E display the percentage of the indicated cell type as a function of time post-dose. Panels F-J display the concentration (x105) of the indicated cell type per μL of blood as a function of time post-dose. Panel K displays the ratio of CD8+ T cells to CD4+ T cells as a function of time post-dose. [Figure 11D] Panels A-J are a series of graphs summarizing in vivo pharmacodynamic (PD) data from non-GLP cynomolgus monkey studies. Panels A-E display the percentage of the indicated cell type as a function of time post-dose. Panels F-J display the concentration (x105) of the indicated cell type per μL of blood as a function of time post-dose. Panel K displays the ratio of CD8+ T cells to CD4+ T cells as a function of time post-dose. [Figure 11E] Panels A-J are a series of graphs summarizing in vivo pharmacodynamic (PD) data from non-GLP cynomolgus monkey studies. Panels A-E display the percentage of the indicated cell type as a function of time post-dose. Panels F-J display the concentration (x105) of the indicated cell type per μL of blood as a function of time post-dose. Panel K displays the ratio of CD8+ T cells to CD4+ T cells as a function of time post-dose. [Figure 11F]Panels A-J are a series of graphs summarizing in vivo pharmacodynamic (PD) data from non-GLP cynomolgus monkey studies. Panels A-E display the percentage of the indicated cell type as a function of time post-dose. Panels F-J display the concentration (x105) of the indicated cell type per μL of blood as a function of time post-dose. Panel K displays the ratio of CD8+ T cells to CD4+ T cells as a function of time post-dose. [Figure 11G] Panels A-J are a series of graphs summarizing in vivo pharmacodynamic (PD) data from non-GLP cynomolgus monkey studies. Panels A-E display the percentage of the indicated cell type as a function of time post-dose. Panels F-J display the concentration (x105) of the indicated cell type per μL of blood as a function of time post-dose. Panel K displays the ratio of CD8+ T cells to CD4+ T cells as a function of time post-dose. [Figure 11H] Panels A-J are a series of graphs summarizing in vivo pharmacodynamic (PD) data from non-GLP cynomolgus monkey studies. Panels A-E display the percentage of the indicated cell type as a function of time post-dose. Panels F-J display the concentration (x105) of the indicated cell type per μL of blood as a function of time post-dose. Panel K displays the ratio of CD8+ T cells to CD4+ T cells as a function of time post-dose. [Figure 11I] Panels A-J are a series of graphs summarizing in vivo pharmacodynamic (PD) data from non-GLP cynomolgus monkey studies. Panels A-E display the percentage of the indicated cell type as a function of time post-dose. Panels F-J display the concentration (x105) of the indicated cell type per μL of blood as a function of time post-dose. Panel K displays the ratio of CD8+ T cells to CD4+ T cells as a function of time post-dose. [Figure 11J]Panels A-J are a series of graphs summarizing in vivo pharmacodynamic (PD) data from non-GLP cynomolgus monkey studies. Panels A-E display the percentage of the indicated cell type as a function of time post-dose. Panels F-J display the concentration (x105) of the indicated cell type per μL of blood as a function of time post-dose. Panel K displays the ratio of CD8+ T cells to CD4+ T cells as a function of time post-dose. [Figure 11K] Panels A-J are a series of graphs summarizing in vivo pharmacodynamic (PD) data from non-GLP cynomolgus monkey studies. Panels A-E display the percentage of the indicated cell type as a function of time post-dose. Panels F-J display the concentration (x105) of the indicated cell type per μL of blood as a function of time post-dose. Panel K displays the ratio of CD8+ T cells to CD4+ T cells as a function of time post-dose. [Figure 12A] Panel A is a graph showing serum concentration as a function of time (days) for the indicated bispecific antibodies. Panel B is a table showing molecule, dose and half-life (t1 / 2) information. [Figure 12B] Panel A is a graph showing serum concentration as a function of time (days) for the indicated bispecific antibodies. Panel B is a table showing molecule, dose and half-life (t1 / 2) information. [Figure 13] FIG. 1 is a graph showing body weight (%) as a function of time (study days) for animals in the accelerated GVHD model. [Figure 14] 1 is a graph showing the probability of survival for animals in the accelerated GVHD model as a function of time (study day). [Figure 15A] Panels AC are a panel of graphs showing cell proliferation of the indicated cell types separated into treatment groups measured on day 5 after treatment. Panel A shows results for CD8+ T cells, Panel B shows results for CD4+ T cells, and Panel C shows results for NK cells. [Figure 15B]Panels AC are a panel of graphs showing cell proliferation of the indicated cell types separated into treatment groups measured on day 5 after treatment. Panel A shows results for CD8+ T cells, Panel B shows results for CD4+ T cells, and Panel C shows results for NK cells. [Figure 15C] Panels AC are a panel of graphs showing cell proliferation of the indicated cell types separated into treatment groups measured on day 5 after treatment. Panel A shows results for CD8+ T cells, Panel B shows results for CD4+ T cells, and Panel C shows results for NK cells. [Figure 16A] Panels A-L are a collection of graphs showing cell proliferation and absolute cell concentration as a function of time for the indicated cell types under the indicated dosing conditions. [Figure 16B] Panels A-L are a collection of graphs showing cell proliferation and absolute cell concentration as a function of time for the indicated cell types under the indicated dosing conditions. [Figure 16C] Panels A-L are a collection of graphs showing cell proliferation and absolute cell concentration as a function of time for the indicated cell types under the indicated dosing conditions. [Figure 16D] Panels A-L are a collection of graphs showing cell proliferation and absolute cell concentration as a function of time for the indicated cell types under the indicated dosing conditions. [Figure 16E] Panels A-L are a collection of graphs showing cell proliferation and absolute cell concentration as a function of time for the indicated cell types under the indicated dosing conditions. [Figure 16F] Panels A-L are a collection of graphs showing cell proliferation and absolute cell concentration as a function of time for the indicated cell types under the indicated dosing conditions. [Figure 16G] Panels A-L are a collection of graphs showing cell proliferation and absolute cell concentration as a function of time for the indicated cell types under the indicated dosing conditions. [Figure 16H] Panels A-L are a collection of graphs showing cell proliferation and absolute cell concentration as a function of time for the indicated cell types under the indicated dosing conditions. [Figure 16I] Panels A-L are a collection of graphs showing cell proliferation and absolute cell concentration as a function of time for the indicated cell types under the indicated dosing conditions. [Figure 16J] Panels A-L are a collection of graphs showing cell proliferation and absolute cell concentration as a function of time for the indicated cell types under the indicated dosing conditions. [Figure 16K] Panels A-L are a collection of graphs showing cell proliferation and absolute cell concentration as a function of time for the indicated cell types under the indicated dosing conditions. [Figure 16L] Panels A-L are a collection of graphs showing cell proliferation and absolute cell concentration as a function of time for the indicated cell types under the indicated dosing conditions. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0565] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. Such techniques are described in the literature, such as “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook et al., 1989); “Oligonucleotide Synthesis” (MJ Gait, ed., 1984); “Animal Cell Culture” (RIFreshney, ed., 1987); Biology” (FMAusubel et al., eds., 1987, and periodic updates); “PCR: The Polymerase Chain Reaction”, (Mullis et al., ed., 1994); “A Practical Guide to Molecular Cloning” (Perbal Bernard V., 1988); “Phage Display: A Laboratory Manual” (Barbas et al., 2001); Harlow, Lane and Harlow, Using Antibodies:A Laboratory Manual:Portable Protocol No.I,Cold Spring Harbor Laboratory (1998); and Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory; (1988).

