Combination therapy comprising T cell redirection therapy and an agonist anti-IL-2R antibody or fragment thereof

Abstract

Methods for treating cancer in a subject in need thereof or enhancing the anti-cancer effect associated with administration of T cell redirecting therapy, comprising administering to the subject an anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof in combination with T cell redirecting therapy, are disclosed, along with uses of anti-IL-2Rβγ heavy chain-only antibodies and antigen-binding fragments thereof and / or T cell redirecting therapy in the manufacture of medicaments adapted for use in the methods described herein; pharmaceutical compositions comprising anti-IL-2Rβγ heavy chain-only antibodies and antigen-binding fragments thereof and / or T cell redirecting therapy for use in the methods described herein; and combination products.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 413,339, filed October 5, 2022, which is incorporated herein by reference in its entirety.

[0002] Sequence Listing The computer-readable amino acid sequence listing submitted herewith is incorporated by reference in its entirety and is identified as follows: 100 kilobyte XML file entitled 10235-WO01-SEC_ST26; created October 3, 2023.

[0003] Disclosed herein are methods for treating cancer in a subject in need thereof or enhancing the anti-cancer effect associated with the administration of T-cell redirecting therapy, comprising administering to the subject an anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof in combination with a T-cell redirecting therapy. Also disclosed herein are uses of anti-IL-2Rβγ heavy chain-only antibodies and antigen-binding fragments thereof and / or T-cell redirecting therapy in the manufacture of medicaments adapted for use in the methods described herein; pharmaceutical compositions comprising anti-IL-2Rβγ heavy chain-only antibodies and antigen-binding fragments thereof and / or T-cell redirecting therapy for use in the methods described herein; and combination products. [Background technology]

[0004] Bispecific T cell-attracting molecules are a new class of immunotherapies being developed to treat various cancers. These molecules are designed to target a patient's T cells to cancer cells, causing the T cells to attack and kill the cancer cells. Bispecific T cell-attracting molecules typically have at least one binding domain specific for a cell surface antigen expressed on cancer cells and at least another binding domain specific for CD3, a subunit of the T cell receptor complex expressed on T cells. First-generation bispecific T cell-attracting molecules (see, e.g., WO 99 / 54440, WO 2005 / 040220, and WO 2008 / 119567) are typically administered by continuous intravenous infusion due to their short half-lives of less than one day. Second generation bispecific T cell engaging molecules (see, for example, WO 2013 / 128027, WO 2014140358, WO 2014 / 144722, WO 2014 / 151910, WO 2017 / 134140, WO 2017 / 223111, and WO 2018 / 052503) have been designed, at least in part, to increase the serum half-life of the molecules, allowing for dosing paradigms that allow for intervals between administrations.

[0005] Chimeric antigen receptor (CAR) T-cell therapy is another novel class of immunotherapy in which autologous or allogeneic T cells are engineered to express a CAR specific for a cell surface antigen expressed on cancer cells, redirecting the patient's T cells to attack and kill the cancer cells that express the cell surface antigen. CAR-T cells have shown great promise for the treatment of hematological malignancies such as leukemia, lymphoma, and myeloma, and are also under investigation for the treatment of solid tumors.

[0006] Although CAR-T cell therapy and bispecific T cell-redirecting molecules have significantly improved clinical outcomes in some patients diagnosed with cancer, the usefulness of these T cell-redirecting therapies has been limited to patients with "non-inflammatory tumors," characterized by low T cell infiltration. Thus, there remains a need in the art for methods to effectively enhance the therapeutic efficacy of T cell-redirecting therapy in patients with non-inflammatory tumors.

[0007] Interleukin-2 (IL-2) therapy offers a potential approach for converting non-inflammatory tumors into treatment-responsive inflammatory tumors by expanding the effector T cell population that can be recruited to the tumor site. IL-2 is a key regulator of immune cells, inducing the proliferation of both T cells and natural killer (NK) cells. The ability to harness the immune system against tumors is well established, and IL-2 was one of the first recombinant proteins successfully used as a cancer treatment approximately 40 years ago. (Lotze et al., Journal of Immunology 135(4), 2865-75 (1985); Rosenberg, SAJ Immunol 192, 5451-58 (2014)) Specifically, 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, D.F. et al., J Clin Oncol 23, 133-141 (2004); Payne, R. et al., J Immunother Cancer 2, 13 (2014); Atkins, M.B. et al., J Clin Oncol 17, 2105-2105 (1999); Rosenberg, S.A. et al., Ann Surg 228, 307-319 (1998). However, despite its potent immunostimulatory effects and the potential to induce durable tumor regression in cancer patients, the success of IL-2 as an immunotherapeutic agent has been limited by adverse events. Specifically, Proleukin® has severe dose-limiting toxicities, including vascular leak syndrome, hypotension, and hepatotoxicity, limiting its use in cancer immunotherapy. These adverse events are primarily attributable to the proliferation of cells expressing the high-affinity trimeric receptor IL-2Rαβγ, such as regulatory T (T regThis is due to the preferential uptake of IL-2 by IL-2-deficient cells and endothelial cells. In contrast, the intermediate-affinity receptor, which consists only of IL-2Rβ and IL-2Rγ, 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). The IL-2Rα subunit is not required for downstream JAK-STAT signaling, but its binding to IL-2Rβ and IL-2Rγ confers a 100-fold higher affinity for IL-2 compared to the heterodimeric receptor composed only of IL-2Rβ and IL-2Rγ. Summary of the Invention [Means for solving the problem]

[0008] The present disclosure provides anti-IL-2Rβγ heavy chain-only antibodies and antigen-binding fragments thereof 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, these anti-IL-2Rβγ heavy chain-only antibodies and antigen-binding fragments thereof suppress the preferential T cell response of native IL-2 while maintaining their potent stimulatory effects on other T cell subsets, as well as NK cells. regIL-2Rβγ heavy chain-only antibodies and antigen-binding fragments described herein eliminate IL-2Rβγ heavy chain-only activation. In addition, the presence of an Fc region in certain anti-IL-2Rβγ heavy chain-only antibodies and antigen-binding fragments described herein significantly extends their in vivo half-life compared to recombinant IL-2, potentially allowing for more convenient therapeutic administration schedules. In vivo studies in both mice and cynomolgus monkeys have confirmed the in vivo biological activity, broad pharmacokinetics, and improved safety profile of the anti-IL-2Rβγ heavy chain-only antibodies and antigen-binding fragments thereof described herein. Additionally, in vitro studies using a bispecific T cell-engaging molecule that specifically binds both human CD3 and human EpCAM demonstrated that combination therapy with the disclosed anti-IL-2Rβγ heavy chain-only antibodies improved maximal T cell killing at low E:T ratios in multiple cell lines. Furthermore, in the SHP77 Luc model in female NSG mice, combination therapy with the disclosed anti-IL-2Rβγ heavy chain-only antibodies and a bispecific T cell-engaging molecule that specifically binds both human CD3 and human EpCAM resulted in robust tumor regression. Taken together, these results indicate that the anti-IL-2Rβγ heavy chain-only antibodies and antigen-binding fragments thereof described herein can improve the therapeutic efficacy of T cell redirection therapy when used in combination therapies and combination products.

[0009] Disclosed herein is a method of treating cancer in a subject in need thereof, comprising administering to the subject an anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof in combination with T cell redirecting therapy, wherein the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof is A first heavy chain variable (VH) region that specifically binds to IL-2Rβ, a VH complementarity-determining region 1 (CDR1) sequence having up to two amino acid alterations relative to any one of SEQ ID NOs: 1 to 3; and a VH CDR2 sequence having up to two amino acid alterations relative to any one of SEQ ID NOs: 4 to 6; a VH CDR3 sequence having up to two amino acid alterations relative to any one of SEQ ID NOs: 7 to 10; and a first VH region comprising: a second VH region that specifically binds IL-2Rγ, a VH CDR1 sequence having up to two amino acid alterations relative to any one of SEQ ID NOs: 15 to 16; a VH CDR2 sequence having up to two amino acid alterations relative to any one of SEQ ID NOs: 17 to 19; a VH CDR3 sequence having up to two amino acid alterations relative to any one of SEQ ID NOs: 20 to 21; and a second VH region comprising:

[0010] In some embodiments, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof is an anti-IL-2Rβγ heavy chain-only antibody. In some embodiments, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof is an antigen-binding fragment of an anti-IL-2Rβγ heavy chain-only antibody.

[0011] Also disclosed herein is a method for enhancing the anti-cancer effect associated with the administration of T cell redirecting therapy in a subject diagnosed with cancer, comprising administering to the subject an anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof in combination with T cell redirecting therapy, wherein the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof is A first heavy chain variable (VH) region that specifically binds to IL-2Rβ, a VH complementarity-determining region 1 (CDR1) sequence having up to two amino acid alterations relative to any one of SEQ ID NOs: 1 to 3; and a VH CDR2 sequence having up to two amino acid alterations relative to any one of SEQ ID NOs: 4 to 6; a VH CDR3 sequence having up to two amino acid alterations relative to any one of SEQ ID NOs: 7 to 10; and a first VH region comprising: a second VH region that specifically binds IL-2Rγ, a VH CDR1 sequence having up to two amino acid alterations relative to any one of SEQ ID NOs: 15 to 16; a VH CDR2 sequence having up to two amino acid alterations relative to any one of SEQ ID NOs: 17 to 19; a VH CDR3 sequence having up to two amino acid alterations relative to any one of SEQ ID NOs: 20 to 21; and a second VH region comprising:

[0012] In some embodiments, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof is an anti-IL-2Rβγ heavy chain-only antibody. In some embodiments, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof is an antigen-binding fragment of an anti-IL-2Rβγ heavy chain-only antibody.

[0013] In some embodiments, the VH CDR1 sequence of the first VH region has up to two amino acid modifications relative to SEQ ID NO: 1 or SEQ ID NO: 2; the VH CDR2 sequence of the first VH region has up to two amino acid modifications relative to SEQ ID NO: 4 or SEQ ID NO: 5; and the VH CDR3 sequence of the first VH region has up to two amino acid modifications relative to any one of SEQ ID NOs: 7 to 9.

[0014] In some embodiments, the VH CDR1 sequence of the first VH region has up to two amino acid modifications relative to SEQ ID NO:3; the VH CDR2 sequence of the first VH region has up to two amino acid modifications relative to SEQ ID NO:6; and the VH CDR3 sequence of the first VH region has up to two amino acid modifications relative to SEQ ID NO:10.

[0015] In some embodiments, the VH CDR1 sequence of the first VH region has at most one amino acid alteration relative to any one of SEQ ID NOs: 1-3. In some embodiments, the VH CDR2 sequence of the first VH region has at most one amino acid alteration relative to any one of SEQ ID NOs: 4-6. In some embodiments, the VH CDR3 sequence of the first VH region has at most one amino acid alteration relative to any one of SEQ ID NOs: 7-10.

[0016] In some embodiments, the VH CDR1 sequence of the first VH region has at most one amino acid alteration relative to SEQ ID NO: 1 or SEQ ID NO: 2; the VH CDR2 sequence of the first VH region has at most one amino acid alteration relative to SEQ ID NO: 4 or SEQ ID NO: 5; and the VH CDR3 sequence of the first VH region has at most one amino acid alteration relative to any one of SEQ ID NOs: 7 to 9.

[0017] In some embodiments, the VH CDR1 sequence of the first VH region has at most one amino acid modification relative to SEQ ID NO:3; the VH CDR2 sequence of the first VH region has at most one amino acid modification relative to SEQ ID NO:6; and the VH CDR3 sequence of the first VH region has at most one amino acid modification relative to SEQ ID NO:10.

[0018] In some embodiments, the VH CDR1 sequence of the second VH region has up to two amino acid modifications relative to SEQ ID NO: 15 or SEQ ID NO: 16; the VH CDR2 sequence of the second VH region has up to two amino acid modifications relative to SEQ ID NO: 17 or SEQ ID NO: 18; and the VH CDR3 sequence of the second VH region has up to two amino acid modifications relative to SEQ ID NO: 20.

[0019] In some embodiments, the VH CDR1 sequence of the second VH region has up to two amino acid modifications relative to SEQ ID NO: 15; the VH CDR2 sequence of the second VH region has up to two amino acid modifications relative to SEQ ID NO: 19; and the VH CDR3 sequence of the second VH region has up to two amino acid modifications relative to SEQ ID NO: 21.

[0020] In some embodiments, the VH CDR1 sequence of the second VH region has at most one amino acid alteration relative to any one of SEQ ID NOs: 15-16. In some embodiments, the VH CDR2 sequence of the second VH region has at most one amino acid alteration relative to any one of SEQ ID NOs: 17-19. In some embodiments, the VH CDR3 sequence of the second VH region has at most one amino acid alteration relative to any one of SEQ ID NOs: 20-21.

[0021] In some embodiments, the VH CDR1 sequence of the second VH region has at most one amino acid modification relative to SEQ ID NO: 15 or SEQ ID NO: 16; the VH CDR2 sequence of the second VH region has at most one amino acid modification relative to SEQ ID NO: 17 or SEQ ID NO: 18; and the VH CDR3 sequence of the second VH region has at most one amino acid modification relative to SEQ ID NO: 20.

[0022] In some embodiments, the VH CDR1 sequence of the second VH region has at most one amino acid modification relative to SEQ ID NO: 15; the VH CDR2 sequence of the second VH region has at most one amino acid modification relative to SEQ ID NO: 19; and the VH CDR3 sequence of the second VH region has at most one amino acid modification relative to SEQ ID NO: 21.

[0023] In some embodiments, at most one amino acid modification is an amino acid substitution. In some embodiments, at most one amino acid modification is a conservative amino acid substitution. In some embodiments, at most one amino acid modification is an amino acid deletion. In some embodiments, at most one amino acid modification is an amino acid addition.

[0024] In some embodiments, each amino acid modification, if any, is a conservative amino acid substitution.

[0025] In some embodiments, the first VH region comprises a VH CDR1 comprising a sequence selected from SEQ ID NOs: 1-3. In some embodiments, the first VH region comprises a VH CDR2 comprising a sequence selected from SEQ ID NOs: 4-6. In some embodiments, the first VH region comprises a VH CDR3 comprising a sequence selected from SEQ ID NOs: 7-10.

[0026] In some embodiments, the first VH region comprises: VH CDR1 comprising a sequence selected from SEQ ID NOs: 1 to 3; and / or VH CDR2 comprising a sequence selected from SEQ ID NOs: 4 to 6; and / or It comprises a VH CDR3 comprising a sequence selected from SEQ ID NOs: 7 to 10.

[0027] In some embodiments, the first VH region comprises: VH CDR1 comprising a sequence selected from SEQ ID NOs: 1 to 3; and VH CDR2 comprising a sequence selected from SEQ ID NOs: 4 to 6; and It comprises a VH CDR3 comprising a sequence selected from SEQ ID NOs: 7 to 10.

[0028] In some embodiments, the first VH region comprises a VH CDR1 comprising the sequence of SEQ ID NO: 1 or SEQ ID NO: 2, a VH CDR2 comprising the sequence of SEQ ID NO: 4 or SEQ ID NO: 5, and a VH CDR3 comprising a sequence selected from SEQ ID NOs: 7 to 9.

[0029] In some embodiments, the first VH region comprises a VH CDR1, a VH CDR2, and a VH CDR3 comprising the sequences of SEQ ID NOs: 1, 4, and 7, respectively.

[0030] In some embodiments, the first VH region comprises a VH CDR1, a VH CDR2, and a VH CDR3 comprising the sequences of SEQ ID NOs: 1, 4, and 8, respectively.

[0031] In some embodiments, the first VH region comprises a VH CDR1, a VH CDR2, and a VH CDR3 comprising the sequences of SEQ ID NOs: 2, 5, and 9, respectively.

[0032] In some embodiments, the first VH region comprises a VH CDR1, a VH CDR2, and a VH CDR3 comprising the sequences of SEQ ID NOs: 3, 6, and 10, respectively.

[0033] In some embodiments, the second VH region comprises a VH CDR1 comprising a sequence selected from SEQ ID NOs: 15-16. In some embodiments, the second VH region comprises a VH CDR2 comprising a sequence selected from SEQ ID NOs: 17-19. In some embodiments, the second VH region comprises a VH CDR3 comprising a sequence selected from SEQ ID NOs: 20-21.

[0034] In some embodiments, the second VH region is VH CDR1 comprising a sequence selected from SEQ ID NOs: 15 to 16; and / or VH CDR2 comprising a sequence selected from SEQ ID NOs: 17 to 19; and / or It comprises a VH CDR3 comprising a sequence selected from SEQ ID NOs: 20 to 21.

[0035] In some embodiments, the second VH region is VH CDR1 comprising a sequence selected from SEQ ID NOs: 15 to 16; and VH CDR2 comprising a sequence selected from SEQ ID NOs: 17 to 19; and It comprises a VH CDR3 comprising a sequence selected from SEQ ID NOs: 20 to 21.

[0036] In some embodiments, the second VH region comprises a VH CDR1 comprising the sequence of SEQ ID NO: 15 or SEQ ID NO: 16, a VH CDR2 comprising the sequence of SEQ ID NO: 17 or SEQ ID NO: 18, and a VH CDR3 comprising the sequence of SEQ ID NO: 20.

[0037] In some embodiments, the second VH region comprises a VH CDR1, a VH CDR2, and a VH CDR3 comprising the sequences of SEQ ID NOs: 15, 17, and 20, respectively.

[0038] In some embodiments, the second VH region comprises a VH CDR1, a VH CDR2, and a VH CDR3 comprising the sequences of SEQ ID NOs: 15, 18, and 20, respectively.

[0039] In some embodiments, the second VH region comprises a VH CDR1, a VH CDR2, and a VH CDR3 comprising the sequences of SEQ ID NOs: 16, 18, and 20, respectively.

[0040] In some embodiments, the second VH region comprises a VH CDR1, a VH CDR2, and a VH CDR3 comprising the sequences of SEQ ID NOs: 15, 19, and 21, respectively.

[0041] In some embodiments, the first VH region comprises VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 1, 4, and 7, respectively; and the second VH region comprises VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 15, 17, and 20, respectively.

[0042] In some embodiments, the first VH region comprises VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 1, 4, and 8, respectively; and the second VH region comprises VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 15, 18, and 20, respectively.

[0043] In some embodiments, the first VH region comprises VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 1, 4, and 8, respectively; and the second VH region comprises VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 16, 18, and 20, respectively.

[0044] In some embodiments, the first VH region comprises VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 1, 4, and 8, respectively; and the second VH region comprises VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 15, 19, and 21, respectively.

[0045] In some embodiments, the first VH region comprises VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 2, 5, and 9, respectively; and the second VH region comprises VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 15, 18, and 20, respectively.

[0046] In some embodiments, the first VH region comprises VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 3, 6, and 10, respectively; and the second VH region comprises VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 15, 17, and 20, respectively.

[0047] Also disclosed herein is a method of treating cancer in a subject in need thereof, comprising administering to the subject an anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof in combination with T cell redirecting therapy, wherein the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof is A first heavy chain variable (VH) region that specifically binds to IL-2Rβ, (1)(a) a VH complementarity-determining region 1 (CDR1) comprising the following sequence: GFTFS X1Y G (SEQ ID NO: 29) (wherein X1 is S or T); and (b) a VH CDR2 comprising the following sequence: ISYDGSN X2 (SEQ ID NO: 30) (wherein X2 is K or R); and (c) a VH CDR3 comprising the following sequence: ARDLDYD X3L TGDPVGGFDI (SEQ ID NO: 31) wherein X3 is V or I; or (2)(a) a VH CDR1 comprising the following sequence: GGSISSS X1 W (SEQ ID NO: 26) (wherein X1 is D or N); (b) a VH CDR2 comprising the following sequence: I X2H SGST (SEQ ID NO: 27) (wherein X2 is D or S); and (c) a VH CDR3 comprising the following sequence: X3R G X4W EL X5D AFDI (SEQ ID NO: 28) (wherein X3 is G or A; X4 is S or Q; and X5 is S or T). a first VH region comprising: a second VH region that specifically binds IL-2Rγ, (1)(a) a VH CDR1 comprising the following sequence: GF X1X2X3X4Y Y (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); and (b) a VH CDR2 comprising the following sequence: IS X5S G X6X7I (SEQ ID NO: 33) wherein X5 is S or N; X6 is D, S, G, or N; and X7 is T or I; and (c) a VH CDR3 comprising the sequence ARGDAVSITGDY (SEQ ID NO: 20); or (2) VH CDR1 comprising the sequence GFTFSDYY (SEQ ID NO: 15); VH CDR2 comprising the sequence ISSSGTTT (SEQ ID NO: 19); and VH CDR3 comprising the sequence ARGAAVAPGFDS (SEQ ID NO: 21) and a second VH region comprising:

[0048] In some embodiments, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof is an anti-IL-2Rβγ heavy chain-only antibody. In some embodiments, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof is an antigen-binding fragment of an anti-IL-2Rβγ heavy chain-only antibody.

[0049] Also disclosed herein is a method for enhancing the anti-cancer effect associated with the administration of T cell redirecting therapy in a subject diagnosed with cancer, comprising administering to the subject an anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof in combination with T cell redirecting therapy, wherein the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof is A first heavy chain variable (VH) region that specifically binds to IL-2Rβ, (1)(a) a VH complementarity-determining region 1 (CDR1) comprising the following sequence: GFTFS X1Y G (SEQ ID NO: 29) (wherein X1 is S or T); and (b) a VH CDR2 comprising the following sequence: ISYDGSN X2 (SEQ ID NO: 30) (wherein X2 is K or R); and (c) a VH CDR3 comprising the following sequence: ARDLDYD X3L TGDPVGGFDI (SEQ ID NO: 31) wherein X3 is V or I; or (2)(a) a VH CDR1 comprising the following sequence: GGSISSS X1 W (SEQ ID NO: 26) (wherein X1 is D or N); (b) a VH CDR2 comprising the following sequence: I X2H SGST (SEQ ID NO: 27) (wherein X2 is D or S); and (c) a VH CDR3 comprising the following sequence: X3R G X4W EL X5D AFDI (SEQ ID NO: 28) (wherein X3 is G or A; X4 is S or Q; and X5 is S or T). a first VH region comprising: a second VH region that specifically binds IL-2Rγ, (1)(a) a VH CDR1 comprising the following sequence: GF X1X2X3X4Y Y (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); and (b) a VH CDR2 comprising the following sequence: IS X5S G X6X7I (SEQ ID NO: 33) wherein X5 is S or N; X6 is D, S, G, or N; and X7 is T or I; and (c) a VH CDR3 comprising the sequence ARGDAVSITGDY (SEQ ID NO: 20); or (2) VH CDR1 comprising the sequence GFTFSDYY (SEQ ID NO: 15); VH CDR2 comprising the sequence ISSSGTTT (SEQ ID NO: 19); and VH CDR3 comprising the sequence ARGAAVAPGFDS (SEQ ID NO: 21) and a second VH region comprising:

[0050] In some embodiments, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof is an anti-IL-2Rβγ heavy chain-only antibody. In some embodiments, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof is an antigen-binding fragment of an anti-IL-2Rβγ heavy chain-only antibody.

[0051] In some embodiments, the first VH region comprises a VH CDR1 comprising the sequence of SEQ ID NO: 29, a VH CDR2 comprising the sequence of SEQ ID NO: 30, and a VH CDR3 comprising the sequence of SEQ ID NO: 31. In other embodiments, the first VH region comprises a VH CDR1 comprising the sequence of SEQ ID NO: 26, a VH CDR2 comprising the sequence of SEQ ID NO: 27, and a VH CDR3 comprising the sequence of SEQ ID NO: 28.

[0052] In some embodiments, the second VH region comprises a VH CDR1 comprising the sequence of SEQ ID NO: 32, a VH CDR2 comprising the sequence of SEQ ID NO: 33, and a VH CDR3 comprising the sequence of SEQ ID NO: 20. In other embodiments, the second VH region comprises a VH CDR1 comprising the sequence of SEQ ID NO: 15, a VH CDR2 comprising the sequence of SEQ ID NO: 19, and a VH CDR3 comprising the sequence of SEQ ID NO: 21.

[0053] In some embodiments, the first VH region comprises a VH CDR1 comprising the sequence of SEQ ID NO: 29, a VH CDR2 comprising the sequence of SEQ ID NO: 30, and a VH CDR3 comprising the sequence of SEQ ID NO: 31, and the second VH region comprises a VH CDR1 comprising the sequence of SEQ ID NO: 32, a VH CDR2 comprising the sequence of SEQ ID NO: 33, and a VH CDR3 comprising the sequence of SEQ ID NO: 20.

[0054] In some embodiments, the first VH region comprises a VH CDR1 comprising the sequence of SEQ ID NO: 29, a VH CDR2 comprising the sequence of SEQ ID NO: 30, and a VH CDR3 comprising the sequence of SEQ ID NO: 31, and the second VH region comprises a VH CDR1 comprising the sequence of SEQ ID NO: 15, a VH CDR2 comprising the sequence of SEQ ID NO: 19, and a VH CDR3 comprising the sequence of SEQ ID NO: 21.

[0055] In some embodiments, the first VH region comprises a VH CDR1 comprising the sequence of SEQ ID NO: 26, a VH CDR2 comprising the sequence of SEQ ID NO: 27, and a VH CDR3 comprising the sequence of SEQ ID NO: 28, and the second VH region comprises a VH CDR1 comprising the sequence of SEQ ID NO: 32, a VH CDR2 comprising the sequence of SEQ ID NO: 33, and a VH CDR3 comprising the sequence of SEQ ID NO: 20.

[0056] In some embodiments, the first VH region comprises a VH CDR1 comprising the sequence of SEQ ID NO: 26, a VH CDR2 comprising the sequence of SEQ ID NO: 27, and a VH CDR3 comprising the sequence of SEQ ID NO: 28, and the second VH region comprises a VH CDR1 comprising the sequence of SEQ ID NO: 15, a VH CDR2 comprising the sequence of SEQ ID NO: 19, and a VH CDR3 comprising the sequence of SEQ ID NO: 21.

[0057] Also disclosed herein is a method of treating cancer in a subject in need thereof, comprising administering to the subject an anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof in combination with T cell redirecting therapy, wherein the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof is a first heavy chain variable (VH) complementarity determining region (CDR) 1, 2, and 3 (combined) of the entire set of VH CDR 1, 2, and 3 have at least 80% (e.g., at least 85%, at least 90%, at least 95%; 80%, 85%, 90%, 95%) sequence identity with VH CDR 1, 2, and 3 of any one of SEQ ID NOS: 11-14; and The entire set of VH complementarity determining regions (CDRs) 1, 2, and 3 (combined) comprises a second VH region that specifically binds to IL-2Rγ, having at least 80% (e.g., at least 85%, at least 90%, at least 95%; 80%, 85%, 90%, 95%) sequence identity with VH CDRs 1, 2, and 3 of any one of SEQ ID NOs: 22 to 25.

[0058] In some embodiments, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof is an anti-IL-2Rβγ heavy chain-only antibody. In some embodiments, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof is an antigen-binding fragment of an anti-IL-2Rβγ heavy chain-only antibody.

[0059] Also disclosed herein is a method for enhancing the anti-cancer effect associated with the administration of T cell redirecting therapy in a subject diagnosed with cancer, comprising administering to the subject an anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof in combination with T cell redirecting therapy, wherein the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof is a first heavy chain variable (VH) complementarity determining region (CDR) 1, 2, and 3 (combined) of the entire set of VH CDR 1, 2, and 3 have at least 80% (e.g., at least 85%, at least 90%, at least 95%; 80%, 85%, 90%, 95%) sequence identity with VH CDR 1, 2, and 3 of any one of SEQ ID NOS: 11-14; and The entire set of VH complementarity determining regions (CDRs) 1, 2, and 3 (combined) comprises a second VH region that specifically binds to IL-2Rγ, having at least 80% (e.g., at least 85%, at least 90%, at least 95%; 80%, 85%, 90%, 95%) sequence identity with VH CDRs 1, 2, and 3 of any one of SEQ ID NOs: 22 to 25.

[0060] In some embodiments, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof is an anti-IL-2Rβγ heavy chain-only antibody. In some embodiments, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof is an antigen-binding fragment of an anti-IL-2Rβγ heavy chain-only antibody.

[0061] In some embodiments, the entire set of VH CDR1, 2, and 3 (combined) of the first VH region has at least 85% sequence identity to VH CDR1, 2, and 3 of any one of SEQ ID NOs: 11-14. In some embodiments, the entire set of VH CDR1, 2, and 3 (combined) of the second VH region has at least 85% sequence identity to CDR1, 2, and 3 of any one of SEQ ID NOs: 22-25. In some embodiments, the entire set of VH CDR1, 2, and 3 (combined) of the first VH region has at least 85% sequence identity to VH CDR1, 2, and 3 of any one of SEQ ID NOs: 11-14, and the entire set of VH CDR1, 2, and 3 (combined) of the second VH region has at least 85% sequence identity to VH CDR1, 2, and 3 of any one of SEQ ID NOs: 22-25.

[0062] In some embodiments, the entire set of VH CDR1, 2, and 3 (combined) of the first VH region has at least 90% sequence identity to VH CDR1, 2, and 3 of any one of SEQ ID NOs: 11-14. In some embodiments, the entire set of VH CDR1, 2, and 3 (combined) of the second VH region has at least 90% sequence identity to CDR1, 2, and 3 of any one of SEQ ID NOs: 22-25. In some embodiments, the entire set of VH CDR1, 2, and 3 (combined) of the first VH region has at least 90% sequence identity to VH CDR1, 2, and 3 of any one of SEQ ID NOs: 11-14, and the entire set of VH CDR1, 2, and 3 (combined) of the second VH region has at least 90% sequence identity to VH CDR1, 2, and 3 of any one of SEQ ID NOs: 22-25.

[0063] In some embodiments, the entire set of VH CDR1, 2, and 3 (combined) of the first VH region has at least 95% sequence identity to VH CDR1, 2, and 3 of any one of SEQ ID NOs: 11-14. In some embodiments, the entire set of VH CDR1, 2, and 3 (combined) of the second VH region has at least 95% sequence identity to CDR1, 2, and 3 of any one of SEQ ID NOs: 22-25. In some embodiments, the entire set of VH CDR1, 2, and 3 (combined) of the first VH region has at least 95% sequence identity to VH CDR1, 2, and 3 of any one of SEQ ID NOs: 11-14, and the entire set of VH CDR1, 2, and 3 (combined) of the second VH region has at least 95% sequence identity to VH CDR1, 2, and 3 of any one of SEQ ID NOs: 22-25.

[0064] In some embodiments, the first VH region comprises a VH CDR1, a VH CDR2, and a VH CDR3 of any one of SEQ ID NOs: 11-14. In some embodiments, the second VH region comprises a VH CDR1, a VH CDR2, and a VH CDR3 of any one of SEQ ID NOs: 22-25. In some embodiments, the first VH region comprises a VH CDR1, a VH CDR2, and a VH CDR3 of any one of SEQ ID NOs: 11-14, and the second VH region comprises a VH CDR1, a VH CDR2, and a VH CDR3 of any one of SEQ ID NOs: 22-25.

