Cytokine receptor agonist

Abstract

The application relates to cytokine receptor agonists comprising: i) a first PD-1 binding domain capable of binding a first epitope on PD-1, ii) a second PD-1 binding domain capable of binding a second epitope on PD-1, iii) an IL2Rγ binding domain, iv) an IL2Rβ binding domain, and v) an Fc region, and wherein the first and second PD-1 binding domains do not compete for binding on PD-1. The PD1-binding domains simultaneously bind the PD-1 and the IL-2 receptor-binding domains bind subunits of an IL-2 receptor complex. Biparatopic assembly of the IL-2 receptor-binding domains in the presence of PD-1 allows to selectively activate IL-2 receptors and effectively mimic cytokine activity in a targeted manner.

Description

[0001] Cytokine receptor agonist

[0002] Field of the Invention

[0003] The application relates to cytokine receptor agonists comprising a pair of biparatopic PD-1 binding domains, a pair of cytokine receptor-binding domains and an Fc region, wherein the PD-1 binding domains simultaneously bind PD-1 and the cytokine receptor-binding domains bind subunits of a cytokine receptor complex. Biparatopic assembly of the cytokine receptor-binding domains in presence of PD-1 allows to selectively activate cytokine receptors and effectively mimic cytokine activity in a targeted manner.

[0004] Background

[0005] In recent years, immunotherapy treatments for cancer have grown dramatically, and cancer immunotherapy is becoming a major strategy for combating disease. For many cancer types, immune checkpoint modulators, including anti-PD-1 , have become the standard of care. However, despite all the advances made in the field of cancer immunotherapy treatment in recent years, a significant proportion of patients still fail to respond to available immunotherapies because of intrinsic or adaptive mechanisms of resistance. Cancer immunotherapy patients with non-inflamed immune conditions are more likely to not respond to immunotherapies. Immune cell infiltration in tumors has been shown to correlate with the ability of patients to respond to immunotherapy treatments. Developing new therapies aimed at increasing immune cell infiltration and enhancing immunogenicity is essential for patients.

[0006] In parallel with the above developments, cytokines have gained great interest as potential cancer treatments. Targeting the Interleukin 2 (IL-2) pathway as a cancer immunotherapy strategy has a long and ramified history, serving as testament to both the triumphs and the complexities of this clinical approach. IL-2 is a cytokine that activates lymphocytes and natural killer (NK) cells. Efficacy in the clinic is often overshadowed by reports of IL-2-associated toxicity linked to peripheral T cell activity and CD25- mediated complications.

[0007] Cytokines are powerful immune modulators that initiate signalling through receptor dimerization, but natural cytokines have some limitations as therapeutics. Low stability and difficulties in the production process are just some of them. It is known for some time that antibodies can induce signalling on cells, and therefore substitute a natural ligand.

[0008] In the recent literature there is growing evidence that bringing together heterodimeric cytokine receptors using both antibody and non-antibody based protein scaffolds is a viable strategy to mimic the activity of native cytokines. For instance, Moraga and colleagues provide an early example of using diabodies as surrogate ligands for Erythropoietin receptor (EpoR) (Moraga et al. Cell 160, 1196-1208 (2015)). Researchers at Teneobio combined heavy chain-only antibodies (VHHs fused to an Fc domain) against different epitopes on the lnterleukin-2 receptors (IL-2R0 and I L-2Ry) into a bispecific effectorless lgG4 Fc (CH1 deleted) using knob-in-hole technology. While monospecific anti-IL-2Rp or anti-IL-2Ry alone or in mixture did not induce STAT5 phosphorylation on human CD8+T cells, bispecific anti-IL-2Rpy antibodies showed varying levels of agonist activity (Harris, K. E., et al. Sci Rep 11 (1): 10592 (2021)). A similar approach as described by Teneobio, was undertaken by scientists at Synthekine, which described the functional induction of signaling of the two interleukin receptors (IL-2Rp and IL-2Ry) via single domain antibodies (sdAbs; WO 2022 / 032040 A1), and reviewed by Saxton and colleagues (Saxton, R. A., et al. Nat Rev Drug Discov. 22, 21-37 (2022)). This approach was further expanded by researchers at Stanford University where the authors presented a strategy to discover cytokine surrogate agonists by using modular ligands like VHH or scFv human for interleukin-2 / 15, type-l interferon, and interleukin-10 receptors. Interestingly, they also identified functional, non-natural assemblies like the IL-2Rp / IL-1 ORp heterodimer (Yen, M., et al. Cell 185(8): 1414-1430 e1419 (2022)). The same authors also discuss a structure-based approach for the decoupling of the pro- and anti-inflammatory functions of interleukin-10 (Saxton, R. A., et al. Science 371 (6535) (2021)). Two academic research groups from Czech Republic and Israel reported together the discovery of non-antibody based scaffolds that mimic the cytokine IFNA. Combinatorial libraries derived from several established small protein scaffolds were used to identify variants capable of binding to the IFNAR1 or IL-1 ORp and induce functional signaling (Kolarova, L., et al. FEBS J 289(9): 2672-2684 (2022)). Reducing the size of the agonistic modules was addressed by researchers at Medikine. They obtained molecules selected from peptide libraries by screens designed to identify molecules binding simultaneously to the Ra and yc subunits of the human IL-7 receptor. Those peptides, with a molecular weight of less than 5 kDa fused to an IgGi-Fc domain exhibits biological properties similar to those of IL-7 in vitro, and when administered to non-human primates (Dower, W., et al. Journal for Immuno Therapy of Cancer 8 (Suppl 3): A341-A342 (2020)).

[0009] Due to the pleiotropic effects of cytokines, there is a need for novel approaches to selectively activate cytokine receptors and effectively mimic cytokine activity.

[0010] Summary of the Invention

[0011] The present invention relates to cytokine receptor agonists comprising cytokine receptor-binding domains, which, under the desired conditions of biparatopic assembly on PD-1 , serve as cytokine mimetics selectively activating the cytokine receptor pathway.

[0012] In a general sense, the invention provides a cytokine receptor agonist comprising: i) a first PD-1 binding domain capable of binding a first epitope on PD-1 , ii) a second PD-1 binding domain capable of binding a second epitope on PD-1 , iii) an IL2Ry binding domain, iv) an IL2Rp binding domain, and v) an Fc region, wherein the first and second PD-1 binding domains do not compete for binding on PD-1 . The invention thus provides a cytokine receptor agonist antibody. Biparatopic assembly of the IL2Ry binding domain and the IL2Rp binding domain on PD-1 selectively activates the IL2R pathway.

[0013] In a first format, the cytokine receptor agonist comprises the IL2Ry binding domain or the IL2Rp binding domain, the first PD-1 binding domain, and a first Fc region, which form a first antigen binding molecule, and the other of the IL2Ry binding domain or the IL2Rp binding domain, the second PD-1 binding domain, and a second Fc region, which form a separate and different second antigen binding molecule. This is referred to herein as the “split” format.

[0014] Alternatively, in a second format, the IL2Ry binding domain, the first PD-1 binding domain, the Fc region, the second PD-1 binding domain, and the IL2Rp binding domain all form part of the same antigen binding molecule. This is referred to herein as the “all-in-one” format.

[0015] In a first aspect, the invention provides a cytokine receptor agonist comprising: i) a first PD-1 binding domain capable of binding a first epitope on PD-1 , ii) a second PD-1 binding domain capable of binding a second epitope on PD-1 , iii) an IL2Ry binding domain, iv) an IL2Rp binding domain, and v) an Fc region, wherein the first PD-1 binding domain comprises: a heavy chain complementarity determining region 1 (HCDR1) of SEQ ID NO: 1 ; a heavy chain complementarity determining region 2 (HCDR2) of SEQ ID NO: 2; a heavy chain complementarity determining region 3 (HCDR3) of SEQ ID NO: 3; a light chain complementarity determining region 1 (LCDR1) of SEQ ID NO: 4; a light chain complementarity determining region 2 (LCDR2) of SEQ ID NO: 5; and a light chain complementarity determining region 3 (LCDR3) of SEQ ID NO: 6, and wherein the first and second PD-1 binding domains do not compete for binding on PD-1 .

[0016] The present inventors identified specific PD-1 binding domains that assemble biparatopically on PD-1 such that the cytokine receptor agonist of the invention, which comprises said PD-1 binding domains, exhibits IL2R mimetic activity with limited or minimal PD-1 -independent IL-2R activation. Through an extensive development programme involving rounds of mutagenesis, screening and selection, the inventors isolated a specific PD-1 binding domain that not only binds to human PD-1 with a good affinity but also exhibits a surprisingly good affinity to cynomolgus PD-1 , thus exhibiting surprisingly good crossreactivity to the human and cynomolgus receptors and allowing for in vivo preclinical testing. This approach allowed the present inventors to identify specific PD-1 binding domains that could be included in the cytokine receptor agonists of the invention and selectively activate cytokine receptors in the presence of PD-1 .

[0017] The following embodiments may apply to cytokine receptor agonists having the “split” format and to cytokine receptor agonists having the “all-in-one” format.

[0018] In the first aspect, the first PD-1 binding domain (also referred to herein as a “non-blocking” binding domain) comprises the HCDR1-3 and LCDR1-3 of SEQ ID NOs: 1-6. In other aspects of the invention, the first PD-1 binding domain may comprise a different set of 6 CDRs.

[0019] In some embodiments, the first PD-1 binding domain comprises a heavy chain variable domain (VH) comprising an amino acid sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 7. Preferably, the first PD-1 binding domain comprises a heavy chain variable domain (VH) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 7. In some embodiments, the first PD-1 binding domain comprises a heavy chain variable domain (VH) comprising an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 7. In some embodiments, the first PD-1 binding domain comprises a heavy chain variable domain (VH) comprising an amino acid sequence that is at least about 96% identical to the amino acid sequence of SEQ ID NO: 7. In some embodiments, the first PD-1 binding domain comprises a heavy chain variable domain (VH) comprising an amino acid sequence that is at least about 97% identical to the amino acid sequence of SEQ ID NO: 7. In some embodiments, the first PD-1 binding domain comprises a heavy chain variable domain (VH) comprising an amino acid sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO: 7. In some embodiments, the first PD-1 binding domain comprises a heavy chain variable domain (VH) comprising an amino acid sequence that is at least about 99% identical to the amino acid sequence of SEQ ID NO: 7.

[0020] In some embodiments, the first PD-1 binding domain comprises a light chain variable domain (VL) comprising an amino acid sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence of SEQ ID NO: 8. Preferably, the first PD-1 binding domain comprises a light chain variable domain (VL) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence of SEQ ID NO: 8. In some embodiments, the first PD-1 binding domain comprises a light chain variable domain (VL) comprising an amino acid sequence that is at least about 95% identical to an amino acid sequence of SEQ ID NO: 8. In some embodiments, the first PD-1 binding domain comprises a light chain variable domain (VL) comprising an amino acid sequence that is at least about 96% identical to an amino acid sequence of SEQ ID NO: 8. In some embodiments, the first PD-1 binding domain comprises a light chain variable domain (VL) comprising an amino acid sequence that is at least about 97% identical to an amino acid sequence of SEQ ID NO: 8. In some embodiments, the first PD-1 binding domain comprises a light chain variable domain (VL) comprising an amino acid sequence that is at least about 98% identical to an amino acid sequence of SEQ ID NO: 8. In some embodiments, the first PD-1 binding domain comprises a light chain variable domain (VL) comprising an amino acid sequence that is at least about 99% identical to an amino acid sequence of SEQ ID NO: 8.

[0021] In a particular embodiment, the first PD-1 binding domain comprises (a) a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 7 and / or (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 8.

[0022] In some embodiments, the first PD-1 binding domain binds to human PD-1 (SEQ ID NO: 454) with a KD equal or less than about 1 x 108or 109as measured by surface plasmon resonance at 25 °C and / or to cynomolgus PD-1 (SEQ ID NO: 457) with a KD equal or less than about 1 x 106, 107, or 108as measured by surface plasmon resonance at 25 °C. Preferably, KD is measured using the method described in Example 1.3. In preferred embodiments, the first PD-1 binding domain binds to human PD1 (SEQ ID NO: 454) with a KD equal or less than about 1 x 109as measured by surface plasmon resonance at 25 °C and / or to cynomolgus PD-1 (SEQ ID NO: 457) with a KD equal or less than about 1 x IO8as measured by surface plasmon resonance at 25 °C. Preferably, KD is measured using the method described in Example 1.3.

[0023] The second PD-1 binding domain may comprise: a heavy chain complementarity determining region 1 (HCDR1) of SEQ ID NO: 9; a heavy chain complementarity determining region 2 (HCDR2) of SEQ ID NO: 10; a heavy chain complementarity determining region 3 (HCDR3) of SEQ ID NO: 11 ; a light chain complementarity determining region 1 (LCDR1) of SEQ ID NO: 12; a light chain complementarity determining region 2 (LCDR2) of SEQ ID NO: 13; and a light chain complementarity determining region 3 (LCDR3) of SEQ ID NO: 14.

[0024] In some embodiments, the second PD-1 binding domain comprises a heavy chain variable domain (VH) comprising an amino acid sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 15. Preferably, the second PD-1 binding domain comprises a heavy chain variable domain (VH) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:

[0025] 15. In some embodiments, the second PD-1 binding domain comprises a heavy chain variable domain (VH) comprising an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 15. In some embodiments, the second PD-1 binding domain comprises a heavy chain variable domain (VH) comprising an amino acid sequence that is at least about 96% identical to the amino acid sequence of SEQ ID NO: 15. In some embodiments, the second PD-1 binding domain comprises a heavy chain variable domain (VH) comprising an amino acid sequence that is at least about 97% identical to the amino acid sequence of SEQ ID NO: 15. In some embodiments, the second PD-1 binding domain comprises a heavy chain variable domain (VH) comprising an amino acid sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO: 15. In some embodiments, the second PD-1 binding domain comprises a heavy chain variable domain (VH) comprising an amino acid sequence that is at least about 99% identical to the amino acid sequence of SEQ ID NO: 15.

[0026] In some embodiments, the second PD-1 binding domain comprises a light chain variable domain (VL) comprising an amino acid sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence of SEQ ID NO: 16. Preferably, the second PD-1 binding domain comprises a light chain variable domain (VL) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence of SEQ ID NO:

[0027] 16. In some embodiments, the second PD-1 binding domain comprises a light chain variable domain (VL) comprising an amino acid sequence that is at least about 95% identical to an amino acid sequence of SEQ ID NO: 16. In some embodiments, the second PD-1 binding domain comprises a light chain variable domain (VL) comprising an amino acid sequence that is at least about 96% identical to an amino acid sequence of SEQ ID NO: 16. In some embodiments, the second PD-1 binding domain comprises a light chain variable domain (VL) comprising an amino acid sequence that is at least about 97% identical to an amino acid sequence of SEQ ID NO: 16. In some embodiments, the second PD-1 binding domain comprises a light chain variable domain (VL) comprising an amino acid sequence that is at least about 98% identical to an amino acid sequence of SEQ ID NO: 16. In some embodiments, the second PD-1 binding domain comprises a light chain variable domain (VL) comprising an amino acid sequence that is at least about 99% identical to an amino acid sequence of SEQ ID NO: 16.

[0028] In a particular embodiment, the second PD-1 binding domain comprises (a) a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 15 and / or (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 16.

[0029] Preferably, the second PD-1 binding domain is a “blocking” binding domain. Without wishing to be bound by theory, it is believed that a cytokine receptor agonist of the invention that includes a blocking PD-1 binding domain will inhibit PD-1 / PD-L1 signalling as well as selectively inducing IL-2R signalling, contributing to a synergistic activation of the host immune system which is beneficial in the treatment of cancer.

[0030] The first PD-1 binding domain and / or the second PD-1 binding domain may be an antibody fragment, such as a Fv, Fab, scFv, scFab molecule or single domain antibody. The first PD-1 binding domain and / or the second PD-1 binding domain may be a Fab molecule. The first PD-1 binding domain and / or the second PD-1 binding domain may comprise a VH, VL, a heavy chain constant domain (CH1), and a light chain constant domain (CL). The first PD-1 binding domain and / or the second PD-1 binding domain may be a cross-Fab molecule.

[0031] In some embodiments, the first PD-1 binding domain is a Fab molecule, and the second PD-1 binding domain is a Fab molecule. In some embodiments, the first PD-1 binding domain is a Fab molecule, and the second PD-1 binding domain is a cross-Fab molecule. In some embodiments, the first PD-1 binding domain is a cross-Fab molecule, and the second PD-1 binding domain is a Fab molecule.

[0032] In some embodiments, the first PD-1 binding domain and / or the second PD-1 binding domain comprise charged modifications.

[0033] In some embodiments, the first PD-1 binding domain is a cross-Fab and the second PD-1 binding domain comprises charged modifications. In some embodiments, the second PD-1 binding domain is a cross-Fab and the first PD-1 binding domain comprises charged modifications.

[0034] The cytokine receptor agonist of the invention is an IL-2R agonist. In particular, the cytokine receptor agonist is an agonist of the intermediate-affinity IL-2R. This is because it comprises an IL2Ry binding domain and a IL2Rp binding domain. Signalling through the IL-2 receptor can be determined using, for example, commercially available reporter assays such as those reported in the Examples.

[0035] In some embodiments, the IL2Ry binding domain and / or the IL2Rp binding domain are antibody fragments, such as a Fv, Fab, scFv, scFab molecules or single domain antibodies. In some embodiments, the IL2Ry binding domain and / or the IL2Rp binding domain are Fab molecules. In some embodiments, the IL2Ry binding domain and / or the IL2Rp binding domain comprise a VH, VL, a heavy chain constant domain (CH1), and a light chain constant domain (CL). In some embodiments, the IL2Ry binding domain and / or the IL2Rp binding domain are cross-Fab molecules. Preferably, the IL2Ry binding domain and the IL2Rp binding domain are VHH domains.

[0036] The IL2Ry binding domain may comprise a heavy chain complementarity determining region 1 (HCDR1) of SEQ ID NO: 27, a heavy chain complementarity determining region 2 (HCDR2) of SEQ ID NO: 72, 74, 76, 79, 85 or 87, and a heavy chain complementarity determining region 3 (HCDR3) of SEQ ID NO: 29.

[0037] Where the IL2Ry binding domain comprises a heavy chain complementarity determining region 1 (HCDR1) of SEQ ID NO: 27, a heavy chain complementarity determining region 2 (HCDR2) of SEQ ID NO: 72, and a heavy chain complementarity determining region 3 (HCDR3) of SEQ ID NO: 29, in some cases, the IL2Ry binding domain is a VHH domain comprising an amino acid sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 73. Preferably, the IL2Ry binding domain is a VHH domain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 73. In some embodiments, the IL2Ry binding domain is a VHH domain comprising an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 73. In some embodiments, the IL2Ry binding domain is a VHH domain comprising an amino acid sequence that is at least about 96% identical to the amino acid sequence of SEQ ID NO: 73. In some embodiments, the IL2Ry binding domain is a VHH domain comprising an amino acid sequence that is at least about 97% identical to the amino acid sequence of SEQ ID NO: 73. In some embodiments, the IL2Ry binding domain is a VHH domain comprising an amino acid sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO: 73. In some embodiments, the IL2Ry binding domain is a VHH domain comprising an amino acid sequence that is at least about 99% identical to the amino acid sequence of SEQ ID NO: 73. In a particular embodiment, the IL2Ry binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 73.

[0038] Where the IL2Ry binding domain comprises a heavy chain complementarity determining region 1 (HCDR1) of SEQ ID NO: 27, a heavy chain complementarity determining region 2 (HCDR2) of SEQ ID NO: 72, and a heavy chain complementarity determining region 3 (HCDR3) of SEQ ID NO: 29, in some cases, the IL2Ry binding domain is a VHH domain comprising an amino acid sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 78. Preferably, the IL2Ry binding domain is a VHH domain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 78. In some embodiments, the IL2Ry binding domain is a VHH domain comprising an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 78. In some embodiments, the IL2Ry binding domain is a VHH domain comprising an amino acid sequence that is at least about 96% identical to the amino acid sequence of SEQ ID NO: 78. In some embodiments, the IL2Ry binding domain is a VHH domain comprising an amino acid sequence that is at least about 97% identical to the amino acid sequence of SEQ ID NO: 78. In some embodiments, the IL2Ry binding domain is a VHH domain comprising an amino acid sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO: 78. In some embodiments, the IL2Ry binding domain is a VHH domain comprising an amino acid sequence that is at least about 99% identical to the amino acid sequence of SEQ ID NO: 78. In a particular embodiment, the IL2Ry binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 78.

[0039] Where the IL2Ry binding domain comprises a heavy chain complementarity determining region 1 (HCDR1) of SEQ ID NO: 27, a heavy chain complementarity determining region 2 (HCDR2) of SEQ ID NO: 72, and a heavy chain complementarity determining region 3 (HCDR3) of SEQ ID NO: 29, in some cases, the IL2Ry binding domain is a VHH domain comprising an amino acid sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 82. Preferably, the IL2Ry binding domain is a VHH domain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 82. In some embodiments, the IL2Ry binding domain is a VHH domain comprising an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 82. In some embodiments, the IL2Ry binding domain is a VHH domain comprising an amino acid sequence that is at least about 96% identical to the amino acid sequence of SEQ ID NO: 82. In some embodiments, the IL2Ry binding domain is a VHH domain comprising an amino acid sequence that is at least about 97% identical to the amino acid sequence of SEQ ID NO: 82. In some embodiments, the IL2Ry binding domain is a VHH domain comprising an amino acid sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO: 82. In some embodiments, the IL2Ry binding domain is a VHH domain comprising an amino acid sequence that is at least about 99% identical to the amino acid sequence of SEQ ID NO: 82. In a particular embodiment, the IL2Ry binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 82.

[0040] Where the IL2Ry binding domain comprises a heavy chain complementarity determining region 1 (HCDR1) of SEQ ID NO: 27, a heavy chain complementarity determining region 2 (HCDR2) of SEQ ID NO: 74, and a heavy chain complementarity determining region 3 (HCDR3) of SEQ ID NO: 29, in some cases, the IL2Ry binding domain is a VHH domain comprising an amino acid sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 75. Preferably, the IL2Ry binding domain is a VHH domain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 75. In some embodiments, the IL2Ry binding domain is a VHH domain comprising an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 75. In some embodiments, the IL2Ry binding domain is a VHH domain comprising an amino acid sequence that is at least about 96% identical to the amino acid sequence of SEQ ID NO: 75. In some embodiments, the IL2Ry binding domain is a VHH domain comprising an amino acid sequence that is at least about 97% identical to the amino acid sequence of SEQ ID NO: 75. In some embodiments, the IL2Ry binding domain is a VHH domain comprising an amino acid sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO: 75. In some embodiments, the IL2Ry binding domain is a VHH domain comprising an amino acid sequence that is at least about 99% identical to the amino acid sequence of SEQ ID NO: 75. In a particular embodiment, the IL2Ry binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 75.

[0041] Where the IL2Ry binding domain comprises a heavy chain complementarity determining region 1 (HCDR1) of SEQ ID NO: 27, a heavy chain complementarity determining region 2 (HCDR2) of SEQ ID NO: 74, and a heavy chain complementarity determining region 3 (HCDR3) of SEQ ID NO: 29, in some cases, the IL2Ry binding domain is a VHH domain comprising an amino acid sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 81 . Preferably, the IL2Ry binding domain is a VHH domain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 81 . In some embodiments, the IL2Ry binding domain is a VHH domain comprising an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 81 . In some embodiments, the IL2Ry binding domain is a VHH domain comprising an amino acid sequence that is at least about 96% identical to the amino acid sequence of SEQ ID NO: 81 . In some embodiments, the IL2Ry binding domain is a VHH domain comprising an amino acid sequence that is at least about 97% identical to the amino acid sequence of SEQ ID NO: 81. In some embodiments, the IL2Ry binding domain is a VHH domain comprising an amino acid sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO: 81 . In some embodiments, the IL2Ry binding domain is a VHH domain comprising an amino acid sequence that is at least about 99% identical to the amino acid sequence of SEQ ID NO: 81. In a particular embodiment, the IL2Ry binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 81.

[0042] Where the IL2Ry binding domain comprises a heavy chain complementarity determining region 1 (HCDR1) of SEQ ID NO: 27, a heavy chain complementarity determining region 2 (HCDR2) of SEQ ID NO: 74, and a heavy chain complementarity determining region 3 (HCDR3) of SEQ ID NO: 29, in some cases, the IL2Ry binding domain is a VHH domain comprising an amino acid sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 89. Preferably, the IL2Ry binding domain is a VHH domain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 89. In some embodiments, the IL2Ry binding domain is a VHH domain comprising an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 89. In some embodiments, the IL2Ry binding domain is a VHH domain comprising an amino acid sequence that is at least about 96% identical to the amino acid sequence of SEQ ID NO: 89. In some embodiments, the IL2Ry binding domain is a VHH domain comprising an amino acid sequence that is at least about 97% identical to the amino acid sequence of SEQ ID NO: 89. In some embodiments, the IL2Ry binding domain is a VHH domain comprising an amino acid sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO: 89. In some embodiments, the IL2Ry binding domain is a VHH domain comprising an amino acid sequence that is at least about 99% identical to the amino acid sequence of SEQ ID NO: 89. In a particular embodiment, the IL2Ry binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 89. Where the IL2Ry binding domain comprises a heavy chain complementarity determining region 1 (HCDR1) of SEQ ID NO: 27, a heavy chain complementarity determining region 2 (HCDR2) of SEQ ID NO: 76, and a heavy chain complementarity determining region 3 (HCDR3) of SEQ ID NO: 29, in some cases, the IL2Ry binding domain is a VHH domain comprising an amino acid sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 77. Preferably, the IL2Ry binding domain is a VHH domain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 77. In some embodiments, the IL2Ry binding domain is a VHH domain comprising an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 77. In some embodiments, the IL2Ry binding domain is a VHH domain comprising an amino acid sequence that is at least about 96% identical to the amino acid sequence of SEQ ID NO: 77. In some embodiments, the IL2Ry binding domain is a VHH domain comprising an amino acid sequence that is at least about 97% identical to the amino acid sequence of SEQ ID NO: 77. In some embodiments, the IL2Ry binding domain is a VHH domain comprising an amino acid sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO: 77. In some embodiments, the IL2Ry binding domain is a VHH domain comprising an amino acid sequence that is at least about 99% identical to the amino acid sequence of SEQ ID NO: 77. In a particular embodiment, the IL2Ry binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 77.

[0043] Where the IL2Ry binding domain comprises a heavy chain complementarity determining region 1 (HCDR1) of SEQ ID NO: 27, a heavy chain complementarity determining region 2 (HCDR2) of SEQ ID NO: 76, and a heavy chain complementarity determining region 3 (HCDR3) of SEQ ID NO: 29, in some cases, the IL2Ry binding domain is a VHH domain comprising an amino acid sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 83. Preferably, the IL2Ry binding domain is a VHH domain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 83. In some embodiments, the IL2Ry binding domain is a VHH domain comprising an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 83. In some embodiments, the IL2Ry binding domain is a VHH domain comprising an amino acid sequence that is at least about 96% identical to the amino acid sequence of SEQ ID NO: 83. In some embodiments, the IL2Ry binding domain is a VHH domain comprising an amino acid sequence that is at least about 97% identical to the amino acid sequence of SEQ ID NO: 83. In some embodiments, the IL2Ry binding domain is a VHH domain comprising an amino acid sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO: 83. In some embodiments, the IL2Ry binding domain is a VHH domain comprising an amino acid sequence that is at least about 99% identical to the amino acid sequence of SEQ ID NO: 83. In a particular embodiment, the IL2Ry binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 83.

[0044] Where the IL2Ry binding domain comprises a heavy chain complementarity determining region 1 (HCDR1) of SEQ ID NO: 27, a heavy chain complementarity determining region 2 (HCDR2) of SEQ ID NO: 79, and a heavy chain complementarity determining region 3 (HCDR3) of SEQ ID NO: 29, in some cases, the IL2Ry binding domain is a VHH domain comprising an amino acid sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 80. Preferably, the IL2Ry binding domain is a VHH domain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 80. In some embodiments, the IL2Ry binding domain is a VHH domain comprising an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 80. In some embodiments, the IL2Ry binding domain is a VHH domain comprising an amino acid sequence that is at least about 96% identical to the amino acid sequence of SEQ ID NO: 80. In some embodiments, the IL2Ry binding domain is a VHH domain comprising an amino acid sequence that is at least about 97% identical to the amino acid sequence of SEQ ID NO: 80. In some embodiments, the IL2Ry binding domain is a VHH domain comprising an amino acid sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO: 80. In some embodiments, the IL2Ry binding domain is a VHH domain comprising an amino acid sequence that is at least about 99% identical to the amino acid sequence of SEQ ID NO: 80. In a particular embodiment, the IL2Ry binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 80.

[0045] Where the IL2Ry binding domain comprises a heavy chain complementarity determining region 1 (HCDR1) of SEQ ID NO: 27, a heavy chain complementarity determining region 2 (HCDR2) of SEQ ID NO: 79, and a heavy chain complementarity determining region 3 (HCDR3) of SEQ ID NO: 29, in some cases, the IL2Ry binding domain is a VHH domain comprising an amino acid sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 84. Preferably, the IL2Ry binding domain is a VHH domain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 84. In some embodiments, the IL2Ry binding domain is a VHH domain comprising an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 84. In some embodiments, the IL2Ry binding domain is a VHH domain comprising an amino acid sequence that is at least about 96% identical to the amino acid sequence of SEQ ID NO: 84. In some embodiments, the IL2Ry binding domain is a VHH domain comprising an amino acid sequence that is at least about 97% identical to the amino acid sequence of SEQ ID NO: 84. In some embodiments, the IL2Ry binding domain is a VHH domain comprising an amino acid sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO: 84. In some embodiments, the IL2Ry binding domain is a VHH domain comprising an amino acid sequence that is at least about 99% identical to the amino acid sequence of SEQ ID NO: 84. In a particular embodiment, the IL2Ry binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 84.

[0046] Where the IL2Ry binding domain comprises a heavy chain complementarity determining region 1 (HCDR1) of SEQ ID NO: 27, a heavy chain complementarity determining region 2 (HCDR2) of SEQ ID NO: 85, and a heavy chain complementarity determining region 3 (HCDR3) of SEQ ID NO: 29, in some cases, the IL2Ry binding domain is a VHH domain comprising an amino acid sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 86. Preferably, the IL2Ry binding domain is a VHH domain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 86. In some embodiments, the IL2Ry binding domain is a VHH domain comprising an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 86. In some embodiments, the IL2Ry binding domain is a VHH domain comprising an amino acid sequence that is at least about 96% identical to the amino acid sequence of SEQ ID NO: 86. In some embodiments, the IL2Ry binding domain is a VHH domain comprising an amino acid sequence that is at least about 97% identical to the amino acid sequence of SEQ ID NO: 86. In some embodiments, the IL2Ry binding domain is a VHH domain comprising an amino acid sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO: 86. In some embodiments, the IL2Ry binding domain is a VHH domain comprising an amino acid sequence that is at least about 99% identical to the amino acid sequence of SEQ ID NO: 86. In a particular embodiment, the IL2Ry binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 86.

[0047] Where the IL2Ry binding domain comprises a heavy chain complementarity determining region 1 (HCDR1) of SEQ ID NO: 27, a heavy chain complementarity determining region 2 (HCDR2) of SEQ ID NO: 87, and a heavy chain complementarity determining region 3 (HCDR3) of SEQ ID NO: 29, in some cases, the IL2Ry binding domain is a VHH domain comprising an amino acid sequence that is at least about 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 88. Preferably, the IL2Ry binding domain is a VHH domain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 88. In some embodiments, the IL2Ry binding domain is a VHH domain comprising an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 88. In some embodiments, the IL2Ry binding domain is a VHH domain comprising an amino acid sequence that is at least about 96% identical to the amino acid sequence of SEQ ID NO: 88. In some embodiments, the IL2Ry binding domain is a VHH domain comprising an amino acid sequence that is at least about 97% identical to the amino acid sequence of SEQ ID NO: 88. In some embodiments, the IL2Ry binding domain is a VHH domain comprising an amino acid sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO: 88. In some embodiments, the IL2Ry binding domain is a VHH domain comprising an amino acid sequence that is at least about 99% identical to the amino acid sequence of SEQ ID NO: 88. In a particular embodiment, the IL2Ry binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 88.

[0048] In some embodiments, position 74 (Kabat numbering) of the IL2Ry binding domain is a serine. In some embodiments, the IL2Ry binding domain binds to human IL2Rpy-ECD (for example, P1 AE2657 comprising SEQ ID NOs: 400 and 401) with a KD equal or less than about 1 x 107, 108, or 109as measured by surface plasmon resonance at 25 °C and / or to cynomolgus IL2Rpy-ECD (for example, P1AF8881 comprising SEQ ID NOs: 402 and 403) with a KD equal or less than about 1 x 107, 108, or 10_9measured by surface plasmon resonance at 25 °C. In some embodiments, the IL2Ry binding domain binds to human IL2Rpy-ECD (for example, P1AE2657 comprising SEQ ID NOs: 400 and 401) with a KD between (i) about 1 x 107and about 1 x 108, (ii) about 1 x 108and about 1 x 109, or (iii) about 1 x 109and about 1 x I O10, as measured by surface plasmon resonance at 25 °C. In some embodiments, the IL2Ry binding domain binds to cynomolgus IL2Rpy-ECD (for example, P1AF8881 comprising SEQ ID NOs: 402 and 403) with a KD between (i) about 1 x 107and about 1 x 108, (ii) about 1 x 108and about 1 x 10’9, or (iii) about 1 x 109and about 1 x 10’10, as measured by surface plasmon resonance at 25 °C. Preferably, KD is measured using the method described in Example 5.3.

[0049] The IL2Rp binding domain may comprise a heavy chain complementarity determining region 1 (HCDR1) of SEQ ID NO: 17, a heavy chain complementarity determining region 2 (HCDR2) of SEQ ID NO: 18, and a heavy chain complementarity determining region 3 (HCDR3) of SEQ ID NO: 19.