[0566] Unless otherwise indicated, antibody residues herein are numbered according to the Kabat numbering system (e.g., Kabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0567] In the following description, numerous specific details are set forth to provide a more thorough understanding of the present disclosure. However, it will be apparent to one skilled in the art that the present disclosure may be practiced without one or more of these specific details. In other instances, well-known features and procedures well known to those skilled in the art have not been described to avoid obscuring the present disclosure.

[0568] All references cited throughout this disclosure, including patent applications and publications, are incorporated herein by reference in their entirety. In the event of a discrepancy in definition between a cited reference and a definition provided herein, the definition provided herein shall control.

[0569] Definition: In some embodiments, "about," when used in connection with a measurable numerical variable, refers to the stated value of the variable and all values ​​of the variable that are within the experimental error of the stated value (e.g., within a 95% confidence interval of the mean) or ±10% of the stated value, whichever is greater. In some embodiments, numerical ranges are inclusive of the numbers that define the range.

[0570] Where a range of values ​​is provided, unless the context clearly dictates otherwise, it is understood that each intervening value, to the tenth of the unit of the lower limit between the upper and lower limits of that range, and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges that are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0571] By "comprising," it is meant that the recited elements are required in the composition / method / kit, but other elements can be included to form the composition / method / kit etc. in a claim or embodiment.

[0572] "Consisting essentially of" means limiting the scope of the described composition or method to certain materials or steps that do not materially affect the basic and novel characteristics of the disclosure.

[0573] "Consisting of" means excluding from a composition, method, or kit any element, step, or ingredient not specified in the claim or embodiment.

[0574] Antibody residues herein are numbered according to the Kabat numbering system and the EU numbering system. The Kabat numbering system is generally used when referring to residues in the variable domain (approximately residues 1-113 of the heavy chain) (e.g., Kabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The "EU numbering system" or "EU index" is generally used when referring to residues in the immunoglobulin heavy chain constant region (e.g., the EU index reported in Kabat et al., supra). The "EU index in Kabat" refers to the numbering of residues in the human IgG1 EU antibody. Unless otherwise stated herein, reference to a residue number in the variable domain of an antibody refers to the residue numbering according to the Kabat numbering system. Unless otherwise stated herein, reference to a residue number in the constant domain of an antibody refers to the residue numbering according to the EU numbering system.

[0575] Antibodies, also called immunoglobulins, traditionally comprise at least one heavy chain and one light chain, with the sequences of the amino-terminal domains of the heavy and light chains being variable, and thus commonly referred to as variable region domains or variable heavy (VH) or variable light (VL) domains. The two domains traditionally associate to form a specific binding region, although, as discussed herein, specific binding can also be obtained using the variable sequence of the heavy chain alone, and a variety of non-native structures of antibodies are known and used in the art.

[0576] A "functional" or "biologically active" antibody or antigen-binding molecule (including, for example, heavy chain antibodies and multispecific (e.g., bispecific) antibodies and three-chain antibody-like molecules (TCAs) described herein) is one that is capable of exerting one or more of its native activities in structural, regulatory, biochemical or biophysical events. For example, a functional antibody or other binding molecule, such as a TCA, may have the ability to specifically bind to an antigen, which binding may in turn trigger or alter a cellular or molecular event, such as a signal transduction or enzymatic activity. A functional antibody or other binding molecule, such as a TCA, may block ligand activation of a receptor or may also act as an agonist or antagonist. The ability of an antibody or other binding molecule, such as a TCA, to exert one or more of its native activities depends on several factors, including the proper folding and assembly of the polypeptide chain.

[0577] The term "antibody" herein is used in the broadest sense and specifically includes monoclonal antibodies, polyclonal antibodies, monomers, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), heavy chain antibodies, three-chain antibodies, TCAs, single chain Fvs (scFvs), nanobodies, etc., as well as antibody fragments, so long as they exhibit the desired biological activity. Miller et al (2003) Jour. of Immunology 170:4854-4861. Antibodies can be murine, human, humanized, chimeric, or derived from other species.