[0065] In some embodiments, the VH CDR1, VH CDR2, and VH CDR3 sequences of the first VH region are present within a human VH framework, hi some embodiments, the VH CDR1, VH CDR2, and VH CDR3 sequences of the second VH region are present within a human VH framework.

[0066] Also disclosed herein is a method of treating cancer in a subject in need thereof, comprising administering to the subject an anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof in combination with T cell redirecting therapy, wherein the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof is a first heavy chain variable (VH) region that specifically binds to IL-2Rβ and has at least 80% (e.g., at least 85%, at least 90%, at least 95%; 80%, 85%, 90%, 95%) sequence identity to any one of SEQ ID NOs: 11 to 14; and and a second VH region that specifically binds to IL-2Rγ and has at least 80% (e.g., at least 85%, at least 90%, at least 95%; 80%, 85%, 90%, 95%) sequence identity to any one of SEQ ID NOs: 22 to 25.

[0067] In some embodiments, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof is an anti-IL-2Rβγ heavy chain-only antibody. In some embodiments, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof is an antigen-binding fragment of an anti-IL-2Rβγ heavy chain-only antibody.

[0068] Also disclosed herein is a method for enhancing the anti-cancer effect associated with the administration of T cell redirecting therapy in a subject diagnosed with cancer, comprising administering to the subject an anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof in combination with the T cell redirecting therapy, wherein the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof is a first heavy chain variable (VH) region that specifically binds to IL-2Rβ and has at least 80% (e.g., at least 85%, at least 90%, at least 95%; 80%, 85%, 90%, 95%) sequence identity to any one of SEQ ID NOs: 11 to 14; and and a second VH region that specifically binds to IL-2Rγ and has at least 80% (e.g., at least 85%, at least 90%, at least 95%; 80%, 85%, 90%, 95%) sequence identity to any one of SEQ ID NOs: 22 to 25.

[0069] In some embodiments, the first VH region has at least 85% sequence identity to any one of SEQ ID NOs: 11-14. In some embodiments, the second VH region has at least 85% sequence identity to any one of SEQ ID NOs: 22-25. In some embodiments, the first VH region has at least 85% sequence identity to any one of SEQ ID NOs: 11-14, and the second VH region has at least 85% sequence identity to any one of SEQ ID NOs: 22-25.

[0070] In some embodiments, the first VH region has at least 90% sequence identity to any one of SEQ ID NOs: 11-14. In some embodiments, the second VH region has at least 90% sequence identity to any one of SEQ ID NOs: 22-25. In some embodiments, the first VH region has at least 90% sequence identity to any one of SEQ ID NOs: 11-14, and the second VH region has at least 90% sequence identity to any one of SEQ ID NOs: 22-25.

[0071] In some embodiments, the first VH region has at least 95% sequence identity to any one of SEQ ID NOs: 11-14. In some embodiments, the second VH region has at least 95% sequence identity to any one of SEQ ID NOs: 22-25. In some embodiments, the first VH region has at least 95% sequence identity to any one of SEQ ID NOs: 11-14, and the second VH region has at least 95% sequence identity to any one of SEQ ID NOs: 22-25.

[0072] In some embodiments, the first VH region comprises a sequence selected from any one of SEQ ID NOs: 11-14. In some embodiments, the second VH region comprises a sequence selected from any one of SEQ ID NOs: 22-25. In some embodiments, the first VH region comprises a sequence selected from any one of SEQ ID NOs: 11-14, and the second VH region comprises a sequence selected from any one of SEQ ID NOs: 22-25.

[0073] In some embodiments, the first VH region comprises a sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%; 80%, 85%, 90%, 95%) sequence identity to the sequence of SEQ ID NO: 11, and the second VH region comprises a sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%; 80%, 85%, 90%, 95%) sequence identity to the sequence of SEQ ID NO: 22. In some embodiments, the first VH region comprises the sequence of SEQ ID NO: 11, and the second VH region comprises the sequence of SEQ ID NO: 22.

[0074] In some embodiments, the first VH region comprises a sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%; 80%, 85%, 90%, 95%) sequence identity to the sequence of SEQ ID NO: 12, and the second VH region comprises a sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%; 80%, 85%, 90%, 95%) sequence identity to the sequence of SEQ ID NO: 23. In some embodiments, the first VH region comprises the sequence of SEQ ID NO: 12, and the second VH region comprises the sequence of SEQ ID NO: 23.

[0075] In some embodiments, the first VH region comprises a sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%; 80%, 85%, 90%, 95%) sequence identity to the sequence of SEQ ID NO: 12, and the second VH region comprises a sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%; 80%, 85%, 90%, 95%) sequence identity to the sequence of SEQ ID NO: 24. In some embodiments, the first VH region comprises the sequence of SEQ ID NO: 12, and the second VH region comprises the sequence of SEQ ID NO: 24.

[0076] In some embodiments, the first VH region comprises a sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%; 80%, 85%, 90%, 95%) sequence identity to the sequence of SEQ ID NO: 12, and the second VH region comprises a sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%; 80%, 85%, 90%, 95%) sequence identity to the sequence of SEQ ID NO: 25. In some embodiments, the first VH region comprises the sequence of SEQ ID NO: 12, and the second VH region comprises the sequence of SEQ ID NO: 25.

[0077] In some embodiments, the first VH region comprises a sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%; 80%, 85%, 90%, 95%) sequence identity to the sequence of SEQ ID NO: 13, and the second VH region comprises a sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%; 80%, 85%, 90%, 95%) sequence identity to the sequence of SEQ ID NO: 23. In some embodiments, the first VH region comprises the sequence of SEQ ID NO: 13, and the second VH region comprises the sequence of SEQ ID NO: 23.

[0078] In some embodiments, the first VH region comprises a sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%; 80%, 85%, 90%, 95%) sequence identity to the sequence of SEQ ID NO: 14, and the second VH region comprises a sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%; 80%, 85%, 90%, 95%) sequence identity to the sequence of SEQ ID NO: 22. In some embodiments, the first VH region comprises the sequence of SEQ ID NO: 14, and the second VH region comprises the sequence of SEQ ID NO: 22.

[0079] In some embodiments, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof further comprises a heavy chain constant region sequence that is absent a CH1 sequence.

[0080] In some embodiments, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof further comprises a heavy chain constant region comprising a hinge region, a CH2 domain, and a CH3 domain.

[0081] 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 containing the S228P mutation (SEQ ID NO: 55).

[0082] 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 an F234A mutation, an L235A mutation, or both the F234A and L235A mutations.

[0083] 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 mutation, an L368A mutation, and a Y407V mutation.

[0084] In some embodiments, the anti-IL-2R βγ heavy chain-only antibody or antigen-binding fragment thereof further comprises an Fc region. In some embodiments, the anti-IL-2R βγ heavy chain-only antibody or antigen-binding fragment thereof further comprises a variant Fc region. In some embodiments, the variant Fc region comprises a heterodimerization mutation. In some embodiments, the Fc region is a silenced Fc region.

[0085] In some embodiments, the anti-IL-2Rβγ heavy chain only antibody is an IgG1 antibody. In some embodiments, the anti-IL-2Rβγ heavy chain only antibody is an IgG4 antibody.

[0086] In some embodiments, the anti-IL-2Rβγ heavy chain only antibody or antigen-binding fragment thereof is a first polypeptide comprising a sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%; 80%, 85%, 90%, 95%) sequence identity to the sequence of SEQ ID NO: 53; and and a second polypeptide comprising a sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%; 80%, 85%, 90%, 95%) sequence identity to the sequence of SEQ ID NO:61.

[0087] In some embodiments, the first polypeptide comprises the sequence of SEQ ID NO:53 and the second polypeptide comprises the sequence of SEQ ID NO:61.

[0088] In some embodiments, the anti-IL-2Rβγ heavy chain only antibody or antigen-binding fragment thereof is a first polypeptide comprising a sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%; 80%, 85%, 90%, 95%) sequence identity to the sequence of SEQ ID NO: 62; and and a second polypeptide comprising a sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%; 80%, 85%, 90%, 95%) sequence identity to the sequence of SEQ ID NO:63.

[0089] In some embodiments, the first polypeptide comprises the sequence of SEQ ID NO:62 and the second polypeptide comprises the sequence of SEQ ID NO:63.

[0090] In some embodiments, the anti-IL-2Rβγ heavy chain only antibody or antigen-binding fragment thereof is a first polypeptide comprising a sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%; 80%, 85%, 90%, 95%) sequence identity to the sequence of SEQ ID NO: 64; and and a second polypeptide comprising a sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%; 80%, 85%, 90%, 95%) sequence identity to the sequence of SEQ ID NO:65.

[0091] In some embodiments, the first polypeptide comprises the sequence of SEQ ID NO:64 and the second polypeptide comprises the sequence of SEQ ID NO:65.

[0092] In some embodiments, the anti-IL-2Rβγ heavy chain only antibody or antigen-binding fragment thereof is a first polypeptide comprising a sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%; 80%, 85%, 90%, 95%) sequence identity to the sequence of SEQ ID NO: 66; and and a second polypeptide comprising a sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%; 80%, 85%, 90%, 95%) sequence identity to the sequence of SEQ ID NO:67.

[0093] In some embodiments, the first polypeptide comprises the sequence of SEQ ID NO:66 and the second polypeptide comprises the sequence of SEQ ID NO:67.

[0094] In some embodiments, the anti-IL-2Rβγ heavy chain only antibody or antigen-binding fragment thereof is a first polypeptide comprising a sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%; 80%, 85%, 90%, 95%) sequence identity to the sequence of SEQ ID NO: 34; and and a second polypeptide comprising a sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%; 80%, 85%, 90%, 95%) sequence identity to the sequence of SEQ ID NO:35.

[0095] In some embodiments, the first polypeptide comprises the sequence of SEQ ID NO:34 and the second polypeptide comprises the sequence of SEQ ID NO:35.

[0096] In some embodiments, the anti-IL-2Rβγ heavy chain only antibody or antigen-binding fragment thereof is a first polypeptide comprising a sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%; 80%, 85%, 90%, 95%) sequence identity to the sequence of SEQ ID NO: 36; and and a second polypeptide comprising a sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%; 80%, 85%, 90%, 95%) sequence identity to the sequence of SEQ ID NO: 37.

[0097] In some embodiments, the first polypeptide comprises the sequence of SEQ ID NO:36 and the second polypeptide comprises the sequence of SEQ ID NO:37.

[0098] In some embodiments, the anti-IL-2R βγ heavy chain-only antibody or antigen-binding fragment is about 10 -11 M~about 10 -6 It has affinity for IL2R with a Kd of M.

[0099] In some embodiments, the anti-IL-2R βγ heavy chain-only antibody or antigen-binding fragment is about 10 -8 M ~ approx. 2.5×10 -7 It has affinity for IL2Rβ with a Kd of M.

[0100] In some embodiments, the anti-IL-2R βγ heavy chain-only antibody or antigen-binding fragment is about 10 -9 M ~ approx. 2.5×10 -8 It has affinity for IL2Rγ with a Kd of M.

[0101] In some embodiments, anti-IL-2Rβγ heavy chain only antibodies or antigen-binding fragments function as IL-2Rβγ agonists.

[0102] In some embodiments, the methods of the present disclosure further comprise administering a premedication to the subject prior to administration of the first dose of the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment or the first dose of T cell redirecting therapy. In some embodiments, the premedication is selected from an antihistamine, a glucocorticoid, an IL-6 receptor antagonist, and a tumor necrosis factor α (TNF-α) antagonist.

[0103] In some embodiments of the methods disclosed herein, the T cell redirecting therapy is a bispecific T cell engaging molecule. In some embodiments, the bispecific T cell engaging molecule comprises a first domain that specifically binds to a target cancer cell antigen and a second domain that specifically binds to human CD3. In some embodiments, the target cancer cell antigen is selected from CEA, CD19, CD33, CD70, EGFRvIII, FLT3, GPRC5D, DLL3, BCMA, PSMA, STEAP1, STEAP2, MUC16, MUC17, and CLDN18.2.

[0104] In some embodiments, the bispecific T cell engaging molecule further comprises a half-life prolonging domain. In some embodiments, the half-life prolonging domain provides the bispecific T cell engaging molecule with a half-life of greater than about 24 hours. In some embodiments, the half-life prolonging domain is selected from an immunoglobulin Fc domain, a domain derived from serum albumin (e.g., human serum albumin), an albumin binding domain (e.g., comprising a human albumin binding peptide or an antibody fragment that specifically binds to serum albumin), a peptide that binds to the neonatal Fc receptor (FcRn), and a polyethylene glycol polymer.

[0105] In some embodiments, the bispecific T cell engaging molecule is a three-chain antibody-like molecule.

[0106] In alternative embodiments of the methods disclosed herein, the T cell redirection therapy is a chimeric antigen receptor (CAR)-expressing T cell. In some embodiments, the CAR-expressing T cell comprises a first domain that specifically binds to a target cancer cell antigen, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the target cancer cell antigen is selected from CEA, CD19, CD33, CD70, EGFRvIII, FLT3, GPRC5D, DLL3, BCMA, PSMA, STEAP1, STEAP2, MUC16, MUC17, and CLDN18.2.

[0107] In some embodiments of the methods disclosed herein, at least one dose of an anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof is administered to the subject prior to the first dose of T cell redirecting therapy.

[0108] In some embodiments, the method comprises administering an anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof in combination with T cell redirecting therapy in one or more treatment cycles. In some embodiments, each of the one or more treatment cycles comprises a single dose of the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof and a single dose of T cell redirecting therapy. In some embodiments, each of the one or more treatment cycles comprises multiple doses of the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof and a single dose of T cell redirecting therapy. In some embodiments, each of the one or more treatment cycles comprises a single dose of the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof and multiple doses of T cell redirecting therapy. In some embodiments, each of the one or more treatment cycles comprises multiple doses of the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof and multiple doses of T cell redirecting therapy.

[0109] In some embodiments of the methods disclosed herein, the subject has been diagnosed with a hematological cancer, hi some embodiments, the hematological cancer is selected from acute myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, multiple myeloma, diffuse large B-cell lymphoma, Burkitt's lymphoma, and non-Hodgkin's lymphoma.

[0110] In some embodiments of the methods disclosed herein, the subject has been diagnosed with a cancer selected from prostate cancer, non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, hepatocellular carcinoma, bladder cancer, testicular cancer, colorectal cancer, esophageal cancer, glioblastoma, head and neck cancer, pancreatic cancer, breast cancer, gastric cancer, gastroesophageal junction cancer, bone cancer, ovarian cancer, endometrial cancer, and melanoma.

[0111] In some embodiments, the subject has at least one tumor with low immune infiltration (e.g., low or no T cell infiltration) prior to co-administration. In some embodiments, co-administration increases T cell infiltration of the tumor. In some embodiments, co-administration is associated with at least one anti-tumor effect. In some embodiments, the at least one anti-cancer effect is selected from a reduction in the number of cancer cells, a reduction in the number of metastases, an increase in life expectancy, a reduction in cancer cell proliferation, a reduction in cancer cell survival, and an improvement in various physiological symptoms associated with a cancerous condition.

[0112] In some embodiments of the methods disclosed herein, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof is administered in a pharmaceutical composition adapted for intravenous or subcutaneous delivery.

[0113] In some embodiments of the methods disclosed herein, the T cell redirecting therapy is administered in a pharmaceutical composition adapted for intravenous or subcutaneous delivery. In some embodiments, the T cell redirecting therapy is a bispecific T cell engaging molecule, and the pharmaceutical composition comprises the bispecific T cell engaging molecule, a buffer, a surfactant, and a stabilizer. In some embodiments, the T cell redirecting therapy is a bispecific T cell engaging molecule, and the pharmaceutical composition comprises the bispecific T cell engaging molecule, glutamate buffer, polysorbate 20 or polysorbate 80, and sucrose at a pH of about 4.0 to about 4.4.

[0114] In some embodiments of the methods disclosed herein, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof and the T cell redirecting therapy are administered in separate pharmaceutical compositions, which in some embodiments may be lyophilized and reconstituted prior to administration to a patient.

[0115] In some embodiments of the methods disclosed herein, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof and the T cell redirecting therapy are administered simultaneously. In alternative embodiments of the methods disclosed herein, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof and the T cell redirecting therapy are administered sequentially.

[0116] In some embodiments of the methods disclosed herein, the subject has previously received a first-line therapy for the cancer, hi some embodiments, the subject has previously received a first-line and a second-line therapy for the cancer.

[0117] Also disclosed herein is the use of an anti-IL-2Rβγ heavy chain-only antibody, or antigen-binding fragment thereof, in the manufacture of a medicament adapted for use in the methods disclosed herein. In some embodiments, the method has one or more of the features of the methods described above. In some embodiments, the anti-IL-2Rβγ heavy chain-only antibody, or antigen-binding fragment thereof, is an anti-IL-2Rβγ heavy chain-only antibody. In some embodiments, the anti-IL-2Rβγ heavy chain-only antibody, or antigen-binding fragment thereof, is an antigen-binding fragment of an anti-IL-2Rβγ heavy chain-only antibody.

[0118] Also disclosed herein is the use of T cell redirection therapy in the manufacture of a medicament adapted for use in the methods disclosed herein. In some embodiments, the method has one or more of the features of the methods described above.

[0119] Also disclosed herein is the use of an anti-IL-2Rβγ heavy chain-only antibody, or antigen-binding fragment thereof, and T cell redirection therapy in the manufacture of a medicament adapted for use in the methods disclosed herein. In some embodiments, the method has one or more of the features of the methods described above. In some embodiments, the anti-IL-2Rβγ heavy chain-only antibody, or antigen-binding fragment thereof, is an anti-IL-2Rβγ heavy chain-only antibody. In some embodiments, the anti-IL-2Rβγ heavy chain-only antibody, or antigen-binding fragment thereof, is an antigen-binding fragment of an anti-IL-2Rβγ heavy chain-only antibody.

[0120] Also disclosed herein are pharmaceutical compositions comprising an anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof for use in the methods disclosed herein. In some embodiments, the methods have one or more features of the methods described above. In some embodiments, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof is an anti-IL-2Rβγ heavy chain-only antibody. In some embodiments, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof is an antigen-binding fragment of an anti-IL-2Rβγ heavy chain-only antibody.

[0121] Also disclosed herein is a pharmaceutical composition comprising a T cell redirecting therapy for use in the methods disclosed herein. In some embodiments, the method has one or more of the features of the methods described above. In some embodiments, the T cell redirecting therapy is a T cell-inducing molecule. In some embodiments, the T cell redirecting therapy is a bispecific T cell-inducing molecule.

[0122] Also disclosed herein is a pharmaceutical composition comprising an anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof and a T cell redirecting therapy for use in the methods disclosed herein. In some embodiments, the method has one or more features of the methods described above. In some embodiments, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof is an anti-IL-2Rβγ heavy chain-only antibody. In some embodiments, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof is an antigen-binding fragment of an anti-IL-2Rβγ heavy chain-only antibody. In some embodiments, the T cell redirecting therapy is a T cell attracting molecule. In some embodiments, the T cell redirecting therapy is a bispecific T cell attracting molecule.

[0123] Also disclosed herein is a combination product comprising an anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof and a T cell redirecting therapy. In some embodiments, the combination product is adapted for use in a method disclosed herein. In some embodiments, the method has one or more features of the methods described above. In some embodiments, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof is an anti-IL-2Rβγ heavy chain-only antibody. In some embodiments, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof is an antigen-binding fragment of an anti-IL-2Rβγ heavy chain-only antibody. In some embodiments, the T cell redirecting therapy is a T cell-inducing molecule. In some embodiments, the T cell-redirecting therapy is a bispecific T cell-inducing molecule.

[0124] Also disclosed herein is a combination product for use in a method of treating cancer in a subject in need thereof, comprising an anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof and a T cell redirecting therapy, wherein the anti-IL-2Rβγ heavy chain-only antibody enhances the anti-cancer effect associated with administration of the T cell redirecting therapy. In some embodiments, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof is an anti-IL-2Rβγ heavy chain-only antibody. In some embodiments, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof is an antigen-binding fragment of an anti-IL-2Rβγ heavy chain-only antibody. In some embodiments, the T cell redirecting therapy is a T cell engaging molecule. In some embodiments, the T cell redirecting therapy is a bispecific T cell engaging molecule.

[0125] Also disclosed herein is an anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof for use in treating cancer in combination with T cell redirection therapy, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof comprising: A first heavy chain variable (VH) region that specifically binds to IL-2Rβ, a VH complementarity-determining region 1 (CDR1) sequence having up to two amino acid alterations relative to any one of SEQ ID NOs: 1 to 3; and a VH CDR2 sequence having up to two amino acid alterations relative to any one of SEQ ID NOs: 4 to 6; a VH CDR3 sequence having up to two amino acid alterations relative to any one of SEQ ID NOs: 7 to 10; and a first VH region comprising: a second VH region that specifically binds IL-2Rγ, a VH CDR1 sequence having up to two amino acid alterations relative to any one of SEQ ID NOs: 15 to 16; a VH CDR2 sequence having up to two amino acid alterations relative to any one of SEQ ID NOs: 17 to 19; a VH CDR3 sequence having up to two amino acid alterations relative to any one of SEQ ID NOs: 20 to 21; and a second VH region comprising:

[0126] In some embodiments, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof is an anti-IL-2Rβγ heavy chain-only antibody. In some embodiments, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof is an antigen-binding fragment of an anti-IL-2Rβγ heavy chain-only antibody.

[0127] In some embodiments, the T cell redirecting therapy is a T cell engaging molecule. In some embodiments, the T cell redirecting therapy is a bispecific T cell engaging molecule.

[0128] Also disclosed herein is an anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof for use in treating cancer in combination with T cell redirection therapy, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof comprising: A first heavy chain variable (VH) region that specifically binds to IL-2Rβ, (1)(a) a VH complementarity-determining region 1 (CDR1) comprising the following sequence: GFTFS X1Y G (SEQ ID NO: 29) (wherein X1 is S or T); and (b) a VH CDR2 comprising the following sequence: ISYDGSN X2 (SEQ ID NO: 30) (wherein X2 is K or R); and (c) a VH CDR3 comprising the following sequence: ARDLDYD X3L TGDPVGGFDI (SEQ ID NO: 31) wherein X3 is V or I; or (2)(a) a VH CDR1 comprising the following sequence: GGSISSS X1 W (SEQ ID NO: 26) (wherein X1 is D or N); (b) a VH CDR2 comprising the following sequence: I X2H SGST (SEQ ID NO: 27) (wherein X2 is D or S); and (c) a VH CDR3 comprising the following sequence: X3R G X4W EL X5D AFDI (SEQ ID NO: 28) (wherein X3 is G or A; X4 is S or Q; and X5 is S or T). a first VH region comprising: a second VH region that specifically binds IL-2Rγ, (1)(a) a VH CDR1 comprising the following sequence: GF X1X2X3X4Y Y (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); and (b) a VH CDR2 comprising the following sequence: IS X5S G X6X7I (SEQ ID NO: 33) wherein X5 is S or N; X6 is D, S, G, or N; and X7 is T or I; and (c) a VH CDR3 comprising the sequence ARGDAVSITGDY (SEQ ID NO: 20); or (2) VH CDR1 comprising the sequence GFTFSDYY (SEQ ID NO: 15); VH CDR2 comprising the sequence ISSSGTTT (SEQ ID NO: 19); and VH CDR3 comprising the sequence ARGAAVAPGFDS (SEQ ID NO: 21) and a second VH region comprising:

[0129] In some embodiments, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof is an anti-IL-2Rβγ heavy chain-only antibody. In some embodiments, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof is an antigen-binding fragment of an anti-IL-2Rβγ heavy chain-only antibody.

[0130] In some embodiments, the T cell redirecting therapy is a T cell engaging molecule. In some embodiments, the T cell redirecting therapy is a bispecific T cell engaging molecule.

[0131] Also disclosed herein is an anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof for use in treating cancer in combination with T cell redirection therapy, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof comprising: a first heavy chain variable (VH) complementarity-determining region (CDR) 1, 2, and 3 (combined) of which the entire set has at least 95% sequence identity with VH CDR 1, 2, and 3 of any one of SEQ ID NOS: 11 to 14; and The entire set of VH complementarity determining regions (CDRs) 1, 2, and 3 (combined) comprises a second VH region that specifically binds to IL-2Rγ, the second VH region having at least 95% sequence identity with VH CDRs 1, 2, and 3 of any one of SEQ ID NOs: 22 to 25.

[0132] In some embodiments, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof is an anti-IL-2Rβγ heavy chain-only antibody. In some embodiments, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof is an antigen-binding fragment of an anti-IL-2Rβγ heavy chain-only antibody.

[0133] In some embodiments, the T cell redirecting therapy is a T cell engaging molecule. In some embodiments, the T cell redirecting therapy is a bispecific T cell engaging molecule.

[0134] Also disclosed herein is an anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof for use in treating cancer in combination with T cell redirection therapy, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof comprising: a first heavy chain variable (VH) region that specifically binds to IL-2Rβ, having at least 95% sequence identity to any one of SEQ ID NOs: 11 to 14; and It comprises a second VH region that specifically binds to IL-2Rγ and has at least 95% sequence identity with any one of SEQ ID NOs: 22 to 25.

[0135] In some embodiments, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof is an anti-IL-2Rβγ heavy chain-only antibody. In some embodiments, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof is an antigen-binding fragment of an anti-IL-2Rβγ heavy chain-only antibody.

[0136] In some embodiments, the T cell redirecting therapy is a T cell engaging molecule. In some embodiments, the T cell redirecting therapy is a bispecific T cell engaging molecule.

[0137] Also disclosed herein is an anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof for use in enhancing the anti-cancer effect associated with the administration of T cell redirecting therapy in a subject diagnosed with cancer, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof comprising: A first heavy chain variable (VH) region that specifically binds to IL-2Rβ, a VH complementarity-determining region 1 (CDR1) sequence having up to two amino acid alterations relative to any one of SEQ ID NOs: 1 to 3; and a VH CDR2 sequence having up to two amino acid alterations relative to any one of SEQ ID NOs: 4 to 6; a VH CDR3 sequence having up to two amino acid alterations relative to any one of SEQ ID NOs: 7 to 10; and a first VH region comprising: a second VH region that specifically binds IL-2Rγ, a VH CDR1 sequence having up to two amino acid alterations relative to any one of SEQ ID NOs: 15-16, and / or a VH CDR2 sequence having up to two amino acid alterations relative to any one of SEQ ID NOs: 17-19, and / or a VH CDR3 sequence having up to two amino acid alterations relative to any one of SEQ ID NOs: 20 to 21; and a second VH region comprising:

[0138] In some embodiments, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof is an anti-IL-2Rβγ heavy chain-only antibody. In some embodiments, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof is an antigen-binding fragment of an anti-IL-2Rβγ heavy chain-only antibody.

[0139] Also disclosed herein is an anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof for use in enhancing the anti-cancer effect associated with the administration of T cell redirecting therapy in a subject diagnosed with cancer, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof comprising: A first heavy chain variable (VH) region that specifically binds to IL-2Rβ, (1)(a) a VH complementarity-determining region 1 (CDR1) comprising the following sequence: GFTFS X1Y G (SEQ ID NO: 29) (wherein X1 is S or T); and (b) a VH CDR2 comprising the following sequence: ISYDGSN X2 (SEQ ID NO: 30) (wherein X2 is K or R); and (c) a VH CDR3 comprising the following sequence: ARDLDYD X3L TGDPVGGFDI (SEQ ID NO: 31) wherein X3 is V or I; or (2)(a) a VH CDR1 comprising the following sequence: GGSISSS X1 W (SEQ ID NO: 26) (wherein X1 is D or N); (b) a VH CDR2 comprising the following sequence: I X2H SGST (SEQ ID NO: 27) (wherein X2 is D or S); and (c) a VH CDR3 comprising the following sequence: X3R G X4W EL X5D AFDI (SEQ ID NO: 28) (wherein X3 is G or A; X4 is S or Q; and X5 is S or T). a first VH region comprising: a second VH region that specifically binds IL-2Rγ, (1)(a) a VH CDR1 comprising the following sequence: GF X1X2X3X4Y Y (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); and (b) a VH CDR2 comprising the following sequence: IS X5S G X6X7I (SEQ ID NO: 33) wherein X5 is S or N; X6 is D, S, G, or N; and X7 is T or I; and (c) a VH CDR3 comprising the sequence ARGDAVSITGDY (SEQ ID NO: 20); or (2) VH CDR1 comprising the sequence GFTFSDYY (SEQ ID NO: 15); VH CDR2 comprising the sequence ISSSGTTT (SEQ ID NO: 19); and VH CDR3 comprising the sequence ARGAAVAPGFDS (SEQ ID NO: 21) and a second VH region comprising:

[0140] In some embodiments, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof is an anti-IL-2Rβγ heavy chain-only antibody. In some embodiments, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof is an antigen-binding fragment of an anti-IL-2Rβγ heavy chain-only antibody.

[0141] Also disclosed herein is an anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof for use in enhancing the anti-cancer effect associated with the administration of T cell redirecting therapy in a subject diagnosed with cancer, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof comprising: a first heavy chain variable (VH) complementarity-determining region (CDR) 1, 2, and 3 (combined) of which the entire set has at least 95% sequence identity with VH CDR 1, 2, and 3 of any one of SEQ ID NOS: 11 to 14; and The entire set of VH complementarity determining regions (CDRs) 1, 2, and 3 (combined) comprises a second VH region that specifically binds to IL-2Rγ, the second VH region having at least 95% sequence identity with VH CDRs 1, 2, and 3 of any one of SEQ ID NOs: 22 to 25.

[0142] In some embodiments, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof is an anti-IL-2Rβγ heavy chain-only antibody. In some embodiments, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof is an antigen-binding fragment of an anti-IL-2Rβγ heavy chain-only antibody.

[0143] Also disclosed herein is an anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof for use in enhancing the anti-cancer effect associated with the administration of T cell redirecting therapy in a subject diagnosed with cancer, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof comprising: a first heavy chain variable (VH) region that specifically binds to IL-2Rβ, having at least 95% sequence identity to any one of SEQ ID NOs: 11 to 14; and It comprises a second VH region that specifically binds to IL-2Rγ and has at least 95% sequence identity with any one of SEQ ID NOs: 22 to 25.

[0144] In some embodiments, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof is an anti-IL-2Rβγ heavy chain-only antibody. In some embodiments, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof is an antigen-binding fragment of an anti-IL-2Rβγ heavy chain-only antibody.

[0145] In an alternative aspect of the present disclosure, an NK cell redirecting therapy, such as an NK cell-inducing molecule or CAR-NK cells, may be used in place of the T cell redirecting therapy in the methods, uses, pharmaceutical compositions, or combination products described herein.