[0050] In some embodiments, the IL2Rp binding domain is a VHH domain comprising an amino acid sequence that is at least about 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 20. Preferably, the IL2Rp binding domain is a VHH domain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 20. In some embodiments, the IL2Rp binding domain is a VHH domain comprising an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 20. In some embodiments, the IL2Rp binding domain is a VHH domain comprising an amino acid sequence that is at least about 96% identical to the amino acid sequence of SEQ ID NO: 20. In some embodiments, the IL2Rp binding domain is a VHH domain comprising an amino acid sequence that is at least about 97% identical to the amino acid sequence of SEQ ID NO: 20. In some embodiments, the IL2Rp binding domain is a VHH domain comprising an amino acid sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO: 20. In some embodiments, the IL2Rp binding domain is a VHH domain comprising an amino acid sequence that is at least about 99% identical to the amino acid sequence of SEQ ID NO: 20. In a particular embodiment, the IL2Rp binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 20.

[0051] In some embodiments, the IL2Rp binding domain is a VHH domain comprising an amino acid sequence that is at least about 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 21 . Preferably, the IL2Rp binding domain is a VHH domain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 21 . In some embodiments, the IL2Rp binding domain is a VHH domain comprising an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 21 . In some embodiments, the IL2Rp binding domain is a VHH domain comprising an amino acid sequence that is at least about 96% identical to the amino acid sequence of SEQ ID NO: 21. In some embodiments, the IL2Rp binding domain is a VHH domain comprising an amino acid sequence that is at least about 97% identical to the amino acid sequence of SEQ ID NO: 21 . In some embodiments, the IL2Rp binding domain is a VHH domain comprising an amino acid sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO: 21. In some embodiments, the IL2Rp binding domain is a VHH domain comprising an amino acid sequence that is at least about 99% identical to the amino acid sequence of SEQ ID NO: 21 . In a particular embodiment, the IL2Rp binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 21 . The IL2Rp binding domain may comprise a heavy chain complementarity determining region 1 (HCDR1) of SEQ ID NO: 22 a heavy chain complementarity determining region 2 (HCDR2) of SEQ ID NO: 23, and a heavy chain complementarity determining region 3 (HCDR3) of SEQ ID NO: 24.

[0052] In some embodiments, the IL2Rp binding domain is a VHH domain comprising an amino acid sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 25. Preferably, the IL2Rp binding domain is a VHH domain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 25. In some embodiments, the IL2Rp binding domain is a VHH domain comprising an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 25. In some embodiments, the IL2Rp binding domain is a VHH domain comprising an amino acid sequence that is at least about 96% identical to the amino acid sequence of SEQ ID NO: 25. In some embodiments, the IL2Rp binding domain is a VHH domain comprising an amino acid sequence that is at least about 97% identical to the amino acid sequence of SEQ ID NO: 25. In some embodiments, the IL2Rp binding domain is a VHH domain comprising an amino acid sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO: 25. In some embodiments, the IL2Rp binding domain is a VHH domain comprising an amino acid sequence that is at least about 99% identical to the amino acid sequence of SEQ ID NO: 25. In a particular embodiment, the IL2Rp binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 25.

[0053] In some embodiments, the IL2Rp binding domain is a VHH domain comprising an amino acid sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 26. Preferably, the IL2Rp binding domain is a VHH domain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 26. In some embodiments, the IL2Rp binding domain is a VHH domain comprising an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 26. In some embodiments, the IL2Rp binding domain is a VHH domain comprising an amino acid sequence that is at least about 96% identical to the amino acid sequence of SEQ ID NO: 26. In some embodiments, the IL2Rp binding domain is a VHH domain comprising an amino acid sequence that is at least about 97% identical to the amino acid sequence of SEQ ID NO: 26. In some embodiments, the IL2Rp binding domain is a VHH domain comprising an amino acid sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO: 26. In some embodiments, the IL2Rp binding domain is a VHH domain comprising an amino acid sequence that is at least about 99% identical to the amino acid sequence of SEQ ID NO: 26. In a particular embodiment, the IL2Rp binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 26.

[0054] In some embodiments, the IL2Rp binding domain binds to human IL2Rpy-ECD (for example, P1AE2657 comprising SEQ ID NOs: 400 and 401) with a KD equal or less than about 1 x 10-8or 10-9as measured by surface plasmon resonance at 25 °C and / or to cynomolgus IL2Rpy-ECD (for example, P1 AF8881 comprising SEQ ID NOs: 402 and 403) with a KD equal or less than about 1 x 10-7, 10-8, or 10-9measured by surface plasmon resonance at 25 °C. Preferably, the IL2Rp binding domain binds to human IL2R0Y-ECD (for example, P1 AE2657 comprising SEQ ID NOs: 400 and 401) with a KD equal or less than about 1 x 10-9as measured by surface plasmon resonance at 25 °C. In some embodiments, the IL2Rp binding domain binds to human IL2Rpy-ECD (for example, P1 AE2657 comprising SEQ ID NOs: 400 and 401) with a KD between about 1 x 109and about 1 x I O10, as measured by surface plasmon resonance at 25 °C. In some embodiments, the IL2Rp binding domain binds to cynomolgus IL2Rpy-ECD (for example, P1 AF8881 comprising SEQ ID NOs: 402 and 403) with a KD between (i) about 1 x 108and about 1 x I O7or (ii) about 1 x 109and about 1 x I O10, as measured by surface plasmon resonance at 25 °C. Preferably, KD is measured using the method described in Example 5.3.

[0055] In one embodiment, the IL2Ry binding domain comprises an amino acid sequence selected from SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 81 , SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88 or SEQ ID NO: 89 and the IL2Rp binding domain comprises an amino acid sequence selected from SEQ ID NO: 20, SEQ ID NO: 21 , SEQ ID NO: 25 and SEQ ID NO: 26.

[0056] In a specific embodiment, the cytokine receptor agonist can elicit one or more of the cellular responses selected from the group consisting of: proliferation of activated T lymphocyte cells, differentiation in an activated T lymphocyte cell, cytotoxic T cell (CTL) activity, proliferation of activated B cells, differentiation in an activated B cell, proliferation of natural killer (NK) cells, differentiation in a NK cell, cytokine secretion by an activated T cell or an NK cell, and / or NK / lymphocyte activated killer (LAK) antitumor cytotoxicity. Examples 8.1 .5 and 8.2.5 demonstrate that the cytokine receptor agonist of the invention can elicit such cellular responses.

[0057] In one embodiment the cytokine receptor agonist has a reduced ability to induce IL-2 signalling in regulatory T cells, compared to a wild-type IL-2 polypeptide. In one embodiment the cytokine receptor agonist induces less activation-induced cell death (AICD) in T cells, compared to a wild-type IL-2 polypeptide. In one embodiment the cytokine receptor agonist has a reduced toxicity profile in vivo, compared to a wild-type IL-2 polypeptide. In one embodiment the cytokine receptor agonist has a prolonged serum half-life, compared to a wild-type IL-2 polypeptide.

[0058] The cytokine receptor agonist induces IL-2 signalling via the intermediate-affinity IL-2 receptor. The cytokine receptor agonist does not comprise an IL-2Ra binding domain. In some embodiments, the cytokine receptor agonist has abolished or reduced affinity to the a-subunit of the IL-2 receptor and has substantially similar affinity to the intermediate-affinity IL-2 receptor, each compared to a wild-type IL-2 polypeptide. In some embodiments, the cytokine receptor agonist does not induce IL-2 signalling via the high-affinity IL-2 receptor or induces reduced levels of IL-2 signalling via the high-affinity IL-2 receptor compared to a wild-type IL-2 polypeptide.

[0059] In the cytokine receptor agonist of the invention, the IL2Ry binding domain is fused to the first PD-1 binding domain or the second PD-1 binding domain, and the IL2Rp binding domain is fused to the other of the first PD-1 binding domain or the second PD-1 binding domain. That is, either (i) the IL2Ry binding domain is fused to the first PD-1 binding domain and the IL2Rp binding domain is fused to the second PD-1 binding domain; or

[0060] (ii) the IL2Rp binding domain is fused to the first PD-1 binding domain and the IL2Ry binding domain is fused to the second PD-1 binding domain.

[0061] The IL2Ry binding domain may be fused at its C-terminus to the first PD-1 binding domain or the second PD-1 binding domain. The IL2Ry binding domain may be fused at its C-terminus to the VH of the first PD- 1 binding domain. The IL2Ry binding domain may be fused at its C-terminus to the VL of the second PD- 1 binding domain. Preferably, the IL2Ry binding domain is fused at its C-terminus to the first PD-1 binding domain. Even more preferably, the IL2Ry binding domain is fused at its C-terminus to the VH of the first PD-1 binding domain.

[0062] The IL2Rp binding domain may be fused at its C-terminus to the first PD-1 binding domain or the second PD-1 binding domain. The IL2Rp binding domain may be fused at its C-terminus to the VH of the first PD- 1 binding domain. The IL2Rp binding domain may be fused at its C-terminus to the VL of the second PD- 1 binding domain. Preferably, the IL2Rp binding domain is fused at its C-terminus to the second PD-1 binding domain. Even more preferably, the IL2Rp binding domain is fused at its C-terminus to the VL of the second PD-1 binding domain.

[0063] In some embodiments, the IL2Ry binding domain and / or the IL2Rp binding domain are fused at their respective fusion points on the respective PD-1 binding domains directly or via a peptide linker which consists of between 2 and 30 amino acids. In some embodiments, the IL2Ry binding domain and the IL2Rp binding domain are fused via peptide linkers to their respective fusion points on the respective PD- 1 binding domains. In some embodiments, the peptide linker consists of between 5 and 25 amino acids, for example 5 amino acids, 15 amino acids or 25 amino acids. In some embodiments, the peptide linker is any flexible G-S linker of between 5 and 25 amino acids, for example 5 amino acids, 15 amino acids or 25 amino acids, preferably 5 amino acids. In some embodiments, the peptide linker is a (G4S)i peptide linker (GGGGS; SEQ ID NO: 38), a (G4S)2peptide linker (GGGGSGGGGS; SEQ ID NO: 39), or a (G4S)3peptide linker (GGGGSGGGGSGGGGS; SEQ ID NO: 40) or a (G4S)5 peptide linker (GGGGSGGGGSGGGGSGGGGSGGGGS; SEQ ID NO: 41). Preferably, the peptide linker is a (G2SG2) 1 peptide linker (GGSGG; SEQ ID NO: 33), a (G2SG2)2peptide linker (GGSGGGGSGG; SEQ ID NO: 34), a (G2SG2)3peptide linker (GGSGGGGSGGGGSGG; SEQ ID NO: 35), a (G2SG2)4peptide linker (GGSGGGGSGGGGSGGGGSGG; SEQ ID NO: 36), or a (G2SG2)5peptide linker (GGSGGGGSGGGGSGGGGSGGGGSGG; SEQ ID NO: 37). In some embodiments, the peptide linker is a (G2SG2) 1 peptide linker (SEQ ID NO: 33) or a (G2SG2)5 peptide linker (SEQ ID NO: 37). Most preferably, the peptide linker is a (G2SG2) 1 peptide linker (GGSGG; SEQ ID NO: 33).

[0064] In the cytokine receptor agonist of the invention, a hinge region (made up of two hinge peptides) is typically fused at its C-terminus to the N-terminus of an Fc region (made up of two Fc region subunits). That is, in some embodiments the cytokine receptor agonist further comprises a hinge region made up of a first and second hinge peptide, where the first hinge peptide is fused at its C-terminus to the N-terminus of the first Fc region subunit and the second hinge peptide is fused at its C-terminus to the N-terminus of the second Fc region subunit. In some embodiments, at least one of the hinge peptides (e.g. the first hinge peptide) is fused at its N-terminus to the C-terminus of the first or second PD-1 binding domain (e.g. the first PD-1 binding domain).

[0065] The cytokine receptor agonist of the invention may be provided in at least two different formats. The above embodiments may apply to cytokine receptor agonists having the “split” format and to cytokine receptor agonists having the “all-in-one” format. These two formats are described in turn below, along with embodiments which apply to that format.

[0066] In a first format, the cytokine receptor agonist comprises the IL2Ry binding domain or the IL2Rp binding domain, the first PD-1 binding domain, and a first Fc region, which form a first antigen binding molecule, and the other of the IL2Ry binding domain or the IL2Rp binding domain, the second PD-1 binding domain, and a second Fc region, which form a separate and different second antigen binding molecule. This is referred to herein as the “split” format. An example of the split format is illustrated in Figures 29A- B.

[0067] In other words, the cytokine receptor agonist having the “split” format may comprise (i) the IL2Rp binding domain, the first PD-1 binding domain, and a first Fc region, which form the first antigen binding molecule, and the IL2Ry binding domain, the second PD-1 binding domain, and a second Fc region, which form the separate and different second antigen binding molecule or (ii) the IL2Ry binding domain, the first PD-1 binding domain, and a first Fc region, which form the first antigen binding molecule, and the IL2Rp binding domain, the second PD-1 binding domain, and a second Fc region, which form the separate and different second antigen binding molecule. Configuration (ii) is preferable. As shown in Example 2, pairs of antigen binding molecules in configuration (ii), in which the IL2Ry binding domain is on the same antigen binding molecule as the first PD-1 binding domain having the HCDR1-3 and LCDR1-3 of SEQ ID NOs: 1-6, induced increased levels of IL-2R signalling as compared to pairs of antigen binding molecules in configuration (i), in which the IL2Rp binding domain is on the same antigen binding molecule as the first PD-1 binding domain having the HCDR1-3 and LCDR1-3 of SEQ ID NOs: 1-6.

[0068] The cytokine receptor agonist having the split format described above and herein may incorporate, singly or in combination, any of the features described in the following (unless the context dictates otherwise).

[0069] The Fc regions of the first and second antigen binding molecules (the first Fc region and the second Fc region, respectively) of a cytokine receptor agonist having the split format each comprise a first Fc region subunit and a second Fc region subunit. In some embodiments, the Fc regions of the first and second antigen binding molecules each comprise a first Fc region subunit comprising in order from N-terminus to C-terminus a CH2 domain and a CH3 domain and a second Fc region subunit comprising in order from N-terminus to C-terminus a CH2 domain and a CH3 domain. In particular embodiments, each of the antigen binding molecules comprises not more than one Fc region.

[0070] In some embodiments, the Fc regions of the first and second antigen binding molecules are IgG, particularly lgG1 , Fc regions. In some embodiments, the Fc regions of the first and second antigen binding molecules are human Fc regions. In some embodiments, the Fc regions of the first and second antigen binding molecules comprise a modification promoting the association of the first and the second subunit of the Fc regions. In some embodiments, the Fc regions of the first and second antigen binding molecules comprise one or more amino acid substitution that reduces binding to an Fc receptor and / or effector function.

[0071] In some embodiments, the Fc region of the first antigen binding molecule is a human IgG 1 Fc region. In particular embodiments, the Fc region of the first antigen binding molecule comprises a first Fc region subunit comprising substitutions M252Y, S254T and T256E. In particular embodiments, the Fc region of the first antigen binding molecule comprises a second Fc region subunit comprising substitutions M252Y, S254T and T256E. In particular embodiments, the Fc region of the first antigen binding molecule comprises a first Fc region subunit comprising substitutions M252Y, S254T and T256E and a second Fc region subunit comprising substitutions M252Y, S254T and T256E.

[0072] In some embodiments, the Fc region of the second antigen binding molecule is a human IgG 1 Fc region. In particular embodiments, the Fc region of the second antigen binding molecule comprises a first Fc region subunit comprising substitutions M252Y, S254T and T256E. In particular embodiments, the Fc region of the second antigen binding molecule comprises a second Fc region subunit comprising substitutions M252Y, S254T and T256E. In particular embodiments, Fc region of the second antigen binding molecule comprises a first Fc region subunit comprising substitutions M252Y, S254T and T256E and a second Fc region subunit comprising substitutions M252Y, S254T and T256E.

[0073] In some embodiments, the Fc regions of the first and second antigen binding molecules are human lgG1 Fc regions. In particular embodiments, the Fc regions of the first and second antigen binding molecules each comprise a first Fc region subunit comprising substitutions M252Y, S254T and T256E. In particular embodiments, the Fc regions of the first and second antigen binding molecules each comprise a second Fc region subunit comprising substitutions M252Y, S254T and T256E. In particular embodiments, the Fc regions of the first and second antigen binding molecules each comprise a first Fc region subunit comprising substitutions M252Y, S254T and T256E and a second Fc region subunit comprising substitutions M252Y, S254T and T256E.ln some embodiments of the cytokine receptor agonist of the invention having the split format, in each of the antigen binding molecules, the Fc region is fused at its N- terminus to the C-terminus of a hinge region, and the hinge region is fused at its N-terminus to the C- terminus of the PD-1 binding domain. In some embodiments, both subunits of the Fc region are fused at their N-terminus to an immunoglobulin hinge region, made up of two hinge peptides.

[0074] In some embodiments, in the first and / or second antigen binding molecule, a CH2 domain of an Fc region subunit is fused at its N-terminus to the C-terminus of the hinge peptide and the hinge peptide is fused at its N-terminus to the CH1 domain of the PD-1 binding molecule. The hinge peptide may consist of EPKSCDKTHTCPPCP (SEQ ID NO: 42).

[0075] In some embodiments, both CH2 domains of the Fc region of the first antigen binding molecule are fused at their N-terminus to an immunoglobulin hinge region, made up of two hinge peptides. In one embodiment, the two hinge peptides each consist of the sequence EPKSCDKTHTCPPCP (SEQ ID NO: 42) and one of the hinge peptides is fused at its N-terminus to a CH1 domain of the PD-1 binding molecule. In a further embodiment, the other hinge peptide is fused at its N-terminus to an antigenbinding domain, for example a CH1 domain of a further PD-1 binding molecule. In an alternative further embodiment, the N-terminus of the other hinge peptide is the N-terminus of the polypeptide chain in which it is comprised.

[0076] In some embodiments, where both CH2 domains of the Fc region of the first antigen binding molecule are fused at their N-terminus to an immunoglobulin hinge region, made up of two hinge peptides, one hinge peptide consists of the sequence EPKSCDKTHTCPPCP (SEQ ID NO: 42) and is fused at its N-terminus to a CH1 domain of the PD-1 binding molecule and the other hinge peptide consists of a truncated hinge peptide, for example a truncated hinge peptide consisting of the sequence DKTHTCPPCP (SEQ ID NO: 43). In a further embodiment, the truncated hinge peptide may be fused at its N-terminus to an antigenbinding domain, for example a CL domain of the same PD-1 binding molecule. In an alternative further embodiment, the N-terminus of the truncated hinge peptide is the N-terminus of the polypeptide chain in which it is comprised.

[0077] In some embodiments, both CH2 domains of the Fc region of the second antigen binding molecule are fused at their N-terminus to an immunoglobulin hinge region, made up of two hinge peptides. In one embodiment, the two hinge peptides each consist of the sequence EPKSCDKTHTCPPCP (SEQ ID NO: 42) and one of the hinge peptides is fused at its N-terminus to a CH1 domain of the PD-1 binding molecule. In a further embodiment, the other hinge peptide is fused at its N-terminus to an antigenbinding domain, for example a CH1 domain of a further PD-1 binding molecule. In an alternative further embodiment, the N-terminus of the other hinge peptide is the N-terminus of the polypeptide chain in which it is comprised.

[0078] In some embodiments, where both CH2 domains of the Fc region of the second antigen binding molecule are fused at their N-terminus to an immunoglobulin hinge region, made up of two hinge peptides, one hinge peptide consists of the sequence EPKSCDKTHTCPPCP (SEQ ID NO: 42) and is fused at its N- terminus to a CH1 domain of the PD-1 binding molecule and the other hinge peptide consists of a truncated hinge peptide, for example a truncated hinge peptide consisting of the sequence DKTHTCPPCP (SEQ ID NO: 43). In a further embodiment, the truncated hinge peptide may be fused at its N-terminus to an antigen-binding domain, for example a CL domain of the same PD-1 binding molecule. In an alternative further embodiment, the N-terminus of the truncated hinge peptide is the N- terminus of the polypeptide chain in which it is comprised.

[0079] In some embodiments, both CH2 domains of the Fc region of the first antigen binding molecule are fused at their N-terminus to an immunoglobulin hinge region, made up of two hinge peptides, and both CH2 domains of the Fc region of the second antigen binding molecule are fused at their N-terminus to an immunoglobulin hinge region, made up of a further two hinge peptides. In one embodiment, each of the four hinge peptides consist of the sequence EPKSCDKTHTCPPCP (SEQ ID NO: 42) and one of the hinge peptides on each of the first and second antigen binding molecules is fused at its N-terminus to a CH1 domain of a respective PD-1 binding molecule. In a further embodiment, the other of the hinge peptides on each of the first and second antigen binding molecules is fused at its N-terminus to an antigen-binding domain, for example a CH1 domain of a further PD-1 binding molecule. In an alternative further embodiment, the N-terminus of the other hinge peptide is the N-terminus of the polypeptide chain in which it is comprised.

[0080] In some embodiments, where both CH2 domains of the Fc region of the first antigen binding molecule are fused at their N-terminus to an immunoglobulin hinge region, made up of two hinge peptides, and both CH2 domains of the Fc region of the second antigen binding molecule are fused at their N-terminus to an immunoglobulin hinge region, made up of a further two hinge peptides, one hinge peptide on each of the first and second antigen binding molecules consists of the sequence EPKSCDKTHTCPPCP (SEQ ID NO: 42) and is fused at its N-terminus to a CH1 domain of the respective PD-1 binding molecule and the other of the hinge peptides on each of the first and second antigen binding molecules consists of a truncated hinge peptide, for example a truncated hinge peptide consisting of the sequence DKTHTCPPCP (SEQ ID NO: 43). In a further embodiment, the truncated hinge peptide on each of the first and second antigen binding molecules may be fused at its N-terminus to an antigen-binding domain, for example a CL domain of the same PD-1 respective binding molecule. In an alternative further embodiment, the N-terminus of the truncated hinge peptide on each of the first and second antigen binding molecules is the N-terminus of the polypeptide chain in which it is comprised.

[0081] The C-terminal residue of the CL domain may be C214 (kappa CL domain, Kabat numbering) or S215 (lambda CL domain, Kabat numbering). Thus, in some embodiments, the C214 of the CL domain is fused directly by a peptide bond to the N-terminus of the hinge peptide, for example to the D221 (Kabat numbering) of a truncated hinge peptide consisting of DKTHTCPPCP (SEQ ID NO: 43).

[0082] Where the cytokine receptor agonist has the “split” format, the second antigen binding molecule preferably comprises a third PD-1 binding domain which binds the second epitope. In some embodiments, the second antigen binding molecule bivalently binds the second epitope. In some embodiments, where the second PD-1 binding domain comprises the HCDR1-3 and LCDR1-3 of SEQ ID NOs: 9-14, the third PD-1 binding domain comprises the HCDR1-3 and LCDR1-3 of SEQ ID NOs: 9-14.

[0083] In some embodiments, the second PD-1 binding domain comprises a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 15 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 16 and the third PD-1 binding domain comprises a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 15 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 16.

[0084] Preferably, the third PD-1 binding domain is a “blocking” binding domain. Without wishing to be bound by theory, it is believed that a cytokine receptor agonist of the invention that includes a blocking PD-1 binding domain will inhibit PD-1 / PD-L1 signalling as well as selectively inducing IL-2R signalling, contributing to a synergistic activation of the host immune system which is beneficial in the treatment of cancer.

[0085] Preferably, the second PD-1 binding domain and the third PD-1 binding domain are each a “blocking binding domain. The third PD-1 binding domain may be an antibody fragment, such as Fv, Fab, scFv, scFab molecules or single domain antibodies. The third PD-1 binding domain may be a Fab molecule. The third PD-1 binding domain may comprise a heavy chain variable domain (VH3), a light chain variable domain (VL3), a heavy chain constant domain (CHI3), and a light chain constant domain (CL3). The third PD-1 binding domain may be a cross-Fab molecule.

[0086] In some embodiments, the cytokine receptor agonist having the “split” format does not comprise a further PD-1 binding domain. Such a cytokine receptor agonist binds the first epitope monovalently and the second epitope monovalently. It does not bivalently bind either the first epitope or second epitope.

[0087] As discussed above, a cytokine receptor agonist of the invention may be provided in the “split” format. One type of “split” format binds the first and second PD-1 epitope monovalently. An example of this format is illustrated in Figures 29A-B.

[0088] Exemplary embodiments relating to the split monovalent format are provided below.

[0089] In one embodiment, the cytokine receptor agonist comprises: a first antigen binding molecule comprising

[0090] (a) a first polypeptide comprising in order from N-terminus to C-terminus: the IL2Ry binding domain, a VH and CH1 of the first PD-1 binding domain, and a first subunit of the first Fc region;

[0091] (b) a second polypeptide comprising a second subunit of the first Fc region; and

[0092] (c) a third polypeptide, comprising in order from N-terminus to C-terminus: a VL and CL of the first PD-1 binding domain; and a second antigen binding molecule comprising:

[0093] (d) a fourth polypeptide comprising in order from N-terminus to C-terminus: a VH and CH1 of the second PD-1 binding domain, and a first subunit of the second Fc region;

[0094] (e) a fifth polypeptide comprising a second subunit of the second Fc region; and

[0095] (f) a sixth polypeptide, comprising in order from N-terminus to C-terminus: the IL2Rp binding domain, and a VL and CL of the second PD-1 binding domain.

[0096] In some embodiments, the first subunit of the Fc regions comprises a “knob” modification and the second subunit of the Fc regions comprises a “hole” modification, as described in more detail herein. In some embodiments, the CH1 of the first PD-1 binding domain and the CL of the first PD-1 binding domain comprise charged modifications, as described in more detail herein. In some embodiments, the CH1 of the first PD-1 binding domain and the CL of the first PD-1 binding domain comprise charged modifications, as described in more detail herein.

[0097] In some embodiments, the IL2Ry binding domain is fused to the VH of the first PD-1 binding domain through a peptide linker, such as a (G2SG2) 1 peptide linker (GGSGG; SEQ ID NO: 33). In some embodiments, the IL2Rp binding domain is fused to the VL of the second PD-1 binding domain through a peptide linker, such as a (G2SG2) 1 peptide linker (GGSGG; SEQ ID NO: 33). In preferred embodiments, the IL2Ry binding domain is fused to the VH of the first PD-1 binding domain through a (G2SG2) 1 peptide linker, and the IL2Rp binding domain is fused to the VL of the second PD-1 binding domain through a (G2SG2) i peptide linker.

[0098] In some embodiments, both the CH1 of the first PD-1 binding and the CH1 of the second PD-1 binding domain are fused to their respective Fc region subunits through an immunoglobulin hinge peptide, such as an immunoglobulin hinge peptide consisting of the amino acid sequence EPKSCDKTHTCPPCP (SEQ ID NO: 42).

[0099] In some embodiments the second polypeptide and the fourth polypeptide comprise a truncated immunoglobulin hinge peptide fused directly (via peptide bonds) to the N-terminus of the second subunits of the second Fc regions, such as a truncated immunoglobulin hinge peptide consisting of the amino acid sequence DKTHTCPPCP (SEQ ID NO: 43).

[0100] In one embodiment, the first antigen binding molecule comprises:

[0101] (a) the first polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 90, SEQ ID NO: 91 , SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, or SEQ ID NO: 101 ;

[0102] (b) the second polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 44;

[0103] (c) the third polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 45; and the second antigen binding molecule comprises:

[0104] (d) the fourth polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 49;

[0105] (e) the fifth polypeptide comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 44; and / or

[0106] (f) the sixth polypeptide comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 50, SEQ ID NO: 51 , SEQ ID NO: 52, or SEQ ID NO: 53.

[0107] In one embodiment, the first antigen binding molecule comprises:

[0108] (a) the first polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 90, SEQ ID NO: 91 , SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, or SEQ ID NO: 101 ; (b) the second polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 44;

[0109] (c) the third polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 45; and the second antigen binding molecule comprises:

[0110] (d) the fourth polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 469;

[0111] (e) the fifth polypeptide comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 468; and / or

[0112] (f) the sixth polypeptide comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 50, SEQ ID NO: 51 , SEQ ID NO: 52, or SEQ ID NO: 53.

[0113] In one embodiment, the first antigen binding molecule comprises:

[0114] (a) the first polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 91 ;

[0115] (b) the second polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 44;

[0116] (c) the third polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 45; and the second antigen binding molecule comprises:

[0117] (d) the fourth polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 49;

[0118] (e) the fifth polypeptide comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 44; and / or

[0119] (f) the sixth polypeptide comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 53.

[0120] In one embodiment, the first antigen binding molecule comprises:

[0121] (a) the first polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 91 ;

[0122] (b) the second polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 44;

[0123] (c) the third polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 45; and the second antigen binding molecule comprises:

[0124] (d) the fourth polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 469;

[0125] (e) the fifth polypeptide comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 468; and / or

[0126] (f) the sixth polypeptide comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 53.

[0127] In one embodiment, the cytokine receptor agonist comprises: a first antigen binding molecule comprising

[0128] (a) a first polypeptide comprising an amino acid sequence of SEQ ID NO: 90, SEQ ID NO: 91 , SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO:

[0129] 98, SEQ ID NO: 99, SEQ ID NO: 100, or SEQ ID NO: 101 ;

[0130] (b) a second polypeptide comprising an amino acid sequence of SEQ ID NO: 44;

[0131] (c) a third polypeptide comprising an amino acid sequence of SEQ ID NO: 45; and a second antigen binding domain comprising:

[0132] (d) a fourth polypeptide comprising an amino acid sequence of SEQ ID NO: 49;

[0133] (e) a fifth polypeptide comprising an amino acid sequence of SEQ ID NO: 44; and

[0134] (f) a sixth polypeptide comprising an amino acid sequence of SEQ ID NO: 50, SEQ ID NO: 51 , SEQ ID NO: 52, or SEQ ID NO: 53.

[0135] In one embodiment, the cytokine receptor agonist comprises: a first antigen binding molecule comprising

[0136] (a) a first polypeptide comprising an amino acid sequence of SEQ ID NO: 90, SEQ ID NO: 91 , SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO:

[0137] 98, SEQ ID NO: 99, SEQ ID NO: 100, or SEQ ID NO: 101 ;

[0138] (b) a second polypeptide comprising an amino acid sequence of SEQ ID NO: 44;

[0139] (c) a third polypeptide comprising an amino acid sequence of SEQ ID NO: 45; and a second antigen binding domain comprising:

[0140] (d) a fourth polypeptide comprising an amino acid sequence of SEQ ID NO: 469;

[0141] (e) a fifth polypeptide comprising an amino acid sequence of SEQ ID NO: 468; and

[0142] (f) a sixth polypeptide comprising an amino acid sequence of SEQ ID NO: 50, SEQ ID NO: 51 , SEQ ID NO: 52, or SEQ ID NO: 53. In one embodiment, the first antigen binding molecule comprises:

[0143] (a) a first polypeptide comprising an amino acid sequence of SEQ ID NO: 91 ;

[0144] (b) a second polypeptide comprising an amino acid sequence of SEQ ID NO: 44;

[0145] (c) a third polypeptide comprising an amino acid sequence of SEQ ID NO: 45; and the second antigen binding molecule comprises:

[0146] (d) a fourth polypeptide comprising an amino acid sequence of SEQ ID NO: 49;

[0147] (e) a fifth polypeptide comprises an amino acid sequence of SEQ ID NO: 44; and / or

[0148] (f) a sixth polypeptide comprises an amino acid sequence of SEQ ID NO: 53.

[0149] In one embodiment, the first antigen binding molecule comprises:

[0150] (a) a first polypeptide comprising an amino acid sequence of SEQ ID NO: 91 ;

[0151] (b) a second polypeptide comprising an amino acid sequence of SEQ ID NO: 44;

[0152] (c) a third polypeptide comprising an amino acid sequence of SEQ ID NO: 45; and the second antigen binding molecule comprises:

[0153] (d) a fourth polypeptide comprising an amino acid sequence of SEQ ID NO: 469;

[0154] (e) a fifth polypeptide comprises an amino acid sequence of SEQ ID NO: 468; and / or

[0155] (f) a sixth polypeptide comprises an amino acid sequence of SEQ ID NO: 53.

[0156] Another example of an antibody in the “split” format contains a third PD-1 binding domain which specifically binds the second epitope. In these embodiments, the second antigen binding molecule bivalently binds the second epitope. An example of this format is illustrated in Figures 29A and C.

[0157] Exemplary embodiments relating to the split bivalent format are provided below.

[0158] In one embodiment, the cytokine receptor agonists comprises: a first antigen binding molecule comprising

[0159] (a) a first polypeptide comprising in order from N-terminus to C-terminus: the IL2Ry binding domain, a VH and CH1 of the first PD-1 binding domain, and a first subunit of the first Fc region;

[0160] (b) a second polypeptide comprising a second subunit of the first Fc region; and

[0161] (c) a third polypeptide, comprising in order from N-terminus to C-terminus: a VL and CL of the first PD-1 binding domain; and a second antigen binding molecule comprising:

[0162] (d) a fourth polypeptide comprising in order from N-terminus to C-terminus: a VH and CH1 of the second PD-1 binding domain, and a first subunit of the second Fc region; (e) a fifth polypeptide comprising in order from N-terminus to C-terminus: a VL and CH1 of the third PD-1 binding domain, and a second subunit of the second Fc region;

[0163] (f) a sixth polypeptide, comprising in order from N-terminus to C-terminus: the IL2Rp binding domain, and a VL and CL of the second PD-1 binding domain; and

[0164] (g) a seventh polypeptide comprising in order from N-terminus to C-terminus: a VH and CL of the third PD-1 binding domain.

[0165] In some embodiments, the first subunit of the Fc regions comprises a “knob” modification and the second subunit of the Fc regions comprises a “hole” modification, as described in more detail herein.

[0166] In some embodiments, the IL2Ry binding domain is fused to the VH of the first PD-1 binding domain through a peptide linker, such as a (G2SG2) 1 peptide linker (GGSGG; SEQ ID NO: 33). In some embodiments, the IL2Rp binding domain is fused to the VL of the second PD-1 binding domain through a peptide linker, such as a (G2SG2) 1 peptide linker (GGSGG; SEQ ID NO: 33). In preferred embodiments, the IL2Ry binding domain is fused to the VH of the first PD-1 binding domain through a (G2SG2) 1 peptide linker, and the IL2Rp binding domain is fused to the VL of the second PD-1 binding domain through (G2SG2) i peptide linker.