[0578] For example, the term "antibody" may refer to a full-length heavy chain, a full-length light chain, an intact immunoglobulin molecule, or an immunologically active portion of any of these polypeptide subunits, i.e., a polypeptide comprising an antigen-binding site that immunospecifically binds to an antigen or a portion thereof of a target of interest, including, but not limited to, a cancer cell or a cell that produces an autoimmune antibody associated with an autoimmune disease. The immunoglobulins disclosed herein may be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecule, including modified subclasses with modified Fc portions that reduce or enhance effector cell activity. The light chain of the antibody may be a kappa light chain (Vkappa) or a lambda light chain (Vlambda). The immunoglobulin may be derived from any species. In one aspect, the immunoglobulin is primarily of human origin.

[0579] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies constituting the population are identical except for minor possible naturally occurring mutations. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Moreover, in contrast to conventional (polyclonal) antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. Monoclonal antibodies according to the present disclosure may be produced, for example, by the hybridoma method first described by Kohler et al. (1975) Nature 256:495, or may be produced by recombinant protein production methods (see, for example, U.S. Pat. No. 4,816,567).

[0580] The term "variable", when used in reference to antibodies, refers to the fact that the sequences of certain portions of antibody variable domains vary widely among antibodies and that these portions are used in the binding and specificity of each particular antibody to its particular antigen. However, variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called hypervariable regions in both the light and heavy chain variable domains. The more highly conserved portions of the variable domains are called framework regions (FRs). Natural heavy and light chain variable domains each contain four FRs connected by three hypervariable regions that largely adopt a β-sheet structure and form loop connections and sometimes form part of the β-sheet structure. The hypervariable regions of each chain are held together in close proximity by the FRs and, together with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity (ADCC).

[0581] The term "hypervariable region" as used herein refers to the amino acid residues of an antibody that are responsible for antigen binding. Hypervariable regions generally include amino acid residues from the "complementarity determining regions" or "CDRs" (e.g., residues 31-35 (H1), 50-65 (H2), and 95-102 (H3) of the heavy chain variable domain; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and / or amino acid residues from the "hypervariable loops" (e.g., residues 26-32 (H1), 53-55 (H2), and 96-101 (H3) of the heavy chain variable domain; Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). In some embodiments, "CDR" refers to the complementarity determining regions of an antibody as defined in Lefranc, MP et al., IMGT, the international ImMunoGeneTics database, Nucleic Acids Res., 27:209-212 (1999). "Framework Region" or "FR" residues are those variable domain residues other than the hypervariable region / CDR residues as defined herein.

[0582] Exemplary CDR names are provided herein, however, one of skill in the art will appreciate that several definitions of CDRs are commonly used, including the Kabat definition (see Zhao et al. "A germline knowledge based computational approach for determining antibody complementarity determining regions." Mol Immunol. 2010;47:694-700), which is based on sequence variability and is the most commonly used. The Chothia definition is based on the location of structural loop regions (Chothia et al. "Conformations of immunoglobulin hypervariable regions." Nature. 1989;342:877-883).Alternative CDR definitions of interest include Honegger, “Yet another numbering scheme for immunoglobulin variable domains:an automatic modeling and analysis tool.”J Mol Biol.2001;309:657-670; Ofran et al. “Automated identification of complementarity determining regions (CDRs) reveals peculiar characteristics of CDRs and B-cell epitopes.”J Immunol.2008;181:6230-6235;Almagro“Identification of differences in the specificity-determining residues of antibodies that recognize antigens of different size:implications for the rational design of antibody repertoires.”J Mol Recognit.2004;17:132-143;and Padlanet al.“Identification of specificity-determining residues in antibodies.”Faseb J. 1995;9:133-139, each of which is expressly incorporated herein by reference.

[0583] The terms "heavy chain-only antibody" and "heavy chain antibody" are used interchangeably herein and refer in the broadest sense to an antibody or one or more portions of an antibody, e.g., one or more arms of an antibody, that lack the light chain of a conventional antibody (i.e., a "heavy chain antibody" can consist of an isolated portion of an antibody that lacks a light chain or an antibody format other than a conventional antibody). The term specifically includes, but is not limited to, homodimeric antibodies comprising a VH antigen-binding domain and CH2 and CH3 constant domains in the absence of a CH1 domain; functional (antigen-binding) variants of such antibodies, soluble VH variants, Ig-NARs and functional fragments thereof comprising a homodimer of one variable domain (V-NAR) and five C-like constant domains (C-NAR); and soluble single-domain antibodies (e.g., UniDabs™). In one embodiment, the heavy chain antibody is composed of a variable region antigen-binding domain composed of framework 1, CDR1, framework 2, CDR2, framework 3, CDR3 and framework 4. In another embodiment, the heavy chain antibody is composed of an antigen-binding domain, at least a portion of a hinge region and a CH2 and CH3 domain. In another embodiment, the heavy chain antibody is composed of an antigen-binding domain, at least a portion of a hinge region and a CH2 domain. In a further embodiment, the heavy chain antibody is composed of an antigen-binding domain, at least a portion of a hinge region and a CH3 domain. Also included herein are heavy chain antibodies in which the CH2 and / or CH3 domains are truncated. In a further embodiment, the heavy chain is composed of an antigen-binding domain and at least one CH (CH1, CH2, CH3 or CH4) domain, but does not include the hinge region. The heavy chain antibody may be in the form of a dimer, in which the two heavy chains are disulfide-bonded to each other or otherwise covalently or non-covalently bound to each other. The heavy chain antibody may belong to the IgG subclass, although antibodies belonging to other subclasses, such as the IgM, IgA, IgD and IgE subclasses, are also included herein. In certain embodiments, the heavy chain antibody may belong to the IgG1, IgG2, IgG3 or IgG4 subtype, such as the IgG1 or IgG4 subtype.In one embodiment, the heavy chain antibody is an IgG1 or IgG4 subtype, and one or more of the CH domains are modified to change the effector function of the antibody. In one embodiment, the heavy chain antibody is an IgG4 subtype, and one or more of the CH domains are modified to change the effector function of the antibody. In one embodiment, the heavy chain antibody is an IgG1 subtype, and one or more of the CH domains are modified to change the effector function of the antibody. Modifications of the CH domains that change the effector function are further described herein. Non-limiting examples of heavy chain antibodies are described, for example, in WO 2018 / 039180, the disclosure of which is incorporated herein by reference in its entirety.