[0146] Some exemplary embodiments of the present disclosure include, but are not limited to: E1. A method of treating cancer in a subject in need thereof, comprising administering to the subject an anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof in combination with T cell redirecting therapy, wherein the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof: A first heavy chain variable (VH) region that specifically binds to IL-2Rβ, a VH complementarity-determining region 1 (CDR1) sequence having up to two amino acid alterations relative to any one of SEQ ID NOs: 1 to 3; and a VH CDR2 sequence having up to two amino acid alterations relative to any one of SEQ ID NOs: 4 to 6; a VH CDR3 sequence having up to two amino acid alterations relative to any one of SEQ ID NOs: 7 to 10; and a first VH region comprising: a second VH region that specifically binds IL-2Rγ, a VH CDR1 sequence having up to two amino acid alterations relative to any one of SEQ ID NOs: 15 to 16; a VH CDR2 sequence having up to two amino acid alterations relative to any one of SEQ ID NOs: 17 to 19; a VH CDR3 sequence having up to two amino acid alterations relative to any one of SEQ ID NOs: 20 to 21; and a second VH region comprising: E2. A method of enhancing the anti-cancer effect associated with the administration of T cell redirecting therapy in a subject diagnosed with cancer, comprising administering to the subject an anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof in combination with T cell redirecting therapy, wherein the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof is: A first heavy chain variable (VH) region that specifically binds to IL-2Rβ, a VH complementarity-determining region 1 (CDR1) sequence having up to two amino acid alterations relative to any one of SEQ ID NOs: 1 to 3; and a VH CDR2 sequence having up to two amino acid alterations relative to any one of SEQ ID NOs: 4 to 6; a VH CDR3 sequence having up to two amino acid alterations relative to any one of SEQ ID NOs: 7 to 10; and a first VH region comprising: a second VH region that specifically binds IL-2Rγ, a VH CDR1 sequence having up to two amino acid alterations relative to any one of SEQ ID NOs: 15 to 16; a VH CDR2 sequence having up to two amino acid alterations relative to any one of SEQ ID NOs: 17 to 19; a VH CDR3 sequence having up to two amino acid alterations relative to any one of SEQ ID NOs: 20 to 21; and a second VH region comprising: E3. The method of E1 or E2, wherein the VH CDR1 sequence of the first VH region has at most one amino acid alteration relative to any one of SEQ ID NOs: 1-3. E4. The method of any one of E1-E3, wherein the VH CDR2 sequence of the first VH region has at most one amino acid alteration relative to any one of SEQ ID NOs: 4-6. E5. The method of any one of E1-E4, wherein the VH CDR3 sequence of the first VH region has at most one amino acid alteration relative to any one of SEQ ID NOs: 7-10. E6. The method of any one of E1-E5, wherein the VH CDR1 sequence of the second VH region has at most one amino acid alteration relative to any one of SEQ ID NOs: 15-16. E7. The method of any one of E1-E6, wherein the VH CDR2 sequence of the second VH region has at most one amino acid alteration relative to any one of SEQ ID NOs: 17-19. E8. The method of any one of E1-E7, wherein the VH CDR3 sequence of the second VH region has at most one amino acid alteration relative to any one of SEQ ID NOs: 20-21. E9. The method of any one of E3-E8, wherein at most one amino acid modification is an amino acid substitution. E10. The method of any one of E3 to E9, wherein at most one amino acid modification is a conservative amino acid substitution. E11. The method of any one of E3 to E8, wherein at most one amino acid modification is an amino acid deletion. E12. The method of any one of E3 to E8, wherein at most one amino acid modification is an amino acid addition. E13. The method of any one of E1-E8, wherein each amino acid modification, if any, is a conservative amino acid substitution. E14. The method of any one of E1 to E13, wherein the first VH region comprises a VH CDR1 comprising a sequence selected from SEQ ID NOs: 1 to 3. E15. The method of any one of E1 to E14, wherein the first VH region comprises a VH CDR2 comprising a sequence selected from SEQ ID NOs: 4 to 6. E16. The method of any one of E1 to E15, wherein the first VH region comprises a VH CDR3 comprising a sequence selected from SEQ ID NOs: 7 to 10. E17. The first VH region is VH CDR1 comprising a sequence selected from SEQ ID NOs: 1 to 3; and VH CDR2 comprising a sequence selected from SEQ ID NOs: 4 to 6; and The method according to any one of E1 to E16, comprising a VH CDR3 comprising a sequence selected from SEQ ID NOs: 7 to 10. E18. The first VH region is (a) a VH CDR1, a VH CDR2, and a VH CDR3 comprising the sequences of SEQ ID NOs: 1, 4, and 7, respectively; or (b) VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 1, 4, and 8, respectively; or (c) VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 2, 5, and 9, respectively; or (d) VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 3, 6, and 10, respectively. The method according to any one of E1 to E17, comprising: E19. The method of any one of E1 to E18, wherein the second VH region comprises a VH CDR1 comprising a sequence selected from SEQ ID NOs: 15-16. E20. The method of any one of E1 to E19, wherein the second VH region comprises a VH CDR2 comprising a sequence selected from SEQ ID NOs: 17-19. E21. The method of any one of E1 to E20, wherein the second VH region comprises a VH CDR3 comprising a sequence selected from SEQ ID NOs: 20-21. E22. The second VH region is VH CDR1 comprising a sequence selected from SEQ ID NOs: 15 to 16; and VH CDR2 comprising a sequence selected from SEQ ID NOs: 17 to 19; and The method according to any one of E1 to E21, comprising a VH CDR3 comprising a sequence selected from SEQ ID NOs: 20 to 21. E23. The second VH region is (a) a VH CDR1, a VH CDR2, and a VH CDR3 comprising the sequences of SEQ ID NOs: 15, 17, and 20, respectively; or (b) VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 15, 18, and 20, respectively; or (c) VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 16, 18, and 20, respectively; or (d) VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 15, 19, and 21, respectively. The method according to any one of E1 to E22, comprising: E24. The method according to any one of E1 to E23, the first VH region comprises a VH CDR1, a VH CDR2, and a VH CDR3 comprising the sequences of SEQ ID NOs: 1, 4, and 7, respectively; and The method wherein the second VH region comprises a VH CDR1, a VH CDR2, and a VH CDR3 comprising the sequences of SEQ ID NOs: 15, 17, and 20, respectively. E25. The method according to any one of E1 to E23, the first VH region comprises a VH CDR1, a VH CDR2, and a VH CDR3 comprising the sequences of SEQ ID NOs: 1, 4, and 8, respectively; and The method wherein the second VH region comprises a VH CDR1, a VH CDR2, and a VH CDR3 comprising the sequences of SEQ ID NOs: 15, 18, and 20, respectively. E26. The method according to any one of E1 to E23, the first VH region comprises a VH CDR1, a VH CDR2, and a VH CDR3 comprising the sequences of SEQ ID NOs: 1, 4, and 8, respectively; and The method wherein the second VH region comprises a VH CDR1, a VH CDR2, and a VH CDR3 comprising the sequences of SEQ ID NOs: 16, 18, and 20, respectively. E27. The method according to any one of E1 to E23, the first VH region comprises a VH CDR1, a VH CDR2, and a VH CDR3 comprising the sequences of SEQ ID NOs: 1, 4, and 8, respectively; and The method wherein the second VH region comprises a VH CDR1, a VH CDR2, and a VH CDR3 comprising the sequences of SEQ ID NOs: 15, 19, and 21, respectively. E28. The method according to any one of E1 to E23, the first VH region comprises a VH CDR1, a VH CDR2, and a VH CDR3 comprising the sequences of SEQ ID NOs: 2, 5, and 9, respectively; and The method wherein the second VH region comprises a VH CDR1, a VH CDR2, and a VH CDR3 comprising the sequences of SEQ ID NOs: 15, 18, and 20, respectively. E29. The method according to any one of E1 to E23, the first VH region comprises a VH CDR1, a VH CDR2, and a VH CDR3 comprising the sequences of SEQ ID NOs: 3, 6, and 10, respectively; and The method wherein the second VH region comprises a VH CDR1, a VH CDR2, and a VH CDR3 comprising the sequences of SEQ ID NOs: 15, 17, and 20, respectively. E30. A method of treating cancer in a subject in need thereof, comprising administering to the subject an anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof in combination with T cell redirecting therapy, wherein the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof: A first heavy chain variable (VH) region that specifically binds to IL-2Rβ, (1)(a) a VH complementarity-determining region 1 (CDR1) comprising the following sequence: GFTFS X1Y G (SEQ ID NO: 29) (wherein X1 is S or T); and (b) a VH CDR2 comprising the following sequence: ISYDGSN X2 (SEQ ID NO: 30) (wherein X2 is K or R); and (c) a VH CDR3 comprising the following sequence: ARDLDYD X3L TGDPVGGFDI (SEQ ID NO: 31) wherein X3 is V or I; or (2)(a) a VH CDR1 comprising the following sequence: GGSISSS X1W (SEQ ID NO: 26) (wherein X1 is D or N); (b) a VH CDR2 comprising the following sequence: I X2H SGST (SEQ ID NO: 27) (wherein X2 is D or S); and (c) a VH CDR3 comprising the following sequence: X3R G X4W EL X5D AFDI (SEQ ID NO: 28) (wherein X3 is G or A; X4 is S or Q; and X5 is S or T). a first VH region comprising: a second VH region that specifically binds IL-2Rγ, (1)(a) a VH CDR1 comprising the following sequence: GF X1X2X3X4Y Y (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); and (b) a VH CDR2 comprising the following sequence: IS X5S G X6X7I (SEQ ID NO: 33) wherein X5 is S or N; X6 is D, S, G, or N; and X7 is T or I; and (c) a VH CDR3 comprising the sequence ARGDAVSITGDY (SEQ ID NO: 20); or (2) VH CDR1 comprising the sequence GFTFSDYY (SEQ ID NO: 15); VH CDR2 comprising the sequence ISSSGTTT (SEQ ID NO: 19); and VH CDR3 comprising the sequence ARGAAVAPGFDS (SEQ ID NO: 21) and a second VH region comprising: E31. A method of enhancing the anti-cancer effect associated with the administration of T cell redirecting therapy in a subject diagnosed with cancer, comprising administering to the subject an anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof in combination with T cell redirecting therapy, wherein the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof is: A first heavy chain variable (VH) region that specifically binds to IL-2Rβ, (1)(a) a VH complementarity-determining region 1 (CDR1) comprising the following sequence: GFTFS X1Y G (SEQ ID NO: 29) (wherein X1 is S or T); and (b) a VH CDR2 comprising the following sequence: ISYDGSN X2 (SEQ ID NO: 30) (wherein X2 is K or R); and (c) a VH CDR3 comprising the following sequence: ARDLDYD X3L TGDPVGGFDI (SEQ ID NO: 31) wherein X3 is V or I; or (2)(a) a VH CDR1 comprising the following sequence: GGSISSS X1W (SEQ ID NO: 26) (wherein X1 is D or N); (b) a VH CDR2 comprising the following sequence: I X2H SGST (SEQ ID NO: 27) (wherein X2 is D or S); and (c) a VH CDR3 comprising the following sequence: X3R G X4W EL X5D AFDI (SEQ ID NO: 28) (wherein X3 is G or A; X4 is S or Q; and X5 is S or T). a first VH region comprising: a second VH region that specifically binds IL-2Rγ, (1)(a) a VH CDR1 comprising the following sequence: GF X1X2X3X4Y Y (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); and (b) a VH CDR2 comprising the following sequence: IS X5S G X6X7I (SEQ ID NO: 33) wherein X5 is S or N; X6 is D, S, G, or N; and X7 is T or I; and (c) a VH CDR3 comprising the sequence ARGDAVSITGDY (SEQ ID NO: 20); or (2) VH CDR1 comprising the sequence GFTFSDYY (SEQ ID NO: 15); VH CDR2 comprising the sequence ISSSGTTT (SEQ ID NO: 19); and VH CDR3 comprising the sequence ARGAAVAPGFDS (SEQ ID NO: 21) and a second VH region comprising: E32. A method of treating cancer in a subject in need thereof, comprising administering to the subject an anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof in combination with T cell redirecting therapy, wherein the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof: a first heavy chain variable (VH) complementarity determining region (CDR) 1, 2, and 3 (combined) of the entire set of VH CDR 1, 2, and 3 have at least 80% (e.g., at least 85%, at least 90%, at least 95%; 80%, 85%, 90%, 95%) sequence identity with VH CDR 1, 2, and 3 of any one of SEQ ID NOS: 11-14; and The method comprises a second VH region that specifically binds to IL-2Rγ, wherein the entire set of VH complementarity-determining regions (CDRs) 1, 2, and 3 (combined) has at least 80% (e.g., at least 85%, at least 90%, at least 95%; 80%, 85%, 90%, 95%) sequence identity to VH CDRs 1, 2, and 3 of any one of SEQ ID NOs: 22 to 25. E33. A method of enhancing the anti-cancer effect associated with the administration of T cell redirecting therapy in a subject diagnosed with cancer, comprising administering to the subject an anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof in combination with T cell redirecting therapy, wherein the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof is: a first heavy chain variable (VH) complementarity determining region (CDR) 1, 2, and 3 (combined) of the entire set of VH CDR 1, 2, and 3 have at least 80% (e.g., at least 85%, at least 90%, at least 95%; 80%, 85%, 90%, 95%) sequence identity with VH CDR 1, 2, and 3 of any one of SEQ ID NOS: 11-14; and The method comprises a second VH region that specifically binds to IL-2Rγ, wherein the entire set of VH complementarity-determining regions (CDRs) 1, 2, and 3 (combined) has at least 80% (e.g., at least 85%, at least 90%, at least 95%; 80%, 85%, 90%, 95%) sequence identity to VH CDRs 1, 2, and 3 of any one of SEQ ID NOs: 22 to 25. E34. The method according to E32 or E33, the entire set of VH CDR1, 2, and 3 (combined) of the first VH region has at least 85% sequence identity with VH CDR1, 2, and 3 of any one of SEQ ID NOs: 11-14; and / or A method in which the entire set of VH CDR1, 2, and 3 (together) of the second VH region has at least 85% sequence identity with VH CDR1, 2, and 3 of any one of SEQ ID NOs: 22 to 25. E35. The method according to any one of E32 to E34, the entire set of VH CDR1, 2, and 3 (combined) of the first VH region has at least 90% sequence identity with VH CDR1, 2, and 3 of any one of SEQ ID NOs: 11-14; and / or A method in which the entire set of VH CDR1, 2, and 3 (together) of the second VH region has at least 90% sequence identity with VH CDR1, 2, and 3 of any one of SEQ ID NOs: 22 to 25. E36. The method according to any one of E32 to E35, the entire set of VH CDR1, 2, and 3 (combined) of the first VH region has at least 95% sequence identity with VH CDR1, 2, and 3 of any one of SEQ ID NOs: 11-14; and / or A method in which the entire set of VH CDR1, 2, and 3 (together) of the second VH region has at least 95% sequence identity with VH CDR1, 2, and 3 of any one of SEQ ID NOs: 22 to 25. E37. The method according to any one of E32 to E36, the entire set of VH CDR1, 2, and 3 (combined) of the first VH region has at least 98% sequence identity with VH CDR1, 2, and 3 of any one of SEQ ID NOs: 11-14; and / or A method in which the entire set of VH CDR1, 2, and 3 (together) of the second VH region has at least 98% sequence identity with VH CDR1, 2, and 3 of any one of SEQ ID NOs: 22 to 25. E38. The method of any one of E32 to E37, wherein the first VH region comprises a VH CDR1, a VH CDR2, and a VH CDR3 of any one of SEQ ID NOs: 11 to 14. E39. The method of any one of E32 to E38, wherein the second VH region comprises a VH CDR1, a VH CDR2, and a VH CDR3 of any one of SEQ ID NOs: 22 to 25. E40. The method according to any one of E32 to E37, the first VH region comprises a VH CDR1, a VH CDR2, and a VH CDR3 of any one of SEQ ID NOs: 11 to 14; and A method in which the second VH region comprises a VH CDR1, a VH CDR2, and a VH CDR3 of any one of SEQ ID NOs: 22 to 25. E41. The method of any one of E1-E40, wherein the VH CDR1, VH CDR2, and VH CDR3 sequences of the first VH region are present within a human VH framework. E42. The method of any one of E1-E41, wherein the VH CDR1, VH CDR2, and VH CDR3 sequences of the second VH region are present within a human VH framework. E43. A method of treating cancer in a subject in need thereof, comprising administering to the subject an anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof in combination with T cell redirecting therapy, wherein the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof: a first heavy chain variable (VH) region that specifically binds to IL-2Rβ and has at least 80% (e.g., at least 85%, at least 90%, at least 95%; 80%, 85%, 90%, 95%) sequence identity to any one of SEQ ID NOs: 11 to 14; and The method includes a second VH region that specifically binds to IL-2Rγ and has at least 80% (e.g., at least 85%, at least 90%, at least 95%; 80%, 85%, 90%, 95%) sequence identity to any one of SEQ ID NOs: 22 to 25. E44. A method for enhancing the anti-cancer effect associated with the administration of T cell redirecting therapy in a subject diagnosed with cancer, comprising administering to the subject an anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof in combination with T cell redirecting therapy, wherein the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof is: a first heavy chain variable (VH) region that specifically binds to IL-2Rβ and has at least 80% (e.g., at least 85%, at least 90%, at least 95%; 80%, 85%, 90%, 95%) sequence identity to any one of SEQ ID NOs: 11 to 14; and The method includes a second VH region that specifically binds to IL-2Rγ and has at least 80% (e.g., at least 85%, at least 90%, at least 95%; 80%, 85%, 90%, 95%) sequence identity to any one of SEQ ID NOs: 22 to 25. E45. The method according to E43 or E44, the first VH region has at least 85% sequence identity to any one of SEQ ID NOs: 11 to 14; and / or A method wherein the second VH region has at least 85% sequence identity with any one of SEQ ID NOs: 22 to 25. E46. The method according to any one of E43 to E45, the first VH region has at least 90% sequence identity to any one of SEQ ID NOs: 11 to 14; and / or A method wherein the second VH region has at least 90% sequence identity with any one of SEQ ID NOs: 22 to 25. E47. The method according to any one of E43 to E46, the first VH region has at least 95% sequence identity to any one of SEQ ID NOs: 11 to 14; and / or A method wherein the second VH region has at least 95% sequence identity with any one of SEQ ID NOs: 22 to 25. E48. The method of any one of E43 to E47, wherein the first VH region comprises a sequence selected from any one of SEQ ID NOs: 11 to 14. E49. The method of any one of E43 to E48, wherein the second VH region comprises a sequence selected from any one of SEQ ID NOs: 22 to 25. E50. The method according to any one of E43 to E47, the first VH region comprises a sequence selected from any one of SEQ ID NOs: 11 to 14; and A method wherein the second VH region comprises a sequence selected from any one of SEQ ID NOs: 22 to 25. E51. The method according to any one of E43 to E50, the first VH region comprises a sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%; 80%, 85%, 90%, 95%) sequence identity to the sequence of SEQ ID NO: 11; and The method wherein the second VH region comprises a sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%; 80%, 85%, 90%, 95%) sequence identity to the sequence of SEQ ID NO: 22. E52. The method according to any one of E43 to E50, the first VH region comprises the sequence of SEQ ID NO: 11; and The method wherein the second VH region comprises the sequence of SEQ ID NO: 22. E53. The method according to any one of E43 to E50, the first VH region comprises a sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%; 80%, 85%, 90%, 95%) sequence identity to the sequence of SEQ ID NO: 12; and The method wherein the second VH region comprises a sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%; 80%, 85%, 90%, 95%) sequence identity to the sequence of SEQ ID NO: 23. E54. The method according to any one of E43 to E50, the first VH region comprises the sequence of SEQ ID NO: 12; and A method wherein the second VH region comprises the sequence of SEQ ID NO: 23. E55. The method according to any one of E43 to E50, the first VH region comprises a sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%; 80%, 85%, 90%, 95%) sequence identity to the sequence of SEQ ID NO: 12; and The method wherein the second VH region comprises a sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%; 80%, 85%, 90%, 95%) sequence identity to the sequence of SEQ ID NO: 24. E56. The method according to any one of E43 to E50, the first VH region comprises the sequence of SEQ ID NO: 12; and The method wherein the second VH region comprises the sequence of SEQ ID NO: 24. E57. The method according to any one of E43 to E50, the first VH region comprises a sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%; 80%, 85%, 90%, 95%) sequence identity to the sequence of SEQ ID NO: 12; and The method wherein the second VH region comprises a sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%; 80%, 85%, 90%, 95%) sequence identity to the sequence of SEQ ID NO: 25. E58. The method according to any one of E43 to E50, the first VH region comprises the sequence of SEQ ID NO: 12; and The method wherein the second VH region comprises the sequence of SEQ ID NO: 25. E59. The method according to any one of E43 to E50, the first VH region comprises a sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%; 80%, 85%, 90%, 95%) sequence identity to the sequence of SEQ ID NO: 13; and The method wherein the second VH region comprises a sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%; 80%, 85%, 90%, 95%) sequence identity to the sequence of SEQ ID NO: 23. E60. The method according to any one of E43 to E50, the first VH region comprises the sequence of SEQ ID NO: 13; and A method wherein the second VH region comprises the sequence of SEQ ID NO: 23. E61. The method according to any one of E43 to E50, the first VH region comprises a sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%; 80%, 85%, 90%, 95%) sequence identity to the sequence of SEQ ID NO: 14; and The method wherein the second VH region comprises a sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%; 80%, 85%, 90%, 95%) sequence identity to the sequence of SEQ ID NO: 22. E62. The method according to any one of E43 to E50, the first VH region comprises the sequence of SEQ ID NO: 14; and The method wherein the second VH region comprises the sequence of SEQ ID NO: 22. E63. The method of any one of E1 to E62, wherein the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof further comprises a heavy chain constant region sequence that is absent of a CH1 sequence. E64. The method of any one of E1 to E63, wherein the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof further comprises a heavy chain constant region comprising a hinge region, a CH2 domain, and a CH3 domain. E65. The method according to E64, wherein the hinge region is wild-type human IgG4 hinge region sequence (SEQ ID NO: 54); or The method comprises a variant human IgG4 hinge region sequence comprising the S228P mutation (SEQ ID NO: 55). E66. The method according to E64 or E65, wherein the CH2 domain is Wild-type human IgG4 CH2 domain sequence (SEQ ID NO: 56); or The method comprises a variant human IgG4 CH2 domain comprising a F234A mutation, a L235A mutation, or both the F234A and L235A mutations. E67. The method according to any one of E64 to E66, wherein the CH3 domain is Wild-type human IgG4 CH3 domain sequence (SEQ ID NO: 58); or a variant human IgG4 CH3 domain sequence containing the T366W mutation; or The method comprises a variant human IgG4 CH3 domain sequence comprising a T366S mutation, a L368A mutation, and a Y407V mutation. E68. The method of any one of E1 to E63, wherein the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof further comprises an Fc region. E69. The method of any one of E1 to E63, wherein the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof further comprises a variant Fc region. E70. The method of E69, wherein the variant Fc region comprises a heterodimerization mutation. E71. The method according to any one of E68 to E70, wherein the Fc region is a silenced Fc region. E72. The method according to any one of E1 to E64, wherein the anti-IL-2R βγ heavy chain only antibody is an IgG1 antibody. E73. The method according to any one of E1 to E64, wherein the anti-IL-2R βγ heavy chain only antibody is an IgG4 antibody. E74. The method according to any one of E1, E2, E30 to E33, E43, or E44, wherein the anti-IL-2R βγ heavy chain-only antibody or antigen-binding fragment thereof is a first polypeptide comprising a sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%; 80%, 85%, 90%, 95%) sequence identity to the sequence of SEQ ID NO: 53; and The method includes a second polypeptide comprising a sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%; 80%, 85%, 90%, 95%) sequence identity to the sequence of SEQ ID NO: 61. E75. The method according to E74, the first polypeptide comprises the sequence of SEQ ID NO: 53; and The method wherein the second polypeptide comprises the sequence of SEQ ID NO:61. E76. The method according to any one of E1, E2, E30 to E33, E43, or E44, wherein the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof is a first polypeptide comprising a sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%; 80%, 85%, 90%, 95%) sequence identity to the sequence of SEQ ID NO: 62; and The method includes a second polypeptide comprising a sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%; 80%, 85%, 90%, 95%) sequence identity to the sequence of SEQ ID NO: 63. E77. The method according to E76, The first polypeptide comprises the sequence of SEQ ID NO: 62; and The method wherein the second polypeptide comprises the sequence of SEQ ID NO:63. E78. The method according to any one of E1, E2, E30 to E33, E43, or E44, wherein the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof is a first polypeptide comprising a sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%; 80%, 85%, 90%, 95%) sequence identity to the sequence of SEQ ID NO: 64; and The method includes a second polypeptide comprising a sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%; 80%, 85%, 90%, 95%) sequence identity to the sequence of SEQ ID NO: 65. E79. The method according to E78, The first polypeptide comprises the sequence of SEQ ID NO: 64; and The method wherein the second polypeptide comprises the sequence of SEQ ID NO:65. E80. The method according to any one of E1, E2, E30 to E33, E43, or E44, wherein the anti-IL-2R βγ heavy chain-only antibody or antigen-binding fragment thereof is a first polypeptide comprising a sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%; 80%, 85%, 90%, 95%) sequence identity to the sequence of SEQ ID NO: 66; and The method includes a second polypeptide comprising a sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%; 80%, 85%, 90%, 95%) sequence identity to the sequence of SEQ ID NO: 67. E81. The method according to E80, The first polypeptide comprises the sequence of SEQ ID NO: 66; and The method wherein the second polypeptide comprises the sequence of SEQ ID NO:67. E82. The method according to any one of E1, E2, E30 to E33, E43, or E44, wherein the anti-IL-2R βγ heavy chain-only antibody or antigen-binding fragment thereof is a first polypeptide comprising a sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%; 80%, 85%, 90%, 95%) sequence identity to the sequence of SEQ ID NO: 34; and The method includes a second polypeptide comprising a sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%; 80%, 85%, 90%, 95%) sequence identity to the sequence of SEQ ID NO: 35. E83. The method according to E82, The first polypeptide comprises the sequence of SEQ ID NO: 34; and The method wherein the second polypeptide comprises the sequence of SEQ ID NO:35. E84. The method according to any one of E1, E2, E30 to E33, E43, or E44, wherein the anti-IL-2R βγ heavy chain-only antibody or antigen-binding fragment thereof is a first polypeptide comprising a sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%; 80%, 85%, 90%, 95%) sequence identity to the sequence of SEQ ID NO: 36; and The method includes a second polypeptide comprising a sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%; 80%, 85%, 90%, 95%) sequence identity to the sequence of SEQ ID NO: 37. E85. The method according to E84, The first polypeptide comprises the sequence of SEQ ID NO: 36; and The method wherein the second polypeptide comprises the sequence of SEQ ID NO:37. E86. The method according to any one of E1 to E85, Anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment is about 10 -11 M~about 10 -6 has affinity for IL2R with a Kd of M; and / or Anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment is about 10 -8 M ~ approx. 2.5×10 -7 has affinity for IL2Rβ with a Kd of M; and / or Anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment is about 10 -9 M ~ approx. 2.5×10 -8 A method having affinity for IL2Rγ with a Kd of M. E87. The method of any one of E1 to E86, wherein the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment functions as an IL-2Rβγ agonist. E88. The method of any one of E1-E87, further comprising administering a premedication to the subject prior to administration of the initial dose of anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment or the initial dose of T cell redirecting therapy. E89. The method of E88, wherein the premedication is selected from an antihistamine, a glucocorticoid, an IL-6 receptor antagonist, and a tumor necrosis factor alpha (TNF-α) antagonist. E90. The method of any one of E1-E89, wherein the T cell redirecting therapy is a bispecific T cell engaging molecule. E91. The method of E90, wherein the bispecific T cell engaging molecule comprises a first domain that specifically binds to a target cancer cell antigen and a second domain that specifically binds to human CD3. E92. The method of E91, wherein the target cancer cell antigen is selected from EpCAM, CEA, CD19, CD33, CD70, EGFRvIII, FLT3, GPRC5D, DLL3, BCMA, PSMA, STEAP1, STEAP2, MUC16, MUC17, and CLDN18.2. E93. The method of any one of E90-E92, wherein the bispecific T cell engaging molecule further comprises a half-life prolonging domain. E94. The method of E93, wherein the half-life prolonging domain provides the bispecific T cell engaging molecule with a half-life of greater than about 24 hours. E95. The method of E93 or E94, wherein the half-life extending domain is selected from an immunoglobulin Fc domain, a domain derived from serum albumin (e.g., human serum albumin), an albumin binding domain (e.g., comprising a human albumin binding peptide or an antibody fragment that specifically binds to serum albumin), a peptide that binds to the neonatal Fc receptor (FcRn), and a polyethylene glycol polymer. E96. The method of any one of E90 to E92, wherein the bispecific T cell engaging molecule is a three-chain antibody-like molecule. E97. The method of any one of E1 to E89, wherein the T cell redirecting therapy is a chimeric antigen receptor (CAR)-expressing T cell. E98. The method of E97, wherein the CAR-expressing T cell comprises a first domain that specifically binds to a target cancer cell antigen, a transmembrane domain, and an intracellular signaling domain. E99. The method of E98, wherein the target cancer cell antigen is selected from EpCAM, CEA, CD19, CD33, CD70, EGFRvIII, FLT3, GPRC5D, DLL3, BCMA, PSMA, STEAP1, STEAP2, MUC16, MUC17, and CLDN18.2. E100. The method of any one of E1 to E99, wherein at least one dose of an anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof is administered to the subject prior to the first dose of T cell redirecting therapy. E101. The method of any one of E1 to E100, comprising administering an anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof in combination with T cell redirecting therapy in one or more treatment cycles. E102. The method of E101, wherein each of the one or more treatment cycles comprises a single dose of an anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof and a single dose of T cell redirecting therapy. E103. The method of E101, wherein each of the one or more treatment cycles comprises multiple doses of an anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof and a single dose of T cell redirecting therapy. E104. The method of E101, wherein each of the one or more treatment cycles comprises a single dose of an anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof and multiple doses of T cell redirecting therapy. E105. The method of E101, wherein each of the one or more treatment cycles comprises multiple doses of an anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof and multiple doses of T cell redirecting therapy. E106. The method according to any one of E1 to E105, wherein the cancer is a blood cancer. E107. The method of any one of E1-106, wherein the cancer is selected from acute myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, multiple myeloma, diffuse large B-cell lymphoma, Burkitt's lymphoma, and non-Hodgkin's lymphoma. E108. The method of any one of E1-105, wherein the cancer is selected from prostate cancer, non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, hepatocellular carcinoma, bladder cancer, testicular cancer, colorectal cancer, esophageal cancer, glioblastoma, head and neck cancer, pancreatic cancer, breast cancer, gastric cancer, gastroesophageal junction cancer, bone cancer, ovarian cancer, endometrial cancer, and melanoma. E109. The method of E108, wherein the subject has at least one tumor with low immune infiltration (e.g., low or no T cell infiltration) prior to co-administration. E110. The method of E108 or E109, wherein the co-administration increases T cell infiltration of the tumor. E111. The method of any one of E108-E110, wherein the co-administration is associated with at least one anti-tumor effect. E112. The method of E111, wherein the at least one anti-tumor effect is selected from a decrease in tumor volume, a decrease in tumor cell number, a decrease in tumor cell proliferation, and a decrease in tumor cell survival. E113. The method of any one of E1-E112, wherein the co-administration is associated with at least one anti-cancer effect. E114. The method of E113, wherein the at least one anti-cancer effect is selected from a reduction in the number of cancer cells, a reduction in the number of metastases, an increase in life expectancy, a reduction in cancer cell proliferation, a reduction in cancer cell survival, and an improvement in various physiological symptoms associated with the cancerous condition. E115. The method of any one of E1-E114, wherein the anti-IL-2Rβγ heavy chain only antibody or antigen-binding fragment thereof is administered in a pharmaceutical composition adapted for intravenous or subcutaneous delivery. E116. The method of any one of E1-E115, wherein the T cell redirecting therapy is administered in a pharmaceutical composition adapted for intravenous or subcutaneous delivery. E117. The method of E116, wherein the pharmaceutical composition comprises the bispecific T cell engaging molecule, a buffer, a surfactant, and a stabilizer. E118. The method of E116 or E117, wherein the pharmaceutical composition comprises the bispecific T cell engaging molecule, glutamate buffer, polysorbate 20 or polysorbate 80, and sucrose at a pH of about 4.0 to about 4.4. E119. The method of any one of E1 to E118, wherein the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof and the T cell redirecting therapy are administered in separate pharmaceutical compositions. E120. The method of E119, wherein the separate pharmaceutical compositions may be lyophilized and reconstituted prior to administration to the patient. E121. The method of any one of E1 to E120, wherein the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof and the T cell redirecting therapy are administered simultaneously. E122. The method of any one of E1 to E120, wherein the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof and T cell redirecting therapy are administered sequentially. E123. The method of any one of E1-E122, wherein the subject has previously received first-line treatment for cancer. E124. The method of any one of E1-E123, wherein the subject has previously received first-line and second-line therapy for cancer.