[0167] In some embodiments, all three of the CH1 of the first PD-1 binding, the CH1 of the second PD-1 binding domain, and the CH1 of the third PD-1 binding domain are fused to their respective Fc region subunits through an immunoglobulin hinge peptide, such as an immunoglobulin hinge peptide consisting of the amino acid sequence EPKSCDKTHTCPPCP (SEQ ID NO: 42).

[0168] In some embodiments, the CH1 of the second PD-1 binding domain and the CL of the second PD-1 binding domain comprise charged modifications, as described in more detail herein.

[0169] In one embodiment, the first antigen binding molecule comprises:

[0170] (a) the first polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 90, SEQ ID NO: 91 , SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, or SEQ ID NO: 101 ;

[0171] (b) the second polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 44;

[0172] (c) the third polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 45; and the second antigen binding molecule comprises:

[0173] (d) the fourth polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 64;

[0174] (e) the fifth polypeptide comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 61 ; (f) the sixth polypeptide comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 66, or SEQ ID NO: 67; and / or

[0175] (g) the seventh polypeptide comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 62.

[0176] In one embodiment, the first antigen binding molecule comprises:

[0177] (a) the first polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 90, SEQ ID NO: 91 , SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, or SEQ ID NO: 101 ;

[0178] (b) the second polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 44;

[0179] (c) the third polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 45; and the second antigen binding molecule comprises:

[0180] (d) the fourth polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 467;

[0181] (e) the fifth polypeptide comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 466;

[0182] (f) the sixth polypeptide comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 66, or SEQ ID NO: 67; and / or

[0183] (g) the seventh polypeptide comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 62.

[0184] In one embodiment, the first antigen binding molecule comprises:

[0185] (a) the first polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 91 ;

[0186] (b) the second polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 44;

[0187] (c) the third polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 45; and the second antigen binding molecule comprises:

[0188] (d) the fourth polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 64; (e) the fifth polypeptide comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 61 ;

[0189] (f) the sixth polypeptide comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 67; and / or

[0190] (g) the seventh polypeptide comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 62.

[0191] In one embodiment, the first antigen binding molecule comprises:

[0192] (a) the first polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 91 ;

[0193] (b) the second polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 44;

[0194] (c) the third polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 45; and the second antigen binding molecule comprises:

[0195] (d) the fourth polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 467;

[0196] (e) the fifth polypeptide comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 466;

[0197] (f) the sixth polypeptide comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 67; and / or

[0198] (g) the seventh polypeptide comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 62.

[0199] In one embodiment, the cytokine receptor agonist comprises: a first antigen binding molecule comprising

[0200] (a) a first polypeptide comprising an amino acid sequence of SEQ ID NO: 90, SEQ ID NO: 91 , SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO:

[0201] 98, SEQ ID NO: 99, SEQ ID NO: 100, or SEQ ID NO: 101 ;

[0202] (b) a second polypeptide comprising an amino acid sequence of SEQ ID NO: 44;

[0203] (c) a third polypeptide comprising an amino acid sequence of SEQ ID NO: 45; and a second antigen binding domain comprising:

[0204] (d) a fourth polypeptide comprising an amino acid sequence of SEQ ID NO: 64;

[0205] (e) a fifth polypeptide comprising an amino acid sequence of SEQ ID NO: 61 ; (f) a sixth polypeptide comprising an amino acid sequence of SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID

[0206] NO: 66, or SEQ ID NO: 67; and

[0207] (g) a seventh polypeptide comprising an amino acid sequence of SEQ ID NO: 62.

[0208] In one embodiment, the cytokine receptor agonist comprises: a first antigen binding molecule comprising

[0209] (a) a first polypeptide comprising an amino acid sequence of SEQ ID NO: 90, SEQ ID NO: 91 , SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO:

[0210] 98, SEQ ID NO: 99, SEQ ID NO: 100, or SEQ ID NO: 101 ;

[0211] (b) a second polypeptide comprising an amino acid sequence of SEQ ID NO: 44;

[0212] (c) a third polypeptide comprising an amino acid sequence of SEQ ID NO: 45; and a second antigen binding domain comprising:

[0213] (d) a fourth polypeptide comprising an amino acid sequence of SEQ ID NO: 467;

[0214] (e) a fifth polypeptide comprising an amino acid sequence of SEQ ID NO: 466;

[0215] (f) a sixth polypeptide comprising an amino acid sequence of SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 66, or SEQ ID NO: 67; and

[0216] (g) a seventh polypeptide comprising an amino acid sequence of SEQ ID NO: 62.

[0217] In one embodiment, the cytokine receptor agonist comprises: a first antigen binding molecule comprising

[0218] (a) a first polypeptide comprising an amino acid sequence of SEQ ID NO: 91 ;

[0219] (b) a second polypeptide comprising an amino acid sequence of SEQ ID NO: 44;

[0220] (c) a third polypeptide comprising an amino acid sequence of SEQ ID NO: 45; and a second antigen binding domain comprising:

[0221] (d) a fourth polypeptide comprising an amino acid sequence of SEQ ID NO: 64;

[0222] (e) a fifth polypeptide comprising an amino acid sequence of SEQ ID NO: 61 ;

[0223] (f) a sixth polypeptide comprising an amino acid sequence of SEQ ID NO: 67; and

[0224] (g) a seventh polypeptide comprising an amino acid sequence of SEQ ID NO: 62.

[0225] In one embodiment, the cytokine receptor agonist comprises: a first antigen binding molecule comprising

[0226] (a) a first polypeptide comprising an amino acid sequence of SEQ ID NO: 91 ;

[0227] (b) a second polypeptide comprising an amino acid sequence of SEQ ID NO: 44;

[0228] (c) a third polypeptide comprising an amino acid sequence of SEQ ID NO: 45; and a second antigen binding domain comprising:

[0229] (d) a fourth polypeptide comprising an amino acid sequence of SEQ ID NO: 467;

[0230] (e) a fifth polypeptide comprising an amino acid sequence of SEQ ID NO: 466;

[0231] (f) a sixth polypeptide comprising an amino acid sequence of SEQ ID NO: 67; and

[0232] (g) a seventh polypeptide comprising an amino acid sequence of SEQ ID NO: 62.

[0233] In a second format, the IL2Ry binding domain, the first PD-1 binding domain, the Fc region, the second PD-1 binding domain, and the IL2Rp binding domain all form part of the same antigen binding molecule. This is referred to herein as the “all-in-one” format. An example of the all-in-one format is illustrated in Figure 43.

[0234] The cytokine receptor agonist having the all-in-one format described above and herein may incorporate, singly or in combination, any of the features described in the following (unless the context dictates otherwise).

[0235] Preferably, the first PD-1 binding domain and the second PD-1 binding domain of a cytokine receptor agonist having the “all-in-one” format are a Fab molecule and a cross-Fab molecule, respectively, or vice versa. Inclusion of a Fab molecule and a cross-Fab molecule as the first and second PD-1 binding domains, or vice versa, in the “all-in-one” format is believed to reduce mispairing between the peptide chains forming the first PD-1 binding domain and the second PD-1 binding domain.

[0236] In a cytokine receptor agonist having the all-in-one format, the Fc region comprises a first Fc region subunit and a second Fc region subunit. In some embodiments, the Fc region comprises a first Fc region subunit comprising in order from N-terminus to C-terminus a CH2 domain and a CH3 domain and a second Fc region subunit comprising in order from N-terminus to C-terminus a CH2 domain and a CH3 domain. In particular embodiments, the cytokine receptor agonist comprises not more than one Fc region.

[0237] In some embodiments, the Fc region is an IgG, particularly an IgGi, Fc region. In some embodiments, the Fc region is a human Fc region. In some embodiments^ the Fc region comprises a modification promoting the association of the first and the second subunit of the Fc region. In some embodiments, the Fc region comprises one or more amino acid substitution that reduces binding to an Fc receptor and / or effector function.

[0238] In some embodiments, the Fc region is a human lgG1 Fc region. In particular embodiments, the Fc region comprises a first Fc region subunit comprising substitutions M252Y, S254T and T256E. In particular embodiments, the Fc region comprises a second Fc region subunit comprising substitutions M252Y, S254T and T256E. In particular embodiments, the Fc region comprises a first Fc region subunit comprising substitutions M252Y, S254T and T256E and a second Fc region subunit comprising substitutions M252Y, S254T and T256E. In some embodiments of the cytokine receptor agonist having the all-in-one format, the Fc region is fused at its N-terminus to the C-terminus of a hinge region, the hinge region is fused at its N-terminus to the C- terminus of the first PD-1 binding domain and the Fc region is fused at its C-terminus to the N-terminus of the second PD-1 binding domain. In some embodiments, both subunits of the Fc region are fused at their N-terminus to the hinge region, made up of two hinge peptides.

[0239] In some embodiments, a CH2 domain of an Fc region subunit is fused at its N-terminus to the C-terminus of the hinge peptide and the hinge peptide is fused at its N-terminus to the CH1 domain of the first PD-1 binding Fab molecule. In some embodiments, the hinge peptide consists of EPKSCDKTHTCPPCP (SEQ ID NO: 42).

[0240] In some embodiments, both CH2 domains of the Fc region are fused at their N-terminus to an immunoglobulin hinge region, made up of two hinge peptides.

[0241] In some embodiments, a CH2 domain of one of the Fc region subunits is fused at its N-terminus to the C- terminus of a first hinge peptide and the first hinge peptide is fused at its N-terminus to the C-terminus of the CH1 domain of the first PD-1 binding Fab molecule and a CH2 domain of the other of the Fc region subunits is fused at its N-terminus to a second hinge peptide and the second hinge peptide is fused at its N-terminus to the C-terminus of the CL domain of the first PD-1 binding Fab molecule. Preferably, the first hinge peptide consists of EPKSCDKTHTCPPCP (SEQ ID NO: 42) and the second hinge peptide is a truncated hinge peptide, such as DKTHTCPPCP (SEQ ID NO: 43).

[0242] The C-terminal residue of the CL domain may be C214 (kappa CL domain, Kabat numbering) or S215 (lambda CL domain, Kabat numbering). Thus, in some embodiments, the C214 of the CL domain is fused directly by a peptide bond to the N-terminus of the hinge peptide, for example to the D221 (Kabat numbering) of a truncated hinge peptide consisting of DKTHTCPPCP (SEQ ID NO: 43).

[0243] In some embodiments, a CH3 region of an Fc region subunit is fused at its C-terminus to the N-terminus of the second PD-1 binding domain via a peptide linker, preferably to the N-terminus of a VH domain of the second PD-1 binding domain. The peptide linker may consist of 10 amino acids. For example, the peptide linker may be a (G2SG2) 2 peptide linker.

[0244] As discussed above, the cytokine receptor agonist may have the “all-in-one” format. One type of all-in- one format has the CH1 domain of the PD-1 binding molecule (e.g. a PD-1 binding Fab or cross-Fab molecule) fused to one of the first or second Fc region subunits via an immunoglobulin hinge peptide (such as an IgG 1 hinge peptide) and the CL domain of the same PD-1 binding Fab molecule fused to the other of the first or second Fc region subunits via an immunoglobulin hinge peptide (such as an IgG 1 hinge peptide, e.g. a truncated IgG 1 hinge peptide). An example of this format is illustrated in Figure 16C. This format is also referred to as an all-in-one antibody with a rigidified hinge.

[0245] Exemplary embodiments relating to the all-in-one antibody with a rigidified hinge are provided below.

[0246] In one embodiment, the cytokine receptor agonist comprises: (a) a first polypeptide comprising in order from N-terminus to C-terminus: a VL and CH1 of the first PD-1 binding domain, a first subunit of the Fc region, and a VH and CH1 of the second PD-1 binding domain;

[0247] (b) a second polypeptide comprising in order from N-terminus to C-terminus: the IL2Ry binding domain, a VH and CL of the first PD-1 binding domain, and a second subunit of the Fc region; and

[0248] (c) a third polypeptide comprising in order from N-terminus to C-terminus: the IL2Rp binding domain, and a VL and CL of the second PD-1 binding domain.

[0249] In some embodiments, the first subunit of the Fc region comprises a “knob” modification and the second subunit of the Fc region comprises a “hole” modification, as described in detail herein.

[0250] In some embodiments, the CH1 of the second PD-1 binding domain and the CL of the second PD-1 binding domain comprise charged modifications, as described herein.

[0251] In some embodiments, the IL2Ry binding domain is fused to the VH of the first PD-1 binding domain through a peptide linker, such as a (G2SG2) 1 peptide linker (GGSGG; SEQ ID NO: 33). In some embodiments, the IL2Rp binding domain is fused to the VL of the second PD-1 binding domain through a peptide linker, such as a (G2SG2) 1 peptide linker (GGSGG; SEQ ID NO: 33). In some embodiments, the first subunit of the Fc region is fused to the VH of the second PD-1 binding domain through a peptide linker, such as a (G2SG2) 2 peptide linker (GGSGGGGSGG; SEQ ID NO: 34). In preferred embodiments, the IL2Ry binding domain is fused to the VH of the first PD-1 binding domain through a (G2SG2) 1 peptide, the IL2Rp binding domain is fused to the VL of the second PD-1 binding domain through a (G2SG2) 1 peptide linker, and the first subunit of the Fc region is fused to the VH of the second PD-1 binding domain through a (G2SG2) 2 peptide linker.

[0252] In some embodiments, the CH1 of the first PD-1 binding domain and the first subunit of the Fc region are fused directly through an immunoglobulin hinge peptide consisting of the amino acid sequence EPKSCDKTHTCPPCP (SEQ ID NO: 42), and the CL of the first PD-1 binding domain and second subunit of the Fc region are fused directly through an immunoglobulin hinge peptide consisting of the amino acid sequence DKTHTCPPCP (SEQ ID NO: 43).

[0253] In one exemplary embodiment, the cytokine receptor agonist comprises:

[0254] (a) a first polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 68,

[0255] (b) a second polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 11 1 , SEQ ID NO: 1 12, or SEQ ID NO: 113; and

[0256] (c) a third polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 67.

[0257] In one exemplary embodiment, the cytokine receptor agonist comprises: (a) a first polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 68,

[0258] (b) a second polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 103; and

[0259] (c) a third polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 67.

[0260] In one embodiment, the cytokine receptor agonist comprises:

[0261] (a) a first polypeptide comprising an amino acid sequence of SEQ ID NO: 68,

[0262] (b) a second polypeptide comprising an amino acid sequence of SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 1 10, SEQ ID NO: 111 , SEQ ID NO: 112, or SEQ ID NO: 113; and

[0263] (c) a third polypeptide comprising an amino acid sequence of SEQ ID NO: 67.

[0264] In one embodiment, the cytokine receptor agonist comprises:

[0265] (a) a first polypeptide comprising an amino acid sequence of SEQ ID NO: 68,

[0266] (b) a second polypeptide comprising an amino acid sequence of SEQ ID NO: 103; and

[0267] (c) a third polypeptide comprising an amino acid sequence of SEQ ID NO: 67.

[0268] In a second aspect, the invention provides a cytokine receptor agonist comprising: i) a first PD-1 binding domain capable of binding a first epitope on PD-1 , ii) a second PD-1 binding domain capable of binding a second epitope on PD-1 , iii) an IL2Ry binding domain, iv) an IL2Rp binding domain, and v) an Fc region comprising a first and a second Fc region subunit, wherein the first and second PD-1 binding domains do not compete for binding on PD-1 , wherein the first and second PD-1 binding domains each comprise a VH, a VL, a CH1 and a CL domain, wherein the first and second Fc region subunit each comprise a CH2 domain and a CH3 domain, wherein the CH1 domain of the first PD-1 binding domain is fused to a CH2 domain of one of the Fc region subunits, and wherein the CL domain of the first PD-1 binding domain is fused to a CH2 domain of the other Fc region subunit.

[0269] In some embodiments, the CH1 domain of the first PD-1 binding domain is fused at its C-terminus to the N-terminus of the CH2 domain of one of the Fc region subunits and the CL domain of the first PD-1 binding domain is fused at its C-terminus to the N-terminus of the CH2 domain of the other Fc region subunit. In some embodiments, the CH1 domain is fused to the CH2 domain of one of the Fc region subunits by a first immunoglobulin hinge peptide. Preferably, the first immunoglobulin hinge peptide consists of the sequence EPKSCDKTHTCPPCP (SEQ ID NO: 42).

[0270] In some embodiments, the CL domain is fused to the CH2 domain of the other Fc region subunit by a second immunoglobulin hinge peptide. Preferably, the second immunoglobulin hinge peptide consists of the sequence DKTHTCPPCP (SEQ ID NO: 43).

[0271] In the second aspect of the invention, the cytokine receptor agonist preferably has the all-in-one format.

[0272] The cytokine receptor agonist according to the second aspect of the invention described above and herein may incorporate, singly or in combination, any of the features described in the preceding or following (unless the context dictates otherwise). The first PD-1 binding domain may optionally comprise the HCDR1-3 and LCDR1-3 of SEQ ID NOs: 1-6, but is not limited thereto. The IL2Ry binding domain may optionally comprise HCDR1 of SEQ ID NO: 27, HCDR2 of SEQ ID NOs: 72, 74, 76, 79, 85, or 87, and HCDR3 of SEQ ID NO: 29, but is not limited thereto. The IL2Rp binding domain may optionally comprise HCDR1-HCDR3 of SEQ ID NOs 17-19 or SEQ ID NOs 22-24, but is not limited thereto.

[0273] In a third aspect, the invention provides a cytokine receptor agonist comprising i) a first PD-1 binding domain capable of binding a first epitope on PD-1 , ii) a second PD-1 binding domain capable of binding a second epitope on PD-1 , iii) an IL2Ry binding domain, iv) an IL2Rp binding domain, and v) an Fc region, wherein the first and second PD-1 binding domains do not compete for binding on PD-1 , wherein the IL2Ry binding domain or the IL2Rp binding domain, the first PD-1 binding domain, and a first Fc region form a first antigen binding molecule, wherein the other of the IL2Ry binding domain or the IL2Rp binding domain, the second PD-1 binding domain, and a second Fc region form a separate and different second antigen binding molecule, wherein (i) the first antigen binding molecule comprises a third PD-1 binding domain capable of binding the first epitope on PD-1 or (ii) the second antigen binding molecule comprises a third PD-1 binding domain capable of binding the second epitope on PD-1 .

[0274] In the third aspect the first PD-1 binding domain may optionally comprise the HCDR1-3 and LCDR1-3 of SEQ ID NOs: 1-6. In some embodiments, the first PD-1 binding domain and the third PD-1 binding domain comprise the HCDR1-3 and LCDR1-3 of SEQ ID NOs: 1-6. The first PD-1 binding domain and the third PD-1 binding domain may comprise the sequences described above for the first PD-1 binding domain.

[0275] In some embodiments, the second PD-1 binding domain comprises the HCDR1-3 and LCDR1-3 of SEQ ID NOs: 9-14. In some embodiments, the second PD-1 binding domain and the third PD-1 binding domain comprise the HCDR1-3 and LCDR1-3 of SEQ ID NOs: 9-14. The second PD-1 binding domain and the third PD-1 binding domain may comprise the sequences described above for the second PD-1 binding domain .

[0276] In the third aspect of the invention, the cytokine receptor agonist preferably has the split format. The cytokine receptor agonist according to the third aspect of the invention described above and herein may incorporate, singly or in combination, any of the features described in the preceding or following (unless the context dictates otherwise). The first PD-1 binding domain may optionally comprise the HCDR1-3 and LCDR1-3 of SEQ ID NOs: 1-6, but is not limited thereto. The IL2Ry binding domain may optionally comprise HCDR1 of SEQ ID NO: 27, HCDR2 of SEQ ID NOs: 72, 74, 76, 79, 85, or 87, and HCDR3 of SEQ ID NO: 29, but is not limited thereto. The IL2Rp binding domain may optionally comprise HCDR1 -HCDR3 of SEQ ID NOs 17-19 or SEQ ID NOs 22-24, but is not limited thereto.

[0277] In a fourth aspect, the invention provides a cytokine receptor agonist comprising: i) a first PD-1 binding domain capable of binding a first epitope on PD-1 , ii) a second PD-1 binding domain capable of binding a second epitope on PD-1 , iii) an IL2Ry binding domain, iv) an IL2Rp binding domain, and v) an Fc region, wherein the first and second PD-1 binding domains do not compete for binding on PD-1 , wherein the IL2Ry binding domain comprises HCDR1 of SEQ ID NO: 27, HCDR2 of SEQ ID NOs: 72, 74, 76, 79, 85, or 87, and HCDR3 of SEQ ID NO: 29.

[0278] The cytokine receptor agonist according to the fourth aspect of the invention described above and herein may incorporate, singly or in combination, any of the features described in the preceding or following (unless the context dictates otherwise). The first PD-1 binding domain may optionally comprise the HCDR1-3 and LCDR1-3 of SEQ ID NOs: 1-6, but is not limited thereto. The IL2Rp binding domain may optionally comprise HCDR1-HCDR3 of SEQ ID NOs 17-19 or SEQ ID NOs 22-24, but is not limited thereto.

[0279] In a fifth aspect, the invention provides a cytokine receptor agonist comprising: i) a first PD-1 binding domain capable of binding a first epitope on PD-1 , ii) a second PD-1 binding domain capable of binding a second epitope on PD-1 , iii) an IL2Ry binding domain, iv) an IL2Rp binding domain, and v) an Fc region, wherein the first and second PD-1 binding domains do not compete for binding on PD-1 , wherein the IL2Rp binding domain comprises

[0280] (i) HCDR1-HCDR3 of SEQ ID NOs 17-19, or

[0281] (ii) HCDR1-HCDR3 of SEQ ID NOs 22-24.

[0282] The cytokine receptor agonist according to the fifth aspect of the invention described above and herein may incorporate, singly or in combination, any of the features described in the preceding or following (unless the context dictates otherwise). The first PD-1 binding domain may optionally comprise the HCDR1-3 and LCDR1-3 of SEQ ID NOs: 1-6, but is not limited thereto. The IL2Ry binding domain may optionally comprise HCDR1 of SEQ ID NO: 27, HCDR2 of SEQ ID NOs: 72, 74, 76, 79, 85, or 87, and HCDR3 of SEQ ID NO: 29, but is not limited thereto.

[0283] The invention further provides isolated polynucleotides encoding a cytokine receptor agonist as described herein, expression vectors comprising said polynucleotides, and host cells comprising the polynucleotides or the expression vectors. Also provided is a method of producing a cytokine receptor agonist as described herein, a pharmaceutical composition comprising a cytokine receptor agonist as described herein and a pharmaceutically acceptable carrier, and methods of using a cytokine receptor agonist as described herein.

[0284] In particular, the invention encompasses a cytokine receptor agonist as described herein for use in the treatment of a disease in an individual in need thereof. In a particular embodiment said disease is cancer. In a particular embodiment the individual is a human.

[0285] Also encompassed by the invention is the use of the cytokine receptor agonist as described herein for the manufacture of a medicament for treating a disease in an individual in need thereof.

[0286] Further provided is a method of treating disease in an individual, comprising administering to said individual a therapeutically effective amount of a composition comprising a cytokine receptor agonist as described herein. Said disease preferably is cancer.

[0287] Also provided is a method of stimulating the immune system of an individual, comprising administering to said individual an effective amount of a composition comprising the cytokine receptor agonist described herein in a pharmaceutically acceptable form.

[0288] The invention includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or expressly avoided.

[0289] Summary of the Figures

[0290] Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures in which:

[0291] Figure 1 . Alignment of PD1 -specific antibody V-domain sequences of 1040 and clone 891 . CDRs are underlined and the symbols between the 2 sequences indicate the degree of similarity of the corresponding amino acids. (A) Alignment of VH-domain sequences of 1040 and clone 891 . (B) Alignment of VL-domain sequences of 1040 and clone 891

[0292] Figure 2. (A) Superimposition of the Fab of 1040 (solid ribbon) with the Fab of clone 891 (line ribbon).

[0293] The bound PD1 ECDs are depicted in the same way. VH-domains are depicted in black ribbons (lower left of figure), VL-domains in light grey (lower right of figure), and the PD1 antigen in a medium grey colour (top of figure). (B) Crystal structures

[0294] Figure 3. Alignment of (A) VH and (B) VL sequence variants generated by rational design. Amino acids which are different to the sequence of the parental 1040 are highlighted in bold.

[0295] Figure 4. Schematic description of antigens used for the selection of affinity-improved 1040 variants: A) human PD1-ECD-AviHis (left) and cynomolgus PD1-ECD-AviHis (right) and B) human PD1-ECD-Fc- AviHis (left) and cynomolgus PD1-ECD-Fc-AviHis (right).

[0296] Figure 5. Randomization strategy for the CDR regions of the parental clone 1040. Shown are the variable domains of the parental clone 1040. (X) represents the randomized positions. Figure 6. Schematic description of the library generation strategy. Shown is the PCR amplification and cloning strategy used for the generation of the 1040 -based affinity maturation library. Respective enzymes used for cloning into the phagemid are indicated.

[0297] Figure 7. Panning strategy for the generation of 1040-derived affinity-matured variants. Shown are the 5 streamlines applied for the generation of affinity-matured binders. Panning rounds conducted with Cynomolgus PD1 are marked in grey. After 4 rounds, 94 clones of each streamline were analysed.

[0298] Figure 8. SPR analysis of the parental clone 1040 and its affinity-matured variants. Shown are the Sensorgrams of the captured Fab fragments clone 1040 and its affinity-matured derivatives that were selected by phage display. Either human or cynomolgus PD1 was used as an analyte in a 2-fold serial dilution series from 3.125 to 400 nM. Clones with significantly improved affinity to cynomolgus PD1 are marked in grey.

[0299] Figure 9. Sequence alignment of the parental clone 1040 with all selected variants. A) Alignment of the VH sequences. B) Alignment of the VL sequences. CDRs of both heavy and light chains are framed.

[0300] Figure 10. Sequence alignment of the parental clone 1040 with all designed variants. A) Alignment of the VH sequences. B) Alignment of the VL sequences

[0301] Figure 11 : Template constructs for optimization of split PD1 -targeted IL-2R agonists. All molecules have the same Fc characteristics, i.e. heterodimerization via knob-into-hole assembly of Fc chains and silencing of Fc effector functions via incorporation of P329G LALA mutations in the CH2 domain. A The previously disclosed P1AI1593 comprising an anti-IL-2Rb VHH fused to the VL of anti-PD1 376 in a one- armed IgG format. B The previously disclosed P1AH6814 comprising an anti-IL-2Rg VHH fused to the VH of anti-PD1 1040 in a one-armed IgG format.

[0302] Figure 12: Optimisation matrix for split PD1 -targeted IL-2R agonists. VHH domains were swapped to alter the specificity pairings between anti-PD1 Fabs and anti-IL2R VHH. Linker lengths were varied between 5 aa and 25 aa. . All molecules have the same Fc characteristics, i.e. heterodimerization via knob-into-hole assembly of Fc chains and silencing of Fc effector functions via incorporation of P329G LALA mutations in the CH2 domain.

[0303] Figure 13: IL-2R signalling of split PD-1-targeted IL-2R agonists with different linker lengths and fusion points. Test compounds were incubated with HEK Blue IL-2 human PD-1 cells for 21 h at 37°C and 5% CO2. Fc-VHH (P1AH1177) was used as reference. A non-binding DP47 antibody (P1 AD3966) was used as negative control. IL-2R signalling was measured via absorbance at 650 nm using QUANTI-Blue reagent. Shown are mean absorbance values + / - SEM of technical duplicates for each tested molecule concentration.

[0304] Figure 14: Optimisation of the ‘all-in-one’ format design. All molecules have the same Fc characteristics, i.e. heterodimerization via knob-into-hole assembly of Fc chains and silencing of Fc effector functions via incorporation of P329G LALA mutations in the CH2 domain. Initial grafting of a VHH-Fab fusion to the C- terminus of the complimetary split concept generated the first iteration of the all-in-one which was further optimised to reduce flexibility. Heavy and light chains of an anti-PD1 Fab were fused to the lgG1 hinge region to tether both chains of the upper Fab.

[0305] Figure 15: IL-2R signaling of all-in-one PD-1-targeted I L-2R agonist with hinge rigidification. Test compounds were incubated with HEK Blue IL-2 wt cells (A) or HEK Blue IL-2 human PD-1 cells (B) for 20h at 37°C and 5% CO2. Fc-VHH (P1 AH1177) was used as reference. A non-binding DP47 antibody (P1 AD3966) was used as negative control. IL-2R signaling was measured via absorbance at 650 nm using QUANTI-Blue reagent. Shown are mean absorbance values + / - SEM of technical duplicates for each tested molecule concentration.

[0306] Figure 16: All-in-one IL-2R agonists with different linker lengths between the IL-2Ry VHH and the VH of the anti-PD1 Fab ‘1040 aff. mat.’ and the IL-2R0 VHH and the VL of the anti-PD1 Fab ‘376 de-risked’. All molecules have the same Fc characteristics, i.e. heterodimerization via knob-into-hole assembly of Fc chains and silencing of Fc effector functions via incorporation of P329G LALA mutations in the CH2 domain. A 25 amino acid glycine-serine linker between the IL-2Ry VHH and the VH of the anti-PD1 Fab ‘1040 aff. mat.’ and 25 amino acid glycine-serine linker between the IL-2R0 VHH and the VL of the anti- PD1 Fab ‘376 de-risked’. B 15 amino acid glycine-serine linker between the IL-2Ry VHH and the VH of the anti-PD1 Fab ‘1040 aff. mat.’ and 15 amino acid glycine-serine linker between the IL-2R0 VHH and the VL of the anti-PD1 Fab ‘376 de-risked’. C 5 amino acid glycine-serine linker between the IL-2Ry VHH and the VH of the anti-PD1 Fab ‘1040 aff. mat.’ and 5 amino acid glycine-serine linker between the IL- 2Rp VHH and the VL of the anti-PD1 Fab ‘376 de-risked’.

[0307] Figure 17: IL-2R signaling of all-in-one PD-1 -targeted IL-2R agonist with different linker lengths. Test compounds were incubated with HEK Blue IL-2 wt cells (A) or HEK Blue IL-2 human PD-1 cells (B) for 18h at 37°C and 5% CO2. Fc-VHH (P1 AH1177) was used as reference. A non-binding DP47 antibody (P1 AD3966) was used as negative control. IL-2R signaling was measured via absorbance at 650 nm using QUANTI-Blue reagent. Shown are mean absorbance values + / - SEM of technical duplicates for each tested molecule concentration.

[0308] Figure 18: Schematic representation of antigens for the isolation of human and cynomolgus cross- reactive IL-2Rp-specific VHH domains. A The extracellular domain (ECD) of human IL-2R0 and human IL-2Ry fused to biotinylated Fc-knob and Fc-hole respectively was generated for llama immunization (P1 AE2657). B The ECD of cynomolgus IL-2R0 fused to biotinylated Fc knob-into-hole was generated for phage display and screening (P1 AJ5008) . C The ECD of human IL-2R0 fused to biotinylated Fc knob- into-hole was generated for screening (P1AF1104). D Biotinylated human lgG1 Fc was generated for screening (P1 AE6073). E Antibody of irrelevant specificity containing human IgG 1 Fc for Fc-competitive soluble selections (P1AD4935).

[0309] Figure 19: Specificity screening for human and cynomolgus cross-reactive IL-2R0 binders by ELISA. Soluble VHH domains enriched for cynomolgus IL-2R0 specificity were randomly selected and screened for binding to immobilized human IL-2Rp-Fc, immobilized cynomolgus IL-2Rp-Fc and immobilized human Fc. A Following three rounds of phage display, VHH were tested for antigen specificity. B Following four rounds, enriched VHH were also tested for specificity by ELISA. Reference-subtracted absorbance (A450-A650) indicating the binding response to each target is represented as a stacked bar chart.

[0310] Figure 20: Format conversion of VHH domains into bispecific heavy chain antibodies and monospecific monovalent heavy chain antibodies. A Selected VHH with human and cynomolgus IL-2R0 specificity were fused upstream of Fc-knob and cloned into a mammalian cell expression vector using Gibson cloning methods. B Bispecific heavy chain antibodies were generated comprising the selected anti- IL-2R0 VHH fused to Fc-knob paired with a previously identified VHH with IL-2Ry specificity fused to Fc-hole. C Monospecific, monovalent VHH-Fc fusion format with IL-2R0 specificity. All formats contain flexible 5(GGSGG) linkers and effector functions of the Fc were silenced via incorporation of P329G LALA mutations in the CH2 domain (C).

[0311] Figure 21 : Functional characterization of dose-dependent response for IL-2R agonists. Bispecific heavy chain antibodies comprising IL-2R0 specificity and IL-2Ry were screened for dose-dependent IL-2R agonism in HEK Blue IL-2 cells. Test compounds were incubated with HEK Blue IL-2 human cells for 20h at 37°C and 5% CO2. The previously identified IL-2R agonist (P1AH1177) was used as reference and non-binding DP47 antibody (P1AD3966) was used as negative control. IL-2R activity was quantified by absorbance at 650 nm using QUANTI-Blue reagent. Shown are mean absorbance values + / - SEM of technical duplicates for each tested molecule concentration.

[0312] Figure 22: Cell binding of parental anti-IL2Rp VHHs to primary human and cyno CD8 T cells. Test compounds were incubated for 60min at 4°C with human or cynomolgus monkey PBMCs. Binding strength was measured via PE conjugated anti-PG mutation antibody detected by flow cytometry. Shown are PE MFI values on human (A) and cyno (B) CD8 T cells for each tested molecule concentration.

[0313] Figure 23: Monospecific single domain antibodies comprising IL-2Rp-specific VHH domains. VHH domains with IL-2R0 specificity were fused to Fc-knob. Flexible 5(GGSGG) or 2(GGSGG) linkers were used to fuse VHH moieties to Fc chains. Heterodimerization was achieved via knob-into-hole assembly of Fc chains. Effector functions of the Fc were silenced via incorporation of P329G LALA mutations in the CH2 domain. A Non-cynomolgus cross-reactive llama VHH domain - Fc fusion. B Cynomolgus cross- reactive llama VHH domain - Fc fusions. C Humanized llama VHH domain - Fc fusions.