[0584] In some embodiments, the heavy chain antibodies described herein are used as binding (targeting) domains of chimeric antigen receptors (CARs). This definition specifically includes human heavy chain antibodies produced by human immunoglobulin transgenic rats (e.g., UniRat™), such as UniAb™. The variable regions (VH) of UniAb™, called UniDabs™, are versatile building blocks that can be linked to Fc regions or serum albumin for the development of novel therapeutics with multispecificity, increased potency and extended half-life. Since homodimeric UniAbs™ lack light chains and thus VL domains, the antigen is recognized by one single domain, namely the variable domain of the heavy chain of the heavy chain antibody (VH or VHH).

[0585] As used herein, an "intact antibody chain" is an antibody chain that comprises a full-length variable region and a full-length constant region (Fc). An intact "conventional" antibody comprises an intact light chain and an intact heavy chain, as well as a light chain constant domain (CL) and a heavy chain constant domain, CH1, hinge, CH2 and CH3, in secreted IgG. Other isotypes, such as IgM or IgA, may have different CH domains. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof. An intact antibody may have one or more "effector functions," which refer to biological activities attributable to the Fc constant region (native sequence Fc region or amino acid sequence variant Fc region) of an antibody. Examples of antibody effector functions include C1q binding; complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; and down-regulation of cell surface receptors. Constant region variants include those that alter effector profile, binding to Fc receptors, etc.

[0586] Depending on the amino acid sequence of the Fc (constant domain) of the heavy chain, antibodies and various antigen-binding proteins can be assigned to different "classes". There are five major classes of heavy chain Fc regions: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into "subclasses" (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The Fc constant domains corresponding to the various classes of antibodies can be called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the various classes of immunoglobulins are well known. Ig forms include hinge-modified or hingeless forms. Roux et al (1998) J. Immunol. 161:4083-4090; Lund et al (2000) Eur. J. Biochem. 267:7246-7256; US Patent Application Publication No. 2005 / 0048572; US Patent Application Publication No. 2004 / 0229310. The light chains of antibodies from any vertebrate species can be assigned to one of two types, called κ (kappa) and λ (lambda), based on the amino acid sequences of their constant domains. Antibodies according to embodiments of the present disclosure can comprise a kappa light chain sequence or a lambda light chain sequence.

[0587] A "functional Fc region" has the "effector functions" of a native sequence Fc region. Non-limiting examples of effector functions include C1q binding; CDC; Fc receptor binding; ADCC; ADCP; downregulation of cell surface receptors (e.g., B cell receptors), and the like. Such effector functions generally require the Fc region to interact with receptors, e.g., FcγRI; FcγRIIA; FcγRIIB1; FcγRIIB2; FcγRIIIA; FcγRIIIB receptors and low affinity FcRn receptors, and can be assessed using various assays known in the art. A "dead" or "silenced" Fc is one that has been mutated to retain activity, e.g., with respect to extending serum half-life, but does not activate high affinity Fc receptors or has reduced affinity for Fc receptors.

[0588] A "native sequence Fc region" comprises an amino acid sequence identical to that of an Fc region found in nature. Native sequence human Fc regions include, for example, native sequence human IgG1 Fc region (non-A and A allotypes), native sequence human IgG2 Fc region, native sequence human IgG3 Fc region, and native sequence human IgG4 Fc region, and naturally occurring variants thereof.

[0589] A "variant Fc region" comprises an amino acid sequence that differs from that of a native sequence Fc region by at least one amino acid modification, e.g., one or more (e.g., two or more, three or more, four or more) amino acid substitutions. Illustratively, in some embodiments, a variant Fc region has at least one amino acid substitution, e.g., about one to about ten amino acid substitutions, e.g., about one to about five amino acid substitutions in a native sequence Fc region or an Fc region of a parent polypeptide, compared to a native sequence Fc region or an Fc region of a parent polypeptide. In some embodiments, a variant Fc region herein has at least about 80% homology to a native sequence Fc region and / or an Fc region of a parent polypeptide, e.g., at least about 85% homology thereto, e.g., at least about 90% homology thereto, e.g., at least about 95% homology thereto, e.g., at least about 99% homology thereto.