[0147] These and further aspects are more fully described in the remainder of the disclosure, including the Examples. [Brief explanation of the drawings]

[0148] [Figure 1] Figure 1 shows the binding kinetics of bispecific heavy chain-only antibody constructs BsAb-1 (IL2RB_F09C**IL2RG_F16A), BsAb-2 (IL2RB_F09G**IL2RG_F16B), BsAb-3 (IL2RB_F09G**IL2RG_F16C), BsAb-4 (IL2RB_F09G**IL2RG_F18A), BsAb-5 (IL2RB_F09K**IL2RG_F16B), and BsAb-6 (IL2RB_F18E**IL2RG_F16A) to human and cynomolgus monkey IL2RB and IL2RG. [Figure 2A] Panels A-C are a series of heatmap tables displaying the fold induction of phosphorylated STAT5 (pSTAT5) in CD8+ T cells from human PBMCs treated for 1 hour with 50 nM of an anti-IL2Rβ / γ bispecific heavy chain-only antibody (Panel A); a 1:1 mixture of anti-IL2Rβ monospecific heavy chain-only and anti-IL2Rγ monospecific heavy chain-only antibodies or alone (Panel B); or IL-2 as a control (Panel C). pSTAT5 levels were measured by flow cytometry and reported as geometric mean fluorescence intensity (gMFI) relative to the gMFI of unstimulated cells. [Figure 2B]Panels A-C are a series of heatmap tables displaying the fold induction of phosphorylated STAT5 (pSTAT5) in CD8+ T cells from human PBMCs treated for 1 hour with 50 nM of an anti-IL2Rβ / γ bispecific heavy chain-only antibody (Panel A); a 1:1 mixture of anti-IL2Rβ monospecific heavy chain-only and anti-IL2Rγ monospecific heavy chain-only antibodies or alone (Panel B); or IL-2 as a control (Panel C). pSTAT5 levels were measured by flow cytometry and reported as geometric mean fluorescence intensity (gMFI) relative to the gMFI of unstimulated cells. [Figure 2C] Panels A-C are a series of heatmap tables displaying the fold induction of phosphorylated STAT5 (pSTAT5) in CD8+ T cells from human PBMCs treated for 1 hour with 50 nM of an anti-IL2Rβ / γ bispecific heavy chain-only antibody (Panel A); a 1:1 mixture of anti-IL2Rβ monospecific heavy chain-only and anti-IL2Rγ monospecific heavy chain-only antibodies or alone (Panel B); or IL-2 as a control (Panel C). pSTAT5 levels were measured 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 heavy chain-only antibody constructs. Cell binding was measured 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 heavy chain-only antibody constructs. Cell binding was measured 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 heavy chain-only antibody constructs. Cell binding was measured 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 dose curves of STAT5 phosphorylation in human and cynomolgus monkey PBMCs as a function of concentration for the indicated bispecific heavy chain-only antibody constructs and control molecules (IL-2 and IL-2 variants). Levels of pSTAT5 were measured by flow cytometry and reported as percentages of the indicated cell types. [Figure 4B] Panels A-E are a series of graphs showing dose curves of STAT5 phosphorylation in human and cynomolgus monkey PBMCs as a function of concentration for the indicated bispecific heavy chain-only antibody constructs and control molecules (IL-2 and IL-2 variants). Levels of pSTAT5 were measured by flow cytometry and reported as percentages of the indicated cell types. [Figure 4C] Panels A-E are a series of graphs showing dose curves of STAT5 phosphorylation in human and cynomolgus monkey PBMCs as a function of concentration for the indicated bispecific heavy chain-only antibody constructs and control molecules (IL-2 and IL-2 variants). Levels of pSTAT5 were measured by flow cytometry and reported as percentages of the indicated cell types. [Figure 4D] Panels A-E are a series of graphs showing dose curves of STAT5 phosphorylation in human and cynomolgus monkey PBMCs as a function of concentration for the indicated bispecific heavy chain-only antibody constructs and control molecules (IL-2 and IL-2 variants). Levels of pSTAT5 were measured by flow cytometry and reported as percentages of the indicated cell types. [Figure 4E]Panels A-E are a series of graphs showing dose curves of STAT5 phosphorylation in human and cynomolgus monkey PBMCs as a function of concentration for the indicated bispecific heavy chain-only antibody constructs and control molecules (IL-2 and IL-2 variants). Levels of pSTAT5 were measured by flow cytometry and reported as percentages of the indicated cell types. [Figure 5A] Panels A-D are a series of graphs showing proliferation (Ki67 dose curves) in the indicated human cells as a function of concentration for the indicated bispecific heavy chain-only antibody constructs and control molecules (IL-2 and IL-2 variants). Ki67 levels were measured by flow cytometry and are reported as a percentage of the indicated cell types. [Figure 5B] Panels A-D are a series of graphs showing proliferation (Ki67 dose curves) in the indicated human cells as a function of concentration for the indicated bispecific heavy chain-only antibody constructs and control molecules (IL-2 and IL-2 variants). Ki67 levels were measured by flow cytometry and are reported as a percentage of the indicated cell types. [Figure 5C] Panels A-D are a series of graphs showing proliferation (Ki67 dose curves) in the indicated human cells as a function of concentration for the indicated bispecific heavy chain-only antibody constructs and control molecules (IL-2 and IL-2 variants). Ki67 levels were measured by flow cytometry and are reported as a percentage of the indicated cell types. [Figure 5D] Panels A-D are a series of graphs showing proliferation (Ki67 dose curves) in the indicated human cells as a function of concentration for the indicated bispecific heavy chain-only antibody constructs and control molecules (IL-2 and IL-2 variants). Ki67 levels were measured by flow cytometry and are 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 show internalization data for the indicated bispecific heavy chain-only antibody constructs. Panel A displays the internalization of the indicated anti-IL2Rβ / γ heavy chain-only antibodies by CD8+ T cells derived from human PBMCs as a function of time. Panel B presents this data in tabular format. Surface levels of heavy chain-only antibodies were detected by flow cytometry and reported relative to non-internalized cells. The observed half-lives ranged from 0.27 to 0.81 hours. As observed here, molecules containing the IL2RG_F16B binding sequence were internalized faster and to a greater extent than molecules containing a different anti-IL2RG binding sequence, suggesting that internalization may depend in part on the specific anti-IL2RG arm of the bispecific heavy chain-only antibody. [Figure 7B]Panels A-B show internalization data for the indicated bispecific heavy chain-only antibody constructs. Panel A displays the internalization of the indicated anti-IL2Rβ / γ heavy chain-only antibodies by CD8+ T cells derived from human PBMCs as a function of time. Panel B presents this data in tabular format. Surface levels of heavy chain-only antibodies were detected by flow cytometry and reported relative to non-internalized cells. The observed half-lives ranged from 0.27 to 0.81 hours. As observed here, molecules containing the IL2RG_F16B binding sequence were internalized faster and to a greater extent than molecules containing a different anti-IL2RG binding sequence, suggesting that internalization may depend in part on the specific anti-IL2RG arm of the bispecific heavy chain-only antibody. [Figure 8A] Panels A-B show PK data from the mouse model in graphical (Panel A) and tabular (Panel B) format. BALB / c mice (n=3 per group per time point) were administered 1 mg / kg of the indicated anti-IL2Rβ / γ heavy chain-only antibody via tail vein injection. Serum was collected at six time points over a two-week period and co-tested for human IgG4 by ELISA. Results are shown as a function of time (Panel A) or in tabular format (Panel B). [Figure 8B] Panels A-B show PK data from the mouse model in graphical (Panel A) and tabular (Panel B) format. BALB / c mice (n=3 per group per time point) were administered 1 mg / kg of the indicated anti-IL2Rβ / γ heavy chain-only antibody via tail vein injection. Serum was collected at six time points over a two-week period and co-tested for human IgG4 by ELISA. Results are shown as a function of time (Panel A) or in tabular format (Panel B). [Figure 9]This table summarizes some properties of the indicated bispecific heavy chain-only antibody constructs. All constructs were expressed in the ExpiCHO expression system and purified in two steps. Stability was determined based on percent aggregation by SE-HPLC after heat stress. Tm and Tag were measured using the UNcle platform. For SE-HPLC experiments, 20 μg of protein was run on a TSK gel G3000 5 μm column. [Figure 10A] Panels A–C show summary data from a mouse model of GVHD. Irradiated NSG mice (five per treatment group) were each transplanted with 20 million human PBMCs. Animals were then treated with either vehicle alone (100 μL), 22 μg of rhIL-2 (daily), or one of the two indicated bispecific antibody constructs at 1 mg / kg in 100 μL (twice weekly) until sacrifice (20% weight loss). Panel A shows an overview of the mouse model of GVHD and the subsequent dosing scheme. Panel B shows the body weight of animals in the indicated experimental groups as a function of time. Panel C displays an analysis of cells from the spleens of mice in this study, harvested 5 days after treatment. CD8+ and CD4+ T cell proliferation was compared among the four treatment groups by measuring CSFE staining in different lymphocyte populations. Both of the two bispecific heavy chain-only antibody constructs tested (IL2RB_F09C**IL2RG_F16A (BsAb-1) and IL2RB_F09G**IL2RG_F16B (BsAb-2)) showed significantly greater proliferation of CD8+ T cells compared to rhIL-2 and vehicle controls. CD4+ T cells expanded to a lesser extent; however, a significant increase in proliferation of CD4+ T cells was seen in mice treated with IL2RB_F09G**IL2RG_F16B (BsAb-2) compared to vehicle controls (Panel C). The data demonstrate 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 show summary data from a mouse model of GVHD. Irradiated NSG mice (five per treatment group) were each transplanted with 20 million human PBMCs. Animals were then treated with either vehicle alone (100 μL), 22 μg of rhIL-2 (daily), or one of the two indicated bispecific antibody constructs at 1 mg / kg in 100 μL (twice weekly) until sacrifice (20% weight loss). Panel A shows an overview of the mouse model of GVHD and the subsequent dosing scheme. Panel B shows the body weight of animals in the indicated experimental groups as a function of time. Panel C displays an analysis of cells from the spleens of mice in this study, harvested 5 days after treatment. CD8+ and CD4+ T cell proliferation was compared among the four treatment groups by measuring CSFE staining in different lymphocyte populations. Both of the two bispecific heavy chain-only antibody constructs tested (IL2RB_F09C**IL2RG_F16A (BsAb-1) and IL2RB_F09G**IL2RG_F16B (BsAb-2)) showed significantly greater proliferation of CD8+ T cells compared to rhIL-2 and vehicle controls. CD4+ T cells expanded to a lesser extent; however, a significant increase in proliferation of CD4+ T cells was seen in mice treated with IL2RB_F09G**IL2RG_F16B (BsAb-2) compared to vehicle controls (Panel C). The data demonstrate 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 show summary data from a mouse model of GVHD. Irradiated NSG mice (five per treatment group) were each transplanted with 20 million human PBMCs. Animals were then treated with either vehicle alone (100 μL), 22 μg of rhIL-2 (daily), or one of the two indicated bispecific antibody constructs at 1 mg / kg in 100 μL (twice weekly) until sacrifice (20% weight loss). Panel A shows an overview of the mouse model of GVHD and the subsequent dosing scheme. Panel B shows the body weight of animals in the indicated experimental groups as a function of time. Panel C displays an analysis of cells from the spleens of mice in this study, harvested 5 days after treatment. CD8+ and CD4+ T cell proliferation was compared among the four treatment groups by measuring CSFE staining in different lymphocyte populations. Both of the two bispecific heavy chain-only antibody constructs tested (IL2RB_F09C**IL2RG_F16A (BsAb-1) and IL2RB_F09G**IL2RG_F16B (BsAb-2)) showed significantly greater proliferation of CD8+ T cells compared to rhIL-2 and vehicle controls. CD4+ T cells expanded to a lesser extent; however, a significant increase in proliferation of CD4+ T cells was seen in mice treated with IL2RB_F09G**IL2RG_F16B (BsAb-2) compared to vehicle controls (Panel C). The data demonstrate 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 show the percentage of the indicated cell types as a function of time post-dose. Panels F-J show the concentration (x105) of the indicated cell types per μL of blood as a function of time post-dose. Panel K shows the ratio of CD4+ T cells to CD8+ 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 show the percentage of the indicated cell types as a function of time post-dose. Panels F-J show the concentration (x105) of the indicated cell types per μL of blood as a function of time post-dose. Panel K shows the ratio of CD4+ T cells to CD8+ 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 show the percentage of the indicated cell types as a function of time post-dose. Panels F-J show the concentration (x105) of the indicated cell types per μL of blood as a function of time post-dose. Panel K shows the ratio of CD4+ T cells to CD8+ 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 show the percentage of the indicated cell types as a function of time post-dose. Panels F-J show the concentration (x105) of the indicated cell types per μL of blood as a function of time post-dose. Panel K shows the ratio of CD4+ T cells to CD8+ 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 show the percentage of the indicated cell types as a function of time post-dose. Panels F-J show the concentration (x105) of the indicated cell types per μL of blood as a function of time post-dose. Panel K shows the ratio of CD4+ T cells to CD8+ 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 show the percentage of the indicated cell types as a function of time post-dose. Panels F-J show the concentration (x105) of the indicated cell types per μL of blood as a function of time post-dose. Panel K shows the ratio of CD4+ T cells to CD8+ 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 show the percentage of the indicated cell types as a function of time post-dose. Panels F-J show the concentration (x105) of the indicated cell types per μL of blood as a function of time post-dose. Panel K shows the ratio of CD4+ T cells to CD8+ 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 show the percentage of the indicated cell types as a function of time post-dose. Panels F-J show the concentration (x105) of the indicated cell types per μL of blood as a function of time post-dose. Panel K shows the ratio of CD4+ T cells to CD8+ 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 show the percentage of the indicated cell types as a function of time post-dose. Panels F-J show the concentration (x105) of the indicated cell types per μL of blood as a function of time post-dose. Panel K shows the ratio of CD4+ T cells to CD8+ 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 show the percentage of the indicated cell types as a function of time post-dose. Panels F-J show the concentration (x105) of the indicated cell types per μL of blood as a function of time post-dose. Panel K shows the ratio of CD4+ T cells to CD8+ 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 show the percentage of the indicated cell types as a function of time post-dose. Panels F-J show the concentration (x105) of the indicated cell types per μL of blood as a function of time post-dose. Panel K shows the ratio of CD4+ T cells to CD8+ 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 heavy chain-only 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 heavy chain-only antibodies. Panel B is a table showing molecule, dose, and half-life (t1 / 2) information. [Figure 13] 1 is a graph showing body weight (%) as a function of time (study day) for animals in the accelerated GVHD model. [Figure 14] 1 is a graph showing survival as a function of time (study day) for animals in the accelerated GVHD model. [Figure 15A] Panels A-C are a panel of graphs showing cell proliferation of the indicated cell types measured by treatment group 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 A-C are a panel of graphs showing cell proliferation of the indicated cell types measured by treatment group 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 A-C are a panel of graphs showing cell proliferation of the indicated cell types measured by treatment group 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 treatment 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 treatment 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 treatment 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 treatment 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 treatment 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 treatment 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 treatment 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 treatment 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 treatment 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 treatment 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 treatment 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 treatment conditions. [Figure 17A] Panels A and B show the results of a TDCC assay using H82_Luc cells (E:T = 1:1, 72-hour assay time) investigating the cytotoxicity of a bispecific T cell engaging molecule (TCE-1) that specifically binds human CD3 and human EpCAM, alone and in combination with an anti-IL2Rβ / γ heavy chain-only antibody (BsAb-5). [Figure 17B] Panels A and B show the results of a TDCC assay using H82_Luc cells (E:T = 1:1, 72-hour assay time) investigating the cytotoxicity of a bispecific T cell engaging molecule (TCE-1) that specifically binds human CD3 and human EpCAM, alone and in combination with an anti-IL2Rβ / γ heavy chain-only antibody (BsAb-5). [Figure 18A] Panels A to C show the results of a TDCC assay using SHP77 Luc cells (E:T = 1:1, 1:3, 1:10, 72-hour assay time) investigating the TDCC activity of a bispecific T cell engaging molecule (TCE-1) that specifically binds human CD3 and human EpCAM, alone and in combination with an anti-IL2Rβ / γ heavy chain-only antibody (BsAb-5). [Figure 18B]Panels A to C show the results of a TDCC assay using SHP77 Luc cells (E:T = 1:1, 1:3, 1:10, 72-hour assay time) investigating the TDCC activity of a bispecific T cell engaging molecule (TCE-1) that specifically binds human CD3 and human EpCAM, alone and in combination with an anti-IL2Rβ / γ heavy chain-only antibody (BsAb-5). [Figure 18C] Panels A to C show the results of a TDCC assay using SHP77 Luc cells (E:T = 1:1, 1:3, 1:10, 72-hour assay time) investigating the TDCC activity of a bispecific T cell engaging molecule (TCE-1) that specifically binds human CD3 and human EpCAM, alone and in combination with an anti-IL2Rβ / γ heavy chain-only antibody (BsAb-5). [Figure 19] FIG. 1 is a schematic showing the design of an in vivo combination study in the NSG mouse model. [Figure 20] 1 is a graph showing tumor volume as a function of time from days 11 to 31 after tumor implantation in mice in an in vivo combination study. [Figure 21] 1 is a graph showing relative body weight (%) as a function of time from days 11 to 31 after tumor implantation in mice in an in vivo combination study. [Figure 22A] Panels AC show results from an in vivo tumor pharmacodynamic study. [Figure 22B] Panels AC show results from an in vivo tumor pharmacodynamic study. [Figure 22C] Panels AC show results from an in vivo tumor pharmacodynamic study. DETAILED DESCRIPTION OF THE INVENTION

[0149] 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 a person of ordinary skill in the art. Such techniques are described in “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).

[0150] 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)).

[0151] 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.

[0152] All references cited throughout this disclosure, including patent applications and publications, are incorporated herein by reference in their entirety. In the event of a conflict of definitions between a cited reference and a definition provided herein, the definition provided herein shall prevail.

[0153] Definition: In some embodiments, "about," when used in connection with a measurable, numerical variable, refers to the indicated value of the variable and all values ​​of the variable that are within experimental error of the indicated value (e.g., within a 95% confidence interval of the mean) or ±10% of the indicated value, whichever is greater. In some embodiments, numerical ranges are inclusive of the numbers (i.e., endpoints) that define the range.

[0154] 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 limit 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 be independently included in 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.

[0155] As used herein, the terms "a" and "an" mean "one or more" unless specifically indicated otherwise. Furthermore, "one or more" and "at least one" are used interchangeably herein. Furthermore, unless the context otherwise requires, singular terms include plurals and plural terms include the singular.

[0156] As used herein, the term "polypeptide" refers to a polymer of amino acids containing at least 50 amino acids, such as, for example, at least 100 amino acids.

[0157] As used herein, the term "antibody" generally refers to a tetrameric immunoglobulin protein comprising two light chain polypeptides (e.g., light chain polypeptides each of approximately 25 kDa) and two heavy chain polypeptides (e.g., heavy chain polypeptides each of approximately 50-70 kDa). The terms "light chain" or "immunoglobulin light chain," as used herein, refer to a polypeptide comprising, from the amino terminus to the carboxyl terminus, a single immunoglobulin light chain variable region (VL) and a single immunoglobulin light chain constant domain (CL). The immunoglobulin light chain constant domain (CL) can be a human kappa (κ) constant domain or a human lambda (λ) constant domain. The term "heavy chain" or "immunoglobulin heavy chain" refers to a polypeptide comprising, from amino to carboxyl terminus, a single immunoglobulin heavy chain variable region (VH), immunoglobulin heavy chain constant domain 1 (CH1), immunoglobulin hinge region, immunoglobulin heavy chain constant domain 2 (CH2), immunoglobulin heavy chain constant domain 3 (CH3), and optionally immunoglobulin heavy chain constant domain 4 (CH4). Heavy chains are classified as mu (μ), delta (Δ), gamma (γ), alpha (α), and epsilon (ε), and define antibody isotypes as IgM, IgD, IgG, IgA, and IgE, respectively. IgG-class and IgA-class antibodies are further divided into subclasses, i.e., IgG1, IgG2, IgG3, and IgG4, and IgA1 and IgA2, respectively. The heavy chains of IgG, IgA, and IgD antibodies have three constant domains (CH1, CH2, and CH3), while the heavy chains of IgM and IgE antibodies have four constant domains (CH1, CH2, CH3, and CH4). Immunoglobulin heavy chain constant domains can be derived from any immunoglobulin isotype, including subtypes. Antibody chains are linked to each other via interpolypeptide disulfide bonds between the CL and CH1 domains (i.e., between the light and heavy chains) and between the hinge regions of the two antibody heavy chains. In some embodiments, the antibodies of the present disclosure are human or humanized antibodies and can be of the IgG1, IgG2, IgG3, or IgG4 type.

[0158] The variable regions of immunoglobulin chains generally exhibit the same overall structure, comprising relatively conserved framework regions (FRs) connected by three hypervariable regions (more often called "complementarity-determining regions" or CDRs). The CDRs from the two chains of each heavy / light chain pair are usually aligned by the framework regions to form a structure that specifically binds to a particular epitope of a target protein (e.g., MSLN or CD3). From N- to C-terminus, both naturally occurring light and heavy chain variable regions usually follow the following order of these elements: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Numbering systems have been devised to assign numbers to the amino acids that occupy each position in these domains. This numbering system is defined in Kabat Sequences of Proteins of Immunological Interest (1987 and 1991, NIH, Bethesda, MD), or Chothia & Lesk, 1987, J. Mol. Biol. 196:901-917; Chothia et al., 1989, Nature 342:878-883. This system can be used to identify the CDRs and FRs of a given antibody. Other numbering systems for the amino acids of immunoglobulin chains include IMGT® (the international ImMunoGeneTics information system; Lefranc et al., Dev. Comp. Immunol. 29:185-203; 2005) and AHo (Honegger and Pluckthun, J. Mol. Biol. 309(3):657-670; 2001).

[0159] As used herein, "CDR" refers to an antibody complementarity-determining region as defined in Lefranc, MP et al., IMGT, the International ImMunoGeneTics database, Nucleic Acids Res., 27:209-212 (1999). However, those skilled in the art will understand that several definitions of CDR are commonly used, including the Kabat definition, which is based on sequence variability and is the most commonly used (see, "Zhao et al. A germline knowledge-based computational approach for determining antibody complementarity-determining regions." Mol Immunol. 2010;47:694-700). 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).For alternative purpose CDR definitions, see 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.

[0160] "Framework Region" or "FR" residues are those variable domain residues other than the hypervariable region / CDR residues as herein defined.

[0161] 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 to 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 specified herein, references to residue numbers in the variable domain of an antibody refer to the numbering of residues according to the Kabat numbering system. Unless otherwise stated herein, references to residue numbers in the constant domains of antibodies, single domain antibodies, antibody fragments, etc. refer to the numbering of residues according to the EU numbering system.

[0162] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible minor naturally occurring mutations. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), monoclonal antibodies generally are directed against a single determinant on an antigen. As a non-limiting example, monoclonal antibodies according to the present disclosure can be produced by the hybridoma method first described by Kohler et al. (1975) Nature 256:495, or can also be produced by recombinant protein production methods (see, e.g., U.S. Pat. No. 4,816,567).

[0163] The term "human antibody," as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the present disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody," as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0164] As used herein, the term "chimeric antibody" refers to an antibody that contains amino acid sequences derived from at least two different Ig loci, e.g., a transgenic antibody that contains a portion encoded by a human Ig locus and a portion encoded by a rat Ig locus. Chimeric antibodies include transgenic antibodies with non-human or artificial Fc regions and human idiotypes. Such immunoglobulins can be isolated from animals of the present disclosure that have been engineered to produce such chimeric antibodies.

[0165] As used herein, the term "antibody construct" refers to a molecule whose structure and / or function is based on the structure and / or function of an antibody, e.g., a full-length immunoglobulin molecule. Thus, an antibody construct immunospecifically binds to its target or antigen and / or comprises domains comprising or derived from the heavy chain variable region (VH) and / or light chain variable region (VL) of an antibody.

[0166] As used herein, an "antibody fragment" generally refers to a fragment of a full-length antibody or a heavy chain-only antibody, such as, for example, a VH, VHH, VL, (s)dAb, Fv, light chain (VL-CL), Fd (VH-CH1), heavy chain, Fab, Fab', F(ab')2 or "r IgG" (a "half antibody" consisting of a heavy and light chain), or a fragment of a heavy chain-only antibody, such as, for example, a three-chain antibody-like molecule, a heavy chain-only antibody, a single-chain variable fragment (scFv), di-scFv or bi(s)-scFv, scFv-Fc, scFv-zipper, a single-chain Fab (scFab), Fab2, Fab3, a diabody, a single-chain diabody, a tandem diabody (Tandabs), a tandem di-scFv, a tandem tri-scFv, a "minibody" ((VH-VL-CH1) 3) refers to modified fragments of full-length antibodies, such as multibodies, e.g., triabodies or tetrabodies, and single domain antibodies, e.g., nanobodies or single variable domain antibodies comprising only one variable region which may be a VHH, VH, or VL, that specifically bind to an antigen or target independent of other variable regions or domains.

[0167] As used herein, the term "heavy chain-only antibody" refers to an immunoglobulin protein consisting of two heavy chain polypeptides (e.g., heavy chain polypeptides of approximately 50-70 kDa each). A "heavy chain-only antibody" is an antibody fragment lacking the two light chain polypeptides found in conventional antibodies. Heavy chain antibodies constitute approximately one-quarter of the IgG antibodies produced by camelids, such as camels and llamas (Hamers-Casterman C., et al. Nature. 363, 446-448 (1993)). These antibodies are formed by two heavy chains but lack light chains. The resulting variable antigen-binding portion, referred to as a VHH domain, represents the smallest naturally occurring intact antigen-binding site and is only approximately 120 amino acids in length (Desmyter, A., et al. J. Biol. Chem. 276, 26285-26290 (2001)). Heavy-chain antibodies with high specificity and affinity can be generated against various antigens by 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, LGJ, et al. J. Biotechnol. 78, 11-21 (2000)). Their expression, solubility, and stability levels are significantly higher than those of classical F(ab) or Fv fragments (Ghahroudi, MA et al. FEBS Lett. 414, 521-526 (1997)). Sharks have also been shown to have a single VH-like domain in their antibodies, termed 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)).

[0168] In some embodiments, a "heavy chain-only antibody" is a homodimeric antibody comprising a VH antigen-binding domain and CH2 and CH3 constant domains, with no CH1 domain present. In some embodiments, a heavy chain-only 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 some embodiments, a heavy chain-only antibody is composed of an antigen-binding domain, at least a portion of a hinge region, and CH2 and CH3 domains. In some embodiments, a heavy chain-only antibody is composed of an antigen-binding domain, at least a portion of a hinge region, and a CH2 domain. In some embodiments, a heavy chain-only antibody is composed of an antigen-binding domain, at least a portion of a hinge region, and a CH3 domain. Heavy chain-only antibodies in which the CH2 and / or CH3 domains are truncated are also included herein. The heavy chain-only antibodies described herein may belong to the IgG subclass, although heavy chain-only antibodies belonging to other subclasses, such as the IgM, IgA, IgD, and IgE subclasses, are also encompassed herein. In some embodiments, the heavy chain-only antibodies may belong to the IgG1, IgG2, IgG3, or IgG4 subtype, e.g., the IgG1 or IgG4 subtype. In some embodiments, the heavy chain-only antibodies are of the IgG1 or IgG4 subtype, and one or more of the CH domains are modified to alter the effector function of the antibody. In some embodiments, the heavy chain-only antibodies are of the IgG4 subtype, and one or more of the CH domains are modified to alter the effector function of the antibody. In some embodiments, the heavy chain-only antibodies are of the IgG1 subtype, and one or more of the CH domains are modified to alter the effector function of the antibody. Modifications of CH domains to alter effector function are further described herein. Non-limiting examples of heavy chain-only antibodies are described, for example, in WO 2018 / 039180, the disclosure of which is incorporated herein by reference in its entirety.

[0169] As used herein, a "single-domain antibody" refers to a single-chain polypeptide that contains all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. In some embodiments, a single-domain antibody is a human single-domain antibody.

[0170] As used herein, the term "tri-chain antibody-like molecule" or "TCA" refers to an antibody-like molecule comprising, consisting essentially of, or consisting of three polypeptide subunits, two of which comprise one heavy chain and one light chain of a monoclonal antibody, or an antigen-binding fragment of such antibody chains, each containing an antigen-binding region and at least one CH domain. This heavy / light chain pair has binding specificity for a first antigen. The third polypeptide subunit comprises an Fc portion, absent a CH1 domain, and including CH2, and / or CH3, and / or CH4 domains, and one or more antigen-binding domains (e.g., two antigen-binding domains) that bind to an epitope of a second antigen or a different epitope of the first antigen, such binding domains comprising, essentially consisting of, or consisting of a heavy chain-only antibody derived from or sharing sequence identity with the variable region of an antibody heavy or light chain. Portions of such variable regions include V H and / or V L Gene segments, D and J H Gene segment, or J L The variable region can be encoded by a rearranged V H DJ H , V L DJ H , V H J L , or V L J L It can be encoded by a gene segment.