[0314] Figure 24: Monospecific single domain antibodies comprising IL-2Ry-specific VHH domains. VHH domains with IL-2Ry specificity were fused to Fc-knob. Flexible 2(GGSGG) linkers were used to fuse VHH moieties to Fc chains. Heterodimerization was achieved via knob-into-hole assembly of Fc chains. Effector functions of the Fc were silenced via incorporation of P329G LALA mutations in the CH2 domain. A Cynomolgus cross-reactive llama VHH domain - Fc fusions. B Humanized llama VHH domain - Fc fusions.

[0315] Figure 25: Cell binding of humanized anti-IL2Rg VHH variants to human IL-2Rpy Bioassay cells. Test compounds were incubated with target cells for 60min at 4°C and binding strength measured via PE conjugated anti-Fc secondary antibody by flow cytometry. Shown are PE MFI values for each tested molecule concentration (A) and (B). Figure 26: Cell binding of humanized anti-IL2Rg VHH variants to primary human and cyno CD8 T cells. Test compounds were incubated for 60min at 4°C with human or cynomolgus monkey PBMCs. Binding strength was measured via PE conjugated anti-PG mutation antibody detected by flow cytometry. Shown are PE MFI values on human CD4 T cells (A), human CD8 T cells (B), cyno CD4 T cells (C) and cyno CD8 T cells (D) for each tested molecule concentration.

[0316] Figure 27: Cell binding of humanized anti-IL2Rp VHH variants to primary human and cyno CD8 T cells. Test compounds were incubated for 60min at 4°C with human or cynomolgus monkey PBMCs. Binding strength was measured via PE conjugated anti-PG mutation antibody detected by flow cytometry. Shown are PE MFI values on human CD4 T cells (A), human CD8 T cells (B), cyno CD4 T cells (C) and cyno CD8 T cells (D) for each tested molecule concentration.

[0317] Figure 28: Cell binding of humanized anti-IL2Rp VHH variants to primary human and cyno CD8 T cells. Test compounds were incubated for 60min at 4°C with human or cynomolgus monkey PBMCs. Binding strength was measured via PE conjugated anti-PG mutation antibody detected by flow cytometry. Shown are PE MFI values on human CD4 T cells (A), human CD8 T cells (B), cyno CD4 T cells (C) and cyno CD8 T cells (D) for each tested molecule concentration.

[0318] Figure 29: Lead series of split IL2R agonists. All molecules have the same Fc characteristics, i.e. heterodimerization via knob-into-hole assembly of Fc chains and silencing of Fc effector functions via incorporation of P329G LALA mutations in the CH2 domain. A VHH domains with IL-2Ry specificity were fused to the N-terminus of the VH-domain of the monovalent PD1 binder ‘1040 aff. mat.’. B VHH domains with IL-2R0 specificity were fused to the N-terminus of the VL-domain of the monovalent PD1 binder ‘376 de-risked’. C VHH domains with IL-2R0 specificity were fused to the N-terminus of the VL-domain of one of the two Fabs of the bivalent PD1 binder ‘376 de-risked’. Light chain mispairing was avoided by the introduction of charges in the constant Fab domains of the VHH-fused Fab and a VH-VL domain crossover in the non-fused Fab. D VHH domains with IL-2R0 specificity were fused to the N-terminus of the VL-domain of one of the two Fabs of the bivalent PD1 binder Pembrolizumab. Light chain mispairing was avoided by the introduction of charges in the constant Fab domains of the VHH-fused Fab and a VH-VL domain cross-over in the non-fused Fab.

[0319] Figure 30: Cell binding of humanized split PD-1 targeted IL-2R agonists to human IL-2Rpy Bioassay cells. Test compounds were incubated with target cells for 60min at 4°C and binding strength measured via PE conjugated anti-Fc secondary antibody by flow cytometry. Shown are PE MFI values for each tested molecule concentration of compounds containing monovalent PD-1 binder 376 (A) or bivalent PD-1 binder 376 or pembrolizumab (B).

[0320] Figure 31 : Cell binding of humanized split PD-1 targeted IL-2R agonists to human IL-2Rpy Bioassay cells expressing PD-1 . Test compounds were incubated with target cells for 60min at 4°C and binding strength measured via PE conjugated anti-Fc secondary antibody by flow cytometry. Shown are PE MFI values for each tested molecule concentration of compounds containing monovalent PD-1 binder 376 (A) or bivalent PD-1 binder 376 or pembrolizumab (B). Figure 32: IL-2R signaling of humanized split PD-1-targeted IL-2R with HEK Blue IL-2 wt cells. Test compounds were incubated with HEK Blue IL-2 cells for 19h at 37°C and 5% CO2. Fc-VHH bearing anti- IL2Rp and anti-IL-2Ry VHHs (P1AM1091) was used as reference. A non-binding DP47 antibody was used as negative control. IL-2R signaling was measured via absorbance at 650 nm using QUANTI-Blue reagent. Shown are mean absorbance values + / - SEM of technical duplicates for each tested molecule concentration.

[0321] Figure 33: IL-2R signaling of humanized split PD-1-targeted IL-2R with HEK Blue IL-2 wt cells. Test compounds were incubated with HEK Blue IL-2 cells for 19h at 37°C and 5% CO2. Fc-VHH bearing parental anti-IL2Rp and anti-IL-2Ry VHHs (P1AM1092) was used as reference. A non-binding DP47 antibody was used as negative control. IL-2R signaling was measured via absorbance at 650 nm using QUANTI-Blue reagent. Shown are mean absorbance values + / - SEM of technical duplicates for each tested molecule concentration.

[0322] Figure 34: IL-2R signaling of humanized split PD-1-targeted IL-2R with HEK Blue IL-2 huPD-1 cells. Test compounds were incubated with HEK Blue IL-2 human PD-1 clone 4 cells for 19h at 37°C and 5% CO2. Fc-VHH bearing parental anti-IL2Rp and anti-IL-2Ry VHHs (P1AM1091) was used as reference. A nonbinding DP47 antibody was used as negative control. IL-2R signaling was measured via absorbance at 650 nm using QUANTI-Blue reagent. Shown are mean absorbance values + / - SEM of technical duplicates for each tested molecule concentration.

[0323] Figure 35: IL-2R signaling of humanized split PD-1-targeted IL-2R with HEK Blue IL-2 huPD-1 cells. Test compounds were incubated with HEK Blue IL-2 human PD-1 clone 4 cells for 19h at 37°C and 5% CO2. Fc-VHH bearing parental anti-IL2Rp and anti-IL-2Ry VHHs (P1AM1092) was used as reference. A nonbinding DP47 antibody was used as negative control. IL-2R signaling was measured via absorbance at 650 nm using QUANTI-Blue reagent. Shown are mean absorbance values + / - SEM of technical duplicates for each tested molecule concentration.

[0324] Figure 36: PD-1 / PD-L1 blockade assay of humanized split PD1-targeted IL-2R agonists. Assay was performed according to the manufacturer’s instructions (Promega, J1250). Pembrolizumab (P1 AA6975) and PD1-IL2v (P1AE4422) were used as reference for PD-1 / PD-L1 blockade. A non-binding DP47 antibody (P1AD5108) was used as negative control and P1AH4495 as bivalent anti-PD-1 IgG with no blocking activity. Shown are mean luminescence values + / - SEM of technical duplicates for each tested molecule concentration. (A) monovalent PD1-IL2 mimetics, (B) bivalent PD1-IL2 mimetic and (C) summary table of EC50 and normalized max values.

[0325] Figure 37: NK cell activation and proliferation induced by humanized split PD-1 targeted IL-2R agonists. Frequency of CD25 expressing NK cells (A) and counts of proliferating NK cells (B) were determined by flow cytometry after five days of test compounds co-incubation with freshly isolated CTV-labelled PBMCs. Proliferating cells were identified based on cell division visualized with CTV staining. PD1-IL2v (P1AE4422) and Fc-VHH (P1AM1092) were used as reference. A non-binding DP47 antibody (P1AD5108) was used as negative control. Shown are mean values + / - SEM of two PBMC donors combined and technical duplicates for each tested molecule concentration. Figure 38: CD8 T cell activation and proliferation induced by humanized split PD-1 targeted IL-2R agonists. Frequency of CD25 expressing CD8 T cells (A) and counts of proliferating CD8 T cells (B) were determined by flow cytometry after four days of test compounds co-incubation with PBMCs pre-activated for four days with 1 pg / ml of coated anti-human CD3 antibody and 1 pg / ml of soluble anti-human CD28 antibody. Proliferating cells were identified based Ki-67+ staining. PD1-IL2v (P1AE4422) and Fc-VHH (P1AM1092) were used as reference. A non-binding DP47 antibody (P1AD5108) was used as negative control. Shown are mean values + / - SEM of two PBMC donors combined and technical duplicates for each tested molecule concentration.

[0326] Figure 39: Anti-apoptotic Bcl-2 expression induced by humanized split PD-1 targeted IL-2R agonists. Frequency of Bcl-2 expressing CD8 T cells was determined by flow cytometry after four days of test compounds co-incubation with PBMCs pre-activated for four days with 1 pg / ml of coated anti-human CD3 antibody and 1 pg / ml of soluble anti-human CD28 antibody. PD1-IL2v (P1AE4422) and Fc-VHH (P1AM1092) were used as reference. A non-binding DP47 antibody (P1AD5108) was used as negative control. Shown are mean values + / - SEM of two PBMC donors combined and technical duplicates for each tested molecule concentration.

[0327] Figure 40: STAT-5 phosphorylation induced by humanized split PD-1 targeted IL-2R agonists. Frequency of STAT-5 phosphorylation in CD4 T cells (A) or CD8 T cells (B) was determined by flow cytometry after 60min of test compounds co-incubation with PBMCs pre-activated for four days with 1 pg / ml of coated anti-human CD3 antibody and 1 pg / ml of soluble anti-human CD28 antibody. PD1-IL2v (P1AE4422) and Fc-VHH (P1AM1092) were used as reference. A non-binding DP47 antibody (P1AD5108) was used as negative control. Shown are mean values + / - SEM of two PBMC donors combined and technical duplicates for each tested molecule concentration.

[0328] Figure 41 : STAT-5 phosphorylation induced by humanized split PD-1 targeted IL-2R agonists. Median fluorescence intensity (MFI) on phosphorylated STAT-5 expressing CD4 T cells (A) or CD8 T cells (B) was determined by flow cytometry after 60min of test compounds co-incubation with PBMCs preactivated for four days with 1 pg / ml of coated anti-human CD3 antibody and 1 pg / ml of soluble anti-human CD28 antibody. PD1-IL2v (P1AE4422) and Fc-VHH (P1AM1092) were used as reference. A non-binding DP47 antibody (P1AD5108) was used as negative control. Shown are mean values + / - SEM of two PBMC donors combined and technical duplicates for each tested molecule concentration.

[0329] Figure 42: T cell effector functions (proliferation and Granzyme B production) induced by humanized split PD-1 targeted IL-2R agonists. Frequency of Ki-67+ and GranzB+ cells in PD1 +CD69+ CD8 T cells was determined by flow cytometry after 3 days incubation of test compounds with human pan T cells in plates coated with 50 ng / ml anti-CD3. PD1-IL2v (P1AE4422) and Fc-VHH (P1AM1092) were used as reference. A non-binding DP47 antibody (P1AD5108) was used as negative control. Shown are mean values + / - SEM of two pan T cell donors combined and technical duplicates for each tested molecule concentration.

[0330] Figure 43: Lead series of all-in-one IL2R agonists. All molecules have the same Fc characteristics, i.e. heterodimerization via knob-into-hole assembly of Fc chains and silencing of Fc effector functions via incorporation of P329G LALA mutations in the CH2 domain. In the all-in-one format, the anti-cytokine receptor VHH - anti-PD1 fusions of the split formats were retained and combined into one molecule with the two different PD1 binders ‘1040 aff. mat.’ (with VH-VL domain cross-over) and ‘376 de-risked’ (with charges in the constant Fab domains) linked via knob-into-hole of Fc chains.

[0331] Figure 44: Cell binding of humanized all-in-one PD-1 targeted IL-2R agonists to human IL-2Rpy Bioassay cells. Test compounds were incubated with target cells for 60min at 4°C and binding strength measured via PE conjugated anti-Fc secondary antibody by flow cytometry. Shown are PE MFI values for each tested molecule concentration (A) and (B).

[0332] Figure 45: Cell binding of humanized all-in-one PD-1 targeted IL-2R agonists to human IL-2Rpy Bioassay cells expressing PD-1 . Test compounds were incubated with target cells for 60min at 4°C and binding strength measured via PE conjugated anti-Fc secondary antibody by flow cytometry. Shown are PE MFI values for each tested molecule concentration (A) and (B).

[0333] Figure 46: IL-2R signaling of humanized all-in-one PD-1-targeted IL-2R with human IL-2Rpy Bioassay cells. Test compounds were incubated with effector cells for 5h at 37°C and 5% CO2. PD1-IL2v (P1AE4422) was used as reference. IL-2R signaling was measured via luminescence (100ms integration time) using BioGio NL reagent. Shown are mean relative luminescence unit (RLU) values + / - SEM of technical duplicates for each tested molecule concentration.

[0334] Figure 47: IL-2R signaling of humanized all-in-one PD-1-targeted IL-2R with human IL-2Rpy Bioassay cells expressing PD-1 . Test compounds were incubated with effector cells for 5h at 37°C and 5% CO2. PD1-IL2v (P1AE4422) was used as reference. IL-2R signaling was measured via luminescence (100ms integration time) using BioGio NL reagent. Shown are mean relative luminescence unit (RLU) values + / - SEM of technical duplicates for each tested molecule concentration.

[0335] Figure 48 : PD-1 / PD-L1 blockade assay of humanized all-in-one PD1-targeted IL-2R agonist. Assay was performed according to the manufacturer’s instructions (Promega, J1250). PD1-IL2v (P1AE4422) was used as reference for PD-1 / PD-L1 blockade. A non-binding DP47 antibody (P1AD5108) was used as negative control. Shown are mean luminescence values + / - SEM of technical duplicates for each tested molecule concentration. (A) all-in-one PD1-IL2 mimetic and (B) summary table of EC50 and normalized max values.

[0336] Figure 49: NK cell activation and proliferation induced by humanized all-in-one PD-1 targeted IL-2R agonist. Frequency of CD25 expressing NK cells (A) and counts of proliferating NK cells (B) were determined by flow cytometry after five days of test compounds co-incubation with freshly isolated CTV- labelled PBMCs. Proliferating cells were identified based on cell division visualized with CTV staining. PD1-IL2v (P1AE4422) and Fc-VHH (P1AM1092) were used as reference. A non-binding DP47 antibody (P1AD5108) was used as negative control. Shown are mean values + / - SEM of two PBMC donors combined and technical duplicates for each tested molecule concentration.

[0337] Figure 50: CD8 T cell activation and proliferation induced by humanized all-in-one PD-1 targeted IL-2R agonist. Frequency of CD25 expressing CD8 T cells (A) and counts of proliferating CD8 T cells (B) were determined by flow cytometry after four days of test compounds co-incubation with PBMCs pre-activated for four days with 1 pg / ml of coated anti-human CD3 antibody and 1 pg / ml of soluble anti-human CD28 antibody. Proliferating cells were identified based Ki-67+ staining. PD1-IL2v (P1AE4422) and Fc-VHH (P1AM1092) were used as reference. A non-binding DP47 antibody (P1AD5108) was used as negative control. Shown are mean values + / - SEM of two PBMC donors combined and technical duplicates for each tested molecule concentration.

[0338] Figure 51 : Anti-apoptotic Bcl-2 expression induced by humanized all-in-one PD-1 targeted IL-2R agonists. Frequency of Bcl-2 expressing CD8 T cells was determined by flow cytometry after four days of test compounds co-incubation with PBMCs pre-activated for four days with 1 pg / ml of coated anti-human CD3 antibody and 1 pg / ml of soluble anti-human CD28 antibody. PD1-IL2v (P1AE4422) and Fc-VHH (P1AM1092) were used as reference. A non-binding DP47 antibody (P1AD5108) was used as negative control. Shown are mean values + / - SEM of two PBMC donors combined and technical duplicates for each tested molecule concentration.

[0339] Figure 52: STAT-5 phosphorylation induced by humanized all-in-one PD-1 targeted IL-2R agonist. Frequency of STAT-5 phosphorylation in CD4 T cells (A) or CD8 T cells (B) was determined by flow cytometry after 60min of test compounds co-incubation with PBMCs pre-activated for four days with 1 pg / ml of coated anti-human CD3 antibody and 1 pg / ml of soluble anti-human CD28 antibody. PD1-IL2v (P1AE4422) and Fc-VHH (P1AM1092) were used as reference. A non-binding DP47 antibody (P1AD5108) was used as negative control. Shown are mean values + / - SEM of two PBMC donors combined and technical duplicates for each tested molecule concentration.

[0340] Figure 53: STAT-5 phosphorylation induced by humanized all-in-one PD-1 targeted IL-2R agonist. Median fluorescence intensity (MFI) on phosphorylated STAT-5 expressing CD4 T cells (A) or CD8 T cells (B) was determined by flow cytometry after 60min of test compounds co-incubation with PBMCs preactivated for four days with 1 pg / ml of coated anti-human CD3 antibody and 1 pg / ml of soluble anti-human CD28 antibody. PD1-IL2v (P1AE4422) and Fc-VHH (P1AM1092) were used as reference. A non-binding DP47 antibody (P1AD5108) was used as negative control. Shown are mean values + / - SEM of two PBMC donors combined and technical duplicates for each tested molecule concentration.

[0341] Figure 54: T cell effector functions (proliferation and Granzyme B production) induced by humanized all- in-one PD-1 targeted IL-2R agonists. Frequency of Ki-67+ and GranzB+ cells in PD1 +CD69+ CD8 T cells was determined by flow cytometry after 3 days incubation of test compounds with human pan T cells in plates coated with 50 ng / ml anti-CD3. PD1-IL2v (P1AE4422) and Fc-VHH (P1AM1092) were used as reference. A non-binding DP47 antibody (P1AD5108) was used as negative control. Shown are mean values + / - SEM of two pan T cell donors combined and technical duplicates for each tested molecule concentration.

[0342] Figure 55: Deamidation repair strategy. A Sequence alignment of the anti-IL-2Ry VHH domains of four affinity variants. B VHH sequences of deamidation repair variants compared to their respective parental origin sequence. Figure 56: 12 deamidation site repair variants in different formats. A Monospecific single domain antibodies comprising deamidation site repaired IL-2Rv-specific VHH domains as VHH - Fc fusions. VHH domains with IL-2Ry specificity were fused to Fc-knob. The Fc-hole chain carries a C-terminal His-tag to facilitate purification. Flexible 2(GGSGG) linkers were used to fuse VHH moieties to Fc chains. Heterodimerization was achieved via knob-into-hole assembly of Fc chains. Effector functions of the Fc were silenced via incorporation of P329G LALA mutations in the CH2 domain. B Deamidation site repaired VHH domains with IL-2Ry specificity were fused to the N-terminus of the VH-domain of the monovalent PD1 binder ‘1040 aff. mat.’. C In the all-in-one format, the anti-cytokine receptor VHH - anti- PD1 fusions of the split formats were retained and combined into one molecule with the two different PD1 binders ‘1040 aff. mat.’ (with VH-VL domain cross-over) and ‘376 de-risked’ (with charges in the constant Fab domains) linked via knob-into-hole of Fc chains. The deamidation site repaired IL-2Ry-specific VHH domains were fused to the N-terminus of the VH domain of the PD1 binder ‘1040 aff. mat.’

[0343] Figure 57: Control constructs used in in vitro assays. A Human IgG 1 antibody ‘DP47’ without binding specificity and with silencing of Fc effector functions via incorporation of P329G LALA mutations in the CH2 domain. In contrast to P1AD5108, P1AD3966 carries a C-terminal avi-tag on each heavy chain. B Non-targeted bispecific IL2R mimetics with IL-2Rp-specific VHH and IL-2R / -specific VHH fused to heterodimerized knob-into-hole Fc chains and silencing of Fc effector functions via incorporation of P329G LALA mutations in the CH2 domain. In contrast to P1AM1091 and P1AM1092, P1AH1177 carries a sortase-tag inbetween the VHH and the hinge region of each chain. C PD1 -targeted IL2v with heterodimerized knob-into-hole Fc chains, silencing of Fc effector functions via incorporation of P329G LALA mutations in the CH2 domain, and human IL2v fused to the C-terminus of the Fc knob chain.

[0344] Figure 58: Control constructs used in in vitro assays. A Split constructs comprising VHH domains with IL- 2Rp specificity that were fused to the N-terminus of the VL-domain of the monovalent PD1 binder ‘376 de-risked’. B All-in-one formats with an IL-2Ry-specific VHH fused to the VH-domain of the PD1 binder ‘1040 aff. mat.’ (with VH-VL domain cross-over) and an IL-2R0 VHH fused to the VL domain of the PD1 binder ‘376 de-risked’ (with charges in the constant Fab domains).

[0345] Figure 59: Cell binding of humanized deamidation repair anti-IL-2Ry VHH variants to human IL-2Rpy Bioassay cells. Test compounds were incubated with target cells for 60min at 4°C and binding strength measured via PE conjugated anti-Fc secondary antibody by flow cytometry. A non-binding DP47 antibody (P1AD5108) was used as negative control. Shown are PE MFI values for each tested molecule concentration grouped according to the parental humanized binder P1AK9665 (A), P1AK9662 (B) and P1AK9661 (C).

[0346] Figure 60: IL-2R signalling of humanized split PD-1-targeted IL-2R agonists with selected anti-IL-2Ry VHH deamidation repair variant. Test compounds were incubated with HEK Blue IL-2 wt (A) or HEK Blue IL-2 human PD-1 cells (B) for 20h at 37°C and 5% CO2. Fc-VHH (P1AM1092) and PD1-IL2v (P1AE4422) were used as reference. A non-binding DP47 antibody (P1AD5108) was used as negative control. IL-2R signalling was measured via absorbance at 650 nm using QUANTI-Blue reagent. Shown are mean absorbance values + / - SEM of technical duplicates for each tested molecule concentration. Figure 61 : PD-1 / PD-L1 blockade assay of humanized split PD-1-targeted IL-2R agonists with selected anti-IL-2Ry VHH deamidation repair variant. Assay was performed according to the manufacturer’s instructions (Promega, J1250). pembrolizumab (P1AA6975) and PD1-IL2v (P1AE4422) were used as reference for PD-1 / PD-L1 blockade. A non-binding DP47 antibody (P1AD5108) was used as negative control. Shown are mean luminescence values + / - SEM of technical duplicates for each tested molecule concentration.

[0347] Figure 62: NK cell activation and proliferation induced by humanized split PD-1 -targeted IL-2R agonists with selected anti-IL-2Ry VHH deamidation repair variant. Frequency of CD25 expressing NK cells (A) and counts of proliferating NK cells (B) were determined by flow cytometry after five days of test compounds co-incubation with freshly isolated CTV-labelled PBMCs. Proliferating cells were identified based on cell division visualized with CTV staining. PD1-IL2v (P1AE4422) and Fc-VHH (P1AM1092) were used as reference. A non-binding DP47 antibody (P1AD5108) was used as negative control. Shown are mean values + / - SEM of two PBMC donors combined and technical duplicates for each tested molecule concentration.

[0348] Figure 63: STAT-5 phosphorylation induced by humanized split PD-1-targeted IL-2R agonists with selected anti-IL-2Ry VHH deamidation repair variant. Frequency of STAT-5 phosphorylation in CD4 T cells (A) or CD8 T cells (B) was determined by flow cytometry after 60min of test compounds coincubation with PBMCs pre-activated for four days with 1 pg / ml of coated anti-human CD3 antibody and 1 pg / ml of soluble anti-human CD28 antibody. PD1-IL2v (P1AE4422) and Fc-VHH (P1AM1092) were used as reference. A non-binding DP47 antibody (P1AD5108) was used as negative control. Shown are mean values + / - SEM of two PBMC donors combined and technical duplicates for each tested molecule concentration.

[0349] Figure 64: IL-2R signalling of humanized all-in-one PD-1-targeted IL-2R agonists with selected anti-IL- 2Ry VHH deamidation repair variant. Test compounds were incubated with HEK Blue IL-2 wt (A) or HEK Blue IL-2 human PD-1 cells (B) for 20h at 37°C and 5% CO2. Fc-VHH (P1AM1092) and PD1-IL2v (P1AE4422) were used as reference. A non-binding DP47 antibody (P1AD5108) was used as negative control. IL-2R signalling was measured via absorbance at 650 nm using QUANTI-Blue reagent. Shown are mean absorbance values + / - SEM of technical duplicates for each tested molecule concentration.

[0350] Figure 65: PD-1 / PD-L1 blockade assay of humanized all-in-one PD-1-targeted IL-2R agonists with selected anti-IL-2Ry VHH deamidation repair variant. Assay was performed according to the manufacturer’s instructions (Promega, J1250). pembrolizumab (P1AA6975) and PD1-IL2v (P1AE4422) were used as reference for PD-1 / PD-L1 blockade. A non-binding DP47 antibody (P1AD5108) was used as negative control. Shown are mean luminescence values + / - SEM of technical duplicates for each tested molecule concentration.

[0351] Figure 66: NK cell activation and proliferation induced by humanized all-in-one PD-1 -targeted IL-2R agonists with selected anti-IL-2Ry VHH deamidation repair variant. Frequency of CD25 expressing NK cells (A) and counts of proliferating NK cells (B) were determined by flow cytometry after five days of test compounds co-incubation with freshly isolated CTV-labelled PBMCs. Proliferating cells were identified based on cell division visualized with CTV staining. PD1-IL2v (P1AE4422) and Fc-VHH (P1AM1092) were used as reference. A non-binding DP47 antibody (P1AD5108) was used as negative control. Shown are mean values + / - SEM of two PBMC donors combined and technical duplicates for each tested molecule concentration.

[0352] Figure 67: STAT-5 phosphorylation induced by humanized all-in-one PD-1-targeted IL-2R agonists with selected anti-IL-2Ry VHH deamidation repair variant. Frequency of STAT-5 phosphorylation in CD4 T cells (A) or CD8 T cells (B) was determined by flow cytometry after 60min of test compounds coincubation with PBMCs pre-activated for four days with 1 pg / ml of coated anti-human CD3 antibody and 1 pg / ml of soluble anti-human CD28 antibody. PD1-IL2v (P1AE4422) and Fc-VHH (P1AM1092) were used as reference. A non-binding DP47 antibody (P1AD5108) was used as negative control. Shown are mean values + / - SEM of two PBMC donors combined and technical duplicates for each tested molecule concentration.

[0353] Figure 68: In vivo efficacy of humanized split and all-in-one PD-1-targeted IL-2R agonists. Immunocompetent mice expressing human PD1 , IL2Rp and IL-2Ry receptors were engrafted with Panc02-H7-Fluc tumors subcutaneously. Efficacy of three PD1-IL2 mimetic formats was compared to that of vehicle, pembrolizumab and low dose PD1-IL2v treated groups. Tumor growth curves are depicted as mean tumor volume (mm3) + / -SEM.

[0354] Figure 69: IL-2R signalling of humanized split PD-1-targeted IL-2R agonists with four anti-IL-2Ry VHH deamidation repair variants. Test compounds were incubated with HEK Blue IL-2 wt (A, C) or HEK Blue IL-2 human PD-1 cells (B, D) for 20h at 37°C and 5% CO2. Fc-VHH (P1AM1092) and PD1-IL2v (P1AE4422) were used as reference. A non-binding DP47 antibody (P1AD5108) was used as negative control. IL-2R signalling was measured via absorbance at 650 nm using QUANTI-Blue reagent. Shown are mean absorbance values + / - SEM of technical duplicates for each tested molecule concentration.

[0355] Figure 70: IL-2R signalling of humanized all-in-one PD-1-targeted IL-2R agonists with 4 anti-IL-2Ry VHH deamidation repair variant. Test compounds were incubated with HEK Blue IL-2 wt (A) or HEK Blue IL-2 human PD-1 cells (B) for 20h at 37°C and 5% CO2. Fc-VHH (P1AM1092) and PD1-IL2v (P1AE4422) were used as reference. A non-binding DP47 antibody (P1AD5108) was used as negative control. IL-2R signalling was measured via absorbance at 650 nm using QUANTI-Blue reagent. Shown are mean absorbance values + / - SEM of technical duplicates for each tested molecule concentration.

[0356] Figure 71 : Measurements of huFcRn recycling efficiency for PD-1-targeted IL-2R agonists with and without the YTE mutation in the Fc region using the in vitro ARC assay. Monovalent (P1 AM9643) and bivalent (P1AM9651) PD-1-targeted IL-2R agonists without the YTE mutation were compared to their YTE-mutated counterparts (P1AP5954 and P1AP5953, respectively), alongside AAA non-binding control, wild-type (WT) IgG control, and a strong FcRn-binding YTE control.

[0357] Figure 72: Functional characterization of PD-1 -targeted IL-2R agonists with and without the YTE mutation in their Fc part. Test compounds were incubated with HEK Blue IL-2 wt (A) or HEK Blue IL-2 human PD- 1 cells (B) for 20h at 37°C and 5% CO2. Fc-VHH (P1AP5880) and PD1-IL2v (P1AE4422) were used as reference. A non-binding DIG antibody (P1 AA0468) was used as negative control. After 20h incubation, IL-2R signalling was measured via absorbance at 640 nm using QUANTI-Blue reagent. Shown are mean absorbance values + / - SEM of technical duplicates for each tested molecule concentration.

[0358] Figure 73: PD-1 / PD-L1 blockade assay of PD-1 targeted IL-2 constructs with YTE mutation in their Fc part (P1AP5954, P1 AP5953) versus without (P1AM9646, P1AM9651). The assay was performed according to the manufacturer’s instructions (Promega, J1250). Pembrolizumab (P1AA6975) and PD1- IL2v (P1AE4422) were used as reference for PD-1 / PD-L1 blockade. A non-binding DIG antibody (P1 AA0468) was used as negative control. Shown are normalized luminescence values + / - SEM of technical duplicates for each tested molecule concentration (A). For normalization the value of 500 nM pembrolizumab was set to 100% blocking. (B) Summary table of EC50 and normalized max values.

[0359] Detailed Description of the Invention

[0360] Definitions

[0361] Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The methods and techniques of the present disclosure are generally performed according to conventional methods well known in the art. Generally, nomenclatures used in connection with, and techniques of biochemistry, enzymology, molecular, and cellular biology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art.

[0362] Throughout this specification, including the claims, unless the context requires otherwise, the word “comprise” and “include”, and variations such as “comprises”, “comprising”, and “including” will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0363] It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent “about,” it will be understood that the particular value forms another embodiment. The term “about” in relation to a numerical value is optional and means for example + / - 10%.

[0364] Cytokine receptors are cell-surface glycoproteins that specifically bind cytokines and transduce their signals. Generally, cytokine receptors function as oligomeric complexes consisting of typically two to four receptor chains, also termed subunits, that may be the same or different. Thus, the term “cytokine receptor complex” refers to cytokine receptors composed of at least two subunits. The term “cytokine receptor agonist” or “cytokine agonist” refers to a substance that binds to and activates a cytokine receptor. For example, an IL-2 agonist binds to an IL-2R (for example, an intermediate-affinity IL-2R) and activates the IL-2R, leading to IL-2R signalling.

[0365] The term “interleukin-2” or “IL-2” as used herein, refers to any native IL-2 from any vertebrate source, including mammals such as primates (e.g. humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses unprocessed IL-2 as well as any form of IL-2 that results from processing in the cell. The term also encompasses naturally occurring variants of IL-2, e.g. splice variants or allelic variants.

[0366] The term “IL-2Ra” or “a-subunit of the IL-2 receptor” also known as CD25, as used herein, refers to any native IL-2Ra from any vertebrate source, including mammals such as primates (e.g. humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses “full-length”, unprocessed IL-2Ra as well as any form of IL-2Ra that results from processing in the cell. The term also encompasses naturally occurring variants of IL-2Ra , e.g. splice variants or allelic variants. In certain embodiments IL-2Ra is human IL-2Ra. The amino acid sequence of an exemplary human IL-2Ra is shown in SEQ ID NO: 411 .

[0367] The term “IL-2Rp” or “p-subunit of the IL-2 receptor”, also known as CD122 or p70, refers to any native IL-2Rp from any vertebrate source, including mammals such as primates (e.g. humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses “full-length”, unprocessed IL-2Rp as well as any form of IL-2Rp that results from processing in the cell. The term also encompasses naturally occurring variants of IL-2Rp, e.g. splice variants or allelic variants. In certain embodiments IL-2Rp is human IL-2Rp. The amino acid sequence of an exemplary human IL-2Rp is shown SEQ ID NO: 412.

[0368] The term “IL-2Ry” or “y-subunit of the IL-2 receptor”, also known as common cytokine receptor y-subunit, common y-chain, yc, or CD132, refers to any native IL-2Ry from any vertebrate source, including mammals such as primates (e.g. humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses “full-length”, unprocessed IL-2Ry as well as any form of IL-2Ry that results from processing in the cell. The term also encompasses naturally occurring variants of IL-2Ry, e.g. splice variants or allelic variants. In certain embodiments IL-2Ry is human IL-2Ry. The amino acid sequence of an exemplary human IL-2Ry is shown in SEQ ID NO: 413.

[0369] Different associations of the individual IL-2R subunits IL-2Ra, IL-2Rp and IL-2Ry, can produce three IL- 2R forms that differ in their affinity to IL-2. The term “high-affinity IL-2 receptor” as used herein refers to the heterotri meric form of the IL-2 receptor, consisting of the receptor y-subunit (also known as IL-2Ry, common cytokine receptor y-subunit, yc, or CD132), the receptor p-subunit (also known as IL-2Rp, CD122 or p70) and the receptor a-subunit (also known as IL-2Ra, CD25 or p55). The term “intermediateaffinity IL-2 receptor” by contrast refers to the heterodimeric form of the IL-2 receptor including only the y- subunit and the p-subunit, without the a-subunit (for a review see e.g. Olejniczak and Kasprzak, Med Sci Monit 14, RA179-189 (2008)). The term “low affinity IL-2R” as used herein refers to the monomeric form of the IL-2R, consisting solely of IL-2Ra (for a review see e.g. Olejniczak and Kasprzak, Med Sci Monit 14, RA179-189 (2008)). The term “IL-2 receptor complex” as used herein refers to the high-affinity IL-2 receptor or the intermediate-affinity IL-2 receptor.