[0590] As used herein, "heterodimerization changes" refers to changes in the A and B chains of an Fc region (i.e., the two chains that comprise an Fc region, where one chain is referred to herein as the "A" chain and the other as the "B" chain) that promote the formation of heterodimeric Fc regions, i.e., Fc regions where the A and B chains of the Fc region do not have identical amino acid sequences. In some embodiments, heterodimerization changes can be asymmetric, i.e., an A chain with a particular change can pair with a B chain with a different change. These changes promote heterodimerization and disfavor homodimerization. Whether hetero- or homodimers are formed can be assessed by size difference, for example, as determined by polyacrylamide gel electrophoresis in the situation where one polyethylene chain is a dummy Fc and the other is a scFv-Fc. One non-limiting example of such paired heterodimerization changes is the so-called "knob and hole" substitution. See, e.g., U.S. Patent No. 7,695,936 and U.S. Patent Application Publication No. 2003 / 0078385. As used herein, an Fc region comprising a pair of knob and hole substitutions comprises one substitution in the A chain and another substitution in the B chain. For example, the following knob and hole substitutions in the A and B chains of an IgG1 Fc region have been found to increase heterodimer formation compared to that found in unmodified A and B chains and may be used in non-limiting embodiments of the present disclosure: 1) Y407T in one chain and T366Y in the other chain; 2) Y407A in one chain and T366W in the other chain; 3) F405A in one chain and T394W in the other chain; 4) F405W in one chain and T394S in the other chain; 5) Y407T in one chain and T366Y in the other chain; 6) T366Y and F405A in one chain and T394W and Y407T in the other chain; 7) T366W and F405W in one chain and T394S and Y407A in the other chain; 8) F405W and Y407A in one chain and T366W and T394S in the other chain; and 9) T366W in one polypeptide of Fc and T366S, L368A, and Y407V in the other polypeptide.Alternatively or in addition to such changes, substitutions that create new disulfide bridges can promote heterodimer formation.See, for example, US Patent Application Publication No. 2003 / 0078385.Such changes in the IgG1 Fc region include, but are not limited to, the following substitutions: Y349C in one Fc polypeptide chain and S354C in the other Fc polypeptide chain; Y349C in one Fc polypeptide chain and E356C in the other Fc polypeptide chain; Y349C in one Fc polypeptide chain and E357C in the other Fc polypeptide chain; L351C in one Fc polypeptide chain and S354C in the other Fc polypeptide chain; T394C in one Fc polypeptide chain and E397C in the other Fc polypeptide chain; or D399C in one Fc polypeptide chain and K392C in the other Fc polypeptide chain. Additionally or alternatively, substitutions that alter the charge of one or more residues in the CH3-CH3 interface can enhance heterodimer formation, for example, as described in WO 2009 / 089004, which is incorporated herein by reference. Such substitutions are referred to herein as "charge pair substitutions," and an Fc region that contains one pair of charge pair substitutions will contain one substitution in the A chain and a different substitution in the B chain. Non-limiting examples of charge pair substitutions include: 1) K409D or K409E in one chain + D399K or D399R in the other chain; 2) K392D or K392E in one chain + D399K or D399R in the other chain; 3) K439D or K439E in one chain + E356K or E356R in the other chain; and 4) K370D or K370E in one chain + E357K or E357R in the other chain. Additionally, substitutions R355D, R355E, K360D, or K360R in both chains can stabilize heterodimers when used with other heterodimerization changes. Certain charge pair substitutions can be used alone or with other charge pair substitutions.Specific examples of charge pair substitutions and combinations thereof include: 1) K409E in one chain + D399K in the other chain; 2) K409E in one chain + D399K in the other chain; 3) K409D in one chain + D399K in the other chain; 4) K409D in one chain + D399R in the other chain; 5) K392E in one chain + D399R in the other chain; 6) K392E in one chain + D399K in the other chain; 7) K392D in one chain + D399R in the other chain; 8) K392D in one chain + D399K in the other chain; 9) K409D and K360D in one chain + D399K and E356K in the other chain; 10) K409D in one chain + D399R in the other chain; 11) K409D and K392D on one chain plus D399K, E356K, and E357K on the other chain; 12) K409D and K392D on one chain and D399K on the other chain; 13) K409D and K392D on one chain plus D399K and E357K on the other chain. 6K; 14) K409D and K392D on one chain plus D399K and D357K on the other chain; 15) K409D and K370D on one chain plus D399K and D357K on the other chain; 16) D399K on one chain plus K409D and K360D on the other chain; and 17) K409D and K439D on one chain plus D399K and E356K on the other chain. Any of these heterodimerization changes can be used in a polypeptide comprising a variant Fc region described herein.

[0591] In some non-limiting embodiments, the variant Fc sequence may contain three amino acid substitutions in the CH2 region to reduce FcγRI binding at EU index positions 234, 235, and 237 (see Duncan et al., (1988) Nature 332:563). Two amino acid substitutions in the complement C1q binding site at EU index positions 330 and 331 reduce complement binding (see Tao et al., J. Exp. Med. 178:661 (1993) and Canfield and Morrison, J. Exp. Med. 173:1483 (1991)). Substitutions at positions 233-236 of human IgG1 or IgG2 residues and at positions 327, 330 and 331 of IgG4 residues significantly reduce ADCC and CDC (see, e.g., Armour KL.et al., 1999 Eur J Immunol.29(8):2613-24; and Shields RL et al., 2001.J Biol Chem.276(9):6591-604). The human IgG4 Fc amino acid sequence (UniProtKB number P01861) is provided herein as SEQ ID NO: 76. Silenced IgG1 is described, for example, in Boesch,AW,et al.,“Highly parallel characterization of IgG Fc binding interactions.”MAbs,2014.6(4):p.915-27, the disclosures of which are incorporated herein by reference in their entirety.

[0592] Other Fc variants are possible, including but not limited to those in which regions capable of forming disulfide bonds have been deleted, or specific amino acid residues have been removed at the N-terminus of the native Fc, or a methionine residue has been added. Thus, in some embodiments, one or more Fc portions of an antibody may contain one or more mutations in the hinge region to eliminate disulfide bonds. In yet another embodiment, the hinge region of the Fc may be completely removed. In yet another embodiment, an antibody may comprise an Fc variant.

[0593] Furthermore, Fc variants can be constructed to eliminate or substantially reduce effector functions by substituting (mutating), deleting or adding amino acid residues to effect complement binding or Fc receptor binding. For example, and without limitation, deletions can occur in complement binding sites, such as the C1q binding site. Techniques for preparing such sequence derivatives of immunoglobulin Fc fragments are disclosed in WO 97 / 34631 and WO 96 / 32478. Furthermore, the Fc domain can be modified by phosphorylation, sulfation, acylation, glycosylation, methylation, farnesylation, acetylation, amidation, and the like.