[0171] As used herein, an "antigen-binding fragment" refers to a portion of an antibody or heavy-chain-only antibody that lacks at least some of the amino acids present in a full-length heavy chain (in the case of an antibody or heavy-chain-only antibody) and / or light chain (in the case of an antibody), but is still capable of specifically binding to an antigen. Antigen-binding fragments include, but are not limited to, single-chain variable fragments (scFv), nanobodies (e.g., the VH domain of a camelid heavy-chain antibody; VHH fragments, see Cortez-Retamozo et al., Cancer Research, Vol. 64:2853-57, 2004), Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, Fd fragments, and CDR fragments, and may be derived from any mammalian source, such as human, mouse, rat, rabbit, or camel.

[0172] Digestion of an antibody with papain produces two identical antigen-binding fragments called "Fab" fragments (each of which has a single antigen-binding site) and a residual "Fc" fragment (containing all but the first domain of the immunoglobulin heavy chain constant region). The Fab fragment contains the variable domains from the light and heavy chains, as well as the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Thus, a "Fab fragment" is composed of one immunoglobulin light chain (light chain variable region (VL) and constant region (CL)) and the CH1 domain and variable region (VH) of one immunoglobulin heavy chain. The heavy chain of a Fab molecule cannot form disulfide bonds with another heavy chain molecule. An "Fd fragment" contains the VH domain and CH1 domain from an immunoglobulin heavy chain. The Fd fragment represents the heavy chain component of a Fab fragment.

[0173] An "Fc fragment" or "Fc region" of an immunoglobulin generally comprises two constant domains, a CH2 domain and a CH3 domain, and optionally a CH4 domain. The Fc region may be derived from an IgG1, IgG2, IgG3, or IgG4 immunoglobulin. In some embodiments, the Fc region comprises the CH2 and CH3 domains derived from a human IgG1 or human IgG2 immunoglobulin. The Fc region may retain effector functions such as C1q binding, complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), and phagocytosis. In other embodiments, the Fc region may be modified to reduce or eliminate effector function.

[0174] A "functional Fc region" possesses an "effector function" of a native sequence Fc region. Non-limiting examples of effector functions include C1q binding, CDC; Fc receptor binding, ADCC, ADCP, down-regulation of cell surface receptors (e.g., B cell receptors), and the like. Such effector functions generally require the Fc region to interact with receptors such as FcγRI, FcγRIIA, FcγRIIB1, FcγRIIB2, FcγRIIIA, FcγRIIIB receptors, and the low-affinity FcRn receptor, and can be assessed using various assays known in the art.

[0175] A "dead" or "silenced" Fc is one that has been mutated to retain activity, e.g., with respect to extended serum half-life, but does not activate high affinity Fc receptors or has reduced affinity for Fc receptors.

[0176] 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 regions (non-A and A allotypes), native-sequence human IgG2 Fc regions, native-sequence human IgG3 Fc regions, and native-sequence human IgG4 Fc regions, as well as naturally occurring variants thereof.

[0177] A "variant Fc region" comprises an amino acid sequence that differs from that of a native-sequence Fc region by virtue of 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 comprises 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 the Fc region of a parent polypeptide, compared to the native-sequence Fc region or the 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 the 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.

[0178] As used herein, "heterodimerization mutations" refer to mutations in the A and B chains of an F region (i.e., the two chains that make up an Fc region, one chain referred to herein as the "A" chain and the other referred to herein as the "B" chain) that promote the formation of heterodimeric Fc regions, i.e., Fc regions in which the A and B chains of the Fc region do not have identical amino acid sequences. In some embodiments, heterodimerization mutations may be asymmetric, i.e., an A chain with a particular mutation can pair with a B chain with a different mutation. These mutations favor heterodimerization and disfavor homodimerization. Whether heterodimers or homodimers are formed can be assessed by differences in size, as measured by polyacrylamide gel electrophoresis, for example, where one polyethylene chain is a dummy Fc and the other is an scFv-Fc. One non-limiting example of such paired heterodimerization mutations 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 containing a pair of knob and hole substitutions contains 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 the IgG1 Fc region have been found to improve heterodimer formation compared to that seen with the unmodified A and B chains and can 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 T39 in the other chain. 4S; 5) Y407T in one chain and T366Y in the other; 6) T366Y and F405A in one chain and T394W and Y407T in the other; 7) T366W and F405W in one chain and T394S and Y407A in the other; 8) F405W and Y407A in one chain and T366W and T394S in the other; and 9) T366W in one polypeptide of the Fc and T366S, L368A, and Y407V in the other. Alternatively or in addition to such mutations, substitutions that generate new disulfide bridges can promote heterodimer formation. See, e.g., U.S. Patent Application Publication No. 2003 / 0078385. Such mutations 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, for example, at the CH3-CH3 interface, can enhance heterodimer formation, as described, for example, in WO 2009 / 089004, which is incorporated herein by reference.Such substitutions are referred to herein as "charge-pair substitutions," and an Fc region containing one pair of charge-pair substitutions contains 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 plus D399K or D399R in the other chain; 2) K392D or K392E in one chain plus D399K or D399R in the other chain; 3) K439D or K439E in one chain plus E356K or E356R in the other chain; and 4) K370D or K370E in one chain plus 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 mutations. A particular charge pair substitution can be used alone or in combination with other charge pair substitutions.Specific examples of single pairs of charge pair substitutions and their combinations include: 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 E in the other chain. 356K; 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 in the other chain. 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. Any of these heterodimerization mutations can be used in polypeptides comprising the variant Fc regions described herein.

[0179] 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 in human IgG1 or IgG2 and at positions 327, 330, and 331 in IgG4 significantly reduce ADCC and CDC (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 Fc amino acid sequence of human IgG4 (UniProtKB number P01861) is presented herein as SEQ ID NO: 43. Silenced IgG1 is described, for example, in Boesch, AW, et al., "Highly parallel characterization of IgG Fc binding interactions." MAbs, 2014.6(4):915-27, the disclosures of which are incorporated herein by reference in their entireties.

[0180] 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.

[0181] Furthermore, Fc variants may be constructed to eliminate or substantially reduce effector functions by substituting (mutating), deleting, or adding amino acid residues that affect complement binding or Fc receptor binding. For example, deletions may be made in complement binding sites, such as, but not limited to, 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 may be modified by phosphorylation, sulfation, acylation, glycosylation, methylation, farnesylation, acetylation, amidation, etc.

[0182] Antibodies and antibody fragments 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 mutant with substitutions of residues 265 and 297 according to EU numbering to alanine (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.

[0183] 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., a variant Fc region lacking glycosylation at N297, such as those described in WO 2014 / 153063, which is incorporated herein by reference. Additional mutations that result in reduced or eliminated effector function include K322A and L234A / L235A (LALA). Alternatively, effector function can be reduced or eliminated by production techniques, such as expression in non-glycosylating host cells (e.g., E. coli) or host cells that result 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)).

[0184] In some embodiments, the proline at position 329 (EU numbering) (P329) of the wild-type human Fc region is substituted with glycine or arginine, or an amino acid residue large enough to disrupt the proline sandwich within the Fc / Fcγ receptor interface formed between P329 of the 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 the human IgG1 Fc region or S228P and L235E in the 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).

[0185] In some embodiments, the variant Fc region has P329 of the human IgG Fc region substituted with glycine, and the variant Fc region comprises at least two additional amino acid substitutions at L234A and L235A of the human IgG1 Fc region or S228P and L235E of the human IgG4 Fc region, where residues are numbered according to EU numbering (see, e.g., U.S. Patent No. 8,969,526). In some embodiments, variant Fc regions comprising the P329G, L234A, and L235A (EU numbering) substitutions exhibit reduced affinity for human FcγRIIIA and FcγRIIA.

[0186] In some embodiments, the variant Fc region comprises a triple mutation: amino acid substitutions at positions P329, L234A, and L235A according to EU numbering (P329 / LALA) (see, e.g., U.S. Patent No. 8,969,526). In some embodiments, the variant Fc region comprises the following amino acid substitutions: P329G, L234A, and L235A according to EU numbering.

[0187] In some embodiments, the antibody or antibody fragment comprises a variant human IgG4 CH3 domain sequence comprising a T366W mutation, which may optionally be referred to herein as an IgG4 CH3 knob sequence. In some embodiments, the antibody or antibody fragment comprises a variant human IgG4 CH3 domain sequence comprising a T366S mutation, an L368A mutation, and a Y407V mutation, which may optionally be referred to herein as an IgG4 CH3 hole sequence. The IgG4 CH3 mutations described herein can be utilized in any suitable manner to place a "knob" in the first heavy chain constant region of the first monomer of the antibody dimer and a "hole" in the second heavy chain constant region of the second monomer of the antibody dimer, thereby facilitating proper pairing of the desired pair of heavy chain polypeptide subunits in the antibody (heterodimerization).

[0188] In some embodiments, the antibody or antibody fragment comprises a heavy chain polypeptide subunit comprising a variant human IgG4 Fc region comprising the S228P, F234A, L235A, and T366W mutations (knob). In some embodiments, the antibody or antibody fragment comprises a heavy chain polypeptide subunit comprising a variant human IgG4 Fc region comprising the S228P, F234A, L235A, T366S, L368A, and Y407V mutations (hole).

[0189] A "Fab' fragment" is a Fab fragment that has one or more cysteine ​​residues from the antibody hinge region at the C-terminus of the CH1 domain.

[0190] A "F(ab')2 fragment" is a bivalent fragment containing two Fab' fragments linked by an inter-heavy chain disulfide bridge at the hinge region.

[0191] An "Fv" fragment is the minimum fragment containing a complete antigen-recognition and binding site derived from an antibody. This fragment consists of a dimer of one immunoglobulin heavy chain variable region (VH) and one immunoglobulin light chain variable region (VL) in tight, non-covalent association. In this structure, the three CDRs of each variable region interact to define an antigen-binding site on the surface of the VH-VL dimer. A single light or heavy chain variable region (or half of an Fv fragment containing only three antigen-specific CDRs) retains the ability to recognize and bind antigen, albeit with lower affinity than the entire binding site comprising both the VH and VL.

[0192] A "single-chain variable antibody fragment" or "scFv fragment" comprises the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain, and optionally contain a peptide linker between the VH and VL domains which enables the Fv to form the desired structure for antigen binding (see, e.g., Bird et al., Science, Vol. 242:423-426, 1988; and Huston et al., Proc. Natl. Acad. Sci. USA, Vol. 85:5879-5883, 1988).

[0193] A "nanobody" is the heavy chain variable region of a heavy chain antibody. Such a variable domain is the smallest fully functional antigen-binding fragment of such a heavy chain antibody, with a molecular weight of only 15 kDa. See Cortez-Retamozo et al., Cancer Research 64:2853-57, 2004. Functional heavy chain antibodies lacking light chains naturally occur in certain species of animals, such as nurse sharks and nurse sharks, and members of the Camelidae family, including camels, dromedaries, alpacas, and llamas. In these animals, the antigen-binding site is a single domain, the VHH domain. These antibodies use only the heavy chain variable region to form the antigen-binding region; i.e., these functional antibodies are heavy chain homodimers with only the structure H2L2 (also referred to as "heavy chain antibodies" or "HCAbs"). Camelized VHHs contain hinge, CH2, and CH3 domains and have been reported to recombine with IgG2 and IgG3 constant regions lacking the CH1 domain. Camelized VHH domains have been shown to bind antigens with high affinity (Desmyter et al., J. Biol. Chem., Vol. 276:26285-90, 2001) and have high stability in solution (Ewert et al., Biochemistry, Vol. 41:3628-36, 2002). Methods for generating antibodies with camelized heavy chains are described, for example, in U.S. Patent Application Publication Nos. 2005 / 0136049 and 2005 / 0037421. Alternative scaffolds can be made from human variable-like domains that more closely match the shark V-NAR scaffold and provide a long, transmembrane loop structure in the framework.

[0194] The antibodies and antibody fragments (e.g., heavy-chain-only antibodies and three-chain antibody-like molecules) of the present disclosure include multispecific antibodies and antibody fragments, which are antibodies and antibody fragments with two or more binding specificities. As used herein, the term "multispecific" includes "bispecific" (i.e., two binding specificities) and "trispecific" (i.e., three binding specificities), as well as higher orders of independent specific binding affinities, e.g., higher orders of polyepitopic specificity.

[0195] As used herein, an "isolated" molecule (e.g., an antibody, antibody fragment, single domain antibody, etc.) is one that has been identified and separated and / or recovered from a component of its natural environment. Contaminant components of its natural environment are substances that would interfere with diagnostic or therapeutic uses of the molecule, and may be, for example, enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, an isolated molecule is purified (1) to greater than 95% by weight, such as greater than 99% by weight, of the molecule as measured by the Lowry method; (2) to the extent that at least 15 residues of N-terminal or internal amino acid sequence are sufficient to be obtained using a spinning cup sequencer; or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, for example, silver staining. In some embodiments, an isolated molecule is prepared by a process that includes at least one purification step.

[0196] Embodiments of the present disclosure include antibodies and antibody fragments (e.g., heavy-chain-only antibodies) comprising heavy-chain-only variable regions in a monovalent or bivalent structure. As used herein, the term "monovalent structure," when used with reference to a heavy-chain-only variable region domain, means that only one heavy-chain-only variable region domain is present, with a single binding site. In contrast, the term "bivalent structure," when used with reference to a heavy-chain-only variable region domain, means that two heavy-chain-only variable region domains are present (each with a single binding site), connected by a linker sequence. Non-limiting examples of linker sequences are discussed further herein and include, but are not limited to, GS linker sequences of various lengths. When the heavy-chain-only variable region is in a bivalent structure, each of the two heavy-chain-only variable region domains can bind to the same antigen or different antigens (e.g., different epitopes on the same protein; two different proteins, etc.). However, unless otherwise specified, a heavy chain-only variable region designated as being in a "bivalent structure" is understood to contain two identical heavy chain-only variable region domains joined by a linker sequence, each of which binds to the same target antigen.

[0197] Aspects of the present disclosure also include antibodies and antibody fragments (e.g., heavy chain-only antibodies) with multispecific structures, including but not limited to bispecific, trispecific, etc. A wide variety of methods and protein configurations are known and used for bispecific monoclonal antibodies (BsMABs), trispecific antibodies, etc.

[0198] Various methods have been developed for generating multivalent artificial antibodies by recombinantly fusing the variable domains of two or more antibodies. In some embodiments, the first and second antigen-binding domains on a polypeptide are linked by a polypeptide linker. One non-limiting example of such a polypeptide linker is the GS linker, which has an amino acid sequence of four glycine residues followed by one serine residue, repeated n times, where n is an integer ranging from 1 to about 10 (SEQ ID NO: 68), e.g., 2, 3, 4, 5, 6, 7, 8, or 9. Non-limiting examples of such linkers include GGGGS (SEQ ID NO: 49) (n=1) and GGGGSGGGGS (SEQ ID NO: 50) (n=2). Other suitable linkers can also be used, e.g., as described in Chen et al., Adv Drug Deliv Rev. 2013 October 15;65(10):1357-69, the disclosure of which is incorporated herein by reference in its entirety.

[0199] As used herein, the term "amino acid" or "amino acid residue" refers to an amino acid having an art-recognized definition, such as, for example, an amino acid selected from the group consisting of alanine (Ala or A); arginine (Arg or R); asparagine (Asn or N); aspartic acid (Asp or D); cysteine ​​(Cys or C); glutamine (Gln or Q); glutamic acid (Glu or E); glycine (Gly or G); histidine (His or H); isoleucine (Ile or I); leucine (Leu or L); lysine (Lys or K); methionine (Met or M); phenylalanine (Phe or F); proline (Pro or P); serine (Ser or S); threonine (Thr or T); tryptophan (Trp or W); tyrosine (Tyr or Y); and valine (Val or V), although modified, synthetic, or rare amino acids may be used if desired. In general, amino acids can be classified by the presence of a nonpolar side chain (e.g., Ala, Cys, Ile, Leu, Met, Phe, Pro, Val); a negatively charged side chain (e.g., Asp, Glu); a positively charged side chain (e.g., Arg, His, Lys); or an uncharged polar side chain (e.g., Asn, Cys, Gln, Gly, His, Met, Phe, Ser, Thr, Trp, and Tyr).

[0200] As used herein, "amino acid modification" includes, but is not limited to, deletion from, and / or insertion into, and / or substitution of, residues within the amino acid sequence. Any combination of deletion, insertion, and substitution may be made to arrive at the final construct, provided that the final construct retains the desired characteristics. Changing amino acids may also alter post-translational processing of the antibody construct (e.g., changing the number or location of glycosylation sites). Non-limiting exemplary substitutions (or replacements) are conservative substitutions. However, any substitution (including non-conservative substitutions) is contemplated as long as the final construct retains the ability to bind to the target antigen.

[0201] Those skilled in the art will recognize that conservative variants of the antibodies, antibody fragments, and antigen-binding fragments thereof described herein can be produced. Such conservative variants employed in antibody fragments, such as dsFv or scFv fragments, include those having a V H Area and V L Retain key amino acid residues required for correct folding and stabilization between regions, and maintain the charge characteristics of the residues to preserve the molecule's low pI and low toxicity. In some embodiments, amino acid substitutions (e.g., up to 1, up to 2, up to 3, up to 4, or up to 5 amino acid substitutions) are made to V to increase yield. H Region and / or V L Conservative amino acid substitution tables providing functionally similar amino acids, such as those set forth in Table A1, are well known to those skilled in the art.

[0202] [Table 1]

[0203] [Table 2]

[0204] As used herein, "percent (%) amino acid sequence identity" or "percent (%) sequence identity" to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering conservative substitutions as part of the sequence identity. Alignment to determine percent amino acid sequence identity can be accomplished in a variety of ways within the skill of one in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximum alignment over the entire length of the sequences being compared. However, for purposes of this specification, percent amino acid sequence identity values ​​are generated using the sequence comparison computer program ALIGN-2.

[0205] As used herein, the term "cancer" refers to a variety of conditions caused by the uncontrolled abnormal growth of cells, including neoplasms, primary tumors, secondary tumors, and other metastatic lesions. Cancer can be detected in many ways, including, but not limited to, the presence of a tumor in tissue detected by clinical or radiological means, the detection of cancerous or abnormal cells in a biological sample (e.g., a tissue biopsy), the detection of a biomarker indicative of cancer or a precancerous condition, or the detection of a genotype indicative of cancer or the risk of developing cancer. The term "cancer" encompasses a variety of cancerous conditions, regardless of stage, grade, invasiveness, malignancy, or histological type. Cancers that can be treated by the methods of the present disclosure include leukemia (e.g., myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia), lymphoma (e.g., diffuse large B-cell lymphoma, Burkitt's lymphoma, non-Hodgkin's lymphoma, follicular lymphoma), multiple myeloma, lung cancer (e.g., small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC)), glioma, glioblastoma, melanoma, and the like. These include, but are not limited to, melanoma, prostate cancer (e.g., castration-resistant prostate cancer, neuroendocrine prostate cancer), pancreatic cancer, breast cancer, bone cancer, cervical cancer, colon cancer, colorectal cancer, endometrial cancer, head and neck cancer, liver cancer, ovarian cancer, stomach cancer, gastroesophageal junction cancer, testicular cancer, thyroid cancer, adrenal cancer, kidney cancer, bladder cancer, uterine cancer, esophageal cancer, urothelial cancer, epithelial carcinoma, and non-epithelial carcinoma, as well as metastatic cancers derived from any of the above.

[0206] As used herein, the term "anti-cancer effect" refers to a biological effect that can be manifested by various means, including, but not limited to, a reduction in tumor volume, a reduction in the number of cancer cells, a reduction in the number of metastases, an increase in life expectancy, a reduction in cancer cell proliferation, a reduction in cancer cell survival, or an improvement in various physiological symptoms associated with a cancerous condition. An "anti-cancer effect" can also be manifested by the ability of peptides, polynucleotides, cells, and antibodies in preventing development in the first place. As used herein, the term "anti-tumor effect" refers to a biological effect that can be manifested by various means, including, but not limited to, a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in tumor cell proliferation, or a reduction in tumor cell survival.

[0207] The terms "IL2" and "IL-2," used interchangeably herein, refer to interleukin-2, a 15.5-16 kDa cytokine signaling protein molecule that regulates the activity of certain immune cells by binding to the IL2 receptor complex expressed by lymphocytes. The term "IL2" includes IL2 proteins from any human and non-human animal species, specifically human IL2 and IL2 from non-human mammals. The human IL-2 sequence (UniProtKB No. P60568) is provided herein as SEQ ID NO: 41. The term "human IL2," as used herein, includes any variant, isoform, and species homolog of human IL2, regardless of its source or mode of preparation. Thus, "human IL2" includes human IL2 naturally expressed by cells and IL2 expressed in cells transfected with the human IL2 gene.

[0208] The terms "IL2R," "IL-2R," "IL2 receptor," and "IL-2 receptor," when used interchangeably herein, generally refer to the IL2 receptor complex, which is composed of three polypeptide subunits or chains designated as the alpha, A, or α, beta, B, or β, and γ, G, or γ, chain. IL-2R is a heterodimeric protein expressed on the surface of various immune cells that serves as the cognate ligand for interleukin-2 (IL-2). The term "IL2R" includes any IL2R protein or any subunit of the IL2 receptor complex of any human or non-human animal species, specifically including human IL2R and IL2R of non-human mammals. As used herein, the term "human IL2R" includes any variant, isoform, and species homolog of human IL2R, regardless of its source or mode of preparation. Thus, "human IL2R" includes both the human IL2R naturally expressed by cells and the IL2R expressed in cells transfected with the human IL2R gene.

[0209] The term "IL2RA" or "IL2Rα" is also referred to as CD25, and the human IL2RA sequence (UniProtKB number P01589) is provided herein as SEQ ID NO: 38.

[0210] The term "IL2RB" or "IL2Rβ" is also referred to as CD122, and the human IL2RB sequence (UniProtKB number P14784) is provided herein as SEQ ID NO: 39.

[0211] The term "IL2RG" or "IL2Rγ" is also referred to as CD132, and the human IL2RG sequence (UniProtKB number P31785) is provided herein as SEQ ID NO: 40.

[0212] The terms "anti-IL2R heavy chain-only antibody," "IL2R heavy chain-only antibody," "anti-IL2R heavy chain antibody," and "IL2R heavy chain antibody" are used interchangeably herein and refer to heavy chain-only antibodies as defined above that specifically bind to IL2R, including human IL2R, as defined above. This definition includes, but is not limited to, human heavy chain antibodies produced by transgenic animals such as transgenic rats or transgenic mice expressing human immunoglobulins, e.g., UniRat™, which produces human anti-IL2R UniAb™, as defined above. Similarly, as used herein, the term "anti-IL2Rβγ heavy chain-only antibody" refers to a heavy chain-only antibody that specifically binds to IL2Rβ and IL2Rγ.

[0213] As used herein, the term "agonist" refers to a molecule that causes an increase in function or activity compared to the same function or activity in the absence of the molecule. Thus, an "agonist" of a signaling pathway is a molecule whose presence causes an increase in the function or activity of the signaling pathway. As used herein, the term "agonize" refers to causing an increase in function or activity. In some embodiments, the agonistic function of an antibody, antibody fragment, or antigen-binding fragment thereof can be determined using the assays described herein.

[0214] An "epitope" is a site on the surface of an antigen molecule to which a single antibody molecule binds. Generally, an antigen has several or many different epitopes and reacts with many different antibodies. The term specifically includes linear and conformational epitopes.

[0215] As used herein, the term "valency" refers to the specific number of binding sites in an antibody molecule.

[0216] A "monovalent" antibody has one binding site and is therefore also monospecific.

[0217] A "multivalent" antibody has two or more binding sites. Thus, the terms "bivalent," "trivalent," and "tetravalent" refer to the presence of two binding sites, three binding sites, and four binding sites, respectively. Thus, bispecific antibodies according to the present disclosure are at least bivalent and may be trivalent, tetravalent, or otherwise multivalent. Bivalent antibodies according to embodiments of the present disclosure may have two binding sites for the same epitope (i.e., bivalent monoparatopic) or two different epitopes (i.e., bivalent biparatopic).

[0218] A wide variety of methods and protein structures are known and have been used to prepare bispecific monoclonal antibodies (BsMABs), trispecific antibodies, and the like.

[0219] As used herein, the term "effector cell" refers to an immune cell that is involved in the effector phase of an immune response, as opposed to the recognition and activation phases of the immune response. Some effector cells express specific Fc receptors and carry out specific immune functions. In some embodiments, effector cells, such as natural killer cells, are capable of inducing antibody-dependent cellular cytotoxicity (ADCC). For example, FcR-expressing monocytes and macrophages are involved in specifically killing target cells and presenting antigens to other components of the immune system or binding to cells that present antigens. In some embodiments, effector cells can phagocytose target antigens or target cells.

[0220] "Human effector cells" are leukocytes that express a receptor, such as a T cell receptor or FcR, and perform effector function. For example, in some embodiments, the cells express at least FcγRIII and perform ADCC effector function. Non-limiting examples of human leukocytes that mediate ADCC include natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils. Effector cells can be isolated from their native source, e.g., blood or PBMCs, as described herein.

[0221] The term "immune cell" is used herein in the broadest sense and includes, but is not limited to, cells of myeloid or lymphoid origin, such as lymphocytes (B cells and T cells, e.g., cytolytic (CTL) cells), killer cells, natural killer (NK) cells, macrophages, monocytes, eosinophils, polymorphonuclear cells, e.g., neutrophils, granulocytes, mast cells, and basophils.

[0222] Antibody "effector functions" refer to the biological activities attributable to the Fc region of an antibody (either a native sequence Fc region or an amino acid sequence variant Fc region). Examples of antibody effector functions include C1q binding, complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, and down-regulation of cell surface receptors (e.g., B cell receptor, BCR).

[0223] "Antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to a cell-mediated reaction in which nonspecific cytotoxic cells expressing Fc receptors (FcR), such as natural killer (NK) cells, neutrophils, and macrophages, recognize bound antibodies on target cells and subsequently cause lysis of the target cells. NK cells, the primary cells for mediating ADCC, express FcγRIII only, whereas monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3, page 464, of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in U.S. Pat. No. 5,500,362 or U.S. Pat. No. 5,821,337, may be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest can be assessed in vivo in an animal model such as that disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).

[0224] "Complement-dependent cytotoxicity" or "CDC" refers to the ability of a molecule to lyse a target in the presence of complement. The complement activation pathway is initiated by the binding of the first component of the complement system (C1q) to a molecule (e.g., an antibody) complexed with a cognate antigen. To assess complement activation, a CDC assay, e.g., as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), may be performed.

[0225] "Binding affinity" refers to the overall strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, "binding affinity" as used herein refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art. Low-affinity antibodies generally bind antigens slowly and tend to dissociate easily, while high-affinity antibodies generally bind antigens more quickly and tend to remain bound.

[0226] As used herein, "Kd" or "Kd value" refers to the dissociation constant measured by biolayer interferometry using an Octet QK384 instrument (Fortebio Inc., Menlo Park, CA) in kinetics mode. For example, an antigen fused to mouse Fc is loaded onto an anti-mouse Fc sensor, which is then immersed in an antibody-containing well to measure the concentration-dependent binding rate (k). In the final step, the sensor is immersed in a well containing only buffer, and the antibody dissociation rate (koff) is measured. Kd is the ratio of koff / koff. (For further details, see Concepcion, J, et al., Comb Chem High Throughput Screen, 12(8), 791-800, 2009.)

[0227] "K DThe term "(M)" as used herein refers to the equilibrium dissociation constant of a specific antigen-binding interaction as determined by biolayer interferometry using an Octet QK384 instrument (Fortebio Inc., Menlo Park, CA) in kinetics mode. For example, an anti-mouse Fc sensor was loaded with a mouse Fc fusion antigen and then immersed in a well containing the antibody, and the concentration-dependent binding rate (k on In a final step, the sensor is immersed in a well containing buffer only, and the antibody dissociation rate (k off ) is measured. D is k off / k on (For further details, see Concepcion, J, et al., Comb Chem High Throughput Screen, 12(8), 791-800, 2009).

[0228] As used herein, a molecule (e.g., a molecule, antibody, antibody fragment, or antigen-binding fragment, etc.) "specifically binds" to a target antigen if, under similar binding assay conditions, it has a significantly higher binding affinity for the target antigen compared to its affinity for other unrelated proteins, and is therefore able to discriminate against the target antigen. A molecule that specifically binds to an antigen has a binding affinity of ≦1×10 -6 The equilibrium dissociation constant (K D ) can bind to its antigen. D is ≦1×10 -8 M. In some embodiments, the molecules described herein specifically bind to a target antigen with a binding affinity of ≦5×10 -7 K of M D In some embodiments, the molecules described herein bind to the target antigen at ≦1×10 -7 K of M D In some embodiments, the molecules described herein bind to the target antigen at ≦5×10 -8 K of M D In some embodiments, the molecules described herein bind to the target antigen at a density of ≦2×10 -8 K of M DIn some embodiments, the molecules described herein bind to the target antigen at ≦1×10 -8 K of M D In some embodiments, the molecules described herein bind to the target antigen at ≦1×10 -9 K of M D Combine with.

[0229] Affinity may be determined using a variety of techniques, non-limiting examples of which include affinity ELISA assays. In some embodiments, affinity is determined by a surface plasmon resonance assay (e.g., a BIAcore®-based assay). Using this methodology, the binding rate constant (k a , M -1 s -1 ) and dissociation rate constant (k d , s -1 ) can then be measured. D , M) is the ratio of reaction rate constants (k d / k a In some embodiments, affinity is determined by a kinetic method, such as the equilibrium exclusion binding assay (KExA) as described by Rathanaswami et al. Analytical Biochemistry, Vol. 373:52-60, 2008. The KinExA assay can be used to determine the equilibrium dissociation constant (K D , M) and association rate constant (k a , M -1 s -1 From these values, the dissociation rate constant (k d , s -1 ) can be calculated (K D ×k a In another embodiment, affinity is determined by biolayer interferometry, such as that described in Kumaraswamy et al., Methods Mol. Biol., Vol. 1278:165-82, 2015, and used in the Octet® system (Pall ForteBio). The reaction rate constant (k a and k d ) and affinity constant (KD ) can be calculated in real time using biolayer interferometry.

[0230] As used herein, the term "administer" and its cognates (e.g., "administering") include both self-administration and administration to a patient by another person (e.g., a medical professional or caregiver).