[0370] As used herein, the terms “first”, “second”, “third” or “fourth” with respect to antigen binding molecules, epitopes, polypeptides etc., are used for convenience of distinguishing when there is more than one of each type of moiety. Use of these terms is not intended to confer a specific order or orientation of the moiety unless explicitly so stated.

[0371] The term “antigen binding molecule” as used herein refers to a polypeptide molecule (composed of one or more polypeptide chains) that is capable of binding to an antigen. A binding molecule may be derived from an antibody, and typically comprises an antigen binding domain. An “antigen binding molecule according to the invention” refers to an antigen binding molecule comprised in the cytokine receptor agonist according to the invention.

[0372] The term “antibody” herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g. bispecific antibodies), heavy-chain antibodies, antibody fragments and antigen binding molecules so long as they exhibit the desired antigen-binding activity.

[0373] The term “heavy chain antibody” and “heavy chain-only antibody” and “HCAb” as used herein refer to antibodies devoid of light chains.

[0374] The terms “full-length antibody,” “intact antibody,” and “whole antibody” are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure.

[0375] An ’’antibody fragment” refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, cross-Fab, Fab', Fab’-SH, F(ab')2, diabodies, linear antibodies, single-chain antibody molecules (e.g. scFv and scFab), single-domain antibodies, and multispecific antibodies formed from antibody fragments. For a review of certain antibody fragments, see Hollinger and Hudson, Nature Biotechnology 23:1126-1136 (2005).

[0376] A "single-domain antibody" refers to an antibody fragment consisting of a single monomeric antibody variable domain such as VHHs, nanobodies, VNARs derived from sharks, autonomous VH domains or autonomous VL domains. Single-domain antibodies provide an antigen-binding site which specifically binds to an epitope, i.e. the antigen binding-site is formed solely by the single-domain antibody.

[0377] A “VHH” or “VHH domain” or “nanobody” refers to a single-domain antibody derived from the variable domains of heavy chain antibodies from camelids, e.g. camel, dromedary, llama, alpaca, etc. (See Nguyen V.K. et al., 2000, The EMBO Journal, 19, 921-930; Muyldermans S., 2001 , J Biotechnol., 74, 277-302 and for review Vanlandschoot P. et al., 2011 , Antiviral Research 92, 389-407). The antigenbinding site of VHHs is devoid of light chain variable domain. A VHH domain may be humanized.

[0378] An “antigen binding domain” as used herein refers to a domain that specifically binds to a target antigen. For example, a “PD-1 binding domain” refers to a domain that specifically binds to PD-1 , an “IL2Ry- binding domain” refers to a domain that specifically binds to IL2Ry, and an “IL2Rp binding domain” refers to a domain that specifically binds to IL2Rp. The term in particular refers to an antigen binding domain of an antibody, i.e. the part that comprises the area which binds to and is complementary to part or all of an antigen. Accordingly, in particular aspects, an antigen binding domain herein is an antigen binding domain of an antibody. Such an antigen binding domain may be provided by an antibody fragment, for example by a Fab molecule, a single-chain antibody molecule or single-domain antibodies, such as a VHH domain. The term “antigen binder” is used interchangeably with “antigen binding domain”.

[0379] As used herein, “specifically binds to a target antigen” is used to mean the antigen binding domain binds the target antigen with a higher affinity than a non-specific partner such as Bovine Serum Albumin (BSA, Genbank accession no. CAA76847, version no. CAA76847.1 Gl:3336842, record update date: Jan 7, 2011 02:30 PM). In some embodiments, the antigen binding domain binds the target antigen with an association constant (Ka) at least 2, 3, 4, 5, 10, 20, 50, 100, 200, 500, 1000, 2000, 5000, 104, 105or 106- fold higher than the antigen binding domain’s association constant for BSA, when measured at physiological conditions. The antigen binding domain may bind the target antigen with a high affinity. For example, in some embodiments the antibody can bind the target antigen with a KD equal to or less than about 10-6M, such as 1 x 10-6, 10-7, 10-8, 10-9,10-10, 10-11, 10-12, 10-13or 10-14.

[0380] The term “variable region” or “variable domain” refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to the antigen. The term includes VHH domains. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and complementarity determining regions (CDRs). See, e.g., Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman & Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624- 628 (1991). As used herein in connection with variable region sequences, "Kabat numbering" refers to the numbering system set forth by Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0381] As used herein, the amino acid positions of all constant regions and domains of the heavy and light chain are numbered according to the Kabat numbering system described in Kabat, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), referred to as “numbering according to Kabat” or “Kabat numbering” herein.

[0382] Specifically the Kabat numbering system (see pages 647-660 of Kabat, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991)) is used for the light chain constant domain CL of kappa and lambda isotype and the Kabat EU index numbering system (see pages 661-723) is used for the heavy chain constant domains (CH1 , CH2 and CH3) and for the hinge peptide, which is herein further clarified by referring to “numbering according to Kabat EU index” or “Kabat EU index numbering” in this case. The terms “binding site” or “antigen-binding site” as used herein refers to the site, i.e. one or more amino acid residues, of a binding molecule which provides interaction with the antigen. For example, the antigen binding site of an antigen binding domain comprises amino acid residues from the complementarity determining regions (CDRs). An antigen-binding site may be provided by, for example, one or more variable domains (also called variable regions). In single domain antibodies the antigen-binding site is provided by a single variable domain. In a Fab fragment the antigen-binding site is provided by the VH and VL domains.

[0383] The term “hypervariable region” or “HVR”, as used herein, refers to each of the regions of an antigen binding domain which are hypervariable in sequence and which determine antigen binding specificity, for example “complementarity determining regions” (“CDRs”). Generally, variable domains comprise three CDRs. Thus antibodies comprising a VH and a VL comprise six CDRs; three in the VH (HCDR1 , HCDR2, HCDR3), and three in the VL (LCDR1 , LCDR2, LCDR3). Exemplary CDRs herein include:

[0384] (a) hypervariable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987));

[0385] (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); and

[0386] (c) antigen contacts occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)).

[0387] Unless otherwise indicated, the CDRs are determined according to Kabat et al., supra. One of skill in the art will understand that the CDR designations can also be determined according to Chothia, supra, McCallum, supra, or any other scientifically accepted nomenclature system.

[0388] "Framework" or "FR" refers to variable domain residues other than complementarity determining regions (CDRs). The FR of a variable domain generally consists of four FR domains: FR1 , FR2, FR3, and FR4. Accordingly, the HVR and FR sequences generally appear in the following order in VH and VHH domains (or VL): FR1-HCDR1 (LCDR1)-FR2-HCDR2 (LCDR2)-FR3-HCDR3 (LCDR3)-FR4. Unless otherwise indicated, CDR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al., supra.

[0389] The term “immunoglobulin molecule” herein refers to a protein having the structure of a naturally occurring antibody. For example, immunoglobulins of the IgG class are heterotetrameric glycoproteins of about 150,000 daltons, composed of two light chains and two heavy chains that are disulfide-bonded. From N- to C-terminus, each heavy chain has a variable domain (VH), also called a variable heavy domain or a heavy chain variable region, followed by three constant domains (CH1 , CH2, and CH3), also called a heavy chain constant region. Similarly, from N- to C-terminus, each light chain has a variable domain (VL), also called a variable light domain or a light chain variable region, followed by a constant light (CL) domain, also called a light chain constant region. The heavy chain of an immunoglobulin may be assigned to one of five types, called a (IgA), 5 (IgD), e (IgE), y (IgG), or p (IgM), some of which may be further divided into subtypes, e.g. y1 (IgG 1 ), y2 (lgG2), y3 (lgG3), y4 (lgG4), a1 (lgA1) and a2 (lgA2). The light chain of an immunoglobulin may be assigned to one of two types, called kappa (K) and lambda (A), based on the amino acid sequence of its constant domain. An immunoglobulin essentially consists of two Fab molecules and an Fc region, linked via the immunoglobulin hinge region.

[0390] A “heavy chain constant region” or “CH region” comprises CH1 , hinge, CH2 and CH3. A CH region may be, or may be derived from, the heavy chain constant sequence of an IgG (e.g. IgG 1 , lgG2, lgG3, lgG4), IgA (e.g. lgA1 , lgA2), IgD, IgE or IgM. An example of a CH region is human immunoglobulin G1 constant (IGHG1 ; UniProt: P01857-1 , v1 ; SEQ ID NO: 450). Positions 1 to 98 of SEQ ID NO: 450 (EU numbering 118 to 215) form the CH1 domain (SEQ ID NO: 451). Positions 99 to 113 of SEQ ID NO: 450 (EU numbering 216 to 230) form a hinge peptide between CH1 and CH2 domain (SEQ ID NO: 42). Positions 114 to 223 of SEQ ID NO: 450 (EU numbering 231 to 340) form the CH2 domain (SEQ ID NO: 452). Positions 224 to 330 of SEQ ID NO: 450 (EU numbering 341 to 447) form the CH3 domain (SEQ ID NO: 453).

[0391] The term “Fc region” as used herein refers to a molecule comprising two Fc region subunits. An “Fc region subunit” as used herein refers to one of the two polypeptides forming the dimeric Fc region, i.e. a polypeptide comprising the C-terminal constant domains (CH2 and CH3) of an immunoglobulin heavy chain, capable of stable self-association. An Fc region thus comprises a first Fc region subunit comprising a CH2 domain and a CH3 domain and a second Fc region subunit comprising a CH2 domain and a CH3 domain, capable of stable self-association. For example, an IgG Fc region may comprise a first Fc region subunit comprising an IgG CH2 domain and an IgG CH3 domain and a second Fc region subunit comprising an IgG CH2 domain and an IgG CH3 domain, capable of stable self-association. Where an “Fc region subunit” is comprised within a polypeptide described from N-terminus to C-terminus, the Fc region subunit is included in its conventional orientation: the CH2 domain will be N-terminal to the CH3 domain. Similarly, where an “Fc region” is comprised within a molecule described from N-terminus to C- terminus, the Fc region will be included in its conventional orientation: the CH2 domains of each Fc region subunit will be N-terminal to the CH3 domains of each Fc region subunit. The term “Fc region subunit” as used herein does not include the immunoglobulin hinge peptide and the term “Fc region” as used herein does not include the immunoglobulin hinge region. In some embodiments, the Fc region subunit consists of a CH2 domain and CH3 domain. The term “Fc region” includes native Fc regions and variant Fc regions.

[0392] A “modification promoting the association of the first and the second subunit of the Fc region” is a manipulation of the peptide backbone or the post-translational modifications of an Fc region subunit that reduces or prevents the association of a polypeptide comprising the Fc region subunit with an identical polypeptide to form a homodimer. A modification promoting association as used herein preferably includes separate modifications made to each of the two Fc region subunits desired to associate (i.e. the first and the second subunit of the Fc region), wherein the modifications are complementary to each other so as to promote association of the two Fc region subunits. For example, a modification promoting association may alter the structure or charge of one or both of the Fc region subunits so as to make their association sterically or electrostatically favourable, respectively. Thus, (hetero)dimerization occurs between a polypeptide comprising the first Fc region subunit and a polypeptide comprising the second Fc region subunit, which may be non-identical in the sense that further components fused to each of the subunits (e.g. antigen binding domains) are not the same. In one embodiment, the modification promoting the association of the first and the second subunit of the Fc region comprises an amino acid mutation in the Fc region, specifically an amino acid substitution. In particular embodiments, the modification promoting the association of the first and the second region of the Fc region comprises a separate amino acid mutation, specifically an amino acid substitution, in each of the two subunits of the Fc region.

[0393] One heterodimerization approach known in the art is the so-called “knobs-into-holes” technology, which is described in detail providing several examples in e.g. WO 96 / 027011 , Ridgway, J.B., et al., Protein Eng. 9 (1996) 617-621 ; Merchant, A.M., et al., Nat. Biotechnol. 16 (1998) 677-681 ; and W098 / 050431 . In the “knobs-into-holes” technology, within the interface formed between two CH3 domains in the tertiary structure of the antibody, particular amino acids on each CH3 domain are engineered to produce a protuberance (“knob”) in one of the CH3 domains and a cavity (“hole”) in the other one of the CH3 domains, respectively. In the tertiary structure of the antibody the introduced protuberance in the one CH3 domain is positionable in the introduced cavity in the other CH3 domain.

[0394] The term “effector functions” refers to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell- mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen presenting cells, down regulation of cell surface receptors (e.g. B-cell receptor), and B-cell activation.

[0395] An “activating Fc receptor” is an Fc receptor that following engagement by an Fc region of an antibody elicits signaling events that stimulate the receptor-bearing cell to perform effector functions. Human activating Fc receptors include FcyRllla (CD16a), FcyRI (CD64), FcyRlla (CD32), and FcaRI (CD89).

[0396] Antibody-dependent cell-mediated cytotoxicity (ADCC) is an immune mechanism leading to the lysis of antibody-coated target cells by immune effector cells. The target cells are cells to which antibodies or derivatives thereof comprising an Fc region specifically bind, generally via the protein part that is N- terminal to the Fc region. As used herein, the term “reduced ADCC” is defined as either a reduction in the number of target cells that are lysed in a given time, at a given concentration of antibody in the medium surrounding the target cells, by the mechanism of ADCC defined above, and / or an increase in the concentration of antibody in the medium surrounding the target cells, required to achieve the lysis of a given number of target cells in a given time, by the mechanism of ADCC. The reduction in ADCC is relative to the ADCC mediated by the same antibody produced by the same type of host cells, using the same standard production, purification, formulation and storage methods (which are known to those skilled in the art), but that has not been engineered. For example, the reduction in ADCC mediated by an antibody comprising in its Fc region an amino acid substitution that reduces ADCC, is relative to the ADCC mediated by the same antibody without this amino acid substitution in the Fc region. Suitable assays to measure ADCC are well known in the art (see e.g. PCT publication no. WO 2006 / 082515 or PCT publication no. WO 2012 / 130831).

[0397] “Reduced binding”, for example reduced binding to an Fc receptor, refers to a decrease in affinity for the respective interaction, as measured for example by SPR. For clarity, the term includes also reduction of the affinity to zero (or below the detection limit of the analytic method), i.e. complete abolishment of the interaction. Conversely, “increased binding” refers to an increase in binding affinity for the respective interaction.

[0398] An “immunoglobulin hinge region” or “hinge region” as used herein refers to the region located between the CH1 and CH2 domains in a conventional antibody, or a region corresponding thereto in a non- conventional antibody (i.e. the region N-terminal to the CH2 domain of the Fc region). As used herein, the hinge region is not a part of the Fc region. In a conventional antibody, the hinge region links the CH1 domain to the CH2 domain. In a conventional antibody, the immunoglobulin hinge region typically comprises two immunoglobulin hinge peptides, one in each heavy chain of the antibody, each fusing a respective Fc region subunit to its respective antigen binding arm. Unless otherwise defined, the immunoglobulin hinge region of an antigen binding molecule according to the invention comprises two immunoglobulin hinge peptides, one in each heavy chain of the antibody, fusing a respective Fc region subunit to its respective antigen binding arm (insofar as the antigen binding arm is present).

[0399] An “immunoglobulin hinge peptide” or “hinge peptide” is derived from the hinge region found in a conventional antibody. In a conventional antibody, the two heavy chains are connected in the hinge region by a number of disulfide bonds. In both lgG1 and lgG4 antibodies, the two heavy chains are connected in the hinge region by two inter-chain disulfide bonds: 2 for IgG 1 , 2 for lgG4, 4 for lgG2 and 11 for lgG3. (see Liu H, May K. Disulfide bond structures of IgG molecules: structural variations, chemical modifications and possible impacts to stability and biological function. MAbs. 2012 Jan-Feb;4(1):17-23. doi: 10.4161 / mabs.4.1.18347. PMID: 22327427; PMCID: PMC3338938.) That is, the hinge peptide of an lgG1 antibody and of an lgG4 antibody comprises 2 cysteine residues capable of forming an inter-chain disulfide bond, the hinge peptide of an lgG2 antibody comprises 4 cysteine residues capable of forming an inter-chain disulfide bond, and the hinge peptide an lgG3 antibody comprises 11 cysteine residues capable of forming an inter-chain disulfide bond.

[0400] An exemplary hinge peptide consists of the sequence EPKSCDKTHTCPPCP (SEQ ID NO: 42), which corresponds to the hinge peptide between the CH1 and CH2 domains in the human immunoglobulin G1 constant region (IGHG1 ; UniProt: P01857-1 , v1) at positions 216-230 (EU numbering). A exemplary hinge peptides include the hinge peptide between the CH1 and CH2 domains in the human immunoglobulin G2 constant region at positions 216-230 (EU numbering), in the human immunoglobulin G3 constant region at positions 216-228 (EU numbering) or in the human immunoglobulin G4 constant region at positions 216-230 (EU numbering).

[0401] Other exemplary hinge peptides, also referred to herein as “truncated immunoglobulin hinge peptides” or “truncated hinge peptides”, lack one or more amino acids from the N-terminal hinge peptide found in the hinge region of a conventional antibody. For example, a truncated hinge peptide may consist of DKTHTCPPCP (SEQ ID NO: 43).

[0402] As used herein, the “N-terminus” or “C-terminus” of a “region” or a “molecule” refers to the N-terminus or C-terminus, respectively, of at least one (poly)peptide comprised in that region or molecule. For example, the “N-terminus” or “C-terminus” of a “hinge region” refers to the N-terminus or C-terminus, respectively, of at least one of the two hinge peptides making up that hinge region. The “N-terminus” or “C-terminus” of a “Fc region” refers to the N-terminus or C-terminus, respectively, of at least one of the two Fc region subunits that make up the Fc region. The “N-terminus” of a PD-1 binding Fab molecule refers to the N- terminus of at least one of the VH domain and VL domain comprised in that molecule. The “C-terminus” of a PD-1 binding Fab molecule refers to the C-terminus of at least one of the CH1 domain and CL domain comprised in that molecule. For example, reference to a hinge region being fused at its N- terminus to the C-terminus of a PD-1 binding Fab means that at least one of the two hinge peptides that make up the hinge region is fused at its N-terminus to the C-terminus of at least one of the CH1 domain or CL domain in the PD-1 binding Fab. Similarly, refence to Fc region being fused at its N-terminus to the C-terminus of a hinge region means that at least one of two Fc region subunits that make up the Fc region is fused at its N-terminus to the C-terminus of at least one of the two hinge peptides that make up the hinge region.

[0403] The “class” of an antibody or immunoglobulin refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., lgG1 , lgG2, lgG3, lgG4, lgA1 , and lgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called a, 5, e, y, and p, respectively.

[0404] A “Fab molecule” or “Fab” or “Fab fragment” refers to a protein consisting of the VH and CH1 domain of the heavy chain (the “Fab heavy chain”) and the VL and CL domain of the light chain (the “Fab light chain”) of an immunoglobulin.

[0405] A “cross-Fab molecule” or “cross-Fab” or “crossover Fab molecule” refers to a Fab molecule, wherein either the variable regions or the constant regions of the heavy and light chain are exchanged. Cross-Fab engineering enables two different chain compositions of a cross-Fab molecules. On the one hand, the variable regions of the Fab heavy and light chain are exchanged, i.e. the cross-Fab molecule comprises a peptide chain composed of the light chain variable region (VL) and the heavy chain constant region (CH1), wherein the CH1 may be fused to an Fc region (for example, via an immunoglobulin hinge region), and a peptide chain composed of the heavy chain variable region (VH) and the light chain constant region (CL). On the other hand, when the constant regions of the Fab heavy and light chain are exchanged, the cross-Fab molecule comprises a peptide chain composed of the heavy chain variable region (VH) and the light chain constant region (CL), wherein the CL may be fused to an Fc region (for example, via an immunoglobulin hinge region), and a peptide chain composed of the light chain variable region (VL) and the heavy chain constant region (CH1). The term “conventional Fab molecule” refers to a Fab molecule composed of a Fab heavy chain comprising a VH and CH1 domain and a Fab light chain comprising a VL and CL domain.

[0406] A “single-chain variable fragment” or “scFv” molecule is a fusion protein of the variable domains of the heavy (VH) and light chain (VL) of an antibody, connected by a linker. In particular, the linker is a short polypeptide of 10 to 25 amino acids and is usually rich in glycine for flexibility, as well as serine or threonine for solubility, and can either connect the N-terminus of the VH with the C-terminus of the VL, or vice versa. This protein retains the specificity of the original antibody, despite removal of the constant regions and the introduction of the linker.

[0407] The term “multispecific” means that a binding molecule (e.g. an antibody, an antigen binding molecule according to the invention, the cytokine receptor agonist according to the invention, etc.) is able to specifically bind to at least two distinct antigens. A multispecific binding molecule (e.g. antibody) can be, for example, a bispecific binding molecule. Typically, a bispecific binding molecule comprises two antigen binding sites, each of which is specific for a different antigens. In certain aspects, the multispecific (e.g. bispecific) binding molecule is capable of simultaneously binding two antigens, particularly two antigens expressed on the same cell, on neighbouring cells, or cells in the same tissue. Both the cytokine receptor agonist according to the invention having the split format and the cytokine receptor agonist according to the invention having the all-in-one format are multispecific.

[0408] The term “valent” as used herein denotes the presence of a specified number of antigen binding sites in a binding molecule. As such, the term “monovalent binding to an antigen” denotes the presence of one (and not more than one) antigen binding site specific for the antigen in the binding molecule.

[0409] As used herein, the term “antigen” refers to a molecule, such as a protein, to which an antigen binding molecule binds. Useful antigens can be found, for example, on the surfaces of tumor cells, on the surfaces of tumor stroma cells, on the surfaces of virus-infected cells, on the surfaces of other diseased cells, on the surface of immune cells, free in blood serum, and / or in the extracellular matrix (ECM). In particular aspects, the antigen is a human protein, for example human PD-1 .

[0410] As used herein, the term “epitope” refers to a site on an antigen to which an antigen-binding site binds. Epitopes can be formed from a contiguous stretch of amino acids or a conformational configuration made up of different regions of non-contiguous amino acids. Epitopes often include chemically active surface groupings of antigens such as amino acids, glycan side chains, phosphoryl, or sulfonyl, and may have specific three dimensional structural characteristics, and / or specific charge characteristics. Two distinct antigen-binding domains capable of binding the same antigen may bind different epitopes of said antigen. Such distinct antigen-binding domains are said to not compete for binding if both antigen-binding domains can simultaneously bind to the antigen. In this case two distinct antigen binding domains are noncompeting. Alternatively, antigen-binding domains may compete for binding, i.e. show competitive binding. This may be due to partially overlapping epitopes or simultaneous binding of the two antigen binding domains is hampered due to steric hindrance. Assays to determine whether antigen-binding domains show non-competitive or competitive binding are well known in the art, for example competitive binding analysis using ELISA, RIA, surface plasmon resonance, flow cytometry or any other quantitative or qualitative antibody-binding assay available in the art.

[0411] “Affinity” refers to the strength of the sum total 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 indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which 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 well- established methods known in the art, including those described herein. A preferred method for measuring affinity is Surface Plasmon Resonance (SPR). Unless indicated otherwise, the “affinity” of a cytokine receptor binding domain (e.g. an IL2Ry binding domain or a IL2Rp binding domain) refers to the affinity of that molecule when tested in an isolated molecule comprising only one cytokine receptor binding domain (rather than as part of the cytokine receptor agonist of the invention). For example, the “affinity” of an IL2Ry binding VHH or a IL2Rp binding VHH may refer to the affinity of that molecule when tested in an anti-L2Ry-Fc molecule or an anti-IL2Rp-Fc molecule, as performed in the examples.

[0412] As used herein, the terms “engineer, engineered, engineering”, are considered to include any manipulation of the peptide backbone or the post-translational modifications of a naturally occurring or recombinant polypeptide or fragment thereof. Engineering includes modifications of the amino acid sequence, of the glycosylation pattern, or of the side chain group of individual amino acids, as well as combinations of these approaches.

[0413] The term “amino acid mutation” as used herein is meant to encompass amino acid substitutions, deletions, insertions, and modifications. Any combination of substitution, deletion, insertion, and modification can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., reduced binding to an Fc receptor, or increased association with another peptide. Amino acid sequence deletions and insertions include amino- and / or carboxy-terminal deletions and insertions of amino acids. Preferred amino acid mutations are amino acid substitutions. For the purpose of altering e.g. the binding characteristics of an Fc region, nonconservative amino acid substitutions, i.e. replacing one amino acid with another amino acid having different structural and / or chemical properties, are particularly preferred. Amino acid substitutions include replacement by non- naturally occurring amino acids or by naturally occurring amino acid derivatives of the twenty standard amino acids (e.g. 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). Amino acid mutations can be generated using genetic or chemical methods well known in the art. Genetic methods may include site directed mutagenesis, PCR, gene synthesis and the like. It is contemplated that methods of altering the side chain group of an amino acid by methods other than genetic engineering, such as chemical modification, may also be useful. Various designations may be used herein to indicate the same amino acid mutation. For example, a substitution from proline at position 329 of the Fc region to glycine can be indicated as 329G, G329, G329, P329G, or Pro329Gly.

[0414] By “fused” is meant that a first and a second component (e.g. a Fab molecule and a hinge peptide, or a hinge peptide and an Fc region subunit) are linked by peptide bonds, either directly or via one or more peptide linkers. The term “fusion point” refers to the site on one component at which said component is fused to another component. For example, an IL2Ry binding domain may be fused at its C-terminus to the N-terminus of the VH domain of the first PD-1 binding domain. Thus, the fusion point on the IL2Ry binding domain is the C-terminus and the fusion point on the first PD-1 binding domain is the N-terminus.

[0415] The term “linker” or “peptide linker” refers to a peptide comprising one or more amino acids, typically about 2 to 30 amino acids. Peptide linkers are known in the art or are described herein. Suitable, non- immunogenic linker peptides are, for example, (G4S)n peptide linkers and (G2SG2)n peptide linkers, wherein G=glycine, S=serine, and “n” is generally a number between 1 and 6. The term “linker” and “peptide linker” does not encompass an antibody domain (e.g. a VH, CH1 , CH2, CH3, VL or CL domain). Thus, where a first domain is “fused” to a second domain via one or more peptide linkers, the first domain and the second domain do not comprise a further domain therebetween.

[0416] “Percent (%) amino acid sequence identity” with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software or the FASTA program package. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. Alternatively, the percent identity values can be generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087 and is described in WO 2001 / 007611.

[0417] Unless otherwise indicated, for purposes herein, % amino acid sequence identity values are generated using the ggsearch program of the FASTA package version 36.3.8c or later with a BLOSUM50 comparison matrix. The FASTA program package was authored by W. R. Pearson and D. J. Lipman (“Improved Tools for Biological Sequence Analysis”, PNAS 85 (1988) 2444-2448), W. R. Pearson (“Effective protein sequence comparison” Meth. Enzymol. 266 (1996) 227-25 258), and Pearson et. al. (Genomics 46 (1997) 24-36) and is publicly available from www.fasta.bioch.virginia.edu / fasta_www2 / fasta_down.shtml or www.ebi.ac.uk / Tools / sss / fasta.

[0418] Alternatively, a public server accessible at fasta.bioch.virginia.edu / fasta_www2 / index.cgi can be used to compare the sequences, using the ggsearch (global protein :protein) program and default options (BLOSUM50; open: -10; ext: -2; Ktup = 2) to ensure a global, rather than local, alignment is performed. Percent amino acid identity is given in the output alignment header.

[0419] A “target antigen” as used herein refers to an antigen presented on the surface of a target cell, for example antigen on a T cell. A “T cell antigen” as used herein refers to any antigen present on the surface of a T lymphocyte. T cell antigens include PD-1 and LAG-3. The target antigen of the first and the second target-binding domains of the cytokine receptor agonist of the invention is PD-1.

[0420] The term “PD-1”, also known as CD279, PD1 or programmed cell death protein 1 , refers to any native PD-1 from any vertebrate source, including mammals such as primates (e.g. humans) non-human primates (e.g. cynomolgus monkeys) and rodents (e.g. mice and rats), in particular to the human protein PD-1 with the amino acid sequence as shown in UniProt (www.uniprot.org) accession no. Q15116.

[0421] A “blocking” PD-1 binding domain as used herein is a binding domain which, when tested as an isolated, monospecific, and monovalent binding molecule, substantially inhibits PD-1 / PD-L1 signalling. For example, levels of PD-1 / PD-L1 signalling may be reduced by at least 2-fold, at least 5-fold, or at least 10- fold in the presence of a “blocking” PD-1 binding domain in its isolated form as compared to in the absence of the “blocking” PD-1 binding domain. A “non-blocking” PD-1 binding domain as used herein is a binding domain which, when tested as an isolated, monospecific, and monovalent binding molecule, does not substantially inhibit PD-1 / PD-L1 signalling. For example, levels of PD-1 / PD-L1 signalling may be substantially unchanged in the presence and absence of a “non-blocking” PD-1 binding domain in its isolated form. PD-1 / PD-L1 signalling may be measured by any suitable assay known in the art, for example the PD-1 / PD-L1 blockade reporter assay (Promega, J1250) used in examples 6.1 .4 and 6.2.4.

[0422] The present inventors have found that a pair of antigen binding domains capable of binding different cytokine receptor subunits of a cytokine receptor complex, hereinafter referred to as cytokine receptorbinding domains, can act as cytokine receptor agonists, i.e. mimic a naturally occurring cytokine. Cytokine receptor agonists are also termed cytokine mimetics. The antibody format of cytokine receptor agonists may provide advantageous properties compared to natural or recombinant cytokines.

[0423] To achieve conditional activation of a cytokine receptor, the pair of cytokine receptor-binding domains were split in two distinct antigen binding molecules, i.e. split molecules or split antibodies. These antigen binding molecules each further comprise an antigen binding domain capable of binding a target antigen (PD-1), hereinafter referred to as PD-1-binding domain. By target-dependent assembly of the pair of cytokine receptor-binding domains, the molecules can act as cytokine mimetics. Using a targetdependent approach further allows to direct the agonistic activity to a site of interest, such as cells or tissues expressing the target. The target-binding domains of the molecules are capable of binding the same target antigen simultaneously, i.e. they bind different epitopes of said antigen and are noncompeting. Such a pair of target-binding domains are also referred to as biparatopic target-binding domains. The assembly of the cytokine receptor-binding domains by the biparatopic target-binding domains allows to target the cytokine receptor-binding domains specifically to a site of interest, i.e. a cell or environment where the target antigen is expressed, to act as cytokine mimetics. The pair of split antigen binding molecules are thus a biparatopic pair of antigen binding molecules. The split format of the cytokine receptor agonist according to the invention provides two separate and different antigen binding molecules, wherein the first antigen binding molecule comprises a IL2Ry binding domain or the IL2Rp binding domain, a first PD-1 binding domain, and a first Fc region, wherein the second antigen binding molecule comprises the other of the IL2Ry binding domain or the IL2Rp binding domain, a second PD-1 binding domain, and a second Fc region, and wherein the first and second PD-1 binding domains do not compete for binding on PD-1 .

[0424] Separately, the pair of cytokine receptor-binding domains and target-binding domains were incorporated into a single molecule, hereinafter referred to as the “all-in-one” format. Target-independent assembly of the cytokine receptor-binding domains, i.e. assembly in absence of the target antigen, is prevented by distancing the cytokine receptor-binding domains from each other. While in presence of the target antigen, biparatopic assembly of the cytokine receptor-binding domains results in activation of the cytokine receptor. The all-in-one format of the cytokine receptor agonist according to the invention provides a single antigen binding molecule comprising: i) a first PD-1 binding domain capable of binding a first epitope on PD-1 , ii) a second PD-1 binding domain capable of binding a second epitope on PD-1 , iii) an IL2Ry binding domain, iv) an IL2Rp binding domain, and v) an Fc region, wherein the first and second PD-1 binding domains do not compete for binding on PD-1 .

[0425] IL2-R binding domains

[0426] According to the invention, the cytokine receptor agonist comprises a IL2Ry binding domain and a IL2Rp binding domain, which each bind a distinct IL-2 receptor subunit of an IL-2 receptor complex. Thus, the cytokine receptor agonist comprises IL2Ry binding domain capable of binding to IL2Ry and a IL2Rp binding domain capable of binding to IL2Rp. In the “split” format, the IL2Ry binding domain and IL2Rp binding domain are comprised in separate and different antigen binding molecules, whereas in the “all-in- one” format the IL2Ry binding domain and IL2Rp binding domain are comprised in a single antigen binding molecule. IL2Ry and IL2Rp are subunits of an IL-2 receptor complex.

[0427] In some embodiments, the IL2Ry binding domain is an antibody fragment, such as Fv, Fab, scFv, scFab or single-domain antibody. In some embodiments, the IL2Ry binding domain is a Fab molecule. In some embodiments, the IL2Ry binding domain is a single-domain antibody. Preferably, the IL2Ry binding domain is a VHH domain.

[0428] In some embodiments, the IL2Rp binding domain is an antibody fragment, such as Fv, Fab, scFv, scFab or single-domain antibody. In some embodiments, the IL2Rp binding domain is a Fab molecule. In some embodiments, the IL2Rp binding domain is a single-domain antibody. Preferably, the IL2Rp binding domain is a VHH domain.

[0429] In some embodiments, the IL2Ry binding domain comprises a heavy chain complementarity determining region 1 (HCDR1) of SEQ ID NO: 27, a heavy chain complementarity determining region 2 (HCDR2) of SEQ ID NO: 72, 74, 76, 79, 85 or 87, and a heavy chain complementarity determining region 3 (HCDR3) of SEQ ID NO: 29 and the IL2Rp binding domain comprises: a) a heavy chain complementarity determining region 1 (HCDR1) of SEQ ID NO: 17, a heavy chain complementarity determining region 2 (HCDR2) of SEQ ID NO: 18, and a heavy chain complementarity determining region 3 (HCDR3) of SEQ ID NO: 19; or b) a heavy chain complementarity determining region 1 (HCDR1) of SEQ ID NO: 22, a heavy chain complementarity determining region 2 (HCDR2) of SEQ ID NO: 23, and a heavy chain complementarity determining region 3 (HCDR3) of SEQ ID NO: 24.