[0594] Antibodies with reduced effector function include, but are not limited to, those with one or more substitutions at Fc region residues 238, 265, 269, 270, 297, 327, and 329 according to EU numbering (see, e.g., U.S. Pat. No. 6,737,056). In some embodiments, variant Fc regions with reduced effector function comprise substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327 according to EU numbering, including the so-called "DANA" Fc variant with substitutions of residues 265 and 297 to alanine according to EU numbering (i.e., D265A and N297A according to EU numbering) (see, e.g., U.S. Pat. No. 7,332,581). In some embodiments, variant Fc regions with reduced effector function comprise the following two amino acid substitutions: D265A and N297A.

[0595] In some embodiments, effector function is reduced by a mutation in the constant region that eliminates glycosylation, e.g., an "effectorless mutation." In some embodiments, the effectorless mutation is an N297A or DANA mutation (D265A+N297A) in the CH2 region. Shields et al., J. Biol. Chem. 276(9):6591-6604 (2001). In some embodiments, the effectorless mutation is an N297G or DANG mutation (D265A+N297G) in the CH2 region. In some embodiments, the variant Fc region lacks glycosylation at N297, e.g., the variant Fc region is a variant Fc region lacking glycosylation at N297, as described in WO 2014 / 153063, which is incorporated herein by reference. Further mutations that result in reduced or eliminated effector function include K322A and L234A / L235A (LALA). Alternatively, effector function can be reduced or eliminated through production techniques, such as expression in a host cell that does not glycosylate (e.g., E. coli) or in a host cell that results in an altered glycosylation pattern that is ineffective or less effective in promoting effector function (e.g., Shinkawa et al., J. Biol. Chem. 278(5):3466-3473 (2003)).

[0596] In some embodiments, the proline at position 329 (EU numbering) of the wild-type human Fc region (P329) is substituted with glycine or arginine or an amino acid residue large enough to disrupt the proline sandwich in the Fc / Fcγ receptor interface formed between P329 of Fc and tryptophan residues W87 and W110 of FcgRIII (Sondermann et al., Nature 406, 267-273 (20 Jul. 2000)). In some further embodiments, the at least one additional amino acid substitution in the Fc variant region is S228P, E233P, L234A, L235A, L235E, N297A, N297D, or P331S. In some embodiments, the at least one additional amino acid substitution is L234A and L235A in a human IgG1 Fc region or S228P and L235E in a human IgG4 Fc region, all according to EU numbering (see, e.g., U.S. Pat. No. 8,969,526, incorporated by reference in its entirety).

[0597] In some embodiments, the variant Fc regi...

Claims

1. （１） (a) Formula: G G S I S S S X1 W (SEQ ID NO: 26) wherein X1 is D or N; (b) Formula: I X2 H S G S T (SEQ ID NO: 27) wherein X2 is D or S; and (c) Formula: X3 R G X4 W E L X5 D A F D I (SEQ ID NO: 28) (Wherein, X3 is G or A; X4 is S or Q; X5 is S or T; or （２） (a) Formula: G F T F S X1 Y G (SEQ ID NO: 29) wherein X1 is S or T; (b) Formula: I S Y D G S N X2 (SEQ ID NO: 30) wherein X2 is K or R; and (c) Formula: A R D L D Y D X3 L T G D P V G G F D I (SEQ ID NO: 31) wherein X3 is V or I. a first heavy chain variable region that binds to IL2RB, comprising: （１） (a) Formula: G F X1 X2 X3 X4 Y Y (SEQ ID NO: 32) (Wherein, X1 is T or I; X2 is F or V; X3 is S, N or G; X4 is D or N; (b) Formula: I S X5 S G X6 X7 I (SEQ ID NO: 33) (Wherein, X5 is S or N; X6 is D, S, G or N; X7 is T or I; and (c) a CDR3 sequence comprising the sequence ARGDAVSITGDY (SEQ ID NO: 20); or （２） CDR1 sequence of SEQ ID NO: 15, CDR2 sequence of SEQ ID NO: 19, and CDR3 sequence of SEQ ID NO: 21 A second heavy chain variable region that binds to IL2RG, comprising: A heavy chain antibody comprising:

2. The first heavy chain variable region comprises a CDR1 sequence comprising SEQ ID NO:26, a CDR2 sequence comprising SEQ ID NO:27, and a CDR3 sequence comprising SEQ ID NO:28, and the second heavy chain variable region comprises a CDR1 sequence comprising SEQ ID NO:32, a CDR2 sequence comprising SEQ ID NO:33, and a CDR3 sequence comprising SEQ ID NO:20; or the first heavy chain variable region comprises a CDR1 sequence comprising SEQ ID NO:26, a CDR2 sequence comprising SEQ ID NO:27, and a CDR3 sequence comprising SEQ ID NO:28; and the second heavy chain variable region comprises a CDR1 sequence comprising SEQ ID NO:15, a CDR2 sequence comprising SEQ ID NO:19, and a CDR3 sequence comprising SEQ ID NO:21; The heavy chain antibody of claim 1.

3. A first heavy chain variable region comprises a CDR1 sequence comprising SEQ ID NO:29, a CDR2 sequence comprising SEQ ID NO:30, and a CDR3 sequence comprising SEQ ID NO:31, and a second heavy chain variable region comprises a CDR1 sequence comprising SEQ ID NO:32, a CDR2 sequence comprising SEQ ID NO:33, and a CDR3 sequence comprising SEQ ID NO:20; or the first heavy chain variable region comprises a CDR1 sequence comprising SEQ ID NO:29, a CDR2 sequence comprising SEQ ID NO:30, and a CDR3 sequence comprising SEQ ID NO:31; and the second heavy chain variable region comprises a CDR1 sequence comprising SEQ ID NO:15, a CDR2 sequence comprising SEQ ID NO:19, and a CDR3 sequence comprising SEQ ID NO:21; The heavy chain antibody of claim 1.