[0231] As used herein, the terms "in combination with," in the context of administration, as well as "co-administration," "administration in combination," and their cognates (e.g., "co-administering") refer to the administration of two or more therapeutic agents to a single subject in a coordinated manner, including, but not limited to, concomitant administration. Specifically, "co-administration" encompasses not only administration of a combined agent or simultaneous administration of separate therapeutic compositions, but also sequential or sequential administration, provided that the administration of one therapeutic agent is coordinated in some way with the administration of another therapeutic agent. Therapeutic agents are not necessarily administered simultaneously and / or by the same route of administration. Illustratively, one therapeutic agent may be administered only after a different therapeutic agent has been administered and allowed to act for a predetermined period of time. Additionally, in some embodiments, co-administered therapeutic agents are present (PK) or otherwise induce an effect (PD) in a subject for similar, identical, or partially overlapping periods of time.

[0232] "Before" in the context of a first therapeutic agent administered before a second therapeutic agent is intended to mean within about 72 hours, about 48 hours, about 36 hours, about 24 hours, about 18 hours, about 16 hours, about 12 hours, about 6 hours, about 5 hours, about 4 hours, or about 3 hours, e.g., within about 120 minutes, about 90 minutes, about 60 minutes, or about 30 minutes before starting administration of the second therapeutic agent.

[0233] In some embodiments, the combination partners may be administered entirely separately or may be in entirely separate pharmaceutical dosage forms. Additionally, in some embodiments, the combination partners may also be pharmaceutical compositions sold independently of each other, with instructions for their combined use provided in packaging materials, such as a leaflet, or in other information (e.g., verbal communication, written communication, etc.) provided to, for example, physicians and medical staff.

[0234] In some embodiments, the combination partners may be incorporated into the combination therapy (i) prior to delivery of the combination partner product to the physician (e.g., in the case of a kit containing the combination partners); (ii) by the physician (or under the physician's guidance) immediately prior to administration; or (iii) by the patient themselves, e.g., during sequential administration of the combination partners.

[0235] As used herein, a "combination product" refers to a pharmaceutical product that results from mixing or combining two or more active ingredients and contains both fixed and non-fixed combinations of the active ingredients, which may be combined. Combination products include kits of components for combined administration.

[0236] As used herein, the term "non-fixed combination" refers to therapeutic agents administered to a patient as separate entities simultaneously, concurrently, or sequentially without specific time restrictions, whereby therapeutically effective levels of both therapeutic agents are provided. The latter also applies to cocktail therapy, e.g., the administration of three or more active ingredients. In non-fixed combinations, the combination partners can be administered independently of each other or by using different fixed combinations with distinct amounts of the combination partners.

[0237] As used herein, the term "fixed combination" refers to at least two therapeutic agents that are both administered to a patient simultaneously in the form of a single entity or dosage (i.e., the therapeutic agents are present in a single dosage form).

[0238] As used herein, the term "treatment" and its cognates (e.g., "treating") encompass any improvement in a disease in a subject, including slowing or halting the progression of the disease in a patient, reducing the number or severity of symptoms of the disease, or increasing the frequency and duration when the subject is symptom-free of the disease.

[0239] As used herein, a "therapeutically effective amount" refers to an amount of an active agent that confers a therapeutic effect on a subject. For example, a "therapeutically effective amount" is an amount that induces, ameliorates, or causes an improvement in pathological symptoms associated with a disease, disease progression, or physiological condition, or improves resistance to a disorder.

[0240] The terms "subject," "individual," and "patient" are used interchangeably herein and refer to a mammal being evaluated for and / or treated for therapy. The subject can be a human, but also includes other mammals, particularly mammals useful as experimental models for human disease, e.g., mice, rats, and the like. In some embodiments, the mammal is a human.

[0241] The term "pharmaceutical composition" refers to a formulation that is in a form that allows the biological activity of the active ingredient to be effective and that does not contain additional components that are unacceptably toxic to the subject to which the formulation is administered. Such compositions are sterile. "Pharmaceutically acceptable" excipients (e.g., vehicles, additives) are those that can reasonably be administered to a mammalian subject to provide an effective dose of the active ingredient employed.

[0242] As used herein, a "sterile" composition is sterile or free or essentially free of all living microorganisms and their spores. As used herein, a "frozen" composition is one at a temperature below 0°C.

[0243] As used herein, a "stable" composition is one in which the protein therein essentially retains its physical stability, chemical stability, and / or biological activity upon storage. In some embodiments, the composition essentially retains its physical and chemical stability and biological activity upon storage. The storage period is generally selected based on the intended shelf life of the composition. Various analytical techniques for measuring protein stability are available in the art and are reviewed, for example, in Peptide and Protein Drug Delivery, 247-301. Vincent Lee Ed., Marcel Dekker, Inc., New York, NY, Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10:29-90) (1993). Stability can be measured at a selected temperature for a selected period of time. Stability can be qualitatively and / or quantitatively assessed in a variety of different ways, including assessing aggregate formation (e.g., by measuring turbidity using size exclusion chromatography and / or by visual inspection); assessing charge heterogeneity using cation exchange chromatography, imaging capillary isoelectric focusing (icIEF), or capillary zone electrophoresis; analysis of amino- or carboxy-terminal sequences; mass spectrometry; SDS-PAGE analysis to compare reduced and intact antibodies; peptide map (e.g., trypsin or LYS-C) analysis; assessment of antibody biological activity or antigen-binding function, etc. Instability can involve any one or more of aggregation, deamidation (e.g., Asn deamidation), oxidation (e.g., Met oxidation), isomerization (e.g., Asp isomerization), clipping / hydrolysis / fragmentation (e.g., hinge region fragmentation), succinimide formation, unpaired cysteines, N-terminal extensions, C-terminal processing, differential glycosylation, etc.

[0244] As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector, a "plasmid," refers to a circular double-stranded DNA loop to which additional DNA segments may be ligated. Another type of vector is a viral vector, in which additional DNA segments may be ligated to the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) may be integrated into the genome of the host cell upon introduction into the host cell, and thereby replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors." In some embodiments, expression vectors used in recombinant DNA techniques are in the form of plasmids.

[0245] As used herein, the term "host cell" refers to a cell into which an expression vector has been introduced. "Host cell" is intended to refer not only to the particular subject cell but also to the progeny of such a cell. Because certain modifications may occur in successive generations due to either mutation or environmental influences, such progeny may not be, in fact, identical to the parent cell, but are still included within the scope of the term "host cell" as used herein. Exemplary recombinant host cells include, but are not limited to, transfectomas such as CHO cells, HEK293 cells, NS / 0 cells, and lymphocytic cells.

[0246] As used herein, the term "autologous" refers to any material that originates from an individual to whom the material is intended to be reintroduced.

[0247] As used herein, the term "allogeneic" refers to any material derived from a different animal of the same species as the individual to which the material is introduced. Two or more individuals are said to be allogeneic to one another if the genes at one or more loci are not identical. In some embodiments, allogeneic material derived from individuals of the same species may be sufficiently genetically different to interact antigenically.

[0248] As used herein, the term "chimeric antigen receptor," or alternatively, "CAR," refers to a recombinant polypeptide construct comprised of at least an extracellular antigen-binding domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as an "intracellular signaling domain") that includes a functional signaling domain derived from a stimulatory molecule. In some embodiments, the domains of a CAR polypeptide construct are in the same polypeptide chain, e.g., comprised of a chimeric fusion protein. In some embodiments, the domains of a CAR polypeptide construct are not contiguous with each other, e.g., are in different polypeptide chains.

[0249] In some embodiments, the stimulatory molecule is a zeta chain associated with the T cell receptor complex. In some embodiments, the cytoplasmic signaling domain comprises a primary signaling domain (e.g., a primary signaling domain of CD3 zeta). In some embodiments, the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one costimulatory molecule. In some embodiments, the costimulatory molecule is selected from 4-1BB (i.e., CD137), CD27, ICOS, and CD28. In some embodiments, the CAR comprises a chimeric fusion protein comprised of an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a stimulatory molecule. In some embodiments, the CAR comprises a chimeric fusion protein comprised of an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a costimulatory molecule and a functional signaling domain derived from a stimulatory molecule. In some embodiments, the CAR comprises a chimeric fusion protein comprised of an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain comprising two functional signaling domains derived from one or more costimulatory molecules and a functional signaling domain derived from a stimulatory molecule. In some embodiments, the CAR comprises a chimeric fusion protein composed of an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain comprising at least two functional signaling domains derived from one or more costimulatory molecules and a functional signaling domain derived from a stimulatory molecule. In some embodiments, the CAR comprises an optional leader sequence at the amino-terminus (N-ter) of the CAR fusion protein. In some embodiments, the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen-binding domain, which is optionally cleaved from the antigen recognition domain (e.g., scFv) during cellular processing and localization of the CAR to the cell membrane.

[0250] As used herein, the term "signaling domain" refers to a functional portion of a protein that acts by transmitting information within a cell to regulate cellular activity through a defined signaling pathway by generating a second messenger or by functioning as an effector in response to such a messenger. In some embodiments, the signaling domain of a CAR described herein is derived from a stimulatory molecule or a costimulatory molecule, or is a synthetic or engineered signaling domain.

[0251] As used herein, "intracellular signaling domain" refers to the intracellular portion of a molecule. The intracellular signaling domain generates a signal that promotes immune effector function of a CAR-expressing cell, such as a CAR-T cell or a CAR-expressing NK cell. For example, non-limiting examples of immune effector function in a CAR-T cell or a CAR-expressing NK cell include cytolytic activity and helper activity, including cytokine secretion. While the entire intracellular signaling domain can be used, it is often not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such a truncated portion can be used in place of the intact chain, provided that it transmits a signal for effector function. Thus, the term intracellular signaling domain is meant to include any truncated portion of the intracellular signaling domain sufficient to transmit a signal for effector function.

[0252] In some embodiments, the intracellular signaling domain can comprise a primary intracellular signaling domain. Exemplary primary intracellular signaling domains include, but are not limited to, those derived from molecules involved in primary or antigen-dependent stimulation. In some embodiments, the intracellular signaling domain comprises a costimulatory intracellular domain. Exemplary costimulatory intracellular signaling domains include, but are not limited to, those derived from molecules involved in costimulatory signals or antigen-independent stimulation. In some embodiments, the intracellular signaling domain is synthetic or engineered. For example, in the case of CAR-expressing immune effector cells, e.g., CAR-T cells or CAR-expressing NK cells, the primary intracellular signaling domain can comprise the cytoplasmic sequence of a T cell receptor, the primary intracellular signaling domain can comprise the cytoplasmic sequence of a T cell receptor, and the costimulatory intracellular signaling domain can comprise the cytoplasmic sequence from a co-receptor or costimulatory molecule.

[0253] In some embodiments, the primary intracellular signaling domain comprises a signaling motif known as an immunoreceptor tyrosine-based activation motif, or ITAM. Examples of ITAMs containing primary cytoplasmic signaling sequences include, but are not limited to, those derived from CD3 zeta, common FcR gamma (FCER1G), Fc gamma RIIa, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CDS, CD22, CD79a, CD79b, CD278 ("ICOS"), FcεRI CD66d, DAP10, and DAP12.

[0254] As used herein, the term "costimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by the T cell, including, but not limited to, proliferation. Costimulatory molecules are cell surface molecules, other than antigen receptors or their ligands, that are required for an effective immune response. Costimulatory molecules include MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), activating NK cell receptors, BTLA, Toll ligand receptors, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD11a / CD18), 4-1BB (CD137), B7 -H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 alpha, CD8 beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, C D49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1 , ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRT These include, but are not limited to, ligands that specifically bind to AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, and CD83.

[0255] In some embodiments, the costimulatory intracellular signaling domain can be the intracellular portion of a costimulatory molecule. The intracellular signaling domain can include the entire intracellular portion of the molecule from which it is derived, or the entire native intracellular signaling domain, or a functional fragment thereof.

[0256] As used herein, the terms "zeta," or alternatively "zeta chain" or "CD3 zeta," are defined as the protein provided under GenBank Accession No. BAG36664.1, or equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape, etc., and a "zeta stimulatory domain," or alternatively, a "CD3 zeta stimulatory domain," are defined as the amino acid residues from the cytoplasmic domain of the zeta chain sufficient to functionally transmit the initial signal required for T cell activation. In some embodiments, the zeta cytoplasmic domain comprises residues 52-164 of GenBank Accession No. BAG36664.1, or equivalent residues from a non-human species that is a functional ortholog, e.g., mouse, rodent, monkey, ape, etc.

[0257] As used herein, the term "4-1BB" refers to a member of the TNFR superfamily containing the amino acid sequence provided as GenBank Accession No. AAA62478.2, or equivalent residues from a non-human species, such as mouse, rodent, monkey, ape, etc., and the "4-1BB costimulatory domain" is defined as amino acid residues 214 to 255 of GenBank Accession No. AAA62478.2, or equivalent residues from a non-human species, such as mouse, rodent, monkey, ape, etc.

[0258] As used herein, the term "T cell redirection therapy" refers to a therapeutic agent, such as a T cell-directing molecule or CAR T cells, that is capable of recruiting T cells to a target cell or tissue.

[0259] As used herein, the term "T cell inducing molecule" refers to a molecule whose structure comprises at least one domain derived from or including the minimal structural features of an antibody, e.g., a full-length immunoglobulin molecule, capable of specifically binding to an antigen on the surface of a T cell, such as CD3. Thus, a T cell inducing molecule according to the present disclosure generally comprises one or more binding domains, each of which typically comprises the minimal structural requirements of an antibody that enable specific target binding. This minimum requirement may be defined, for example, by the presence of at least three light chain "complementarity-determining regions" or CDRs (i.e., CDRL1, CDRL2, and CDRL3 of the VL region) and / or three heavy chain CDRs (i.e., CDRH1, CDRH2, and CDRH3 of the VH region), e.g., a total of six CDRs derived from both the light chain variable region and the heavy chain variable region. A T cell inducing molecule according to the present disclosure may comprise domains or regions (e.g., CDRs or variable regions) derived from a monoclonal antibody, a chimeric antibody, a humanized antibody, and a human antibody. In some embodiments, the T cell inducing molecule used in the methods of the present disclosure is a protein and comprises one or more polypeptide chains. In some embodiments, the T cell inducing molecule administered in accordance with the methods of the present disclosure is a single-chain polypeptide. In other embodiments, the T cell inducing molecule administered in accordance with the methods of the present disclosure comprises two or more polypeptide chains, e.g., a polypeptide dimer or multimer. In certain embodiments, the T cell inducing molecule administered in accordance with the methods of the present disclosure comprises four polypeptide chains and may have the format of, for example, an antibody or immunoglobulin protein.

[0260] As used herein, the term "bispecific T cell attracting molecule" refers to a molecule capable of specifically binding to two different antigens. In the context of the present disclosure, a bispecific T cell attracting molecule specifically binds to a cancer cell antigen (e.g., a human cancer cell antigen) on the cell surface of a target cell and to CD3 (e.g., human CD3) on the cell surface of a T cell. In some embodiments, a T cell attracting molecule can bind to two or more cancer cell antigens (e.g., a human cancer cell antigen) on the cell surface of a target cell as well as CD3 (e.g., human CD3) on the cell surface of a T cell. Thus, in such embodiments, the T cell attracting molecule is "multitargeted" in that it specifically binds to two or more different cancer cell antigens and can attract T cells to two or more types of cancer cells or cancer cells expressing two or more antigens.

[0261] A T cell inducing molecule or a binding domain thereof "specifically binds" to a target antigen if, under similar binding assay conditions, it has a significantly higher binding affinity compared to its affinity for other unrelated proteins, such that it can distinguish between the antigens. A T cell inducing molecule or a binding domain thereof that specifically binds to an antigen has an equilibrium dissociation constant (K D )≦1×10 -6 In one embodiment, the T cell engaging molecule or binding domain thereof used in the methods of the present disclosure can bind to its antigen with a M of ≦5×10 -7 K of M D In another embodiment, the T cell engaging molecule or binding domain thereof used in the methods of the present disclosure binds to a human cancer cell antigen and / or human CD3 at a concentration of ≦1×10 -7 K of M D In yet another embodiment, the T cell engaging molecule or binding domain thereof used in the methods of the present disclosure binds to a human cancer cell antigen and / or human CD3 at a concentration of ≦5×10 -8 K of M D In another embodiment, the T cell engaging molecule or binding domain thereof used in the methods of the present disclosure binds to a human cancer cell antigen and / or human CD3 at a concentration of ≦2×10 -8 K of M DIn certain embodiments, the T cell engaging molecule or binding domain thereof used in the methods of the disclosure binds to a human cancer cell antigen and / or human CD3 at a concentration of ≦1×10 -8 K of M D In other embodiments, the T cell engaging molecule or binding domain thereof used in the methods of the present disclosure binds to a human cancer cell antigen and / or human CD3 at a concentration of ≦1×10 -9 K of M D It binds to human cancer cell antigens and / or human CD3.

[0262] In some embodiments, the T cell engaging molecules or binding domains thereof described herein are administered in a concentration of about 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 , 10 -10 s -1 or less k for human cancer cell antigens and / or human CD3 d Binding affinity as measured by the dissociation rate constant (dissociation rate constant) (lower values ​​indicate greater binding affinity) and / or approximately 10 -7 , 10 -8 , 10 -9 , 10 -10 , 10 -11 M or less K for human cancer cell antigens and / or human CD3 D It exhibits desirable properties such as binding affinity as measured by the equilibrium dissociation constant (equilibrium dissociation constant), with smaller values ​​indicating higher binding affinity.

[0263] In some embodiments, the bispecific T cell engaging molecule used in the methods of the present disclosure is an antibody and may have the general structure of a full-length immunoglobulin. For example, the bispecific T cell engaging molecule may comprise two full-length antibody heavy chains and two full-length antibody light chains. In certain embodiments, the bispecific T cell engaging molecule is a heterodimeric antibody (used interchangeably herein as "heteroimmunoglobulin" or "hetero-Ig"), which refers to an antibody comprising two different light chains and two different heavy chains. For example, in some embodiments, the heterodimeric antibody comprises a light chain and a heavy chain derived from an antibody that specifically binds to a cancer cell antigen, such as a cancer cell antigen described further herein, and a light chain and a heavy chain derived from an antibody that specifically binds to CD3.

[0264] Bispecific T cell engaging molecules used in the methods of the present disclosure may also comprise fragments of full-length antibodies, such as VH, VHH, VL, (s)dAb, Fv, light chain (VL-CL), Fd (VH-CH1), heavy chain, Fab, Fab', F(ab')2 or "rIgG" (a "half antibody" consisting of a heavy and light chain). Bispecific T cell engaging molecules according to the present disclosure may also comprise engineered fragments of antibodies. Examples of such engineered fragments include, but are not limited to, single-chain variable fragments (scFv), di-scFv or bi(s)-scFv, scFv-Fc, scFv-zipper, single-chain Fab (scFab), Fab2, Fab3, diabodies, single-chain diabodies, tandem diabodies (Tandab), tandem di-scFv, tandem tri-scFv, "minibodies" (exemplified by structures such as (VH-VL-CH3)2, (scFv-CH3)2, ((scFv)2-CH3+CH3), ((scFv)2-CH3) or (scFv-CH3-scFv)2), multibodies such as triabodies or tetrabodies, and single domain antibodies, e.g., nanobodies or single variable domain antibodies comprising only one variable region which may be VHH, VH or VL, which specifically bind to an antigen or target independent of other variable regions or domains.

[0265] In certain embodiments, the bispecific T cell inducing molecules used in the methods of the present disclosure are multivalent. The valency of a T cell inducing molecule refers to the number of individual antigen-binding domains within the T cell inducing molecule. In the context of the present disclosure, for example, the terms "monovalent," "bivalent," and "tetravalent" with respect to a T cell inducing molecule refer to T cell inducing molecules having one, two, and four antigen-binding domains, respectively. Thus, a multivalent T cell inducing molecule contains two or more antigen-binding domains. A T cell inducing molecule can have more antigen-binding domains (e.g., higher valency) than specificities. For example, a T cell inducing molecule having two antigen-binding domains for a first target (e.g., a cancer cell antigen) and one antigen-binding domain for a second target (CD3) (or vice versa) is considered trivalent (three antigen-binding domains) and bispecific (binds to two antigens). In certain embodiments, the bispecific T cell inducing molecules used in the methods of the present disclosure are bivalent. Thus, such bispecific, bivalent T cell engaging molecules contain two antigen-binding domains: one antigen-binding domain for a cancer cell antigen (e.g., a human cancer cell antigen) and one antigen-binding domain for CD3 (e.g., human CD3). In other embodiments, the T cell engaging molecules used in the methods of the present disclosure are trivalent, trispecific T cell engaging molecules, comprising three antigen-binding domains: one antigen-binding domain for a first cancer cell antigen, another antigen-binding domain for a second cancer cell antigen, and a third binding domain for CD3. In yet other embodiments, the T cell engaging molecules used in the methods of the present disclosure are tetravalent, trispecific T cell engaging molecules, comprising four antigen-binding domains: one antigen-binding domain for a first cancer cell antigen, another antigen-binding domain for a second cancer cell antigen, and two antigen-binding domains for CD3.

[0266] In some embodiments, a bispecific T cell engaging molecule used in the methods of the present disclosure comprises a first binding domain that specifically binds to a target cancer cell antigen (e.g., a human target cancer cell antigen) and a second binding domain that specifically binds to CD3 (e.g., human CD3). As used herein, the term "antigen-binding domain," used interchangeably with "binding domain," refers to a region of a T cell engaging molecule that contains amino acid residues that interact with an antigen and confer specificity and affinity for that antigen to the T cell engaging molecule. In certain embodiments, one or more binding domains of a T cell engaging molecule may be derived from an antibody or antigen-binding fragment thereof. For example, the binding domain of a bispecific T cell engaging molecule used in the methods of the present disclosure may comprise one or more CDRs derived from the light and heavy chain variable regions of an antibody that specifically binds to a human target cancer cell antigen and / or human CD3. In some embodiments, the anti-cancer cell antigen binding domain of the bispecific T cell attracting molecule comprises all six CDRs from the heavy and light chain variable regions of an antibody that specifically binds to its human target cancer cell antigen, and the anti-CD3 binding domain of the bispecific T cell attracting molecule comprises all six CDRs from the heavy and light chain variable regions of an anti-CD3 antibody. In some embodiments, the binding domains (anti-cancer cell antigen binding domain, anti-CD3 binding domain, or both) of the bispecific T cell attracting molecule used in the methods of the present disclosure comprise a Fab, a Fab', a F(ab')2, an Fv, a single-chain variable fragment (scFv), or a nanobody. In one embodiment, both binding domains of the bispecific T cell attracting molecule are Fab fragments. In another embodiment, one binding domain of the bispecific T cell attracting molecule is a Fab fragment and the other binding domain is an scFv. In yet another embodiment, both binding domains of the bispecific T cell attracting molecule are scFvs.

[0267] Anti-IL-2Rβγ heavy chain only antibody and its antigen-binding fragment 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, and 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.

[0268] 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 heavy chains (heavy-chain-only antibodies or HCAbs).The heavy-chain-only 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-only antibodies lack the first domain (CH1) of the constant region, which is present in the genome but spliced ​​out during mRNA processing. Because the CH1 domain is located at the anchor position of the light-chain constant domain, the absence of this domain explains the absence of a light chain in heavy-chain-only antibodies. Such heavy-chain-only antibodies have naturally evolved to confer antigen-binding specificity and high affinity through three CDRs derived from conventional antibodies or their fragments. (Muyldermans, 2001; J Biotechnol 74:277-302; Revets et al., 2005; Expert Opin Biol Ther 5:111-124). Cartilaginous fish, such as sharks, have also evolved a unique type of immunoglobulin, termed IgNAR, which lacks light chain polypeptides and is composed entirely of heavy chains. IgNAR molecules can be engineered to generate 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).

[0269] The ability of heavy-chain-only antibodies lacking light chains to bind antigens was established in the 1960s (Jaton et al. (1968) Biochemistry, 7, 4185-4195). Heavy-chain immunoglobulins physically separated from light chains retained 80% of their antigen-binding activity compared to tetrameric antibodies. Sitia et al. (1990) Cell, 60, 781-790 demonstrated that removal of the CH1 domain from a rearranged mouse μ gene resulted in the production of heavy-chain-only antibodies lacking light chains in mammalian cell culture. The antibodies produced retained VH binding specificity and effector function.

[0270] Heavy-chain antibodies with high specificity and affinity can be generated against various antigens by 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, LGJ, et al. J. Biotechnol. 78, 11-21 (2000)). Their expression, solubility, and stability levels are significantly higher than those of classical F(ab) or Fv fragments. Ghahroudi, MA et al. FEBS Lett. 414, 521-526 (1997).

[0271] Mice in which the λ (lambda) light chain (L) locus and / or the λ and κ (kappa) light chain loci are functionally silenced 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-only antibodies in mice and rats has been reported, for example, in WO 2006008548; U.S. Patent Application Publication No. 20100122358; Nguyen et al., 2003, Immunology; 109(1), 93-101; Brueggemann 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-only antibodies containing heterologous heavy chain loci and transgenic rodents producing such antibodies are described in U.S. Patent Nos. 8,883,150 and 9,365,655. CAR-T constructs 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.

[0272] The present disclosure provides various families of heavy chain-only antibodies and antigen-binding fragments thereof that bind to human IL2Rβγ for use in the methods disclosed herein. In some embodiments, anti-IL-2Rβγ heavy chain-only antibodies are used in the methods disclosed herein. In some embodiments, antigen-binding fragments of anti-IL-2Rβγ heavy chain-only antibodies are used in the methods disclosed herein.

[0273] In some embodiments, an anti-IL-2R βγ heavy chain-only antibody or antigen-binding fragment thereof comprises CDR sequences having the following sequence formula: X represents a variable amino acid, which in some embodiments can be a specific amino acid listed below: CDR1(IL2RB_F09) GGSISSS X1W (SEQ ID NO: 26) (Wherein X1 is D or N); CDR2(IL2RB_F09) I X2H SGST (SEQ ID NO: 27) (wherein X2 is D or S); and CDR3(IL2RB_F09) X3R G X4W EL X5D AFDI (SEQ ID NO: 28) (In the formula, X3 is G or A; X4 is S or Q; and X5 is S or T).

[0274] In some embodiments, the anti-IL-2Rβγ heavy chain-only antibodies or antigen-binding fragments thereof comprise any combination of CDR1, CDR2, and CDR3 sequences comprising the sequence formulae of SEQ ID NOs: 26, 27, and 28, respectively. Heavy chain-only antibodies of this family may be referred to herein as IL2RB_F09 antibodies.

[0275] In some embodiments, an anti-IL-2R βγ heavy chain-only antibody or antigen-binding fragment thereof comprises CDR sequences having the following sequence formula: X represents a variable amino acid, which in some embodiments can be a specific amino acid listed below: CDR1(IL2RB_F18) GFTFS X1Y G (SEQ ID NO: 29) (Wherein X1 is S or T); CDR2(IL2RB_F18) ISYDGSN X2 (SEQ ID NO: 30) (wherein X2 is K or R); and CDR3(IL2RB_F18) ARDLDYD X3L TGDPVGGFDI (SEQ ID NO: 31) wherein X3 is V or I.

[0276] In some embodiments, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof comprises any combination of CDR1, CDR2, and CDR3 sequences comprising the sequence formulae of SEQ ID NOs: 29, 30, and 31, respectively. Heavy chain-only antibodies of this family may be referred to herein as IL2RB_F18 antibodies.

[0277] Heavy chain-only antibodies and antigen-binding fragments thereof according to embodiments of the present disclosure that bind to IL2RB can comprise a set of CDR sequences defined herein and exemplified by the provided heavy chain CDR1, CDR2, and CDR3 sequences set forth in Table 1, and a heavy chain variable region (VH) sequence set forth in Table 2. These heavy chain-only antibodies and antigen-binding fragments thereof offer many benefits that contribute to their usefulness as clinical therapeutics. Heavy chain-only antibodies and antigen-binding fragments include members with a range of binding affinities, allowing for the selection of specific sequences with the desired binding affinity.

[0278] [Table 3]

[0279] [Table 4]

[0280] [Table 5]

[0281] In some embodiments, an anti-IL-2R βγ heavy chain-only antibody or antigen-binding fragment thereof comprises CDR sequences having the following sequence formula: X represents a variable amino acid, which in some embodiments can be a specific amino acid listed below: CDR1(IL2RG_F16) GF X1X2X3X4Y Y (SEQ ID NO: 32) (In the formula, X1 is T or I; X2 is F or V; X3 is S, N, or G; and X4 is D or N; CDR2(IL2RG_F16) IS X5S G X6X7I (SEQ ID NO: 33) (In the formula, X5 is S or N; X6 is D, S, G, or N; and X7 is T or I; and CDR3(IL2RG_F16) ARGDAVSITGDY (SEQ ID NO: 20).

[0282] In some embodiments, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof comprises any combination of CDR1, CDR2, and CDR3 sequences comprising the sequence formulae of SEQ ID NOs: 32, 33, and 34, respectively. Heavy chain-only antibodies of this family may be referred to herein as IL2RG_F16 antibodies.

[0283] In some embodiments, an anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof comprises a CDR1 sequence comprising GFTFSDYY (SEQ ID NO: 15), a CDR2 (IL2RG_F18) sequence comprising ISSSGTTT (SEQ ID NO: 19), and a CDR3 (IL2RG_F18) sequence comprising ARGAAVAPGFDS (SEQ ID NO: 21). Heavy chain-only antibodies of this family may be referred to herein as IL2RG_F18 antibodies.

[0284] Heavy chain-only antibodies and antigen-binding fragments thereof according to embodiments of the present disclosure that bind to IL2RG comprise a set of CDR sequences defined herein and exemplified by the provided heavy chain CDR1, CDR2, and CDR3 sequences set forth in Table 3, and a heavy chain variable region (VH) sequence set forth in Table 4. This family of heavy chain-only antibodies and antigen-binding fragments thereof offers many benefits that contribute to their usefulness as clinical therapeutics. Heavy chain-only antibodies and antigen-binding fragments thereof include members with a range of binding affinities, allowing for the selection of specific sequences with desired binding affinities.

[0285] [Table 6]

[0286] [Table 7]

[0287] [Table 8]

[0288] Affinity measurements for anti-IL-2Rβγ heavy chain-only antibodies or antigen-binding fragments thereof can be performed using methods known in the art, such as, for example, Biacore measurements. Members of the heavy chain-only antibodies and antigen-binding fragments described herein have affinity of about 10 -6 ~about 10 -10 ;about 10 -6 ~about 10 -9 ;about 10 -6 ~about 10 -8 ;about 10 -8 ~about 10 -11 ;about 10 -8 ~about 10 -10 ;about 10 -8 ~about 10 -9 ;about 10 -9 ~about 10 -11 ;about 10 -9 ~about 10 -10 or any value within these ranges, including but not limited to, about 10-6 ~about 10 -11 The affinity selection may have an affinity for IL2R with a Kd of 0.05 or less. Affinity selection may be confirmed by biological evaluation to modulate, e.g., agonize, IL2R biological activity, including in vitro assays, preclinical models, and clinical trials, as well as evaluation of potential toxicity.