[0430] In some embodiments, the IL2Ry binding domain is a VHH domain comprising the amino acid sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 81 , SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, or SEQ ID NO: 89 and the IL2Rp binding domain is a VHH domain comprising the amino acid sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 20, SEQ ID NO: 21 , SEQ ID NO: 25 or SEQ ID NO: 26. In some embodiments, the IL2Ry binding domain is a VHH domain comprising the amino acid sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 81 , SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, or SEQ ID NO: 89 and the IL2Rp binding domain is a VHH domain comprising the amino acid sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 20, SEQ ID NO: 21 , SEQ ID NO: 25 or SEQ ID NO: 26. In some embodiments, the IL2Ry binding domain is a VHH domain comprising the amino acid sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 81 , SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, or SEQ ID NO: 89 and the IL2Rp binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 20, SEQ ID NO: 21 , SEQ ID NO: 25 or SEQ ID NO: 26. In some embodiments, the IL2Ry binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 81 , SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, or SEQ ID NO: 89 and the IL2Rp binding domain is a VHH domain comprising the amino acid sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 20, SEQ ID NO: 21 , SEQ ID NO: 25 or SEQ ID NO: 26. In some embodiments, the IL2Ry binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 81 , SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, or SEQ ID NO: 89 and the IL2Rp binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 20, SEQ ID NO: 21 , SEQ ID NO: 25 or SEQ ID NO: 26.

[0431] In some embodiments, the IL2Ry binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 73and the IL2Rp binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, the IL2Ry binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 73and the IL2Rp binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 21. In some embodiments, the IL2Ry binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 73and the IL2Rp binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 25. In some embodiments, the IL2Ry binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 73and the IL2Rp binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 26.

[0432] In some embodiments, the IL2Ry binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 75and the IL2Rp binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, the IL2Ry binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 75and the IL2Rp binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 21. In some embodiments, the IL2Ry binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 75and the IL2Rp binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 25. In some embodiments, the IL2Ry binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 75and the IL2Rp binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 26.

[0433] In some embodiments, the IL2Ry binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 77and the IL2Rp binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, the IL2Ry binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 77and the IL2Rp binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 21. In some embodiments, the IL2Ry binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 77and the IL2Rp binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 25. In some embodiments, the IL2Ry binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 77and the IL2Rp binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 26.

[0434] In some embodiments, the IL2Ry binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 78 and the IL2Rp binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, the IL2Ry binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 78 and the IL2Rp binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 21 . In some embodiments, the IL2Ry binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 78 and the IL2Rp binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 25. In some embodiments, the IL2Ry binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 78 and the IL2Rp binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 26.

[0435] In some embodiments, the IL2Ry binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 80 and the IL2Rp binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, the IL2Ry binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 80 and the IL2Rp binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 21 . In some embodiments, the IL2Ry binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 80 and the IL2Rp binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 25. In some embodiments, the IL2Ry binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 80 and the IL2Rp binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, the IL2Ry binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 81 and the IL2Rp binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, the IL2Ry binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 81 and the IL2Rp binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 21 . In some embodiments, the IL2Ry binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 81 and the IL2Rp binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 25. In some embodiments, the IL2Ry binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 81 and the IL2Rp binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 26.

[0436] In some embodiments, the IL2Ry binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 82 and the IL2Rp binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, the IL2Ry binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 82 and the IL2Rp binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 21 . In some embodiments, the IL2Ry binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 82 and the IL2Rp binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 25. In some embodiments, the IL2Ry binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 82 and the IL2Rp binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 26.

[0437] In some embodiments, the IL2Ry binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 83 and the IL2Rp binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, the IL2Ry binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 83 and the IL2Rp binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 21 . In some embodiments, the IL2Ry binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 83 and the IL2Rp binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 25. In some embodiments, the IL2Ry binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 83 and the IL2Rp binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 26.

[0438] In some embodiments, the IL2Ry binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 84 and the IL2Rp binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, the IL2Ry binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 84 and the IL2Rp binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 21 . In some embodiments, the IL2Ry binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 84 and the IL2Rp binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 25. In some embodiments, the IL2Ry binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 84 and the IL2Rp binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 26.

[0439] In some embodiments, the IL2Ry binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 86 and the IL2Rp binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, the IL2Ry binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 86 and the IL2Rp binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 21 . In some embodiments, the IL2Ry binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 86 and the IL2Rp binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 25. In some embodiments, the IL2Ry binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 86 and the IL2Rp binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 26.

[0440] In some embodiments, the IL2Ry binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 88 and the IL2Rp binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, the IL2Ry binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 88 and the IL2Rp binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 21 . In some embodiments, the IL2Ry binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 88 and the IL2Rp binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 25. In some embodiments, the IL2Ry binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 88 and the IL2Rp binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 26.

[0441] In some embodiments, the IL2Ry binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 89 and the IL2Rp binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, the IL2Ry binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 89 and the IL2Rp binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 21 . In some embodiments, the IL2Ry binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 89 and the IL2Rp binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 25. In some embodiments, the IL2Ry binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 89 and the IL2Rp binding domain is a VHH domain comprising the amino acid sequence of SEQ ID NO: 26.

[0442] In some embodiments, the IL2Ry binding domain binds to human IL2Rpy-ECD (for example, P1 AE2657 comprising SEQ ID NOs: 400 and 401) with a KD equal or less than about 1 x 10-7, 10-8, or 10-9as measured by surface plasmon resonance at 25 °C and / or the IL2Rp binding domain binds to human IL2Rpy-ECD (for example, P1 AE2657 comprising SEQ ID NOs: 400 and 401) with a KD equal or less than about 1 x 10-8or 10-9as measured by surface plasmon resonance at 25 °C. Preferably, KD is measured using the method described in Example 5.3.

[0443] In some embodiments, the IL2Ry binding domain binds to cynomolgus IL2Rpy-ECD (for example, P1AF8881 comprising SEQ ID NOs: 402 and 403) with a KD equal or less than about 1 x 10-7, 10-8, or 10-9measured by surface plasmon resonance at 25 °C and / or the IL2Rp binding domain binds cynomolgus IL2Rpy-ECD (for example, P1 AF8881 comprising SEQ ID NOs: 402 and 403) with a KD equal or less than about 1 x 10-7, 10-8, or 10-9(preferably, with a KD equal or less than about 1 x 10-9) measured by surface plasmon resonance at 25 °C. Preferably, KD is measured using the method described in Example 5.3.

[0444] Advantageously, the cytokine receptor agonist of the invention has improved properties for immunotherapy as compared to a wild-type IL-2 polypeptide. In particular, IL-2 demonstrates pharmacological properties that contribute to toxicity but are not essential for efficacy of IL-2. As discussed above, different forms of the IL-2 receptor consist of different subunits and exhibit different affinities for IL-2. The intermediate-affinity IL-2 receptor, consisting of the p and y receptor subunits, is expressed on resting effector cells and is sufficient for IL-2 signalling. The high-affinity IL-2 receptor, additionally comprising the a-subunit of the receptor, is mainly expressed on regulatory T (Treg) cells as well as on activated effector cells where its engagement by IL-2 can promote Treg cell-mediated immunosuppression or activation-induced cell death (AICD), respectively. Thus, without wishing to be bound by theory, reducing or abolishing the affinity of an IL-2R agonist to the a-subunit of the IL-2 receptor as compared to a wild-type IL-2 polypeptide should reduce IL-2R induced downregulation of effector cell function by regulatory T cells and development of tumor tolerance by the process of AICD. On the other hand, maintaining substantially similar affinity to the intermediate-affinity IL-2 receptor, as compared to a wild-type IL-2 polypeptide, should preserve the induction of proliferation and activation of effector cells like NK and T cells.

[0445] In a general sense, the invention provides a cytokine receptor agonist wherein the IL2Ry binding domain and IL2Rp binding domain induce IL-2 signalling via the intermediate-affinity IL-2 receptor. The cytokine receptor agonist does not comprise an IL2Ra binding domain. In some embodiments, the cytokine receptor agonist has abolished or reduced affinity to the a-subunit of the IL-2 receptor, as compared to a wild-type IL-2 polypeptide. In some embodiments, cytokine receptor agonist has abolished or reduced affinity to the a-subunit of the IL-2 receptor, as compared to a wild-type IL-2 polypeptide. In some embodiments, the cytokine receptor agonist does not induce IL-2 signalling via the high-affinity IL-2 receptor. In some embodiments, the cytokine receptor agonist induces reduced levels of IL-2R signalling via the high-affinity IL-2 receptor as compared to a wild-type IL-2 polypeptide.

[0446] PD-1 binding domains

[0447] According to the invention, the cytokine receptor agonist comprises a first PD-1 binding domain and a second PD-1 binding domain. Both the first and the second PD-1 binding domains specifically bind the same target (i.e. the same antigen: PD-1). Yet, the first and the second PD-1 binding domains specifically bind distinct epitopes of said target. The first PD-1 binding domain is capable of binding a first epitope on PD-1 and the second PD-1 binding domain is capable of binding a second epitope on PD-1 . The first and the second target-binding domain are capable of binding the target simultaneously, i.e. the target-binding domains are non-competing domains. The first target-binding domain and the second target-binding domain do not compete for specific binding on the target antigen. In the “split” format, the first PD-1 binding domain and second PD-1 binding domain are comprised in separate and different antigen binding molecules, whereas in the “all-in-one” format the first PD-1 binding domain and second PD-1 binding domain are comprised in a single antigen binding molecule.

[0448] The first PD-1 binding domain and / or second PD-1 binding domain may be an antibody fragment. In one embodiment, the first PD-1 binding domain is an antibody fragment and the second PD-1 binding domain is an antibody fragment. The first PD-1 binding domain and / or second PD-1 binding domain may be a Fv, Fab, scFv, scFab molecule or a single domain antibody. In one embodiment, the first PD-1 binding domain is a Fv, Fab, scFv, scFab molecule or a single domain antibody, and the second PD-1 binding domain is a Fv, Fab, scFv, scFab molecule or a single domain antibody.

[0449] In one embodiment, first PD-1 binding domain and / or second PD-1 binding domain is a Fab molecule. In one embodiment, the first PD-1 binding domain is a Fab molecule and the second PD-1 binding domain is a Fab molecule. In one embodiment, the first PD-1 binding domain comprises a heavy chain variable domain (VHi), a light chain variable domain (VLi), a heavy chain constant domain (CH1 i) and a light chain constant domain (CLi). In one embodiment, the second PD-1 binding domain comprises a heavy chain variable domain (VH2), a light chain variable domain (VL2), a heavy chain constant domain (CHI2) and a light chain constant domain (CL2).

[0450] The first PD-1 binding domain or second PD-1 binding domain may be a cross-Fab. In one embodiment, the first PD-1 binding domain is a cross-Fab. In another embodiment, the second PD-1 binding domain is a cross-Fab. In one embodiment, the first PD-1 binding domain is a Fab molecule, wherein the Fab molecule is a cross-Fab, and the second PD-1 binding domain is a Fab molecule, wherein the Fab molecule is in not a cross-Fab, i.e. a conventional Fab molecule. In one embodiment, the first PD-1 binding domain is Fab molecule, wherein the Fab molecule is a conventional Fab molecule, and the second PD-1 binding domain is a Fab molecule, wherein the Fab molecule is a cross-Fab.

[0451] In one embodiment, the Fab molecule of the first PD-1 binding domain and / or second PD-1 binding domain comprises charged modifications. To reduce mispairing of heavy and light chains from the different Fab molecules and thus to increase the purity and yield of the desired cytokine receptor agonist, in particular the cytokine receptor agonist having the all-in-one format, Fab molecules can contain different charged amino acid substitutions (so-called “charged modifications”). These modifications are introduced in the cross-Fab CH1 and CL domains or in the conventional Fab CH1 and CL domains. In a particular aspect, the Fab molecule is one wherein in the CL domain the amino acid at position 123 (EU numbering) has been replaced by arginine (R) and / or wherein the amino acid at position 124 (EU numbering) has been substituted by lysine (K) and wherein in the CH1 domain the amino acids at position 147 (EU numbering) and / or at position 213 (EU numbering) have been substituted by glutamic acid (E). Preferably, the charged modifications are made in a conventional Fab molecule.

[0452] The charged modifications may be introduced in the CH1 and CL domains of the Fab molecule of the first PD-1 binding domain and / or the Fab molecule of the second PD-1 binding domain. In one embodiment, the first PD-1 binding domain comprises charged modifications. In one embodiment, the second PD-1 binding domain comprises charged modifications. In another embodiment, the first PD-1 binding domain comprises charged modifications and the second PD-1 binding domain comprises a cross-Fab. In a further embodiment, the first PD-1 binding domain comprises a cross-Fab and the second PD-1 binding domain comprises charged modifications.

[0453] In one embodiment, the first PD-1 binding domain comprises a VH of SEQ ID NO: 7 and a VL of SEQ ID NO: 8 and the second PD-1 binding domain comprises a VH of SEQ ID NO: 15 and a VL of SEQ ID NO: 16.

[0454] In some embodiments of an antigen binding molecule according to the invention, both subunits of an Fc region are fused at their N-terminus to an immunoglobulin hinge region, made up of two hinge peptides. Where a cytokine receptor agonist comprises two immunoglobulin hinge peptides, without wishing to be bound by theory, it is expected that cysteine residues at corresponding positions on each linker are capable of forming an inter-chain disulfide bond.

[0455] In embodiments where a CH1 domain of a PD-1 binding Fab molecule is fused to the N-terminus of the CH2 domain of an Fc region subunit, it may be fused via an immunoglobulin hinge peptide. In some embodiments. In some embodiments, the hinge peptide consists of the sequence EPKSCDKTHTCPPCP (SEQ ID NO: 42).

[0456] In some embodiments of an antigen binding molecule according to the invention having the all-in-one format, the CL of a PD-1 binding Fab molecule is fused to the CH2 of the Fc region by a hinge peptide. In some embodiments, the hinge peptide consists of the sequence DKTHTCPPCP (SEQ ID NO: 43).

[0457] In some embodiments of an antigen binding molecule according to the invention having the all-in-one format, the CH1 domain and CL domain of a PD-1 binding Fab molecule are each fused to the N- terminus of the CH2 domain in an Fc region subunit by a hinge peptide. Thus, each subunit of the Fab molecule is fused to each subunit of the Fc region by a hinge peptide. In some embodiments, the hinge peptide between the CH1 domain and the CH2 domain is EPKSCDKTHTCPPCP (SEQ ID NO: 42) and the hinge peptide between the CL domain and the CH2 domain is a truncated hinge peptide, such as DKTHTCPPCP (SEQ ID NO: 43).

[0458] The immunoglobulin hinge peptides used in the cytokine receptor agonist of the invention preferably comprise cysteine residues capable of forming an inter-chain disulfide bond, such that the immunoglobulin hinge region used in the cytokine receptor agonist of the invention connects the heavy chains comprising each hinge peptide by one or more disulfide bonds. For example, where a cytokine receptor agonist having the all-in-one format comprises an immunoglobulin hinge peptide and a truncated immunoglobulin hinge peptide, without wishing to be bound by theory, it is expected that cysteine residues at corresponding positions on each peptide are capable of forming an inter-chain disulfide bond. For example, where the cytokine receptor agonist comprises an IgG 1 hinge peptide consisting of the sequence EPKSCDKTHTCPPCP (SEQ ID NO: 42) and a truncated human lgG1 hinge peptide consisting of the sequence DKTHTCPPCP (SEQ ID NO: 43), the cysteine residues at positions 226 and 229 (EU numbering) of the hinge peptide and the truncated hinge peptide are capable of forming an inter-chain disulfide bond.

[0459] Fc region

[0460] According to the invention, the cytokine receptor agonist comprises one or more Fc regions. The cytokine receptor agonist having the all-in-one format comprises a single antigen binding molecule comprising one Fc region, whereas the cytokine receptor agonist having the split format comprises a first and second antigen binding molecule, each comprising one Fc region (the first and second Fc region, respectively). The term “the Fc region of the antigen binding molecule according to the invention” used hereinbelow refers to (i) the Fc region of the cytokine receptor agonist having the all-in-one format, (ii) the Fc region on the first antigen binding molecule of the cytokine receptor agonist having the split format, and / or (iii) the Fc region on the second antigen binding molecule of the cytokine receptor agonist having the split format. Thus, the below embodiments are applicable to cytokine receptor agonists having the all-in-one format (comprising a single antigen binding molecule) and having the split format (comprising two separate and different antigen binding molecules).

[0461] The Fc region of the antigen binding molecule according to the invention consists of a pair of polypeptide chains: a first Fc region subunit and a second Fc region subunit. The first and second Fc region subunits comprise the CH2 and CH3 domains of an immunoglobulin molecule. For example, the Fc region of an immunoglobulin G (IgG) molecule is a dimer, each subunit of which comprises the CH2 and CH3 IgG heavy chain constant domains. The two subunits of the Fc region are capable of stable association with each other.

[0462] In some embodiments, the Fc region(s) of the antigen binding molecule according to the invention is an IgG Fc region. In particular embodiments, the Fc region is a human IgG Fc region. In particular embodiments, the Fc region is an IgG 1 Fc region, for example a human IgG 1 Fc region, or an lgG4 Fc region, for example a human lgG4 Fc region.

[0463] In more specific embodiments, the Fc region(s) of the antigen binding molecule according to the invention is an lgG4 Fc region comprising an amino acid substitution at position S228 (Kabat EU index numbering), particularly the amino acid substitution S228P. This amino acid substitution reduces in vivo Fab arm exchange of lgG4 antibodies (see Stubenrauch et al., Drug Metabolism and Disposition 38, 84-91 (2010)). In further embodiments, the Fc region is a human Fc region.

[0464] In one embodiment, a human IgG heavy chain Fc region subunit extends from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain. However, antibodies produced by host cells may undergo post-translational cleavage of one or more, particularly one or two, amino acids from the C-terminus of the heavy chain. Therefore, an antibody produced by a host cell by expression of a specific nucleic acid molecule encoding a full-length heavy chain may include the full-length heavy chain, or it may include a cleaved variant of the full-length heavy chain. This may be the case where the final two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, numbering according to Kabat EU index). Therefore, the C-terminal lysine (Lys447), or the C-terminal glycine (Gly446) and lysine (Lys447), of the Fc region subunit may or may not be present. In one embodiment, a heavy chain including an Fc region subunit as specified herein, comprised in a binding molecule according to the invention, comprises an additional C-terminal lysine (K447, numbering according to Kabat EU index). In one embodiment, a heavy chain including an Fc region subunit as specified herein, comprised in a binding molecule according to the invention, comprises a C-terminal glycine residue (G446, numbering according to Kabat EU index) and does not comprise a C-terminal lysine (Lys477). Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991 .

[0465] In some embodiments, the Fc region of the antigen binding molecule according to the invention comprises a modification promoting the association of the first and the second subunit of the Fc region. Fc region modifications promoting heterodimerization are further described hereinbelow.

[0466] In some embodiments, the Fc region of the antigen binding molecule according to the invention comprises one or more amino acid substitution that reduces binding to an Fc receptor and / or effector function. Fc region modifications reducing Fc receptor binding and / or effector function are further described hereinbelow.

[0467] Fc region modifications promoting heterodimerization

[0468] The antigen binding molecule of the cytokine receptor agonist having the all-in-one format comprises two PD-1 binding domains, which may be fused to the first and / or the second Fc region subunit of the Fc region, and which are each fused to one or other of the IL2Ry binding domain and the IL2Rp binding domain. Each of the antigen binding molecules comprised in the cytokine receptor agonist having the split format comprise a PD-1 binding domain which may be fused to the first and / or the second Fc region subunit of the Fc region comprised in said antigen binding molecule and which is fused to one or other of the IL2Ry binding domain and the IL2Rp binding domain. Accordingly, in an antigen binding molecule according to the invention, the two Fc region subunits are typically comprised in two non-identical polypeptide chains.

[0469] Recombinant co-expression of the two non-identical polypeptide chains comprising the two Fc region subunits and subsequent dimerization (to form the Fc region) leads to several possible combinations of the two non-identical polypeptide chains. To improve the yield and purity of an antigen binding molecule according to the invention, it will thus be advantageous to introduce in the Fc region of the antigen binding molecule a modification promoting the association of the desired polypeptides.

[0470] Accordingly, in particular embodiments, the Fc region of an antigen binding molecule according to the invention comprises a modification promoting the association of the first and the second subunit of the Fc region. The site of most extensive protein-protein interaction between the two subunits of a human IgG Fc region is in the CH3 domain. Thus, in one aspect, said modification is in the CH3 domain of the Fc region subunit.

[0471] There exist several approaches for modifications in the CH3 domain of the Fc region in order to enforce heterodimerization, which are well described e.g. in WO 96 / 27011 , WO 98 / 050431 , EP 1870459, WO 2007 / 110205, WO 2007 / 147901 , WO 2009 / 089004, WO 2010 / 129304, WO 2011 / 90754, WO 2011 / 143545, WO 2012058768, WO 2013157954, WO 2013096291 . Typically, in all such approaches the CH3 domain of the first subunit of the Fc region and the CH3 domain of the second subunit of the Fc region are both engineered in a complementary manner so that each CH3 domain (or the heavy chain comprising it) can no longer homodimerize with itself but is forced to heterodimerize with the complementarily engineered other CH3 domain (so that the first and second CH3 domain heterodimerize and no homodimers between the two first or the two second CH3 domains are formed).

[0472] In specific embodiments, said modification promoting the association of the first and the second subunit of the Fc region according to the invention is a so-called “knob-into-hole” modification, comprising a “knob” modification in one of the two subunits of the Fc region and a “hole” modification in the other one of the two subunits of the Fc region. In the following discussion of Fc region modifications promoting heterodimerization, reference is made to a “subunit A” and “subunit B” of the Fc region for brevity. “Subunit A” refers to one of the “first subunit of the Fc region” and “second subunit of the Fc region” and “subunit B” refers to the other of the “first subunit of the Fc region” and “second subunit of the Fc region”.

[0473] The knob-into-hole technology is described e.g. in US 5,731 ,168; US 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001). Generally, the method involves introducing a protuberance (“knob”) at the interface of a first polypeptide and a corresponding cavity (“hole”) in the interface of a second polypeptide, such that the protuberance can be positioned in the cavity so as to promote heterodimer formation and hinder homodimer formation. Protuberances are constructed by replacing small amino acid side chains from the interface of the first polypeptide with larger side chains (e.g. tyrosine or tryptophan).

[0474] Compensatory cavities of identical or similar size to the protuberances are created in the interface of the second polypeptide by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine).

[0475] Accordingly, in preferred embodiments, in the CH3 domain of subunit A of the Fc region of the antigen binding molecule an amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby generating a protuberance within the CH3 domain of subunit A which is positionable in a cavity within the CH3 domain of subunit B of the Fc region, and in the CH3 domain of subunit B an amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby generating a cavity within the CH3 domain of subunit B within which the protuberance within the CH3 domain of subunit A is positionable.

[0476] Preferably said amino acid residue having a larger side chain volume is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W). Preferably said amino acid residue having a smaller side chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T), and valine (V). The protuberance and cavity can be made by altering the nucleic acid encoding the polypeptides, e.g. by site-specific mutagenesis, or by peptide synthesis.

[0477] In specific embodiments, in (the CH3 domain of) the subunit A of the Fc region (the “knobs” subunit) the threonine residue at position 366 is replaced with a tryptophan residue (T366W), and in (the CH3 domain of) subunit B of the Fc region (the “hole” subunit) the tyrosine residue at position 407 is replaced with a valine residue (Y407V). In one case, in subunit B of the Fc region additionally the threonine residue at position 366 is replaced with a serine residue (T366S) and the leucine residue at position 368 is replaced with an alanine residue (L368A) (numberings according to Kabat EU index).

[0478] In yet further embodiments, in subunit A of the Fc region additionally the serine residue at position 354 is replaced with a cysteine residue (S354C) or the glutamic acid residue at position 356 is replaced with a cysteine residue (E356C) (particularly the serine residue at position 354 is replaced with a cysteine residue), and in subunit B of the Fc region additionally the tyrosine residue at position 349 is replaced by a cysteine residue (Y349C) (numberings according to Kabat EU index). Introduction of these two cysteine residues results in formation of a disulfide bridge between the two subunits of the Fc region, further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).

[0479] In particular embodiments, subunit A of the Fc region comprises the amino acid substitutions S354C and T366W, and subunit B of the Fc region comprises the amino acid substitutions Y349C, T366S, L368A and Y407V (numbering according to Kabat EU index).

[0480] Other techniques of CH3-modification for enforcing the heterodimerization are contemplated as alternatives according to the invention and are described e.g. in WO 96 / 27011 , WO 98 / 050431 , EP 1870459, WO 2007 / 110205, WO 2007 / 147901 , WO 2009 / 089004, WO 2010 / 129304, WO 2011 / 90754, WO 2011 / 143545, WO 2012 / 058768, WO 2013 / 157954, WO 2013 / 096291.

[0481] In one case, the heterodimerization approach described in EP 1870459, is used alternatively. This approach is based on the introduction of charged amino acids with opposite charges at specific amino acid positions in the CH3 / CH3 domain interface between the two subunits of the Fc region.

[0482] A particular embodiment for the antigen binding molecule of the invention are amino acid mutations R409D; K370E in one of the two CH3 domains (of the Fc region) and amino acid mutations D399K; E357K in the other one of the CH3 domains of the Fc region (numbering according to Kabat EU index).

[0483] In one embodiment, the antigen binding molecule of the invention comprises amino acid mutation T366W in the CH3 domain of subunit A of the Fc region and amino acid mutations T366S, L368A, Y407V in the CH3 domain of subunit B of the Fc region, and additionally amino acid mutations R409D; K370E in the CH3 domain of subunit A of the Fc region and amino acid mutations D399K; E357K in the CH3 domain of subunit B of the Fc region (numberings according to Kabat EU index).

[0484] In one embodiment, the antigen binding molecule of the invention comprises amino acid mutations S354C, T366W in the CH3 domain of subunit A of the Fc region and amino acid mutations Y349C, T366S, L368A, Y407V in the CH3 domain of subunit B of the Fc region, or said antigen binding molecule comprises amino acid mutations Y349C, T366W in the CH3 domain of subunit A of the Fc region and amino acid mutations S354C, T366S, L368A, Y407V in the CH3 domains of subunit B of the Fc region and additionally amino acid mutations R409D; K370E in the CH3 domain of subunit A of the Fc region and amino acid mutations D399K; E357K in the CH3 domain of subunit B of the Fc region (all numberings according to Kabat EU index).

[0485] In one embodiment, the heterodimerization approach described in WO 2013 / 157953 is used alternatively. In one embodiment, the CH3 domain in subunit A of the Fc region comprises amino acid mutation T366K and the CH3 domain in subunit B of the Fc region comprises amino acid mutation L351 D (numberings according to Kabat EU index). In further aspects, the CH3 domain in subunit A of the Fc region comprises further amino acid mutation L351 K. In further aspects, the CH3 domain in subunit B of the Fc region comprises further an amino acid mutation selected from Y349E, Y349D and L368E (particularly L368E) (numberings according to Kabat EU index).

[0486] In one aspect, the heterodimerization approach described in WO 2012 / 058768 is used alternatively. In one aspect, a CH3 domain in subunit A of the Fc region comprises amino acid mutations L351Y, Y407A and a CH3 domain in subunit B of the Fc region comprises amino acid mutations T366A, K409F. In further aspects, the CH3 domain in subunit B comprises a further amino acid mutation at position T411 , D399, S400, F405, N390, or K392, e.g. selected from a) T411 N, T411 R, T411 Q, T411 K, T411 D, T411 E or T411W, b) D399R, D399W, D399Y or D399K, c) S400E, S400D, S400R, or S400K, d) F405I, F405M, F405T, F405S, F405V or F405W, e) N390R, N390K or N390D, f) K392V, K392M, K392R, K392L, K392F or K392E (numberings according to Kabat EU index). In further aspects, a CH3 domain in subunit A comprises amino acid mutations L351Y, Y407A and a CH3 domain in subunit B comprises amino acid mutations T366V, K409F. In further aspects, a CH3 domain in subunit A comprises amino acid mutation Y407A and a CH3 domain in subunit B comprises amino acid mutations T366A, K409F. In further aspects, the CH3 domain in subunit B further comprises amino acid mutations K392E, T411 E, D399R and S400R (numberings according to Kabat EU index).

[0487] In one embodiment, the heterodimerization approach described in WO 2011 / 143545 is used alternatively, e.g. with the amino acid modification at a position selected from the group consisting of 368 and 409 (numbering according to Kabat EU index).

[0488] In one embodiment, the heterodimerization approach described in WO 2011 / 090762, which also uses the knobs-into-holes technology described above, is used alternatively. In one aspect, a CH3 domain in subunit A of the Fc region comprises amino acid mutation T366W and a CH3 domain in subunit B of the Fc region comprises amino acid mutation Y407A. In one aspect, a CH3 domain in subunit A of the Fc region comprises amino acid mutation T366Y and a CH3 domain in subunit B of the Fc region comprises amino acid mutation Y407T (numberings according to Kabat EU index).

[0489] In one embodiment, the antigen binding molecule or its Fc region is of lgG2 subclass and the heterodimerization approach described in WO 2010 / 129304 is used alternatively. In an alternative embodiment, a modification promoting association of subunits A and B of the Fc region comprises a modification mediating electrostatic steering effects, e.g. as described in PCT publication WO 2009 / 089004. Generally, this method involves replacement of one or more amino acid residues at the interface of the two Fc region subunits by charged amino acid residues so that homodimer formation becomes electrostatically unfavorable but heterodimerization electrostatically favorable. In some such aspects, a CH3 domain in subunit A comprises amino acid substitution of K392 or N392 with a negatively charged amino acid (e.g. glutamic acid (E), or aspartic acid (D), particularly K392D or N392D) and a CH3 domain in subunit B comprises amino acid substitution of D399, E356, D356, or E357 with a positively charged amino acid (e.g. lysine (K) or arginine (R), particularly D399K, E356K, D356K, or E357K, and more particularly D399K and E356K). In further aspects, the CH3 domain in subunit A further comprises amino acid substitution of K409 or R409 with a negatively charged amino acid (e.g. glutamic acid (E), or aspartic acid (D), particularly K409D or R409D). In further aspects, the CH3 domain in subunit A further or alternatively comprises amino acid substitution of K439 and / or K370 with a negatively charged amino acid (e.g. glutamic acid (E), or aspartic acid (D)) (all numberings according to Kabat EU index).

[0490] In one embodiment, the heterodimerization approach described in WO 2007 / 147901 is used alternatively. In one embodiment, a CH3 domain in subunit A of the Fc region comprises amino acid mutations K253E, D282K, and K322D and a CH3 domain in subunit B of the Fc region comprises amino acid mutations D239K, E240K, and K292D (numberings according to Kabat EU index). In one embodiment, the heterodimerization approach described in WO 2007 / 110205 can be used alternatively.

[0491] In one embodiment, subunit A of the Fc region comprises amino acid substitutions K392D and K409D, and subunit B of the Fc region comprises amino acid substitutions D356K and D399K (numbering according to Kabat EU index).

[0492] Fc region modifications reducing Fc receptor binding and / or effector function

[0493] The Fc region confers to the antigen binding molecule according to the invention favourable pharmacokinetic properties, including a long serum half-life which contributes to good accumulation in the target tissue and a favourable tissue-blood distribution ratio. At the same time it may, however, lead to undesirable targeting of the binding molecule to cells expressing Fc receptors rather than to the preferred antigen-bearing cells. Moreover, the co-activation of Fc receptor signalling pathways may lead to cytokine release which may result in excessive activation of cytokine receptors and severe side effects upon systemic administration. Activation of (Fc receptor-bearing) immune cells other than T cells may even reduce efficacy of the pair of antigen binding molecules due to the potential destruction of T cells e.g. by NK cells.

[0494] Accordingly, in particular embodiments, the Fc region of the antigen binding molecule according to the invention exhibits reduced binding affinity to an Fc receptor and / or reduced effector function, as compared to a native IgGi Fc region. In some such embodiments, the Fc region (or the antigen binding molecule comprising said Fc region) exhibits less than 50%, particularly less than 20%, more particularly less than 10% and most particularly less than 5% of the binding affinity to an Fc receptor, as compared to a native IgGi Fc region (or an antigen binding molecule comprising a native IgGi Fc region), and / or less than 50%, particularly less than 20%, more particularly less than 10% and most particularly less than 5% of the effector function, as compared to a native IgGi Fc region (or an antigen binding molecule comprising a native IgGi Fc region). In one embodiment, the Fc region (or the antigen binding molecule comprising said Fc region) does not substantially bind to an Fc receptor and / or induce effector function. In particular embodiments, the Fc receptor is an Fey receptor. In one embodiment, the Fc receptor is a human Fc receptor. In one embodiment, the Fc receptor is an activating Fc receptor. In specific embodiments, the Fc receptor is an activating human Fey receptor, more specifically human FcyRllla, FcyRI or FcyRlla, most specifically human FcyRllla. In one aspect, the effector function is one or more selected from the group of CDC, ADCC, ADCP, and cytokine secretion. In particular embodiments, the effector function is ADCC. In one aspect, the Fc region exhibits substantially similar binding affinity to neonatal Fc receptor (FcRn), as compared to a native IgGi Fc region. Substantially similar binding to FcRn is achieved when the Fc region (or the antigen binding molecule comprising said Fc region) exhibits greater than about 70%, particularly greater than about 80%, more particularly greater than about 90% of the binding affinity of a native IgGi Fc region (or the antigen binding molecule comprising a native IgGi Fc region) to FcRn.