4. The first heavy chain variable region comprises: (a) a CDR1 sequence comprising SEQ ID NO:1 or SEQ ID NO:2, a CDR2 sequence comprising SEQ ID NO:4 or SEQ ID NO:5, and a CDR3 sequence comprising any one of SEQ ID NOs:7-9; or (b) a CDR1 sequence of SEQ ID NO:3, a CDR2 sequence of SEQ ID NO:6, and a CDR3 sequence of SEQ ID NO:10 The heavy chain antibody of claim 1 .

5. The first heavy chain variable region comprises: (a) the CDR1 sequence of SEQ ID NO: 1, the CDR2 sequence of SEQ ID NO: 4, and the CDR3 sequence of SEQ ID NO: 7; or (b) the CDR1 sequence of SEQ ID NO:1, the CDR2 sequence of SEQ ID NO:4, and the CDR3 sequence of SEQ ID NO:8; or (c) the CDR1 sequence of SEQ ID NO:2, the CDR2 sequence of SEQ ID NO:5, and the CDR3 sequence of SEQ ID NO:9; or (d) the CDR1 sequence of SEQ ID NO: 3, the CDR2 sequence of SEQ ID NO: 6, and the CDR3 sequence of SEQ ID NO:

10. The heavy chain antibody of claim 1 .

6. The first heavy chain variable region comprises a heavy chain variable region having at least 95% sequence identity to any one of SEQ ID NOs: 11 to 14, and / or the first heavy chain variable region is selected from SEQ ID NOs: 11-14; The heavy chain antibody of claim 1.

7. The second heavy chain variable region comprises: (a) comprises a CDR1 sequence comprising SEQ ID NO: 15 or SEQ ID NO: 16, a CDR2 sequence comprising SEQ ID NO: 17 or SEQ ID NO: 18, and a CDR3 sequence comprising SEQ ID NO: 20; or (b) a CDR1 sequence comprising SEQ ID NO: 15, a CDR2 sequence comprising SEQ ID NO: 19, and a CDR3 sequence comprising SEQ ID NO: 21 The heavy chain antibody of claim 1 .

8. The second heavy chain variable region comprises: (a) the CDR1 sequence of SEQ ID NO: 15, the CDR2 sequence of SEQ ID NO: 17, and the CDR3 sequence of SEQ ID NO: 20; or (b) the CDR1 sequence of SEQ ID NO: 15, the CDR2 sequence of SEQ ID NO: 18, and the CDR3 sequence of SEQ ID NO: 20; or (c) the CDR1 sequence of SEQ ID NO: 16, the CDR2 sequence of SEQ ID NO: 18, and the CDR3 sequence of SEQ ID NO: 20; or (d) the CDR1 sequence of SEQ ID NO: 15, the CDR2 sequence of SEQ ID NO: 19, and the CDR3 sequence of SEQ ID NO: 21 The heavy chain antibody of claim 1 .

9. The second heavy chain variable region comprises a heavy chain variable region having at least 95% sequence identity to any one of SEQ ID NOs: 22 to 25; and / or the second heavy chain variable region is selected from SEQ ID NOs: 22-25; The heavy chain antibody of claim 1.

10. The heavy chain antibody of claim 1, wherein the first heavy chain variable region comprises a CDR1 sequence of SEQ ID NO:1, a CDR2 sequence of SEQ ID NO:4, and a CDR3 sequence of SEQ ID NO:7, and the second heavy chain variable region comprises a CDR1 sequence of SEQ ID NO:15, a CDR2 sequence of SEQ ID NO:17, and a CDR3 sequence of SEQ ID NO:

20.

11. the first heavy chain variable region has at least 95% sequence identity to SEQ ID NO:11 and the second heavy chain variable region has at least 95% sequence identity to SEQ ID NO:22; and / or the first heavy chain variable region comprises SEQ ID NO: 11, and the second heavy chain variable region comprises SEQ ID NO: 22; The heavy chain antibody of claim 10.

12. 12. The heavy chain antibody of claim 10 or 11, comprising a first polypeptide comprising SEQ ID NO:53 and a second polypeptide comprising SEQ ID NO:

61.

13. The first heavy chain variable region comprises a CDR1 sequence of SEQ ID NO:1, a CDR2 sequence of SEQ ID NO:4, and a CDR3 sequence of SEQ ID NO:8; the second heavy chain variable region comprises the CDR1 sequence of SEQ ID NO: 15, the CDR2 sequence of SEQ ID NO: 18, and the CDR3 sequence of SEQ ID NO: 20; The heavy chain antibody of claim 1.

14. the first heavy chain variable region has at least 95% sequence identity to SEQ ID NO: 12 and the second heavy chain variable region has at least 95% sequence identity to SEQ ID NO: 23; and / or the first heavy chain variable region comprises SEQ ID NO: 12, and the second heavy chain variable region comprises SEQ ID NO: 23; The heavy chain antibody of claim 13.

15. 15. The heavy chain antibody of claim 13 or 14, comprising a first polypeptide comprising SEQ ID NO:62 and a second polypeptide comprising SEQ ID NO:

63.

16. The method of claim 1, wherein the first heavy chain variable region comprises a CDR1 sequence of SEQ ID NO:2, a CDR2 sequence of SEQ ID NO:5, and a CDR3 sequence of SEQ ID NO:9; the second heavy chain variable region comprises the CDR1 sequence of SEQ ID NO: 15, the CDR2 sequence of SEQ ID NO: 18, and the CDR3 sequence of SEQ ID NO: 20; The heavy chain antibody of claim 1.