[0289] Members of the family of heavy chain-only antibodies and antigen-binding fragments described herein are cross-reactive with the IL2R protein of Cynomolgus macaques, and this cross-reactivity facilitates the use of Cynomolgus macaques as an animal model for testing, for example, the mechanism of action, pharmacokinetics, toxicity, and other properties of the heavy chain-only antibodies and antigen-binding fragments described herein.

[0290] In some embodiments, the IL2R-specific heavy chain-only antibodies and antigen-binding fragments described herein comprise a VH domain comprising CDR1, CDR2, and CDR3 sequences within a human VH framework. The CDR sequences can be located, by way of example, in the regions around amino acid residues 26-33; 51-58; and 97-116 (CDR1, CDR2, and CDR3, respectively) of the exemplary variable region sequences provided in SEQ ID NOS: 11-14 and 22-25. It will be understood by those skilled in the art that if different framework sequences are selected, the CDR sequences may be in different positions, although the order of the sequences will generally remain the same.

[0291] In certain embodiments, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof comprises the CDR1 sequence of any one of SEQ ID NOs: 1 to 3. In certain embodiments, the CDR1 sequence comprises SEQ ID NO: 1. In certain embodiments, the CDR1 sequence comprises SEQ ID NO: 2. In certain embodiments, the CDR1 sequence comprises SEQ ID NO: 3.

[0292] In certain embodiments, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof comprises the CDR2 sequence of any one of SEQ ID NOs: 4 to 6. In certain embodiments, the CDR2 sequence comprises SEQ ID NO: 4. In certain embodiments, the CDR2 sequence comprises SEQ ID NO: 5. In certain embodiments, the CDR2 sequence comprises SEQ ID NO: 6.

[0293] In certain embodiments, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof comprises the CDR3 sequence of any one of SEQ ID NOs: 7 to 10. In certain embodiments, the CDR3 sequence comprises SEQ ID NO: 7. In certain embodiments, the CDR3 sequence comprises SEQ ID NO: 8. In certain embodiments, the CDR3 sequence comprises SEQ ID NO: 9. In certain embodiments, the CDR3 sequence comprises SEQ ID NO: 10.

[0294] In a further embodiment, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof comprises 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.

[0295] In a further embodiment, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof comprises 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.

[0296] In a further embodiment, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof comprises 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.

[0297] In a further embodiment, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof comprises 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.

[0298] In a further embodiment, the anti-IL-2R βγ heavy chain only antibody or antigen-binding fragment thereof comprises any of the amino acid sequences of the heavy chain variable region of SEQ ID NOs: 11 to 14 (Table 2).

[0299] In still further embodiments, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof comprises the heavy chain variable region sequence of SEQ ID NO: 11. In still further embodiments, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof comprises the heavy chain variable region sequence of SEQ ID NO: 12. In still further embodiments, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof comprises the heavy chain variable region sequence of SEQ ID NO: 13. In still further embodiments, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof comprises the heavy chain variable region sequence of SEQ ID NO: 14.

[0300] In some embodiments, the CDR sequences of an anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof of the present disclosure comprise one or two amino acid substitutions compared to the CDR1, CDR2, and / or CDR3 sequence, or the set of CDR1, CDR2, and CDR3 sequences in any one of SEQ ID NOs: 1-10 (Table 1).

[0301] In some embodiments, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof comprises a heavy chain variable domain (VH) whose CDR3 sequence has 80% or more, e.g., at least 85%, at least 90%, at least 95%, or at least 99%, sequence identity at the amino acid level to the CDR3 sequence of any one of the antibodies whose CDR3 sequences are provided in Table 1, and which specifically binds to IL2RB.

[0302] In some embodiments, an anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof comprises a heavy chain variable domain (VH) in which the complete set of CDR1, 2, and 3 (combined) has 85 percent (85%) or more (e.g., ≧90%, ≧95%, ≧98%, ≧99%) sequence identity at the amino acid level to CDR1, 2, and 3 (combined) of an antibody whose CDR sequences are provided in Table 1, and which specifically binds to IL2RB.

[0303] In some embodiments, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof comprises a heavy chain variable region sequence that has at least about 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to any of the heavy chain variable region sequences of SEQ ID NOs: 11-14 (shown in Table 2) and specifically binds to IL2RB.

[0304] In some alternative embodiments, an anti-IL-2Rβγ antibody or antigen-binding fragment thereof is used in place of an anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof. In some embodiments, the anti-IL-2Rβγ antibody or antigen-binding fragment thereof comprises a heavy chain variable region sequence as described herein paired with a fixed light chain sequence. In some embodiments, the fixed light chain comprises a CDR1 sequence of SEQ ID NO: 44, a CDR2 sequence of SEQ ID NO: 45, and a CDR3 sequence of SEQ ID NO: 46 in a human VL framework. The anti-IL2RB VH region and the fixed light chain variable region together have binding affinity for IL2RB. In some embodiments, the fixed light chain comprises a light chain variable region sequence of SEQ ID NO: 47. In some embodiments, the fixed light chain comprises a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% percent identity to the light chain variable region sequence of SEQ ID NO: 47. In some embodiments, the fixed light chain further comprises a light chain constant region sequence (CL). In some embodiments, the fixed light chain comprises the sequence of SEQ ID NO:48.

[0305] In certain embodiments, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof comprises the CDR1 sequence of any one of SEQ ID NOs: 15 to 16. In certain embodiments, the CDR1 sequence comprises SEQ ID NO: 15. In certain embodiments, the CDR1 sequence comprises SEQ ID NO: 16.

[0306] In certain embodiments, an anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof comprises the CDR2 sequence of any one of SEQ ID NOs: 17 to 19. In certain embodiments, the CDR2 sequence comprises SEQ ID NO: 17. In certain embodiments, the CDR2 sequence comprises SEQ ID NO: 18. In certain embodiments, the CDR2 sequence comprises SEQ ID NO: 19.

[0307] In certain embodiments, an anti-IL-2R βγ heavy chain-only antibody or antigen-binding fragment thereof comprises the CDR3 sequence of any one of SEQ ID NOs: 20 to 21. In certain embodiments, the CDR3 sequence comprises SEQ ID NO: 20. In certain embodiments, the CDR2 sequence comprises SEQ ID NO: 21.

[0308] In a further embodiment, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof 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.

[0309] In a further embodiment, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof 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.

[0310] In a further embodiment, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof 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.

[0311] In a further embodiment, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof 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.

[0312] In a further embodiment, the anti-IL-2R βγ heavy chain only antibody or antigen-binding fragment thereof comprises any of the amino acid sequences of the heavy chain variable region of SEQ ID NOs: 22 to 25 (Table 4).

[0313] In still further embodiments, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof comprises the heavy chain variable region sequence of SEQ ID NO: 22. In still further embodiments, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof comprises the heavy chain variable region sequence of SEQ ID NO: 23. In still further embodiments, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof comprises the heavy chain variable region sequence of SEQ ID NO: 24. In still further embodiments, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof comprises the heavy chain variable region sequence of SEQ ID NO: 25.

[0314] In some embodiments, the CDR sequences of an anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof of the present disclosure comprise one or two amino acid substitutions compared to the CDR1, CDR2, and / or CDR3 sequences, or the set of CDR1, CDR2, and CDR3 sequences, in any one of SEQ ID NOs: 15-21 (Table 3).

[0315] In some embodiments, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof comprises a heavy chain variable domain (VH) whose CDR3 sequence has 80% or more, e.g., at least 85%, at least 90%, at least 95%, or at least 99%, sequence identity at the amino acid level to the CDR3 sequence of any one of the antibodies whose CDR3 sequences are provided in Table 3, and which specifically binds to IL2RG.

[0316] In some embodiments, an anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof comprises a heavy chain variable domain (VH) in which the complete set of CDR1, 2, and 3 (combined) have eighty-five percent (85%) or greater (e.g., ≥ 90%, ≥ 95%, ≥ 98%, ≥ 99%) sequence identity at the amino acid level to CDR1, 2, and 3 (combined) of an antibody whose CDR sequences are provided in Table 3, and which specifically binds IL2RG.

[0317] In some embodiments, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof comprises a heavy chain variable region sequence that has at least about 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity to any of the heavy chain variable region sequences of SEQ ID NOs: 22-25 (shown in Table 4) and specifically binds to IL2RG.

[0318] In some alternative embodiments, an anti-IL-2Rβγ antibody or antigen-binding fragment thereof is used in place of an anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof. In some embodiments, the anti-IL-2Rβγ antibody or antigen-binding fragment thereof comprises a heavy chain variable region sequence as described herein paired with a fixed light chain sequence. In some embodiments, the fixed light chain comprises a CDR1 sequence of SEQ ID NO: 44, a CDR2 sequence of SEQ ID NO: 45, and a CDR3 sequence of SEQ ID NO: 46 in a human VL framework. The anti-IL2RG VH region and the fixed light chain variable region together have binding affinity for IL2RG. In some embodiments, the fixed light chain comprises a light chain variable region sequence of SEQ ID NO: 47. In some embodiments, the fixed light chain comprises a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% percent identity to the light chain variable region sequence of SEQ ID NO: 47. In some embodiments, the fixed light chain further comprises a light chain constant region sequence (CL). In some embodiments, the fixed light chain comprises the sequence of SEQ ID NO:48.

[0319] In some embodiments, the anti-IL-2Rβγ heavy chain-only antibodies or antigen-binding fragments thereof used in the methods described herein can have any of the configurations discussed herein, including, but not limited to, bispecific, bivalent heavy chain antibody formats comprising two non-identical heavy chain polypeptide subunits that associate with each other via an asymmetric (e.g., knobs-into-holes (KiH)) interface. In certain embodiments, the bispecific, bivalent heavy chain antibody can comprise two non-identical heavy chain polypeptide subunits that associate with each other via an asymmetric interface and may optionally further comprise two identical, fixed light chain polypeptide subunits, each of which associates with one of the two heavy chain polypeptide subunits.

[0320] In some embodiments, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof comprises at least one heavy chain variable region that specifically binds IL2RB and at least one heavy chain variable region that specifically binds IL2RG. In some embodiments, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof further comprises an Fc portion that comprises a CH2 and / or CH3 and / or CH4 domain and is absent of a CH1 domain.

[0321] In some embodiments, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof comprises a first variable region comprising a member of the IL2RB_F09 family consisting of 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 a second variable region comprising a member of the IL2RG_F16 family consisting of 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.

[0322] In some embodiments, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof comprises a first variable region comprising a member of the IL2RB_F09 family consisting of 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 a second variable region comprising a member of the IL2RG_F18 family consisting of 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.

[0323] In some embodiments, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof comprises a first variable region comprising a member of the IL2RB_F18 family consisting of 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 variable region comprising a member of the IL2RG_F16 family consisting of 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.

[0324] In some embodiments, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof comprises a first variable region comprising a member of the IL2RB_F18 family consisting of 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 variable region comprising a member of the IL2RG_F18 family consisting of 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.

[0325] In some embodiments, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof comprises a first variable region that specifically binds to IL2RB, comprising 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, and a second variable region that specifically binds to IL2RG, comprising 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.

[0326] In some embodiments, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof comprises a first variable region that specifically binds to IL2RB, comprising 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, and a second variable region that specifically binds to IL2RG, comprising 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.

[0327] In some embodiments, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof comprises a first variable region that specifically binds to IL2RB, comprising 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, and a second variable region that specifically binds to IL2RG, comprising 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.

[0328] In some embodiments, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof comprises a first variable region that specifically binds to IL2RB, comprising 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, and a second variable region that specifically binds to IL2RG, comprising 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.

[0329] In some embodiments, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof comprises a first variable region that specifically binds to IL2RB, comprising 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, and a second variable region that specifically binds to IL2RG, comprising 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.

[0330] In some embodiments, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof comprises a first variable region that specifically binds to IL2RB, comprising 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, and a second variable region that specifically binds to IL2RG, comprising 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.

[0331] Table 5 summarizes the various CDR combinations of bispecific IL2RBxIL2RG heavy chain-only antibodies and antigen-binding fragments thereof according to certain embodiments of the present disclosure.

[0332] [Table 9]

[0333] [Table 10]

[0334] In some embodiments, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof comprises a first variable region that specifically binds to IL2RB and comprises the heavy chain variable region sequence of SEQ ID NO: 11, and a second variable region that specifically binds to IL2RG and comprises the heavy chain variable region sequence of SEQ ID NO: 22.

[0335] In some embodiments, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof comprises a first variable region that specifically binds to IL2RB and comprises the heavy chain variable region sequence of SEQ ID NO: 12, and a second variable region that specifically binds to IL2RG and comprises the heavy chain variable region sequence of SEQ ID NO: 23.

[0336] In some embodiments, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof comprises a first variable region that specifically binds to IL2RB and comprises the heavy chain variable region sequence of SEQ ID NO: 13, and a second variable region that specifically binds to IL2RG and comprises the heavy chain variable region sequence of SEQ ID NO: 23.

[0337] In some embodiments, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof comprises a first variable region that specifically binds to IL2RB and comprises the heavy chain variable region sequence of SEQ ID NO: 14, and a second variable region that specifically binds to IL2RG and comprises the heavy chain variable region sequence of SEQ ID NO: 22.

[0338] In some embodiments, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof comprises a first variable region that specifically binds to IL2RB and comprises the heavy chain variable region sequence of SEQ ID NO: 12, and a second variable region that specifically binds to IL2RG and comprises the heavy chain variable region sequence of SEQ ID NO: 24.

[0339] In some embodiments, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof comprises a first variable region that specifically binds to IL2RB and comprises the heavy chain variable region sequence of SEQ ID NO: 12, and a second variable region that specifically binds to IL2RG and comprises the heavy chain variable region sequence of SEQ ID NO: 25.

[0340] Table 6 summarizes the various heavy chain variable region combinations of bispecific IL2RBxIL2RG heavy chain-only antibodies according to certain embodiments of the present disclosure.

[0341] [Table 11]

[0342] [Table 12]

[0343] In some embodiments, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof comprises a first and a second polypeptide, i.e., a first and a second polypeptide subunit, each comprising the antigen-binding domain of a heavy chain-only antibody. In some embodiments, each of the first and second polypeptides further comprises a hinge region or at least a portion of a hinge region capable of promoting the formation of at least one disulfide bond between the first and second polypeptides. In some embodiments, each of the first and second polypeptides further comprises at least one heavy chain constant region (CH) domain, e.g., a CH2 domain, a CH3 domain, and / or a CH4 domain. In certain embodiments, the CH domain lacks a CH1 domain. The antigen-binding domain of each of the first and second polypeptides can incorporate any of the CDR sequences and / or variable region sequences described herein to confer antigen-binding capability to the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment. Thus, in certain embodiments, each polypeptide subunit in an anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment may contain an antigen-binding domain that specifically binds to a different IL2R subunit or chain (e.g., IL2RB and IL2RG).

[0344] In some embodiments, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof comprises a variant human IgG4 Fc domain comprising a first heavy chain constant region sequence comprising the S228P, F234A, L235A, and T366W mutations (knob) and a second heavy chain constant region sequence comprising the S228P, F234A, L235A, T366S, L368A, and Y407V mutations (hole). This variant or modified IgG4 Fc domain prevents undesired Fab exchange, reduces effector function of the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment, and promotes heterodimerization of heavy chain polypeptide subunits to form multispecific (e.g., bispecific) antibodies.

[0345] The components of the anti-IL-2Rβγ heavy chain-only antibodies and antigen-binding fragments described herein (i.e., CDR sequences, variable region sequences, and Fc domain sequences (e.g., hinge, CH2 and CH3 domain sequences)) can be combined in various ways to generate heavy chain-only antibodies and antigen-binding fragments that bind to IL2R, e.g., IL2RB and IL2RG, and have beneficial properties, e.g., reduced effector function activity, increased IL2R agonist activity, etc.

[0346] Table 7 shows the sequences of human IgG1 and IgG4 Fc region sequences, as well as versions of these sequences that incorporate additional mutations (variants) that confer additional desirable properties.

[0347] [Table 13]

[0348] [Table 14]

[0349] In some embodiments, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof comprises a first heavy chain polypeptide subunit comprising a variable region that specifically binds IL2RB, the first heavy chain polypeptide subunit comprising the sequence of SEQ ID NO: 53, and a second heavy chain polypeptide subunit comprising a variable region that specifically binds IL2RG, the second heavy chain polypeptide subunit comprising the sequence of SEQ ID NO: 61.

[0350] In some embodiments, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof comprises a first heavy chain polypeptide subunit comprising a variable region that specifically binds IL2RB, the first heavy chain polypeptide subunit comprising the sequence of SEQ ID NO: 62, and a second heavy chain polypeptide subunit comprising a variable region that specifically binds IL2RG, the second heavy chain polypeptide subunit comprising the sequence of SEQ ID NO: 63.

[0351] In some embodiments, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof comprises a first heavy chain polypeptide subunit comprising a variable region that specifically binds IL2RB, the first heavy chain polypeptide subunit comprising the sequence of SEQ ID NO: 64, and a second heavy chain polypeptide subunit comprising a variable region that specifically binds IL2RG, the second heavy chain polypeptide subunit comprising the sequence of SEQ ID NO: 65.

[0352] In some embodiments, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof comprises a first heavy chain polypeptide subunit comprising a variable region that specifically binds to IL2RB, the first heavy chain polypeptide subunit comprising the sequence of SEQ ID NO: 66, and a second heavy chain polypeptide subunit comprising a variable region that specifically binds to IL2RG, the second heavy chain polypeptide subunit comprising the sequence of SEQ ID NO: 67.

[0353] In some embodiments, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof comprises a first heavy chain polypeptide subunit comprising a variable region that specifically binds IL2RB, the first heavy chain polypeptide subunit comprising the sequence of SEQ ID NO: 34, and a second heavy chain polypeptide subunit comprising a variable region that specifically binds IL2RG, the second heavy chain polypeptide subunit comprising the sequence of SEQ ID NO: 35.

[0354] In some embodiments, the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof comprises a first heavy chain polypeptide subunit comprising a variable region that specifically binds to IL2RB, the first heavy chain polypeptide subunit comprising the sequence of SEQ ID NO: 36, and a second heavy chain polypeptide subunit comprising a variable region that specifically binds to IL2RG, the second heavy chain polypeptide subunit comprising the sequence of SEQ ID NO: 37.

[0355] Table 8 summarizes the various heavy chain polypeptide subunit sequence combinations of bispecific IL2RBxIL2RG heavy chain-only antibodies according to certain embodiments of the present disclosure.

[0356] [Table 15]

[0357] [Table 16]

[0358] [Table 17]

[0359] [Table 18]

[0360] Further sequences referred to herein are set out in Tables 9 and 10 for reference.

[0361] [Table 19]

[0362] [Table 20]

[0363] [Table 21]

[0364] Intracellular internalization In some embodiments of the present disclosure, the anti-IL-2Rβγ heavy chain-only antibodies and antigen-binding fragments thereof disclosed herein are internalized within cells upon binding to a binding target (e.g., IL2R), and the internalization is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, or at least about 200% or more compared to one or more control antibodies that do not internalize.

[0365] The results of intracellular translocation are shown in panels A and B of Figure 7. Panel A shows the results of intracellular translocation of CD8 + Internalization of the indicated anti-IL2Rβ / γ heavy chain-only antibodies by T cells is shown as a function of time. Panel B shows this data in tabular format. Surface levels of heavy chain-only antibodies were detected by flow cytometry and reported relative to non-internalized cells. The observed half-lives ranged from 0.27 to 0.81 hours. As observed here, molecules containing the IL2RG_F16B binding sequence were internalized faster and to a greater extent than molecules containing a different anti-IL2RG binding sequence, suggesting that internalization may depend in part on the specific anti-IL2RG arm of the bispecific heavy chain-only antibody.

[0366] Preparation of anti-IL2R heavy chain only antibody The anti-IL2Rβ / γ heavy chain-only antibodies and antigen-binding fragments thereof of the present disclosure can be prepared by methods known in the art. In some embodiments, the anti-IL2Rβ / γ heavy chain-only antibodies and antigen-binding fragments thereof are produced by transgenic animals, e.g., transgenic mice and rats, e.g., transgenic rats, in which endogenous immunoglobulin genes have been knocked out or disabled. In some embodiments, the anti-IL-2Rβγ heavy chain-only antibodies and antigen-binding fragments thereof described herein are produced in UniRat™, which has silenced endogenous immunoglobulin genes and uses a human immunoglobulin heavy chain translocus to express a diverse, naturally optimized repertoire of fully human HCAbs. Various techniques can be used to knock out or silence endogenous immunoglobulin loci in rats. UniRat™ uses zinc finger (endo)nuclease (ZNF) technology to inactivate the endogenous rat heavy chain J locus, light chain Cκ locus, and light chain Cλ locus. IgH and IgL knockout (KO) strains can be generated by microinjecting ZNF constructs into oocytes. For details, see, e.g., Geurts et al., 2009, Science 325:433. The characteristics of Ig heavy chain knockout rats are reported in Menoret et al., 2010, Eur. J. Immunol. 40:2932-2941. An advantage of ZNF technology is that it can also provide target sites for homologous integration by non-homologous end joining, silencing genes or loci by deletion of up to several kb (Cui et al., 2011, Nat Biotechnol 29:64-67). The human heavy chain-only antibodies produced by UniRat™ are called UniAbs™ and can bind to epitopes that cannot be attacked by conventional antibodies. The high specificity, affinity, and small size of UniAbs™ make them ideal for monospecific and multispecific applications.

[0367] In addition to UniAbs™, specifically encompassed herein are heavy chain-only antibodies and antigen-binding fragments thereof lacking camelid VHH frameworks and mutations, as well as functional VH regions thereof. For example, such heavy chain-only antibodies and antigen-binding fragments thereof can be produced in transgenic rats or mice containing a fully human heavy chain-only locus, as described, for example, in WO 2006 / 008548, although other transgenic mammals, such as rabbits, guinea pigs, and rats, can also be used. Heavy chain-only antibodies and antigen-binding fragments (including their VHH or VH functional fragments) can also be produced by recombinant DNA techniques, for example, by expressing encoding nucleic acids in suitable eukaryotic or prokaryotic hosts, including mammalian cells (e.g., CHO cells), E. coli, or yeast.

[0368] Heavy chain-only antibody and antigen-binding fragment domains combine the advantages of antibodies and small molecule drugs, can be monovalent or multivalent, have low toxicity, and are cost-effective to produce. These domains are easily administered, for example orally or topically, due to their small size, and are characterized by high stability, including gastrointestinal stability, and their half-lives can be tailored to the desired use or indication. In addition, VH and VHH domains of HCAbs can be produced cost-effectively.

[0369] In certain embodiments, heavy chain-only antibodies and antigen-binding fragments thereof, including UniAbs™, of the present disclosure have a native amino acid residue at the first position of the FR4 region (amino acid position 101 according to the Kabat numbering system) substituted with another amino acid residue, thereby disrupting a surface-exposed hydrophobic patch comprising or associated with the native amino acid residue at that position. Such a hydrophobic patch is normally buried at the interface with the antibody light chain constant region, but in HCAbs, it is at least partially surface-exposed, potentially contributing to undesirable aggregation of the HCAb and light chain binding. In some embodiments, the substituted amino acid residue is charged. In some embodiments, the substituted amino acid residue is positively charged, e.g., lysine (Lys, K), arginine (Arg, R), or histidine (His, H), e.g., arginine (R). In some embodiments, heavy chain-only antibodies and antigen-binding fragments thereof derived from transgenic animals comprise a Trp to Arg mutation at position 101. In some embodiments, the resulting heavy chain-only antibodies and antigen-binding fragments thereof have high antigen-binding affinity and solubility under physiological conditions, without the absence of aggregation.

[0370] Human IgG anti-IL2R heavy chain-only antibodies and antigen-binding fragments thereof (UniAb™) with unique sequences derived from UniRat™ animals were identified that bind to human IL2R in ELISA protein and cell binding assays. The identified heavy chain variable region (VH) sequences were positive for human IL2R protein binding and / or positive for binding to IL2R+ cells, and all negative for binding to cells that do not express IL2R.

[0371] Heavy chain-only antibodies and antigen-binding fragments, such as UniAbs™, that specifically bind to non-overlapping epitopes on the IL2R protein can be identified by competitive binding assays, such as enzyme-linked immunosorbent assays (ELISA assays) or flow cytometry competitive binding assays. For example, competition between a known antibody that binds to a target antigen and a heavy chain-only antibody or antigen-binding fragment of interest can be utilized. Using this approach, a set of heavy chain-only antibodies and antigen-binding fragments can be separated into those that compete with the reference antibody and those that do not. Non-competing heavy chain-only antibodies and antigen-binding fragments are identified as binding to distinct epitopes that do not overlap with the epitope bound by the reference antibody. Often, one antibody is immobilized to bind the antigen, and the ability of a second, labeled (e.g., biotinylated) antibody to bind to this captured antigen is tested in an ELISA assay. This assay can also be performed using surface plasmon resonance (SPR) platforms including ProteOn XPR36 (BioRad, Inc), Biacore 2000 and Biacore T200 (GE Healthcare Life Sciences), and MX96 SPR Imager (Ibis technologies BV), as well as on biolayer interference platforms such as Octet Red384 and Octet HTX (ForteBio, Pall Inc). For further details, see the Examples herein.

[0372] Typically, a heavy chain-only antibody or antigen-binding fragment thereof "competes" with a reference antibody if it causes about a 15-100% reduction in binding of the reference antibody to the target antigen, as measured by standard techniques, such as the competitive binding assays described above. In some embodiments, competitive binding is measured using an enzyme-linked immunosorbent assay (ELISA assay). In some embodiments, one antibody construct is immobilized to bind the antigen, and the ability of a second, labeled (e.g., biotinylated) antibody to bind to this captured antigen is tested in an ELISA assay. This assay can be performed, for example, using surface plasmon resonance (SPR) platforms such as the ProteOn XPR36 (BioRad, Inc.), Biacore 2000, and Biacore T200 (GE Healthcare Life Sciences), as well as the MX96 SPR Imager (Ibis technologies BV), and on biolayer interferometry platforms such as the Octet Red 384 and Octet HTX (ForteBio, Pall Inc.). In some embodiments, competitive binding is measured using a flow cytometric competitive binding assay.

[0373] In various embodiments, the relative inhibition is at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or more.

[0374] T cell redirection therapy T cell redirecting therapies, such as bispecific T cell engaging molecules and CAR-expressing T cells, represent a promising class of immunotherapies under development for treating a variety of solid and liquid cancers. While any therapeutic agent capable of recruiting T cells to target cells or tissues is contemplated for use in the methods described herein, specific embodiments relating to bispecific T cell engaging molecules and CAR-expressing T cells are provided for illustrative purposes.

[0375] In some embodiments, the T cell redirecting therapy used in the methods described herein is a bispecific T cell-redirecting molecule. The bispecific T cell-redirecting molecule used in the methods of the present disclosure generally comprises a first domain that specifically binds to a target cancer cell antigen (e.g., CEA, CD19, CD33, CD70, EGFRvIII, EpCAM, FLT3, GPRC5D, DLL3, BCMA, PSMA, STEAP1, STEAP2, MUC16, MUC17, or CLDN18.2), a second domain that specifically binds to human CD3, and a half-life-extending domain that provides a molecular half-life of greater than 24 hours. The half-life-extending domain can be an immunoglobulin Fc domain, a domain derived from serum albumin (e.g., human serum albumin), an albumin-binding domain (e.g., including a human albumin-binding peptide or an antibody fragment that specifically binds to serum albumin), a peptide that binds to the neonatal Fc receptor (FcRn), and a polyethylene glycol polymer. In certain embodiments, the bispecific T cell-inducing molecule used in the methods of the present disclosure comprises an immunoglobulin Fc domain. In some such embodiments, the bispecific T cell-inducing molecule may be a bispecific antibody and may have the general structure of a full-length immunoglobulin. For example, in some embodiments, the bispecific T cell-inducing molecule may be a heterodimeric antibody comprising a light chain and a heavy chain derived from an antibody that specifically binds to a target cancer cell antigen and a light chain and a heavy chain derived from an antibody that specifically binds to human CD3. In other embodiments, the bispecific T cell-inducing molecule may be an antibody fragment (e.g., a TCA) comprising a heavy chain derived from a heavy-chain-only antibody that specifically binds to a target cancer cell antigen and a light chain and a heavy chain derived from an antibody that specifically binds to human CD3. In yet another embodiment, the bispecific T cell engaging molecule used in the methods of the present disclosure comprises, in amino to carboxyl order: (i) a first domain that specifically binds to a target cancer cell antigen; (ii) a second domain that specifically binds to human CD3; and (iii) an Fc domain comprising two Fc monomers, each monomer comprising an immunoglobulin hinge region, a CH2 domain, and a CH3 domain, wherein the two monomers are fused to each other via a peptide linker.In such embodiments, the bispecific T cell engaging molecule may be a single chain polypeptide, with all three domains optionally linked together via a peptide linker to form a single polypeptide chain.

[0376] In certain embodiments, the binding domain of the bispecific T cell engaging molecule used in the methods of the present disclosure comprises the immunoglobulin heavy chain variable region (VH) and immunoglobulin light chain variable region (VL) of an antibody or antibody fragment that specifically binds to a desired antigen. For example, the anti-cancer cell antigen binding domain of the bispecific T cell engaging molecule of the present disclosure comprises the VH and VL regions derived from an antibody that specifically binds to a target cancer cell antigen, and the anti-CD3 binding domain comprises the VH and VL regions derived from an antibody that specifically binds to CD3. The binding domain that specifically binds to a human cancer cell antigen or human CD3 can be derived from known antibodies against these antigens or from novel antibodies or antibody fragments obtained by novel immunization methods using antigen proteins or fragments thereof, by phage display, or by other methods known in the art. The antibody from which the binding domain of the bispecific T cell engaging molecule is derived can be a monoclonal antibody, a recombinant antibody, a chimeric antibody, a human antibody, or a humanized antibody. In certain embodiments, the antibody from which the binding domain is derived is a monoclonal antibody. In these and other embodiments, the antibody is a human or humanized antibody and can be of the IgG1, IgG2, IgG3, or IgG4 type.

[0377] The first binding domain of the bispecific T cell engaging molecule used in the methods of the present disclosure specifically binds to a target cancer cell antigen, such as a human target cancer cell antigen. This binding domain is referred to herein as an anti-cancer cell antigen binding domain. The term "target cancer cell antigen" refers to an antigen expressed on the surface of malignant cells, tumor cells, or other types of cancerous cells. A target cancer cell antigen may be expressed only in cancer cells or may be overexpressed in cancer cells compared to normal cells. A target cancer cell antigen may also include a mutant or aberrant form of a protein that is expressed in cancer cells but not in normal cells. Examples of target cancer cell antigens include, but are not limited to, 5T4, AFP, BCMA, beta-catenin, BRCA1, CD19, CD20, CD22, CD33, CD70, CD123, CDH3, CDH19, CDK4, CEA, CLDN18.2, DLL3, DLL4, EGFR, EGFRvIII, EpCAM, EphA2, FLT3, FOLR1, gpA33, GPRC5D, HER2, IGFR, MAGE-1, MAGE-2, MAGE-3, MAGE-4, MAGE-6, MAGE-12, MSLN, MUC1, MUC2, MUC3, MUC4, MUC5, MUC16, MUC17, PSCA, PSMA, RAGE protein, STEAP1, STEAP2, TRP1, and TRP2. In certain embodiments, the first domain of the bispecific T cell engaging molecule used in the methods of the disclosure specifically binds to a target cancer cell antigen selected from MUC17, CLDN18.2, CD19, CD33, FLT3, DLL3, BCMA, and PSMA.