[0495] In certain embodiments, the Fc region is engineered to have reduced binding affinity to an Fc receptor and / or reduced effector function, as compared to a non-engineered Fc region. In particular embodiments, the Fc region of the antigen binding molecule comprises one or more amino acid mutation that reduces the binding affinity of the Fc region to an Fc receptor and / or effector function. Typically, the same one or more amino acid mutation is present in each of the two subunits of the Fc region. In one embodiment, the amino acid mutation reduces the binding affinity of the Fc region to an Fc receptor. In one embodiment, the amino acid mutation reduces the binding affinity of the Fc region to an Fc receptor by at least 2-fold, at least 5-fold, or at least 10-fold. In embodiments where there is more than one amino acid mutation that reduces the binding affinity of the Fc region to the Fc receptor, the combination of these amino acid mutations may reduce the binding affinity of the Fc region to an Fc receptor by at least 10-fold, at least 20-fold, or even at least 50-fold. In one embodiment, the antigen binding molecule(s) comprising an engineered Fc region exhibits less than 20%, particularly less than 10%, more particularly less than 5% of the binding affinity to an Fc receptor as compared to an antigen binding molecule comprising a nonengineered Fc region. In particular embodiments, the Fc receptor is an Fey receptor. In one embodiment, the Fc receptor is a human Fc receptor. In one embodiment, the Fc receptor is an activating Fc receptor. In specific embodiments, the Fc receptor is an activating human Fey receptor, more specifically human FcyRllla, FcyRI or FcyRlla, most specifically human FcyRllla. Preferably, binding to each of these receptors is reduced. In one embodiment, binding affinity to a complement component, specifically binding affinity to C1q, is also reduced. In one embodiment, binding affinity to neonatal Fc receptor (FcRn) is not reduced.

[0496] Substantially similar binding to FcRn, i.e. preservation of the binding affinity of the Fc region to said receptor, is achieved when the Fc region (or the antigen binding molecule comprising said Fc region) exhibits greater than about 70% of the binding affinity of a non-engineered form of the Fc region (or the antigen binding molecule comprising said non-engineered form of the Fc region) to FcRn. The Fc region, or antigen binding molecule(s) of the invention comprising said Fc region, may exhibit greater than about 80% and even greater than about 90% of such affinity. In certain embodiments, the Fc region of the antigen binding molecule(s) is engineered to have reduced effector function, as compared to a nonengineered Fc region. The reduced effector function can include, but is not limited to, one or more of the following: reduced complement dependent cytotoxicity (CDC), reduced antibody dependent cell-mediated cytotoxicity (ADCC), reduced antibody-dependent cellular phagocytosis (ADCP), reduced cytokine secretion, reduced immune complex-mediated antigen uptake by antigen-presenting cells, reduced binding to NK cells, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced direct signalling inducing apoptosis, reduced crosslinking of targetbound antibodies, reduced dendritic cell maturation, or reduced T cell priming. In one embodiment, the reduced effector function is one or more selected from the group of reduced CDC, reduced ADCC, reduced ADCP, and reduced cytokine secretion. In particular embodiments, the reduced effector function is reduced ADCC. In one embodiment, the reduced ADCC is less than 20% of the ADCC induced by a non-engineered Fc region (or a binding molecule comprising a non-engineered Fc region).

[0497] In one embodiment, the amino acid mutation that reduces the binding affinity of the Fc region to an Fc receptor and / or effector function is an amino acid substitution. In one embodiment, the Fc region comprises an amino acid substitution at a position selected from the group of E233, L234, L235, N297, P331 and P329 (numberings according to Kabat EU index). In a more specific embodiment, the Fc region comprises an amino acid substitution at a position selected from the group of L234, L235 and P329 (numberings according to Kabat EU index). In one embodiment, the Fc region comprises the amino acid substitutions L234A and L235A (numberings according to Kabat EU index). In some such embodiments, the Fc region is an IgGi Fc region, particularly a human IgGi Fc region. In one embodiment, the Fc region comprises an amino acid substitution at position P329. In a more specific embodiment, the amino acid substitution is P329A or P329G, particularly P329G (numberings according to Kabat EU index). In one embodiment, the Fc region comprises an amino acid substitution at position P329 and a further amino acid substitution at a position selected from E233, L234, L235, N297 and P331 (numberings according to Kabat EU index). In a more specific embodiment, the further amino acid substitution is E233P, L234A, L235A, L235E, N297A, N297D or P331S. In particular embodiments, the Fc region comprises amino acid substitutions at positions P329, L234 and L235 (numberings according to Kabat EU index). In more particular embodiments, the Fc region comprises the amino acid mutations L234A, L235A and P329G (“P329G LALA”, “PGLALA” or “LALAPG”). Specifically, in particular embodiments, each subunit of the Fc region comprises the amino acid substitutions L234A, L235A and P329G (Kabat EU index numbering), i.e. in each of the first and the second subunit of the Fc region the leucine residue at position 234 is replaced with an alanine residue (L234A), the leucine residue at position 235 is replaced with an alanine residue (L235A) and the proline residue at position 329 is replaced by a glycine residue (P329G) (numbering according to Kabat EU index).

[0498] In some such embodiments, the Fc region is an IgGi Fc region, particularly a human IgGi Fc region. The “P329G LALA” combination of amino acid substitutions almost completely abolishes Fey receptor (as well as complement) binding of a human IgGi Fc region, as described in PCT publication no. WO 2012 / 130831 , which is incorporated herein by reference in its entirety. WO 2012 / 130831 also describes methods of preparing such mutant Fc regions and methods for determining its properties such as Fc receptor binding or effector functions. lgG4 antibodies exhibit reduced binding affinity to Fc receptors and reduced effector functions as compared to IgGi antibodies. Hence, in one embodiment, the Fc region of the binding molecule(s) of the invention is an lgG4 Fc region, particularly a human lgG4 Fc region. In one embodiment, the lgG4 Fc region comprises an amino acid substitution at position S228, specifically the amino acid substitution S228P (numberings according to Kabat EU index). To further reduce its binding affinity to an Fc receptor and / or its effector function, in one embodiment, the lgG4 Fc region comprises an amino acid substitution at position L235, specifically the amino acid substitution L235E (numberings according to Kabat EU index). In one embodiment, the lgG4 Fc region comprises an amino acid substitution at position P329, specifically the amino acid substitution P329G (numberings according to Kabat EU index). In a preferred embodiment, the lgG4 Fc region comprises amino acid substitutions at positions S228, L235 and P329, specifically amino acid substitutions S228P, L235E and P329G (numberings according to Kabat EU index). Such lgG4 Fc region mutants and their Fey receptor binding properties are described in PCT publication no. WO 2012 / 130831 , incorporated herein by reference in its entirety.

[0499] In particular embodiments, the Fc region exhibiting reduced binding affinity to an Fc receptor and / or reduced effector function, as compared to a native IgGi Fc region, is a human IgGi Fc region comprising the amino acid substitutions L234A, L235A and optionally P329G, or a human lgG4 Fc region comprising the amino acid substitutions S228P, L235E and optionally P329G (numberings according to Kabat EU index).

[0500] In certain embodiments, N-glycosylation of the Fc region has been eliminated. In some such embodiments, the Fc region comprises an amino acid mutation at position N297, particularly an amino acid substitution replacing asparagine by alanine (N297A) or aspartic acid (N297D) (numberings according to Kabat EU index).

[0501] In addition to the Fc regions described hereinabove and in PCT publication no. WO 2012 / 130831 , Fc regions with reduced Fc receptor binding and / or effector function also include those with substitution of one or more of Fc region residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Patent No. 6,737,056) (numberings according to Kabat EU index). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called “DANA” Fc mutant with substitution of residues 265 and 297 to alanine (US Patent No. 7,332,581).

[0502] Mutant Fc regions can be prepared by amino acid deletion, substitution, insertion or modification using genetic or chemical methods well known in the art. Genetic methods may include site specific mutagenesis of the encoding DNA sequence, PCR, gene synthesis, and the like. The correct nucleotide changes can be verified for example by sequencing. Binding to Fc receptors can be easily determined e.g. by ELISA, or by Surface Plasmon Resonance (SPR) using standard instrumentation such as a BIAcore instrument (GE Healthcare), and Fc receptors such as may be obtained by recombinant expression. Alternatively, binding affinity of Fc regions or binding molecule(s) comprising an Fc region for Fc receptors may be evaluated using cell lines known to express particular Fc receptors, such as human NK cells expressing Fcyllla receptor.

[0503] Effector function of an Fc region, or a binding molecule comprising an Fc region, can be measured by methods known in the art. Examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Patent No. 5,500,362; Hellstrom et al. Proc Natl Acad Sci USA 83, 7059-7063 (1986) and Hellstrom et al., Proc Natl Acad Sci USA 82, 1499-1502 (1985); U.S. Patent No. 5,821 ,337; Bruggemann et al., J Exp Med 166, 1351-1361 (1987).

[0504] Alternatively, non-radioactive assays may be employed (see, for example, ACTI™ nonradioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA); and CytoTox 96® nonradioactive cytotoxicity assay (Promega, Madison, Wl)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells.

[0505] Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g. in an animal model such as that disclosed in Clynes et al., Proc Natl Acad Sci USA 95, 652-656 (1998).

[0506] In one embodiment, binding of the Fc region to a complement component, specifically to C1q, is reduced. Accordingly, in one embodiment wherein the Fc region is engineered to have reduced effector function, said reduced effector function includes reduced CDC. C1q binding assays may be carried out to determine whether the Fc region, or the binding molecule comprising the Fc region, is able to bind C1q and hence has CDC activity. See e.g., C1q and C3c binding ELISA in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay may be performed (see, for example, Gazzano-Santoro et al., J Immunol Methods 202, 163 (1996); Cragg et al., Blood 101 , 1045-1052 (2003); and Cragg and Glennie, Blood 103, 2738-2743 (2004)).

[0507] FcRn binding and in vivo clearance / half life determinations can also be performed using methods known in the art (see, e.g., Petkova, S.B. et al., Int’l. Immunol. 18(12):1759-1769 (2006); WO 2013 / 120929).

[0508] Fc region modifications increasing binding to human FcRn and / or serum half-life

[0509] In certain embodiments, the Fc region comprises one or more amino acid substitution(s) which increase binding to human FcRn and / or serum half-life of the antibody, e.g. substitutions at positions 252, 254, 256, 428 and / or 434 (Kabat EU numbering of residues). In one aspect, the Fc region comprises substitutions M252Y, S254T and T256E in an Fc region derived from a human IgG 1 Fc region (see, e.g., Dall’Acqua et al. J Biol Chem 281 , 23514-23524 (2006); WO 2002 / 60919). These substitutions may be referred to as “YTE” substitutions and are used in Example 8. In one aspect, the Fc region comprises substitutions M428L and N434S (see e.g. Zalevsky et al. Nat Biotech 28, 157-159 (2010); WO 2009 / 086320). In one aspect, the Fc region comprises substitutions M428L and N434A. of the

[0510] The cytokine receptor agonist according to the present invention may have various molecular configurations, i.e. the domains of the cytokine receptor agonist may be linked to each other in different ways. The cytokine receptor agonist of the invention comprises a first and a second PD-1 binding domain and domains which bind a first and a second IL-2 receptor subunit, wherein the subunits are subunits of the intermediate affinity IL-2 receptor complex (i.e., an IL2Ry binding domain and an IL2Rp binding domain). In the following, in order to distinguish between the domains of the two PD-1 binding domains, the domains of the first PD-1 binding domain are denoted with a subscript 1 (1) and the domains of the second PD-1 binding domain, are denoted with a subscript 2 (2). Unless indicated otherwise, the first and second IL-2 receptor subunits can respectively refer either to (i) the IL2Ry binding domain and the IL2Rp binding domain or (ii) the IL2Rp binding domain and the IL2Ry binding domain.

[0511] Configuration of the antigen binding molecules

[0512] The first and second antigen binding molecules of the cytokine receptor agonist having the split format each comprise a PD-1 binding domain, a IL2 receptor subunit-binding domain and a Fc region. In the following, reference to “the antigen binding molecule(s)” refers to one or both of the first and second antigen binding molecules.

[0513] In some embodiments, the IL2 receptor subunit-binding domain is fused at its C-terminus to the N- terminus of the PD-1 binding domain, and the PD-1 binding domain is fused at its C-terminus to the N- terminus of one of the Fc region subunits. In some embodiments, the PD-1 binding domains of the antigen binding molecules are Fab molecules. Thus, the IL2 receptor subunit-binding domain may be fused to the VH or VL domain of the PD-1 binding domain.

[0514] In one embodiment, the antigen binding molecule(s) comprise a PD-1 binding domain, a IL2 receptor subunit-binding domain and a Fc region, wherein the PD-1 binding domain is a Fab molecule, and wherein the IL2 receptor subunit-binding domain is fused at its C-terminus to the N-terminus of the VH domain of the PD-1 binding domain, and the PD-1 binding domain is fused at its C-terminus to the N- terminus of one of the Fc region subunits. In one embodiment, the antigen binding molecule(s) comprise a PD-1 binding domain, a IL2 receptor subunit-binding domain and a Fc region, wherein the PD-1 binding domain is a Fab molecule, and wherein the IL2 receptor subunit-binding domain is fused at its C-terminus to the N-terminus of the VL domain of the PD-1 binding domain, and the PD-1 binding domain is fused at its C-terminus to the N-terminus of one of the Fc region subunits.

[0515] The IL2 receptor subunit-binding domain may be fused via a peptide linker to the PD-1 binding domain. The peptide linker may consist of 5 amino acids. The peptide linker may consist of an amino acid sequence according to SEQ ID NOs: 33 or 38. Configuration of pairs of antigen binding molecules

[0516] In one embodiment, both antigen binding molecules of the pair of antigen binding molecules comprise a PD-1 binding domain, a IL2 receptor subunit-binding domain and a Fc region, wherein the IL2 receptor subunit-binding domain is fused at its C-terminus to the N-terminus of the PD-1 binding domain, and the PD-1 binding domain is fused at its C-terminus to the N-terminus of one of the Fc region subunits.

[0517] In one embodiment, the pair of antigen binding molecules comprises a first antigen binding molecule comprising a first polypeptide comprising a first Fc region subunit, a second polypeptide comprising in order from the N-terminus to C-terminus a first IL2 receptor subunit-binding domain, VHi, CH1 i and a second Fc region subunit, and a third polypeptide comprising in order from the N-terminus to C-terminus VLi and CLi; and a second antigen binding molecule comprising a first polypeptide comprising a first Fc region subunit, a second polypeptide comprising in order from the N-terminus to C-terminus VH2, CHI2 and a second Fc region subunit; a third polypeptide comprising in order from the N-terminus to C- terminus a second IL2 receptor subunit-binding domain, VL2 and CL2.

[0518] Configuration of the all-in-one format

[0519] The all-in-one antigen binding molecule comprises a first and a second PD-1 binding domain, a first and a second IL2 receptor subunit-binding domain and a first and second Fc region subunit.

[0520] The first and second IL2 receptor subunit-binding domains are distanced from each other. Without wishing to be bound by theory, distancing the first and second IL2 receptor subunit-binding domains from each other prevents PD-1 independent assembly of the IL2 receptor-binding domains, i.e. assembly in absence of PD-1 .

[0521] Preferably, the first and second IL2 receptor subunit-binding domains are separated by an Fc region comprising the first and second Fc region subunit. Without wishing to be bound by theory, the Fc region provides a spacer to distance the first and second IL2 receptor subunit-binding domains and thereby reduces IL2 mimetic activity in the absence of PD-1 assembly. Alternatively, or additionally, the first and second IL2 receptor subunit-binding domains may be separated by, for example, the first PD-1 binding domain and / or the second PD-1 binding domain. In some embodiments, the first and second IL2 receptor subunit-binding domains are distributed to opposite termini of the all-in-one antigen binding molecule.

[0522] In a particular embodiment, one PD-1 binding domain and one IL-2-receptor subunit-binding domain are fused to both of the Fc region subunits and the other PD-1 binding domain and IL-2-receptor subunitbinding domain are fused the other Fc region subunit. The IL2 receptor subunit-binding domains may be fused at their C-terminus to the N-terminus of the PD-1 binding domains. The PD-1 binding domains may be fused at their N-terminus or their C-terminus to the C- or N-terminus, respectively, of one or both of the Fc region subunits.

[0523] In one embodiment the PD-1 binding domains are Fab molecules. Thus, the first PD-1 binding domain may comprise a heavy chain variable domain (VH1), a light chain variable domain (VL1), a heavy chain constant domain (CH11) and a light chain constant domain (CL1) and the second PD-1 binding domain may comprise a heavy chain variable domain (VH2 ), a light chain variable domain (VL2 ), a heavy chain constant domain (CHI 2) and a light chain constant domain (CL2). In one embodiment, the first and / or the second PD-1 binding domain is a cross-Fab molecule. In one embodiment, the first PD-1 binding domain is a cross-Fab molecule. In one embodiment, the second PD-1 binding domain is a cross-Fab molecule. In one embodiment, the antigen binding molecule comprises a first and a second PD-1 binding domain, wherein the first PD-1 binding domain is a cross-Fab molecule and the second PD-1 binding domain is a conventional Fab molecule. In one embodiment, the antigen binding molecule comprises a first and a second PD-1 binding domain, wherein the first PD-1 binding domain is a conventional Fab molecule and the second PD-1 binding domain is a cross-Fab molecule.

[0524] In one embodiment, the antigen binding molecule comprises a first I L-2-receptor subunit-binding domain fused at its C-terminus to the N-terminus of VH2 or VL2 of the second PD-1 binding domain, and the second PD-1 binding domain is fused at its N-terminus of VH2 or VL2 to the C-terminus of the first Fc region subunit, and the second IL2 receptor subunit-binding domain is fused at its C-terminus to the N- terminus of VH1 or VLi of the first PD-1 binding domain, and the first PD-1 binding domain is fused at its C-terminus of CH1 i to the N-terminus of the second Fc region subunit.

[0525] In one embodiment, the antigen binding molecule comprises a first polypeptide comprising in order from the N-terminus to C-terminus a VL1, a CH1 i , a first Fc region subunit, a VH2 and a CHI 2, a second polypeptide comprising in order from the N-terminus to C-terminus a first IL2 receptor subunit-binding domain, a VL2 and a CL2, and a third polypeptide comprising in order from the N-terminus to C-terminus a second IL2 receptor subunit-binding domain, a VH1, a CL1, and a second Fc region subunit.

[0526] The domains of the binding molecule (PD-1 binding domain, IL2 receptor subunit-binding domain and Fc region) may be fused to each other through one or more peptide linker, especially a (G4S)n peptide linker or a (G2SG2)n peptide linker.

[0527] A IL2 receptor subunit-binding domain may be linked to a PD-1-binding domain via a (G4S)i peptide linker (GGGGS; SEQ ID NO: 38), a (G4S)2peptide linker (GGGGSGGGGS; SEQ ID NO: 39), a (G4S)3peptide linker (GGGGSGGGGSGGGGS; SEQ ID NO: 40) or a (G4S)5 peptide linker (GGGGSGGGGSGGGGSGGGGSGGGGS; SEQ ID NO: 41), or via a (G2SG2) 1 peptide linker (GGSGG; SEQ ID NO: 33), a (G2SG2)2peptide linker (GGSGGGGSGG; SEQ ID NO: 34), a (G2SG2) 3 peptide linker (GGSGGGGSGGGGSGG; SEQ ID NO: 35), a (G2SG2)4peptide linker (GGSGGGGSGGGGSGGGGSGG; SEQ ID NO: 36), or a (G2SG2)5peptide linker (GGSGGGGSGGGGSGGGGSGGGGSGG; SEQ ID NO: 37). In some embodiments, the peptide linker is a (G2SG2) 1 peptide linker (SEQ ID NO: 33) or a (G2SG2)5 peptide linker (SEQ ID NO: 37). Most preferably, the peptide linker consists of 5 amino acids, for example the peptide linker is a (G2SG2) 1 peptide linker (GGSGG; SEQ ID NO: 33).

[0528] A PD-1 binding domain may be fused at its N-terminus of its VH or VL to the C-terminus of one of the Fc region subunits via a (G4S)i peptide linker (GGGGS; SEQ ID NO: 38), a (G4S)2 peptide linker (GGGGSGGGGS; SEQ ID NO: 39), a (G4S)3peptide linker (GGGGSGGGGSGGGGS; SEQ ID NO: 40) or a (G4S)5peptide linker (GGGGSGGGGSGGGGSGGGGSGGGGS; SEQ ID NO: 41), or via a (G2SG2) 1 peptide linker (GGSGG; SEQ ID NO: 33), a (G2SG2)2peptide linker (GGSGGGGSGG; SEQ ID NO: 34), a (G2SG2)3peptide linker (GGSGGGGSGGGGSGG; SEQ ID NO: 35), a (G2SG2)4peptide linker (GGSGGGGSGGGGSGGGGSGG; SEQ ID NO: 36), or a (G2SG2)5peptide linker (GGSGGGGSGGGGSGGGGSGGGGSGG; SEQ ID NO: 37). Preferably, the peptide linker consists of 10 amino acids, for example the peptide linker is a (G2SG2) 2 peptide linker (GGSGGGGSGG; SEQ ID NO: 34).

[0529] Compositions, formulations and routes of administration

[0530] In a further aspect, the invention provides compositions comprising any of the cytokine receptor agonists provided herein.

[0531] In a further aspect, the invention provides pharmaceutical compositions comprising any of the cytokine receptor agonists provided herein, e.g., for use in any of the below therapeutic methods. In one embodiment, a pharmaceutical composition comprises any of the cytokine receptor agonists provided herein and a pharmaceutically acceptable carrier. In another embodiment, a pharmaceutical composition comprises any of the cytokine receptor agonists provided herein and at least one additional therapeutic agent, e.g., as described below.

[0532] Further provided is a method of producing a cytokine receptor agonist of the invention in a form suitable for administration in vivo, the method comprising (a) obtaining a cytokine receptor agonist according to the invention, and (b) formulating the cytokine receptor agonist with at least one pharmaceutically acceptable carrier, whereby a preparation of cytokine receptor agonist is formulated for administration in vivo.

[0533] Pharmaceutical compositions of the present invention comprise a therapeutically effective amount of one or more cytokine receptor agonist dissolved or dispersed in a pharmaceutically acceptable carrier. The phrases "pharmaceutical or pharmacologically acceptable" refers to molecular entities and compositions that are generally non-toxic to recipients at the dosages and concentrations employed, i.e. do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate. The preparation of a pharmaceutical composition that contains at least one cytokine receptor agonist and optionally an additional active ingredient will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference. Moreover, for animal (e.g., human) administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards or corresponding authorities in other countries. Preferred compositions are lyophilized formulations or aqueous solutions. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, buffers, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g. antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, antioxidants, proteins, drugs, drug stabilizers, polymers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated.

[0534] The composition may comprise different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether it need to be sterile for such routes of administration as injection. Cytokine receptor agonists of the present invention (and any additional therapeutic agent) can be administered intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrasplenically, intrarenally, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, intratumorally, intramuscularly, intraperitoneally, subcutaneously, subconjunctivally, intravesicularlly, mucosally, intrapericardially, intraumbilically , intraocularally, orally, topically, locally, by inhalation (e.g. aerosol inhalation), injection, infusion, continuous infusion, localized perfusion bathing target cells directly, via a catheter, via a lavage, in cremes, in lipid compositions (e.g. liposomes), or by other method or any combination of the forgoing as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference). Parenteral administration, in particular intravenous injection, is most commonly used for administering polypeptide molecules such as the cytokine receptor agonists of the invention.

[0535] Parenteral compositions include those designed for administration by injection, e.g. subcutaneous, intradermal, intralesional, intravenous, intraarterial intramuscular, intrathecal or intraperitoneal injection. For injection, the cytokine receptor agonists of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer. The solution may contain formulatory agents such as suspending, stabilizing and / or dispersing agents. Alternatively, the cytokine receptor agonists may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use. Sterile injectable solutions are prepared by incorporating the cytokine receptor agonists of the invention in the required amount in the appropriate solvent with various of the other ingredients enumerated below, as required. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and / or the other ingredients. In the case of sterile powders for the preparation of sterile injectable solutions, suspensions or emulsion, the preferred methods of preparation are vacuumdrying or freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered liquid medium thereof. The liquid medium should be suitably buffered if necessary and the liquid diluent first rendered isotonic prior to injection with sufficient saline or glucose. The composition must be stable under the conditions of manufacture and storage, and preserved against the contaminating action of microorganisms, such as bacteria and fungi. It will be appreciated that endotoxin contamination should be kept minimally at a safe level, for example, less that 0.5 ng / mg protein. Suitable pharmaceutically acceptable carriers include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG). Aqueous injection suspensions may contain compounds which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, dextran, or the like. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl cleats or triglycerides, or liposomes.

[0536] Active ingredients may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly- (methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences (18th Ed. Mack Printing Company, 1990). Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the polypeptide, which matrices are in the form of shaped articles, e.g. films, or microcapsules. In particular embodiments, prolonged absorption of an injectable composition can be brought about by the use in the compositions of agents delaying absorption, such as, for example, aluminum monostearate, gelatin or combinations thereof.

[0537] In addition to the compositions described previously, the cytokine receptor agonists may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the cytokine receptor agonists may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

[0538] Pharmaceutical compositions comprising the cytokine receptor agonists of the invention may be manufactured by means of conventional mixing, dissolving, emulsifying, encapsulating, entrapping or lyophilizing processes. Pharmaceutical compositions may be formulated in conventional manner using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries which facilitate processing of the proteins into preparations that can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen. The cytokine receptor agonists may be formulated into a composition in a free acid or base, neutral or salt form. Pharmaceutically acceptable salts are salts that substantially retain the biological activity of the free acid or base. These include the acid addition salts, e.g., those formed with the free amino groups of a proteinaceous composition, or which are formed with inorganic acids such as for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric or mandelic acid. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as for example, sodium, potassium, ammonium, calcium or ferric hydroxides; or such organic bases as isopropylamine, trimethylamine, histidine or procaine. Pharmaceutical salts tend to be more soluble in aqueous and other protic solvents than are the corresponding free base forms.

[0539] Any of the cytokine receptor agonists provided herein may be used in therapeutic methods. Cytokine receptor agonists of the invention can be used as immunotherapeutic agents, for example in the treatment of cancers.

[0540] For use in therapeutic methods, cytokine receptor agonists of the invention would be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners.

[0541] Cytokine receptor agonists of the invention are useful in treating disease states where stimulation of the immune system of the host is beneficial, in particular conditions where an enhanced cellular immune response is desirable. These may include disease states where the host immune response is insufficient or deficient. Disease states for which the cytokine receptor agonists of the invention can be administered comprise, for example, a tumor or infection where a cellular immune response would be a critical mechanism for specific immunity. Specific disease states for which cytokine receptor agonists of the present invention can be employed include cancer, for example renal cell carcinoma or melanoma. The cytokine receptor agonists of the invention may be administered per se or in any suitable pharmaceutical composition.

[0542] In one aspect, cytokine receptor agonists of the invention for use as a medicament are provided. In further aspects, cytokine receptor agonists of the invention for use in treating a disease are provided. In certain embodiments, cytokine receptor agonists of the invention for use in a method of treatment are provided. In one embodiment, the invention provides a cytokine receptor agonist as described herein for use in the treatment of a disease in an individual in need thereof. In certain embodiments, the invention provides a cytokine receptor agonist for use in a method of treating an individual having a disease comprising administering to the individual a therapeutically effective amount of the cytokine receptor agonist. In certain embodiments the disease to be treated is a proliferative disorder. In a preferred embodiment the disease is cancer. In certain embodiments the method further comprises administering to the individual a therapeutically effective amount of at least one additional therapeutic agent, e.g., an anticancer agent if the disease to be treated is cancer. In further embodiments, the invention provides a cytokine receptor agonist for use in stimulating the immune system. In certain embodiments, the invention provides a cytokine receptor agonist for use in a method of stimulating the immune system in an individual comprising administering to the individual an effective amount of the cytokine receptor agonist to stimulate the immune system. An “individual” according to any of the above embodiments is a mammal, preferably a human. “Stimulation of the immune system” according to any of the above embodiments may include any one or more of a general increase in immune function, an increase in T cell function, an increase in B cell function, a restoration of lymphocyte function, an increase in the expression of IL-2 receptors, an increase in T cell responsiveness, an increase in natural killer cell activity or lymphokine-activated killer (LAK) cell activity, and the like.

[0543] In a further aspect, the invention provides for the use of a cytokine receptor agonist of the invention in the manufacture or preparation of a medicament for the treatment of a disease in an individual in need thereof. In one embodiment, the medicament is for use in a method of treating a disease comprising administering to an individual having the disease a therapeutically effective amount of the medicament. In certain embodiments the disease to be treated is a proliferative disorder. In a preferred embodiment the disease is cancer. In one such embodiment, the method further comprises administering to the individual a therapeutically effective amount of at least one additional therapeutic agent, e.g., an anti-cancer agent if the disease to be treated is cancer. In a further embodiment, the medicament is for stimulating the immune system. In a further embodiment, the medicament is for use in a method of stimulating the immune system in an individual comprising administering to the individual an amount effective of the medicament to stimulate the immune system. An “individual” according to any of the above embodiments may be a mammal, preferably a human. “Stimulation of the immune system” according to any of the above embodiments may include any one or more of a general increase in immune function, an increase in T cell function, an increase in B cell function, a restoration of lymphocyte function, an increase in the expression of IL-2 receptors, an increase in T cell responsiveness, an increase in natural killer cell activity or lymphokine-activated killer (LAK) cell activity, and the like.

[0544] In a further aspect, the invention provides a method for treating a disease in an individual, comprising administering to said individual a therapeutically effective amount of a cytokine receptor agonist of the invention. In one embodiment a composition is administered to said individual, comprising the cytokine receptor agonist of the invention in a pharmaceutically acceptable form. In certain embodiments the disease to be treated is a proliferative disorder. In a preferred embodiment the disease is cancer. In certain embodiments the method further comprises administering to the individual a therapeutically effective amount of at least one additional therapeutic agent, e.g., an anti-cancer agent if the disease to be treated is cancer. In a further aspect, the invention provides a method for stimulating the immune system in an individual, comprising administering to the individual an effective amount of a cytokine receptor agonist to stimulate the immune system. An “individual” according to any of the above embodiments may be a mammal, preferably a human. “Stimulation of the immune system” according to any of the above embodiments may include any one or more of a general increase in immune function, an increase in T cell function, an increase in B cell function, a restoration of lymphocyte function, an increase in the expression of IL-2 receptors, an increase in T cell responsiveness, an increase in natural killer cell activity or lymphokine-activated killer (LAK) cell activity, and the like.

[0545] In certain embodiments the disease to be treated is a proliferative disorder, preferably cancer. Nonlimiting examples of cancers include bladder cancer, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, colon cancer, colorectal cancer, rectal cancer, gastric cancer, prostate cancer, blood cancer, skin cancer, squamous cell carcinoma, bone cancer, and kidney cancer. Other cell proliferation disorders that can be treated using a cytokine receptor agonist of the present invention include, but are not limited to neoplasms located in the: abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid), eye, head and neck, nervous system (central and peripheral), lymphatic system, pelvic, skin, soft tissue, spleen, thoracic region, and urogenital system. Also included are pre-cancerous conditions or lesions and cancer metastases. In certain embodiments the cancer is chosen from the group consisting of renal cell cancer, skin cancer, lung cancer, colorectal cancer, breast cancer, brain cancer, head and neck cancer. Similarly, other cell proliferation disorders can also be treated by the cytokine receptor agonist of the present invention. Examples of such cell proliferation disorders include, but are not limited to: hypergammaglobulinemia, lymphoproliferative disorders, paraproteinemias, purpura, sarcoidosis, Sezary Syndrome, Waldenstron's Macroglobulinemia, Gaucher's Disease, histiocytosis, and any other cell proliferation disease, besides neoplasia, located in an organ system listed above A skilled artisan readily recognizes that in many embodiments the cytokine receptor agonist may not provide a cure but may only provide partial benefit. In some embodiments, a physiological change having some benefit is also considered therapeutically beneficial. Thus, in some embodiments, an amount of cytokine receptor agonist that provides a physiological change is considered an "effective amount" or a "therapeutically effective amount". The subject, patient, or individual in need of treatment is typically a mammal, more specifically a human.

[0546] In some embodiments, an effective amount of the cytokine receptor agonist of the invention is administered to a cell. In other embodiments, a therapeutically effective amount of the cytokine receptor agonist of the invention is administered to an individual for the treatment of disease.

[0547] For the prevention or treatment of disease, the appropriate dosage of a cytokine receptor agonist of the invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the route of administration, the body weight of the patient, the type of polypeptide (e.g. cytokine receptor agonist in the split format or the all-in-one format), the severity and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, previous or concurrent therapeutic interventions, the patient's clinical history and response to the cytokine receptor agonist, and the discretion of the attending physician.. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject. Various dosing schedules including but not limited to single or multiple administrations over various time-points, bolus administration, and pulse infusion are contemplated herein.

[0548] A therapeutically effective amount may comprise only a single administration or many administrations over a period of time. Similarly, the cytokine receptor agonist is suitably administered to the patient at one time or over a series of treatments.

[0549] The cytokine receptor agonists of the invention will generally be used in an amount effective to achieve the intended purpose. For use to treat or prevent a disease condition, the cytokine receptor agonists s of the invention, or pharmaceutical compositions thereof, are administered or applied in a therapeutically effective amount. Determination of a therapeutically effective amount is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure provided herein.

[0550] For systemic administration, a therapeutically effective dose can be estimated initially from in vitro assays, such as cell culture assays. A dose can then be formulated in animal models to achieve a circulating concentration range that includes the IC50 as determined in cell culture. Such information can be used to more accurately determine useful doses in humans.

[0551] Initial dosages can also be estimated from in vivo data, e.g., animal models, using techniques that are well known in the art. One having ordinary skill in the art could readily optimize administration to humans based on animal data.

[0552] Dosage amount and interval may be adjusted individually to provide plasma levels of the cytokine receptor agonists which are sufficient to maintain therapeutic effect. Therapeutically effective plasma levels may be achieved by administering multiple doses each day. Levels in plasma may be measured, for example, by HPLC.

[0553] In embodiments of local administration or selective uptake, the effective local concentration of the cytokine receptor agonists may not be related to plasma concentration. One having skill in the art will be able to optimize therapeutically effective local dosages without undue experimentation.