17. the first heavy chain variable region has at least 95% sequence identity to SEQ ID NO: 13 and the second heavy chain variable region has at least 95% sequence identity to SEQ ID NO: 23; and / or the first heavy chain variable region comprises SEQ ID NO: 13, and the second heavy chain variable region comprises SEQ ID NO: 23; The heavy chain antibody of claim 16.

18. 18. The heavy chain antibody of claim 16 or 17, comprising a first polypeptide comprising SEQ ID NO:64 and a second polypeptide comprising SEQ ID NO:

65.

19. The first heavy chain variable region comprises a CDR1 sequence of SEQ ID NO:3, a CDR2 sequence of SEQ ID NO:6, and a CDR3 sequence of SEQ ID NO:10; the second heavy chain variable region comprises the CDR1 sequence of SEQ ID NO: 15, the CDR2 sequence of SEQ ID NO: 17, and the CDR3 sequence of SEQ ID NO: 20; The heavy chain antibody of claim 1.

20. the first heavy chain variable region has at least 95% sequence identity to SEQ ID NO: 14 and the second heavy chain variable region has at least 95% sequence identity to SEQ ID NO: 22; and / or the first heavy chain variable region comprises SEQ ID NO: 14 and the second heavy chain variable region comprises SEQ ID NO: 22; 20. The heavy chain antibody of claim 19.

21. 21. The heavy chain antibody of claim 19 or 20, comprising a first polypeptide comprising SEQ ID NO:66 and a second polypeptide comprising SEQ ID NO:

67.

22. The first heavy chain variable region comprises a CDR1 sequence of SEQ ID NO:1, a CDR2 sequence of SEQ ID NO:4, and a CDR3 sequence of SEQ ID NO:8; the second heavy chain variable region comprises the CDR1 sequence of SEQ ID NO: 16, the CDR2 sequence of SEQ ID NO: 18, and the CDR3 sequence of SEQ ID NO: 20; The heavy chain antibody of claim 1.

23. the first heavy chain variable region has at least 95% sequence identity to SEQ ID NO: 12 and the second heavy chain variable region has at least 95% sequence identity to SEQ ID NO: 24; and / or the first heavy chain variable region comprises SEQ ID NO: 12 and the second heavy chain variable region comprises SEQ ID NO: 24; 23. The heavy chain antibody of claim 22.

24. 24. The heavy chain antibody of claim 22 or 23, comprising a first polypeptide comprising SEQ ID NO:34 and a second polypeptide comprising SEQ ID NO:

35.

25. The method of claim 20, wherein the first heavy chain variable region comprises a CDR1 sequence of SEQ ID NO:1, a CDR2 sequence of SEQ ID NO:4, and a CDR3 sequence of SEQ ID NO:8; the second heavy chain variable region comprises the CDR1 sequence of SEQ ID NO: 15, the CDR2 sequence of SEQ ID NO: 19, and the CDR3 sequence of SEQ ID NO: 21; The heavy chain antibody of claim 1.

26. 26. The heavy chain antibody of claim 25, wherein the first heavy chain variable region has at least 95% sequence identity to SEQ ID NO: 12 and the second heavy chain variable region has at least 95% sequence identity to SEQ ID NO:

25.

27. 27. The heavy chain antibody of claim 25 or 26, comprising a first polypeptide comprising SEQ ID NO:36 and a second polypeptide comprising SEQ ID NO:

37.

28. the CDR1, CDR2 and CDR3 sequences of the first heavy chain variable region are in a VH human framework; and / or the CDR1, CDR2 and CDR3 sequences of the second heavy chain variable region are in a VH human framework; A heavy chain antibody described in any one of claims 1 to 5, 7, 8, 10, 13, 16, 19, 22 or 25.

29. 27. A heavy chain antibody according to any one of claims 1 to 11, 13, 14, 16, 17, 19, 20, 22, 23, 25 or 26, comprising a silenced Fc region.

30. 27. A heavy chain antibody according to any one of claims 1 to 11, 13, 14, 16, 17, 19, 20, 22, 23, 25 or 26, comprising a heavy chain constant region sequence in the absence of a CH1 sequence.

31. The heavy chain antibody of any one of claims 1 to 11, 13, 14, 16, 17, 19, 20, 22, 23, 25 and 26, which is a human antibody and / or an isolated antibody.

32. about 10 －１１ M ~ about 10 －６ and / or has an affinity for IL2R with a Kd of around M about 10 －８ M ~ approx. 2.5 x 10 －７ and / or has an affinity for IL2RB with a Kd of around M; about 10 －９ M ~ approx. 2.5 x 10 －８ The heavy chain antibody according to any one of claims 1 to 11, 13, 14, 16, 17, 19, 20, 22, 23, 25 and 26, having affinity for IL2RG with a Kd of approximately M.

33. 27. A heavy chain antibody according to any one of claims 1 to 11, 13, 14, 16, 17, 19, 20, 22, 23, 25 and 26, which functions as an IL2 receptor beta / gamma agonist.

34. A heavy chain antibody according to any one of claims 1 to 11, 13, 14, 16, 17, 19, 20, 22, 23, 25 and 26, and Pharmaceutically acceptable excipients 23. A pharmaceutical composition comprising:

35. A pharmaceutical composition for treating cancer, comprising a heavy chain antibody of any one of claims 1 to 11, 13, 14, 16, 17, 19, 20, 22, 23, 25 and 26.

36. 36. The pharmaceutical composition of claim 35, wherein the heavy chain antibody is administered in conjunction with a chemotherapy regimen.

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