[0378] The second binding domain of the bispecific T cell-engaging molecule used in the methods of the present disclosure specifically binds to CD3, e.g., human CD3. This binding domain is referred to herein as the anti-CD3 binding domain. "CD3" (cluster of differentiation 3) is a T cell coreceptor composed of four chains. In mammals, the CD3 protein complex contains the CD3γ (gamma) chain, the CD3δ (delta) chain, and two CD3ε (epsilon) chains. These four chains associate with the T cell receptor (TCR) and the so-called ζ (zeta) chain to form the "T cell receptor complex," which generates activation signals in T lymphocytes. The CD3γ (gamma), CD3δ (delta), and CD3ε (epsilon) chains are closely related cell surface proteins of the immunoglobulin superfamily, each containing a single extracellular immunoglobulin domain. The intracellular tail of the CD3 molecule contains a single conserved motif known as an immunoreceptor tyrosine-based activation motif (ITAM), which is essential for the signaling ability of the TCR. The CD3 epsilon molecule is a polypeptide encoded by the CD3E gene located on chromosome 11 in humans.

[0379] Lysis of target cells redirected by recruitment of T cells by T cell inducer molecules that bind CD3 on T cells to target proteins (e.g., cancer cell antigens) on target cells (e.g., tumor cells) typically involves the formation of a cytolytic synapse and the delivery of perforin and granzymes. Induced T cells are capable of continuous target cell lysis and are not subject to immune evasion mechanisms that prevent peptide antigen processing and presentation or clonal T cell differentiation. See, e.g., WO 2007 / 042261.

[0380] In certain embodiments, the second binding domain of the bispecific T cell attracting molecule used in the methods of the present disclosure specifically binds to CD3 on the surface of T cells, e.g., human CD3 on the surface of T cells. In some embodiments, the second binding domain of the bispecific T cell attracting molecule specifically binds to CD3 epsilon, e.g., human CD3 epsilon, e.g., human CD3 epsilon on the surface of T cells. In some embodiments, the second binding domain of the bispecific T cell attracting molecule specifically binds to CD3 delta / epsilon, e.g., human CD3 delta / epsilon, e.g., human CD3 epsilon on the surface of T cells. Examples of anti-CD3 antibodies or anti-CD3 binding domains from which the second binding domain of the bispecific T cell engaging molecules used in the methods of the disclosure can be constructed or derived are described in WO 2007 / 042261, WO 2008 / 119567, WO 2017 / 053856, WO 2017 / 201493, WO 2017 / 223111, WO 2018 / 052503, and WO 2019 / 224717, all of which are incorporated herein by reference in their entireties. In certain embodiments, the second domain of the bispecific T cell engaging molecules used in the methods of the disclosure specifically binds to an epitope within the extracellular domain of human CD3 epsilon.

[0381] Bispecific T cell engaging molecules suitable for use in the methods of the present disclosure may contain additional domains that can, for example, modulate the pharmacokinetic profile of the molecule. For example, bispecific T cell engaging molecules may further contain domains or moieties that increase the elimination half-life of the molecule. The elimination half-life refers to the time it takes for the plasma drug concentration or total amount in the body to decrease by 50%. Thus, after one half-life, the drug concentration in the body will be half of the starting dose. For example, bispecific T cell engaging molecules may contain half-life extending moieties that provide a half-life of the molecule of greater than 24 hours, greater than 48 hours, greater than 72 hours, greater than 5 days, greater than 7 days, greater than 10 days, greater than 14 days, or greater than 21 days. Thus, bispecific T cell engaging molecules suitable for use in the methods of the present disclosure may have a half-life of about 2 days to about 21 days, about 3 days to about 14 days, about 5 days to about 15 days, about 3 days to about 7 days, or about 2 days to about 5 days. Examples of half-life extending moieties that can be incorporated into the bispecific T cell engaging molecules used in the methods of the present disclosure include, but are not limited to, immunoglobulin Fc domains, domains derived from serum albumin (e.g., human serum albumin) or albumin-binding domains (including, for example, human albumin-binding peptides), peptides that bind to the neonatal Fc receptor (FcRn), and polyethylene glycol polymers. Non-limiting examples of domains derived from human serum albumin or variants thereof that can be incorporated into bispecific T cell engaging molecules are described, for example, in WO 2011 / 051489, WO 2012 / 059486, WO 2013 / 075066, WO 2013 / 135896, and WO 2014 / 072481, all of which are incorporated herein by reference in their entireties. In some embodiments, the half-life extending moiety incorporated into the bispecific T cell engaging molecules used in the methods of the present disclosure is an albumin binding domain, such as a domain comprising an albumin binding peptide or antibody fragment that specifically binds to serum albumin (e.g., a single domain antibody or scFv domain).Non-limiting examples of albumin binding domains that can be incorporated into bispecific T cell engaging molecules suitable for use in the methods of the present disclosure are described, for example, in WO 2013 / 128027, WO 2014 / 140358 and WO 2017 / 201488, all of which are incorporated by reference herein in their entireties.

[0382] In certain embodiments, the bispecific T cell engaging molecules used in the methods of the present disclosure comprise an immunoglobulin Fc domain. The immunoglobulin Fc domain may comprise one or more Fc monomers. Each "Fc monomer" typically comprises at least a CH2 domain and a CH3 domain derived from an immunoglobulin molecule. The Fc monomer may comprise a CH2 and a CH3 domain derived from an IgG1, IgG2, IgG3, or IgG4 immunoglobulin. For example, the CH2 domain comprises amino acids 231-340 of IgG1 immunoglobulins, and the CH3 domain comprises amino acids 341-446 of IgG1 immunoglobulins, with amino acid numbering according to the EU numbering system described in Edelman et al., Proc. Natl. Acad. USA, Vol. 63:78-85 (1969) and Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health Publication No. 91-3242, Bethesda, MD (1991). Although the boundaries between the CH2 and CH3 domains may vary slightly between IgG isoforms, the CH2 and CH3 domains of IgG2, IgG3, and IgG4 can be confirmed by aligning them with the CH2 and CH3 domains of IgG1.

[0383] In some embodiments, an Fc monomer can comprise an immunoglobulin hinge region or a portion thereof. An immunoglobulin hinge region is typically the region defined by amino acids 216-231 (according to the EU numbering system) of an IgG immunoglobulin. In certain embodiments, an Fc monomer comprises a hinge region derived from an IgG1 immunoglobulin or a portion thereof. In certain embodiments, an Fc monomer comprises, in amino to carboxyl order, an immunoglobulin hinge region, an immunoglobulin CH2 domain, and an immunoglobulin CH3 domain.

[0384] In certain embodiments, the bispecific T cell attracting molecule comprises an Fc domain having one Fc monomer. In alternative embodiments, the bispecific T cell attracting molecule comprises an Fc domain having two or more Fc monomers. For example, in one embodiment, the bispecific T cell attracting molecule used in the methods of the present disclosure comprises an Fc domain having two Fc monomers. The two Fc monomers are present on separate polypeptide chains and can associate to form a dimer, e.g., via non-covalent interactions and / or disulfide bonds (e.g., between cysteine ​​residues in the hinge regions of the Fc monomers). In another embodiment, the two Fc monomers are fused to each other via a peptide linker, e.g., a linker of sufficient length to allow the Fc monomers to associate and form an intrachain dimer. The fusion of two Fc monomers to form a single polypeptide chain is referred to herein as a single-chain Fc domain (scFc domain) and is described in more detail below.

[0385] The peptide linker that fuses the Fc monomers together to form the single-chain Fc domain can comprise at least 25 amino acid residues (e.g., 25, 26, 27, 28, 29, 30, or more). For example, in some embodiments, the peptide linker comprises at least 30 amino acid residues (e.g., 30, 31, 32, 33, 34, 35, or more). In some embodiments, the linker comprises up to 40 amino acid residues, e.g., up to 35 amino acid residues, e.g., exactly 30 amino acid residues.

[0386] An Fc monomer may contain one or more amino acid substitutions compared to the native CH2 or CH3 immunoglobulin amino acid sequence, for example, to modulate effector function, alter glycosylation, or enhance stability. For example, in one embodiment, the glycosylation site in the CH2 domain at amino acid position 297 according to EU numbering is eliminated by substituting the asparagine residue at this position with a different amino acid. In some embodiments, an N297G substitution is used. Stability-enhancing mutations include substituting one or more amino acids in the CH2 and / or CH3 domains with cysteine ​​residues to promote disulfide bond formation. In some embodiments, specific pairs of residues are substituted with cysteines such that they preferentially form disulfide bonds with each other, thereby suppressing or preventing scrambling of the disulfide bond. Exemplary pairs include, but are not limited to, A287C and L306C, V259C and L306C, R292C and V302C, and V323C and I332C (these amino acid positions are numbered according to the EU numbering system). In one particular embodiment, the Fc monomer incorporated into the Fc domain of the bispecific T cell engaging molecule comprises N297G, R292C, and V302C substitutions (these amino acid positions are numbered according to the EU numbering system).

[0387] In certain embodiments, the bispecific T cell engaging molecules used in the methods of the present disclosure comprise an Fc domain that is a single-chain Fc domain. Thus, in certain such embodiments, the Fc domain comprises two Fc monomers, each monomer comprising an immunoglobulin hinge region, an immunoglobulin CH2 domain, and an immunoglobulin CH3 domain, and the two Fc monomers are fused to each other via a peptide linker as described herein.

[0388] In certain embodiments, the bispecific T cell engaging molecules used in the methods of the disclosure comprise, in amino to carboxyl order: (i) a first domain that specifically binds to a target cancer cell antigen (e.g., a human cancer cell antigen), the first domain comprising a first immunoglobulin heavy chain variable region (VH1) and a first immunoglobulin light chain variable region (VL1); (ii) a second domain that specifically binds to CD3 (e.g., human CD3) comprising a second immunoglobulin heavy chain variable region (VH2) and a second immunoglobulin light chain variable region (VL2); and (iii) an Fc domain comprising two Fc monomers.

[0389] In certain embodiments, the bispecific T cell engaging molecule used in the methods of the present disclosure is a single-chain polypeptide or a single-chain fusion protein. As used herein, a "single-chain polypeptide" or "single-chain fusion protein" refers to a molecule consisting of only one polypeptide chain. That is, all of the domains in the bispecific T cell engaging molecule are linked, optionally via a peptide linker, to form a single polypeptide chain. One non-limiting example of such a single-chain polypeptide or single-chain fusion protein in the context of the present disclosure is a single-chain polypeptide comprising, in amino to carboxyl order, an anti-cancer cell antigen scFv domain, a first peptide linker, an anti-CD3 scFv domain, a second peptide linker, and an scFc domain.

[0390] In one embodiment, the subject treated according to the methods of the present disclosure has been diagnosed with a leukemia or lymphoma, such as diffuse large B-cell lymphoma, Burkitt's lymphoma, follicular lymphoma, non-Hodgkin's lymphoma or acute lymphoblastic leukemia, and the anti-cancer cell antigen-binding domain of the bispecific T cell engaging molecule specifically binds to CD19.

[0391] In another embodiment, the subject treated according to the methods of the present disclosure has been diagnosed with myeloid leukemia, e.g., acute myeloid leukemia, and the anti-cancer cell antigen-binding domain of the bispecific T cell engaging molecule specifically binds to CD33 or FLT3.

[0392] In yet another embodiment, the subject treated according to the methods of the present disclosure has been diagnosed with or has a DLL3-expressing cancer, such as small cell lung cancer, neuroendocrine prostate cancer, melanoma, or glioblastoma, and the anti-cancer cell antigen-binding domain of the bispecific T cell engaging molecule specifically binds to DLL3.

[0393] In certain embodiments, the subject treated according to the methods of the present disclosure has been diagnosed with or has a BCMA-positive cancer, and the anti-cancer cell antigen-binding domain of the bispecific T cell engaging molecule specifically binds to BCMA. In some embodiments, the BCMA-positive cancer is multiple myeloma. The multiple myeloma can be refractory and / or relapsed multiple myeloma.

[0394] In certain other embodiments, the subject treated according to the methods of the present disclosure has been diagnosed with or has a PSMA-expressing cancer, such as prostate cancer, non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, hepatocellular carcinoma, bladder cancer, testicular cancer, colon cancer, glioblastoma, breast cancer, ovarian cancer, endometrial cancer, or melanoma, and the anti-cancer cell antigen-binding domain of the bispecific T cell engaging molecule specifically binds to PSMA. In some embodiments, the PSMA-expressing cancer is prostate cancer. The prostate cancer may be castration-resistant prostate cancer (prostate cancer that is resistant to androgen deprivation therapy). In these and other embodiments, the prostate cancer is metastatic prostate cancer, particularly metastatic castration-resistant prostate cancer.

[0395] In some embodiments, the subject treated according to ...

Claims

1. 1. A method of treating cancer in a subject in need thereof, comprising administering to the subject an anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof in combination with T cell redirecting therapy, wherein the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof: A first heavy chain variable (VH) region that specifically binds to IL-2Rβ, a VH complementarity determining region 1 (CDR1) sequence having up to two amino acid alterations relative to any one of SEQ ID NOs: 1-3; a VH CDR2 sequence having up to two amino acid alterations relative to any one of SEQ ID NOs: 4-6; a VH CDR3 sequence having up to two amino acid alterations relative to any one of SEQ ID NOs: 7-10; a first VH region comprising: a second VH region that specifically binds IL-2Rγ, a VH CDR1 sequence having up to two amino acid alterations relative to any one of SEQ ID NOs: 15-16; a VH CDR2 sequence having up to two amino acid alterations relative to any one of SEQ ID NOs: 17-19; a VH CDR3 sequence having up to two amino acid alterations relative to any one of SEQ ID NOs:20-21; a second VH region comprising A method comprising:

2. 1. A method of enhancing an anti-cancer effect associated with administration of T cell redirecting therapy in a subject diagnosed with cancer, comprising administering to the subject an anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof in combination with the T cell redirecting therapy, wherein the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof is A first heavy chain variable (VH) region that specifically binds to IL-2Rβ, a VH complementarity determining region 1 (CDR1) sequence having up to two amino acid alterations relative to any one of SEQ ID NOs: 1-3; a VH CDR2 sequence having up to two amino acid alterations relative to any one of SEQ ID NOs: 4-6; a VH CDR3 sequence having up to two amino acid alterations relative to any one of SEQ ID NOs: 7-10; a first VH region comprising: a second VH region that specifically binds IL-2Rγ, a VH CDR1 sequence having up to two amino acid alterations relative to any one of SEQ ID NOs: 15-16; and / or a VH CDR2 sequence having up to two amino acid alterations relative to any one of SEQ ID NOs: 17-19; and / or a VH CDR3 sequence having up to two amino acid alterations relative to any one of SEQ ID NOs:20-21; a second VH region comprising A method comprising:

3. 3. The method of claim 1 or 2, wherein each amino acid modification, if any, is a conservative amino acid substitution.

4. the first VH region comprises: a VH CDR1 comprising a sequence selected from SEQ ID NOs: 1-3; and a VH CDR2 comprising a sequence selected from SEQ ID NOs: 4-6; and The method of any one of claims 1 to 3, comprising a VH CDR3 comprising a sequence selected from SEQ ID NOs: 7 to 10.

5. the first VH region comprises: (a) a VH CDR1, a VH CDR2, and a VH CDR3 comprising the sequences of SEQ ID NOs: 1, 4, and 7, respectively; or (b) VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 1, 4, and 8, respectively; or (c) VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 2, 5, and 9, respectively; or (d) VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 3, 6, and 10, respectively. The method according to any one of claims 1 to 4, comprising:

6. the second VH region comprises: a VH CDR1 comprising a sequence selected from SEQ ID NOs: 15-16; and a VH CDR2 comprising a sequence selected from SEQ ID NOs: 17-19; and VH CDR3 comprising a sequence selected from SEQ ID NOs: 20-21 The method according to any one of claims 1 to 5, comprising:

7. the second VH region comprises: (a) a VH CDR1, a VH CDR2, and a VH CDR3 comprising the sequences of SEQ ID NOs: 15, 17, and 20, respectively; or (b) VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 15, 18, and 20, respectively; or (c) VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 16, 18, and 20, respectively; or (d) VH CDR1, VH CDR2, and VH CDR3 comprising the sequences of SEQ ID NOs: 15, 19, and 21, respectively. The method according to any one of claims 1 to 6, comprising:

8. The method according to any one of claims 1 to 7, the first VH region comprises a VH CDR1, a VH CDR2, and a VH CDR3 comprising the sequences of SEQ ID NOs: 1, 4, and 7, respectively; and the second VH region comprises a VH CDR1, a VH CDR2, and a VH CDR3 comprising the sequences of SEQ ID NOs: 15, 17, and 20, respectively; A method comprising:

9. The method according to any one of claims 1 to 7, the first VH region comprises a VH CDR1, a VH CDR2, and a VH CDR3 comprising the sequences of SEQ ID NOs: 1, 4, and 8, respectively; and the second VH region comprises a VH CDR1, a VH CDR2, and a VH CDR3 comprising the sequences of SEQ ID NOs: 15, 18, and 20, respectively; A method comprising:

10. The method according to any one of claims 1 to 7, the first VH region comprises a VH CDR1, a VH CDR2, and a VH CDR3 comprising the sequences of SEQ ID NOs: 1, 4, and 8, respectively; and the second VH region comprises a VH CDR1, a VH CDR2, and a VH CDR3 comprising the sequences of SEQ ID NOs: 16, 18, and 20, respectively; A method comprising:

11. The method according to any one of claims 1 to 7, the first VH region comprises a VH CDR1, a VH CDR2, and a VH CDR3 comprising the sequences of SEQ ID NOs: 1, 4, and 8, respectively; and the second VH region comprises a VH CDR1, a VH CDR2, and a VH CDR3 comprising the sequences of SEQ ID NOs: 15, 19, and 21, respectively; A method comprising:

12. The method according to any one of claims 1 to 7, the first VH region comprises a VH CDR1, a VH CDR2, and a VH CDR3 comprising the sequences of SEQ ID NOs: 2, 5, and 9, respectively; and the second VH region comprises a VH CDR1, a VH CDR2, and a VH CDR3 comprising the sequences of SEQ ID NOs: 15, 18, and 20, respectively; A method comprising:

13. The method according to any one of claims 1 to 7, the first VH region comprises a VH CDR1, a VH CDR2, and a VH CDR3 comprising the sequences of SEQ ID NOs: 3, 6, and 10, respectively; and the second VH region comprises a VH CDR1, a VH CDR2, and a VH CDR3 comprising the sequences of SEQ ID NOs: 15, 17, and 20, respectively; A method comprising:

14. 1. A method of treating cancer in a subject in need thereof, comprising administering to the subject an anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof in combination with T cell redirecting therapy, wherein the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof: A first heavy chain variable (VH) region that specifically binds to IL-2Rβ, (1) (a) a VH complementarity-determining region 1 (CDR1) comprising the following sequence: G F T F S X １ Y G (allocation number 29) (In the formula, X １ is S or T); and (b) a VH CDR2 comprising the following sequence: I S Y D G S N X ２ (SEQ ID NO: 30) (In the formula, X ２ is K or R); and (c) a VH CDR3 comprising the following sequence: A R D L D Y D X ３ L T G D P V G G F D I (SEQ ID NO: 31) (In the formula, X ３ is V or I); or (2) (a) a VH CDR1 comprising the following sequence: G G S I S S S X １ W (SEQ ID NO: 26) (In the formula, X １ is D or N); (b) a VH CDR2 comprising the following sequence: I X ２ H S G S T (Arrangement No. 27) (In the formula, X ２ is D or S); and (c) a VH CDR3 comprising the following sequence: X ３ RGX ４ W E L X ５ D A F D I (SEQ ID NO: 28) (In the formula, X ３ is G or A; X ４ is S or Q; X ５ is S or T) a first VH region comprising: a second VH region that specifically binds IL-2Rγ, (1) (a) a VH CDR1 comprising the following sequence: GFX １ X ２ X ３ X ４ Y Y (SEQ ID NO: 32) (In the formula, X １ is T or I; X ２ is F or V; X ３ is S, N, or G; X ４ is D or N); and (b) a VH CDR2 comprising the following sequence: I S X ５ S G X ６ X ７ I (SEQ ID NO: 33) (In the formula, X ５ is S or N; X ６ is D, S, G, or N; X ７ is T or I; and (c) a VH CDR3 comprising the sequence ARGDAVSITGDY (SEQ ID NO: 20); or (2) VH CDR1 comprising the sequence GFTFSDYY (SEQ ID NO: 15); VH CDR2 comprising the sequence ISSSGTTT (SEQ ID NO: 19); and VH CDR3 comprising the sequence ARGAAVAPGFDS (SEQ ID NO: 21). a second VH region comprising A method comprising:

15. 1. A method of enhancing an anti-cancer effect associated with administration of T cell redirecting therapy in a subject diagnosed with cancer, comprising administering to the subject an anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof in combination with the T cell redirecting therapy, wherein the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof is A first heavy chain variable (VH) region that specifically binds to IL-2Rβ, (1) (a) a VH complementarity-determining region 1 (CDR1) comprising the following sequence: G F T F S X １ Y G (allocation number 29) (In the formula, X １ is S or T); and (b) a VH CDR2 comprising the following sequence: I S Y D G S N X ２ (SEQ ID NO: 30) (In the formula, X ２ is K or R); and (c) a VH CDR3 comprising the following sequence: A R D L D Y D X ３ L T G D P V G G F D I (SEQ ID NO: 31) (In the formula, X ３ is V or I); or (2) (a) a VH CDR1 comprising the following sequence: G G S I S S S X １ W (SEQ ID NO: 26) (In the formula, X １ is D or N); (b) a VH CDR2 comprising the following sequence: I X ２ H S G S T (Arrangement No. 27) (In the formula, X ２ is D or S); and (c) a VH CDR3 comprising the following sequence: X ３ RGX ４ W E L X ５ D A F D I (SEQ ID NO: 28) (In the formula, X ３ is G or A; X ４ is S or Q; X ５ is S or T) a first VH region comprising: a second VH region that specifically binds IL-2Rγ, (1) (a) a VH CDR1 comprising the following sequence: GFX １ X ２ X ３ X ４ Y Y (SEQ ID NO: 32) (In the formula, X １ is T or I; X ２ is F or V; X ３ is S, N, or G; X ４ is D or N); and (b) a VH CDR2 comprising the following sequence: I S X ５ S G X ６ X ７ I (SEQ ID NO: 33) (In the formula, X ５ is S or N; X ６ is D, S, G, or N; X ７ is T or I; and (c) a VH CDR3 comprising the sequence ARGDAVSITGDY (SEQ ID NO: 20); or (2) VH CDR1 comprising the sequence GFTFSDYY (SEQ ID NO: 15); VH CDR2 comprising the sequence ISSSGTTT (SEQ ID NO: 19); and VH CDR3 comprising the sequence ARGAAVAPGFDS (SEQ ID NO: 21). a second VH region comprising A method comprising:

16. 1. A method of treating cancer in a subject in need thereof, comprising administering to the subject an anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof in combination with T cell redirecting therapy, wherein the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof: a first heavy chain variable (VH) complementarity determining region (CDR) 1, 2, and 3 (combined) of which the complete set has at least 95% sequence identity with said VH CDR 1, 2, and 3 of any one of SEQ ID NOs: 11-14; and a second VH region that specifically binds to IL-2Rγ, wherein the entire set of VH complementarity-determining regions (CDRs) 1, 2, and 3 (collectively) have at least 95% sequence identity with said VH CDRs 1, 2, and 3 of any one of SEQ ID NOs: 22-25; A method comprising:

17. 1. A method of enhancing an anti-cancer effect associated with administration of T cell redirecting therapy in a subject diagnosed with cancer, comprising administering to the subject an anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof in combination with the T cell redirecting therapy, wherein the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof is a first heavy chain variable (VH) complementarity determining region (CDR) 1, 2, and 3 (combined) of which the complete set has at least 95% sequence identity with said VH CDR 1, 2, and 3 of any one of SEQ ID NOs: 11-14; and a second VH region that specifically binds to IL-2Rγ, wherein the entire set of VH complementarity-determining regions (CDRs) 1, 2, and 3 (collectively) have at least 95% sequence identity with said VH CDRs 1, 2, and 3 of any one of SEQ ID NOs: 22-25; A method comprising:

18. 18. The method of claim 16 or 17, the first VH region comprises the VH CDR1, VH CDR2, and VH CDR3 of any one of SEQ ID NOs: 11-14; and / or the second VH region comprises the VH CDR1, VH CDR2, and VH CDR3 of any one of SEQ ID NOs: 22 to 25; A method comprising:

19. 19. The method according to any one of claims 1 to 18, the VH CDR1, VH CDR2, and VH CDR3 sequences of the first VH region are present within a human VH framework; and / or wherein the VH CDR1, VH CDR2, and VH CDR3 sequences of the second VH region are present within a human VH framework.

20. 1. A method of treating cancer in a subject in need thereof, comprising administering to the subject an anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof in combination with T cell redirecting therapy, wherein the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof: a first heavy chain variable (VH) region that specifically binds to IL-2Rβ, having at least 95% sequence identity to any one of SEQ ID NOs: 11-14; and A second VH region that specifically binds to IL-2Rγ, having at least 95% sequence identity with any one of SEQ ID NOs: 22 to 25. A method comprising:

21. 1. A method of enhancing an anti-cancer effect associated with administration of T cell redirecting therapy in a subject diagnosed with cancer, comprising administering to the subject an anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof in combination with the T cell redirecting therapy, wherein the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof is a first heavy chain variable (VH) region that specifically binds to IL-2Rβ, having at least 95% sequence identity to any one of SEQ ID NOs: 11-14; and A second VH region that specifically binds to IL-2Rγ, having at least 95% sequence identity with any one of SEQ ID NOs: 22 to 25. A method comprising:

22. 22. The method of claim 20 or 21, the first VH region comprises a sequence selected from any one of SEQ ID NOs: 11-14; and / or The method, wherein the second VH region comprises a sequence selected from any one of SEQ ID NOs: 22-25.

23. The method according to any one of claims 20 to 22, the first VH region comprises the sequence of SEQ ID NO: 11; and The method, wherein the second VH region comprises the sequence of SEQ ID NO:

22.

24. The method according to any one of claims 20 to 22, the first VH region comprises the sequence of SEQ ID NO: 12; and The method, wherein the second VH region comprises the sequence of SEQ ID NO:

23.

25. The method according to any one of claims 20 to 22, the first VH region comprises the sequence of SEQ ID NO: 12; and The method, wherein the second VH region comprises the sequence of SEQ ID NO:

24.

26. The method according to any one of claims 20 to 22, the first VH region comprises the sequence of SEQ ID NO: 12; and The method, wherein the second VH region comprises the sequence of SEQ ID NO:

25.

27. The method according to any one of claims 20 to 22, the first VH region comprises the sequence of SEQ ID NO: 13; and The method, wherein the second VH region comprises the sequence of SEQ ID NO:

23.

28. The method according to any one of claims 20 to 22, the first VH region comprises the sequence of SEQ ID NO: 14; and The method, wherein the second VH region comprises the sequence of SEQ ID NO:

22.

29. The method of any one of claims 1 to 28, wherein the anti-IL-2Rβγ heavy chain only antibody or antigen-binding fragment thereof further comprises an Fc region.

30. 30. The method of any one of claims 1 to 29, wherein the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof further comprises a variant Fc region.

31. 31. The method of claim 30, wherein the variant Fc region comprises a heterodimerization mutation.

32. The method of any one of claims 29 to 31, wherein the Fc region is a silenced Fc region.

33. 33. The method of any one of claims 1 to 32, wherein the T cell redirecting therapy is a bispecific T cell engaging molecule.

34. 34. The method of claim 33, wherein the bispecific T cell engaging molecule comprises a first domain that specifically binds to a target cancer cell antigen and a second domain that specifically binds to human CD3.

35. 35. The method of claim 34, wherein the target cancer cell antigen is selected from EpCAM, CEA, CD19, CD33, CD70, EGFRvIII, FLT3, GPRC5D, DLL3, BCMA, PSMA, STEAP1, STEAP2, MUC16, MUC17, and CLDN18.

2.

36. The method of any one of claims 33 to 35, wherein the bispecific T cell engaging molecule further comprises a half-life prolonging domain.

37. The method of any one of claims 33 to 36, wherein the bispecific T cell engaging molecule is a three-chain antibody-like molecule.

38. 33. The method of any one of claims 1 to 32, wherein the T cell redirecting therapy is chimeric antigen receptor (CAR)-expressing T cells.

39. 39. The method of claim 38, wherein the CAR-expressing T cell comprises a first domain that specifically binds to a target cancer cell antigen, a transmembrane domain, and an intracellular signaling domain.

40. 40. The method of claim 39, wherein the target cancer cell antigen is selected from EpCAM, CEA, CD19, CD33, CD70, EGFRvIII, FLT3, GPRC5D, DLL3, BCMA, PSMA, STEAP1, STEAP2, MUC16, MUC17, and CLDN18.

2.

41. 41. The method according to any one of claims 1 to 40, or administering to the subject at least one dose of the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof prior to the first dose of the T cell redirecting therapy; The method wherein at least one dose of the T cell redirecting therapy is administered to the subject prior to the first dose of the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof.

42. 42. The method of any one of claims 1 to 41, comprising administering the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof in combination with the T cell redirecting therapy in one or more treatment cycles.

43. 43. The method of claim 42, wherein each of the one or more treatment cycles comprises a single dose of the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof and a single dose of the T cell redirecting therapy.

44. 43. The method of claim 42, wherein each of the one or more treatment cycles comprises multiple doses of the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof and a single dose of the T cell redirecting therapy.

45. 43. The method of claim 42, wherein each of the one or more treatment cycles comprises a single dose of the anti-IL-2Rβγ heavy chain-only antibody or antigen-binding fragment thereof and multiple doses of the T cell redirecting therapy.

46. 46. ​​The method of any one of claims 1 to 45, wherein the cancer is a blood cancer.

47. 47. The method of any one of claims 1 to 46, wherein the cancer is selected from acute myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, multiple myeloma, diffuse large B-cell lymphoma, Burkitt's lymphoma, and non-Hodgkin's lymphoma.

48. 46. ​​The method of any one of claims 1 to 45, wherein the cancer is selected from prostate cancer, non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, hepatocellular carcinoma, bladder cancer, testicular cancer, colorectal cancer, esophageal cancer, glioblastoma, head and neck cancer, pancreatic cancer, breast cancer, gastric cancer, gastroesophageal junction cancer, bone cancer, ovarian cancer, endometrial cancer, and melanoma.

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