[0554] A therapeutically effective dose of the cytokine receptor agonists described herein will generally provide therapeutic benefit without causing substantial toxicity. Toxicity and therapeutic efficacy of an cytokine receptor agonist can be determined by standard pharmaceutical procedures in cell culture or experimental animals. Cell culture assays and animal studies can be used to determine the LD50 (the dose lethal to 50% of a population) and the ED50 (the dose therapeutically effective in 50% of a population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio LD50 / ED50. Cytokine receptor agonists that exhibit large therapeutic indices are preferred. In one embodiment, the cytokine receptor agonist according to the present invention exhibits a high therapeutic index. The data obtained from cell culture assays and animal studies can be used in formulating a range of dosages suitable for use in humans. The dosage lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon a variety of factors, e.g., the dosage form employed, the route of administration utilized, the condition of the subject, and the like. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See, e.g., Fingl et al., 1975, In: The Pharmacological Basis of Therapeutics, Ch. 1 , p. 1 , incorporated herein by reference in its entirety).

[0555] The attending physician for patients treated with cytokine receptor agonists of the invention would know how and when to terminate, interrupt, or adjust administration due to toxicity, organ dysfunction, and the like. Conversely, the attending physician would also know to adjust treatment to higher levels if the clinical response were not adequate (precluding toxicity). The magnitude of an administered dose in the management of the disorder of interest will vary with the severity of the condition to be treated, with the route of administration, and the like. The severity of the condition may, for example, be evaluated, in part, by standard prognostic evaluation methods. Further, the dose and perhaps dose frequency will also vary according to the age, body weight, and response of the individual patient.

[0556] Other agents and treatments

[0557] The cytokine receptor agonists according to the invention may be administered in combination with one or more other agents in therapy. For instance, a cytokine receptor agonist of the invention may be coadministered with at least one additional therapeutic agent. The term "therapeutic agent” encompasses any agent administered to treat a symptom or disease in an individual in need of such treatment. Such additional therapeutic agent may comprise any active ingredients suitable for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. In certain embodiments, an additional therapeutic agent is an immunomodulatory agent, a cytostatic agent, an inhibitor of cell adhesion, a cytotoxic agent, an activator of cell apoptosis, or an agent that increases the sensitivity of cells to apoptotic inducers. In a particular embodiment, the additional therapeutic agent is an anti-cancer agent, for example a microtubule disruptor, an antimetabolite, a topoisomerase inhibitor, a DNA intercalator, an alkylating agent, a hormonal therapy, a kinase inhibitor, a receptor antagonist, an activator of tumor cell apoptosis, or an antiangiogenic agent.

[0558] Such other agents are suitably present in combination in amounts that are effective for the purpose intended. The effective amount of such other agents depends on the amount of cytokine receptor ag...

Claims

1. Claims:1 . A cytokine receptor agonist comprising: i) a first PD-1 binding domain capable of binding a first epitope on PD-1 , ii) a second PD-1 binding domain capable of binding a second epitope on PD-1 , iii) an IL2Ry binding domain, iv) an IL2Rp binding domain, and v) an Fc region, wherein the first PD-1 binding domain comprises: a heavy chain complementarity determining region 1 (HCDR1) of SEQ ID NO: 1 ; a heavy chain complementarity determining region 2 (HCDR2) of SEQ ID NO: 2; a heavy chain complementarity determining region 3 (HCDR3) of SEQ ID NO: 3; a light chain complementarity determining region 1 (LCDR1) of SEQ ID NO: 4; a light chain complementarity determining region 2 (LCDR2) of SEQ ID NO: 5; and a light chain complementarity determining region 3 (LCDR3) of SEQ ID NO: 6, and wherein the first and second PD-1 binding domains do not compete for binding on PD-1 .

2. The cytokine receptor agonist according to claim 1 , wherein the first PD-1 binding domain comprises (i) a heavy chain variable domain (VH) comprising an amino acid sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 7 and (ii) a light chain variable domain (VL) comprising an amino acid sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence of SEQ ID NO: 8.

3. The cytokine receptor agonist according to any preceding claim, wherein the first PD-1 binding domain comprises (i) a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 7 and (ii) a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 8.

4. The cytokine receptor agonist according to any preceding claim, wherein the second PD-1 binding domain comprises: a heavy chain complementarity determining region 1 (HCDR1) of SEQ ID NO: 9; a heavy chain complementarity determining region 2 (HCDR2) of SEQ ID NO: 10; a heavy chain complementarity determining region 3 (HCDR3) of SEQ ID NO: 11 ; a light chain complementarity determining region 1 (LCDR1) of SEQ ID NO: 12; a light chain complementarity determining region 2 (LCDR2) of SEQ ID NO: 13; and a light chain complementarity determining region 3 (LCDR3) of SEQ ID NO: 14.

5. The cytokine receptor agonist according to any preceding claim, wherein the second PD-1 binding domain comprises (i) a heavy chain variable domain (VH) that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 15and (ii) a light chain variable domain (VL) that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 16.

6. The cytokine receptor agonist according to any preceding claim, wherein the second PD-1 binding domain comprises (i) a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 15 and (ii) a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 16.

7. The cytokine receptor agonist according to any preceding claim, wherein the IL2Ry binding domain comprises a heavy chain complementarity determining region 1 (HCDR1) of SEQ ID NO: 27, a heavy chain complementarity determining region 2 (HCDR2) of SEQ ID NO: 72, and a heavy chain complementarity determining region 3 (HCDR3) of SEQ ID NO: 29.

8. The cytokine receptor agonist according to any preceding claim, wherein the IL2Ry binding domain is a variable heavy domain of heavy chain (VHH) comprising an amino acid sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 73.

9. The cytokine receptor agonist according to any one of claims 1-7, wherein the IL2Ry binding domain comprises the amino acid sequence of SEQ ID NO: 73.

10. The cytokine receptor agonist according to any one of claims 1-7, wherein the IL2Ry binding domain is a variable heavy domain of heavy chain (VHH) comprising an amino acid sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 78.

11. The cytokine receptor agonist according to any one of claims 1-7, wherein the IL2Ry binding domain comprises the amino acid sequence of SEQ ID NO: 78.

12. The cytokine receptor agonist according to any one of claims 1-7, wherein the IL2Ry binding domain is a variable heavy domain of heavy chain (VHH) comprising an amino acid sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 82.

13. The cytokine receptor agonist according to any one of claims 1-7, wherein the IL2Ry binding domain comprises the amino acid sequence of SEQ ID NO: 82.

14. The cytokine receptor agonist according to any one of claims 1-6, wherein the IL2Ry binding domain comprises a heavy chain complementarity determining region 1 (HCDR1) of SEQ ID NO: 27, a heavy chain complementarity determining region 2 (HCDR2) of SEQ ID NO: 74, and a heavy chain complementarity determining region 3 (HCDR3) of SEQ ID NO: 29.

15. The cytokine receptor agonist according to any one of claims 1-6 or 14, wherein the IL2Ry binding domain is a variable heavy domain of heavy chain (VHH) comprising an amino acid sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 75.

16. The cytokine receptor agonist according to any one of claims 1-6 or 14 , wherein the IL2Ry binding domain comprises the amino acid sequence of SEQ ID NO: 75.

17. The cytokine receptor agonist according to any one of claims 1-6 or 14, wherein the IL2Ry binding domain is a variable heavy domain of heavy chain (VHH) comprising an amino acid sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 81 .

18. The cytokine receptor agonist according to any one of claims 1-6 or 14 , wherein the IL2Ry binding domain comprises the amino acid sequence of SEQ ID NO: 81 .

19. The cytokine receptor agonist according to any one of claims 1-6 or 14, wherein the IL2Ry binding domain is a variable heavy domain of heavy chain (VHH) comprising an amino acid sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 89.

20. The cytokine receptor agonist according to any one of claims 1-6 or 14, wherein the IL2Ry binding domain comprises the amino acid sequence of SEQ ID NO: 89.

21. The cytokine receptor agonist according to any one of claims 1-6, wherein the IL2Ry binding domain comprises a heavy chain complementarity determining region 1 (HCDR1) of SEQ ID NO: 27, a heavy chain complementarity determining region 2 (HCDR2) of SEQ ID NO: 76, and a heavy chain complementarity determining region 3 (HCDR3) of SEQ ID NO: 29.

22. The cytokine receptor agonist according to any one of claims 1-6 or 21 , wherein the IL2Ry binding domain is a variable heavy domain of heavy chain (VHH) comprising an amino acid sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 77.

23. The cytokine receptor agonist according to any one of claims 1-6 or 21 , wherein the IL2Ry binding domain comprises the amino acid sequence of SEQ ID NO: 77.

24. The cytokine receptor agonist according to any one of claims 1-6 or 21 , wherein the IL2Ry binding domain is a variable heavy domain of heavy chain (VHH) comprising an amino acid sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 83.

25. The cytokine receptor agonist according to any one of claims 1-6 or 21 , wherein the IL2Ry binding domain comprises the amino acid sequence of SEQ ID NO: 83.

26. The cytokine receptor agonist according to any one of claims 1-6, wherein the IL2Ry binding domain comprises a heavy chain complementarity determining region 1 (HCDR1) of SEQ ID NO: 27, a heavy chain complementarity determining region 2 (HCDR2) of SEQ ID NO: 79, and a heavy chain complementarity determining region 3 (HCDR3) of SEQ ID NO: 29.

27. The cytokine receptor agonist according to any one of claims 1-6 or 26, wherein the IL2Ry binding domain is a variable heavy domain of heavy chain (VHH) comprising an amino acid sequence that is atleast about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 80.

28. The cytokine receptor agonist according to any one of claims 1-6 or 26, wherein the IL2Ry binding domain comprises the amino acid sequence of SEQ ID NO: 80.

29. The cytokine receptor agonist according to any one of claims 1-6 or 26, wherein the IL2Ry binding domain is a variable heavy domain of heavy chain (VHH) comprising an amino acid sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 84.

30. The cytokine receptor agonist according to any one of claims 1-6 or 26, wherein the IL2Ry binding domain comprises the amino acid sequence of SEQ ID NO: 84.

31. The cytokine receptor agonist according to any one of claims 1-6, wherein the IL2Ry binding domain comprises a heavy chain complementarity determining region 1 (HCDR1) of SEQ ID NO: 27, a heavy chain complementarity determining region 2 (HCDR2) of SEQ ID NO: 85, and a heavy chain complementarity determining region 3 (HCDR3) of SEQ ID NO: 29.

32. The cytokine receptor agonist according to any one of claims 1-6 or 31 , wherein the IL2Ry binding domain is a variable heavy domain of heavy chain (VHH) comprising an amino acid sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 86.

33. The cytokine receptor agonist according to any one of claims 1-6 or 31 , wherein the IL2Ry binding domain comprises the amino acid sequence of SEQ ID NO: 86.

34. The cytokine receptor agonist according to any one of claims 1-6, wherein the IL2Ry binding domain comprises a heavy chain complementarity determining region 1 (HCDR1) of SEQ ID NO: 27, a heavy chain complementarity determining region 2 (HCDR2) of SEQ ID NO: 87, and a heavy chain complementarity determining region 3 (HCDR3) of SEQ ID NO: 29.

35. The cytokine receptor agonist according to any one of claims 1-6 or 34, wherein the IL2Ry binding domain is a variable heavy domain of heavy chain (VHH) comprising an amino acid sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 88.

36. The cytokine receptor agonist according to any one of claims 1-6 or 34, wherein the IL2Ry binding domain comprises the amino acid sequence of SEQ ID NO: 88.

37. The cytokine receptor agonist according to any preceding claim, wherein the IL2Ry binding domain comprises a serine at position 74.

38. The cytokine receptor agonist according to any preceding claim, wherein the IL2Rp binding domain comprises a heavy chain complementarity determining region 1 (HCDR1) of SEQ ID NO: 17, a heavy chain complementarity determining region 2 (HCDR2) of SEQ ID NO: 18, and a heavy chain complementarity determining region 3 (HCDR3) of SEQ ID NO: 19.

39. The cytokine receptor agonist according to any preceding claim, wherein the IL2Rp binding domain is a VHH domain comprising an amino acid sequence that is at least about 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 20.

40. The cytokine receptor agonist according to any one of claims 1-38, wherein the IL2Rp binding domain comprises the amino acid sequence of SEQ ID NO: 20.

41. The cytokine receptor agonist according to any one of claims 1-38, wherein the IL2Rp binding domain is a VHH domain comprising an amino acid sequence that is at least about 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 21 .

42. The cytokine receptor agonist according to any one of claims 1-38, wherein the IL2Rp binding domain comprises the amino acid sequence of SEQ ID NO: 21 .

43. The cytokine receptor agonist according to any one of claims 1-37, wherein the IL2Rp binding domain comprises a heavy chain complementarity determining region 1 (HCDR1) of SEQ ID NO: 22, a heavy chain complementarity determining region 2 (HCDR2) of SEQ ID NO: 23, and a heavy chain complementarity determining region 3 (HCDR3) of SEQ ID NO: 24.

44. The cytokine receptor agonist according to any one of claims 1-37 or 43, wherein the IL2Rp binding domain is a VHH domain comprising an amino acid sequence that is at least about 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 25.

45. The cytokine receptor agonist according to any one of claims 1-37 or 43, wherein the IL2Rp binding domain comprises the amino acid sequence of SEQ ID NO: 25.

46. The cytokine receptor agonist according to any one of claims 1-37 or 43, wherein the IL2Rp binding domain is a VHH domain comprising an amino acid sequence that is at least about 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 26.

47. The cytokine receptor agonist according to any one of claims 1-37 or 43, wherein the IL2Rp binding domain comprises the amino acid sequence of SEQ ID NO: 26.

48. The cytokine receptor agonist according to any preceding claim, wherein the cytokine receptor agonist can elicit one or more of the cellular responses selected from the group consisting of: proliferation of activated T lymphocyte cells, differentiation in an activated T lymphocyte cell, cytotoxic T cell (CTL) activity, proliferation of activated B cells, differentiation in an activated B cell, proliferation of natural killer (NK) cells, differentiation in a NK cell, cytokine secretion by an activated T cell or an NK cell, and / or NK / lymphocyte activated killer (LAK) antitumor cytotoxicity.

49. The cytokine receptor agonist according to any preceding claim, wherein the cytokine receptor agonist:(i) has a reduced ability to induce IL-2 signalling in regulatory T cells, compared to a wild-type IL-2 polypeptide;(ii) induces less activation-induced cell death (AICD) in T cells, compared to a wild-type IL-2 polypeptide;(iii) has a reduced toxicity profile in vivo, compared to a wild-type IL-2 polypeptide; and / or(iv) has a prolonged serum half-life, compared to a wild-type IL-2 polypeptide.

50. The cytokine receptor agonist according to any preceding claim, wherein the cytokine receptor agonist induces IL-2 signalling via the intermediate-affinity IL-2 receptor.

51. The cytokine receptor agonist according to any preceding claim, wherein the cytokine receptor agonist:(i) has abolished or reduced affinity to the a-subunit of the IL-2 receptor, compared to a wild-type IL-2 polypeptide; and / or(ii) has substantially similar affinity to the intermediate-affinity IL-2 receptor, compared to a wild-type IL-2 polypeptide.

52. The cytokine receptor agonist according to any preceding claim, wherein the cytokine receptor agonist:(i) does not induce IL-2 signalling via the high-affinity IL-2 receptor, compared to a wild-type IL-2 polypeptide; or(ii) induces reduced levels of IL-2 signalling via the high-affinity IL-2 receptor compared to a wild-type IL-2 polypeptide.

53. The cytokine receptor agonist according to any preceding claim, wherein the first PD-1 binding domain is a Fab molecule comprising a first chain comprising a VH and CH1 domain and a second chain comprising a VL and CL domain.

54. The cytokine receptor agonist according to any one of claims 1-52, wherein the first PD-1 binding domain is a cross-Fab molecule comprising a first chain comprising a VL and a CH1 domain and a second chain comprising a VH and a CL domain.

55. The cytokine receptor agonist according to any preceding claim, wherein the second PD-1 binding domain is a Fab molecule comprising a first chain comprising a VH and CH1 domain and a second chain comprising a VL and CL domain.

56. The cytokine receptor agonist according to any one of claims 1-54, wherein the second PD-1 binding domain is a cross-Fab molecule comprising a first chain comprising a VL and a CH1 domain and a second chain comprising a VH and a CL domain.

57. The cytokine receptor agonist according to any preceding claim, wherein the IL2Ry binding domain is a VHH domain.

58. The cytokine receptor agonist according to any preceding claim, wherein the IL2Rp binding domain is a VHH domain.

59. The cytokine receptor agonist according to any preceding claim, wherein the C-terminus of the IL2Ry binding domain or of the IL2Rp binding domain is fused to the N-terminus of the first PD-1 binding domainand the C-terminus of the other of the IL2Ry binding domain or the IL2Rp binding domain is fused to the N-terminus of the second PD-1 binding domain.

60. The cytokine receptor agonist according to claim 59, wherein the C-terminus of the IL2Ry binding domain is fused to the N-terminus of the first PD-1 binding domain and the C-terminus of the IL2Rp binding domain is fused to the N-terminus of the second PD-1 binding domain.61 . The cytokine receptor agonist according to claim 60, wherein the C-terminus of the IL2Ry binding domain is fused to the N-terminus of the VH domain of the first PD-1 binding domain and the IL2Rp binding domain is fused to the N-terminus of the VL domain of the second PD-1 binding domain.

62. The cytokine receptor agonist according to any one of claims 59-61 , wherein the C-terminus of the IL2Ry binding domain is fused to the N-terminus of the first PD-1 binding domain via a first peptide linker and wherein the C-terminus of the IL2Rp binding domain is fused to the N-terminus of the second PD-1 binding domain via a second peptide linker.

63. The cytokine receptor agonist according to claim 59, wherein the C-terminus of the IL2Rp binding domain is fused to the N-terminus of the first PD-1 binding domain via a first peptide linker and wherein the C-terminus of the IL2Ry binding domain is fused to the N-terminus of the second PD-1 binding domain via a second peptide linker.

64. The cytokine receptor agonist according to any one of claims 59-63, wherein the first peptide linker and second peptide linker each consist of 5 amino acids.

65. The cytokine receptor agonist according to claim 64, wherein the first peptide linker and second peptide linker are each a GGSGG (SEQ ID NO: 33) peptide linker.

66. The cytokine receptor agonist according to any preceding claim, wherein the Fc region consists of a first Fc region subunit and a second Fc region subunit.

67. The cytokine receptor agonist according to any preceding claim, wherein the first Fc region subunit and the second Fc region subunit each consist of, from N-terminus to C-terminus, a CH2 domain and a CH3 domain.

68. The cytokine receptor agonist according to any preceding claim, wherein the Fc region is an IgG Fc region, optionally wherein the Fc region is an lgG1 Fc region.

69. The cytokine receptor agonist according to any preceding claim, wherein the Fc region is a human Fc region, optionally wherein the human Fc region is(i) a human IgG Fc region wherein the first Fc region subunit comprises substitutions M252Y, S254T and T256E;(ii) a human IgG Fc region wherein the second Fc region subunit comprises substitutions M252Y, S254T and T256E; or(iii) a human IgG Fc region wherein the first Fc region subunit comprises substitutions M252Y, S254T and T256E and the second Fc region subunit comprises substitutions M252Y, S254T and T256E.

70. The cytokine receptor agonist according to any preceding claim, wherein the Fc region comprises:(i) a “knob-into-hole” modification; and / or(ii) one or more amino acid substitution that reduces binding to an Fc receptor and / or effector function.

71. The cytokine receptor agonist according to any preceding claim, wherein the cytokine receptor agonist comprises a hinge region, wherein the hinge region is fused at its C-terminus to the N-terminus of the Fc region and wherein the hinge region is fused at its N-terminus to the C-terminus of the first PD-1 binding domain.

72. The cytokine receptor agonist according to claim 71 , wherein the hinge region comprises a first hinge peptide, wherein the first hinge peptide is fused at its C-terminus to the N-terminus of a CH2 domain of a first Fc region subunit.

73. The cytokine receptor agonist according to claim 72, wherein the first hinge peptide consists of the sequence EPKSCDKTHTCPPCP (SEQ ID NO: 42).

74. The cytokine receptor agonist according to any one of claims 72-73, wherein the first hinge peptide is fused at its N-terminus to a CH1 domain of the first PD-1 binding domain.

75. The cytokine receptor agonist according to any one of claims 71-74, wherein the hinge region comprises a second hinge peptide, wherein the second hinge peptide is fused at its C-terminus to the N- terminus of a CH2 domain of a second Fc region subunit.

76. The cytokine receptor agonist according to claim 75, wherein the second hinge peptide is a truncated hinge peptide.

77. The cytokine receptor agonist according to claim 76, wherein the second hinge peptide consists of the sequence DKTHTCPPCP (SEQ ID NO: 43).

78. The cytokine receptor agonist according to any one of claims 75-77, wherein the N-terminus of the second hinge peptide is the N-terminus of the polypeptide chain in which the second hinge peptide is comprised.

79. The cytokine receptor agonist according to any preceding claim, wherein the cytokine receptor agonist further comprises a second Fc region, wherein the IL2Ry binding domain or the IL2Rp binding domain, the first PD-1 binding domain, and the first Fc region form a first antigen binding molecule, and the other of the IL2Ry binding domain or the IL2Rp binding domain, the second PD-1 binding domain, and the second Fc region, form a separate and different second antigen binding molecule.

80. The cytokine receptor agonist according to claim 79, wherein the first antigen binding molecule comprises the IL2Ry binding domain.

81. The cytokine receptor agonist according to any one of claims 79-80, wherein the first PD-1 binding domain is a Fab molecule.

82. The cytokine receptor agonist according to any one of claims 79-81 wherein the second antigen binding molecule comprises the IL2Rp binding domain.

83. The cytokine receptor agonist according to any one of claims 79-82, wherein the second PD-1 binding domain is a Fab molecule.

84. The cytokine receptor agonist according to any one of claims 79-83, wherein the second antigen molecule further comprises a third PD-1 binding domain which binds the second epitope.

85. The cytokine receptor agonist according to claim 84, wherein the second antigen molecule bivalently binds the second epitope.

86. The cytokine receptor agonist according to any one of claims 84-85, wherein (i) the second PD-1 binding domain comprises the HCDR1-3 and LCDR1-3 of SEQ ID NOs: 9-14 and the third PD-1 binding domain comprises the HCDR1-3 and LCDR1-3 of SEQ ID NOs: 9-14.

87. The cytokine receptor agonist according to any one of claims 84-86, wherein the second PD-1 binding domain comprises the VH and VL of SEQ ID NOs: 15 and 16 and the third PD-1 binding domain comprises the VH and VL of SEQ ID NOs: 15 and 16.

88. The cytokine receptor agonist according to any one of claims 84-87, wherein:(i) second PD-1 binding domain is a Fab molecule, optionally wherein the CH1 domain and CL domain of the second PD-1 binding domain comprise charged modifications; and / or(ii) the third PD-1 binding domain is a cross-Fab molecule.

89. The cytokine receptor agonist according to any one of claims 79-88 wherein the second Fc region consists of a first Fc region subunit and a second Fc region subunit.

90. The cytokine receptor agonist according to any one of claims 79-89, wherein the first Fc region subunit of the second Fc region and the second Fc region subunit of the second Fc region each consist of, from N-terminus to C-terminus, a CH2 domain and a CH3 domain.

91. The cytokine receptor agonist according to any one of claims 79-90, wherein the second Fc region is an IgG Fc region.

92. The cytokine receptor agonist according to any one of claims 79-91 , wherein the second Fc region is an lgG1 Fc region.

93. The cytokine receptor agonist according to any one of claims 79-92, wherein the second Fc region is a human Fc region, optionally wherein the human Fc region is(i) a human IgG Fc region wherein the first Fc region subunit comprises substitutions M252Y, S254T and T256E;(ii) a human IgG Fc region wherein the second Fc region subunit comprises substitutions M252Y, S254T and T256E; or(iii) a human IgG Fc region wherein the first Fc region subunit comprises substitutions M252Y, S254T and T256E and the second Fc region subunit comprises substitutions M252Y, S254T and T256E.

94. The cytokine receptor agonist according to any one of claims 79-93, wherein the second Fc region comprises:(i) a “knob-into-hole” modification; and / or(ii) one or more amino acid substitution that reduces binding to an Fc receptor and / or effector function.

95. The cytokine receptor agonist according to any one of claims 79-94, wherein the cytokine receptor agonist comprises a second hinge region, wherein the second hinge region is fused at its C-terminus to the N-terminus of the second Fc region and wherein the second hinge region is fused at its N-terminus to the C-terminus of the second PD-1 binding domain.

96. The cytokine receptor agonist according to claim 95, wherein the second hinge region comprises a third hinge peptide, wherein the third hinge peptide is fused at its C-terminus to the N-terminus of a CH2 domain of the first Fc region subunit of the second Fc region.

97. The cytokine receptor agonist according to claim 96, wherein the third hinge peptide consists of the sequence EPKSCDKTHTCPPCP (SEQ ID NO: 42).

98. The cytokine receptor agonist according to any one of claims 95-97, wherein the third hinge peptide is fused at its N-terminus to the C-terminus of the CH1 domain of the second PD-1 binding domain.

99. The cytokine receptor agonist according to any one of claims 95-98, wherein the hinge region comprises a fourth hinge peptide, wherein the fourth hinge peptide is fused at its C-terminus to the N- terminus of a CH2 domain of the second Fc region subunit of the second Fc region.

100. The cytokine receptor agonist according to claim 99, wherein the fourth hinge peptide is a truncated hinge peptide.

101. The cytokine receptor agonist according to claim 100, wherein the fourth hinge peptide consists of the sequence DKTHTCPPCP (SEQ ID NO: 43).

102. The cytokine receptor agonist according to claim 99, wherein the fourth hinge peptide consists of the sequence EPKSCDKTHTCPPCP (SEQ ID NO: 42).

103. The cytokine receptor agonist according to any one of claims 99-102, wherein the N-terminus of the fourth hinge peptide is the N-terminus of the polypeptide chain in which it is comprised.

104. The cytokine receptor agonist according to any one of claims 99 or 102, wherein the fourth hinge peptide is fused at its N-terminus to the C-terminus of the CH1 domain of the third PD-1 binding domain.

105. The cytokine receptor agonist according to any one of claims 79-83 or 89-104, wherein the cytokine receptor agonist comprises: a first antigen binding molecule comprising(a) a first polypeptide comprising in order from N-terminus to C-terminus: the IL2Ry binding domain, a VH and CH1 of the first PD-1 binding domain, and a first subunit of the first Fc region;(b) a second polypeptide comprising a second subunit of the first Fc region; and(c) a third polypeptide, comprising in order from N-terminus to C-terminus: a VL and CL of the first PD-1 binding domain; and a second antigen binding molecule comprising:(d) a fourth polypeptide comprising in order from N-terminus to C-terminus: a VH and CH1 of the second PD-1 binding domain, and a first subunit of the second Fc region;(e) a fifth polypeptide comprising a second subunit of the second Fc region; and(f) a sixth polypeptide, comprising in order from N-terminus to C-terminus: the IL2Rp binding domain, and a VL and CL of the second PD-1 binding domain.

106. The cytokine receptor agonist according to claim 105, wherein the cytokine receptor agonist comprises:(I) a first antigen binding molecule comprising(a) a first polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 90, SEQ ID NO: 91 , SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, or SEQ ID NO: 101 ;(b) a second polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 44;(c) a third polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 45; and a second antigen binding molecule comprising:(d) a fourth polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 49;(e) a fifth polypeptide comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 44; and / or(f) a sixth polypeptide comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 50, SEQ ID NO: 51 , SEQ ID NO: 52, or SEQ ID NO: 53; or(II) a first antigen binding molecule comprising(a) a first polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 90, SEQ ID NO: 91 , SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, or SEQ ID NO: 101 ;(b) a second polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 44;(c) a third polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 45; and a second antigen binding molecule comprising:(d) a fourth polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 469;(e) a fifth polypeptide comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 468; and / or(f) a sixth polypeptide comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 50, SEQ ID NO: 51 , SEQ ID NO: 52, or SEQ ID NO: 53; or(III) a first antigen binding molecule comprising:(a) a first polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 91 ;(b) a second polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 44;(c) a third polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 45; and a second antigen binding molecule comprising:(d) a fourth polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 49;(e) a fifth polypeptide comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 44; and / or(f) a sixth polypeptide comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 53; or(IV) a first antigen binding molecule comprising:(a) a first polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 91 ;(b) a second polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 44;(c) a third polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 45; and a second antigen binding molecule comprising:(d) a fourth polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 469;(e) a fifth polypeptide comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 468; and / or(f) a sixth polypeptide comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 53.

107. The cytokine receptor agonist according to claim 105, wherein the cytokine receptor agonist comprises:(I) a first antigen binding molecule comprising:(a) a first polypeptide comprising an amino acid sequence of SEQ ID NO: 90, SEQ ID NO: 91 , SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO:98, SEQ ID NO: 99, SEQ ID NO: 100, or SEQ ID NO: 101 ;(b) a second polypeptide comprising an amino acid sequence of SEQ ID NO: 44;(c) a third polypeptide comprising an amino acid sequence of SEQ ID NO: 45; and a second antigen binding domain comprising:(d) a fourth polypeptide comprising an amino acid sequence of SEQ ID NO: 49;(e) a fifth polypeptide comprising an amino acid sequence of SEQ ID NO: 44; and(f) a sixth polypeptide comprising an amino acid sequence of SEQ ID NO: 50, SEQ ID NO: 51 , SEQ ID NO: 52, or SEQ ID NO: 53; or(I) a first antigen binding molecule comprising:(a) a first polypeptide comprising an amino acid sequence of SEQ ID NO: 90, SEQ ID NO: 91 , SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO:98, SEQ ID NO: 99, SEQ ID NO: 100, or SEQ ID NO: 101 ;(b) a second polypeptide comprising an amino acid sequence of SEQ ID NO: 44;(c) a third polypeptide comprising an amino acid sequence of SEQ ID NO: 45; and a second antigen binding domain comprising:(d) a fourth polypeptide comprising an amino acid sequence of SEQ ID NO: 469;(e) a fifth polypeptide comprising an amino acid sequence of SEQ ID NO: 468; and(f) a sixth polypeptide comprising an amino acid sequence of SEQ ID NO: 50, SEQ ID NO: 51 , SEQ ID NO: 52, or SEQ ID NO: 53; or(III) a first antigen binding molecule comprising:(a) a first polypeptide comprising an amino acid sequence of SEQ ID NO: 91 ;(b) a second polypeptide comprising an amino acid sequence of SEQ ID NO: 44;(c) a third polypeptide comprising an amino acid sequence of SEQ ID NO: 45; and a second antigen binding molecule comprising:(d) a fourth polypeptide comprising an amino acid sequence of SEQ ID NO: 49;(e) a fifth polypeptide comprises an amino acid sequence of SEQ ID NO: 44; and / or(f) a sixth polypeptide comprises an amino acid sequence of SEQ ID NO: 53 or(IV) a first antigen binding molecule comprising:(a) a first polypeptide comprising an amino acid sequence of SEQ ID NO: 91 ;(b) a second polypeptide comprising an amino acid sequence of SEQ ID NO: 44;(c) a third polypeptide comprising an amino acid sequence of SEQ ID NO: 45; and a second antigen binding molecule comprising:(d) a fourth polypeptide comprising an amino acid sequence of SEQ ID NO: 469;(e) a fifth polypeptide comprises an amino acid sequence of SEQ ID NO: 468; and / or(f) a sixth polypeptide comprises an amino acid sequence of SEQ ID NO: 53.

108. The cytokine receptor agonist according to any one of claims 79-99, 102 or 104, wherein the cytokine receptor agonist comprises: a first antigen binding molecule comprising:(a) a first polypeptide comprising in order from N-terminus to C-terminus: the IL2Ry binding domain, a VH and CH1 of the first PD-1 binding domain, and a first subunit of the first Fc region;(b) a second polypeptide comprising a second subunit of the first Fc region; and(c) a third polypeptide, comprising in order from N-terminus to C-terminus: a VL and CL of the first PD-1 binding domain; and a second antigen binding molecule comprising:(d) a fourth polypeptide comprising in order from N-terminus to C-terminus: a VH and CH1 of the second PD-1 binding domain, and a first subunit of the second Fc region;(e) a fifth polypeptide comprising in order from N-terminus to C-terminus: a VL and CH1 of the third PD-1 binding domain, and a second subunit of the second Fc region;(f) a sixth polypeptide, comprising in order from N-terminus to C-terminus: the IL2Rp binding domain, and a VL and CL of the second PD-1 binding domain; and(g) a seventh polypeptide comprising in order from N-terminus to C-terminus: a VH and CL of the third PD-1 binding domain.

109. The cytokine receptor agonist according to claim 108 wherein the cytokine receptor agonist comprises:(I) a first antigen binding molecule comprising:(a) a first polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 90, SEQ ID NO: 91 , SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, or SEQ ID NO: 101 ;(b) a second polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 44;(c) a third polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 45; and a second antigen binding molecule comprising:(d) a fourth polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 64;(e) a fifth polypeptide comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 61 ;(f) a sixth polypeptide comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 66, or SEQ ID NO: 67; and / or(g) a seventh polypeptide comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 62; or(II) a first antigen binding molecule comprising:(a) a first polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 90, SEQ ID NO: 91 , SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, or SEQ ID NO: 101 ;(b) a second polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 44;(c) a third polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 45; and a second antigen binding molecule comprising:(d) a fourth polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 467;(e) a fifth polypeptide comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 466;(f) a sixth polypeptide comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 66, or SEQ ID NO: 67; and / or(g) a seventh polypeptide comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 62; or(III) a first antigen binding molecule comprising:(a) a first polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 91 ;(b) a second polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 44;(c) a third polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 45; and a second antigen binding molecule comprising:(d) a fourth polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 64;(e) a fifth polypeptide comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 61 ;(f) a sixth polypeptide comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 67; and / or(g) a seventh polypeptide comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 62; or(IV) a first antigen binding molecule comprising:(a) a first polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 91 ;(b) a second polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 44;(c) a third polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 45; and a second antigen binding molecule comprising:(d) a fourth polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 467;(e) a fifth polypeptide comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 466;(f) a sixth polypeptide comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 67; and / or(g) a seventh polypeptide comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 62.

110. The cytokine receptor agonist according to claim 108 wherein the cytokine receptor agonist comprises:(I) a first antigen binding molecule comprising:(a) a first polypeptide comprising an amino acid sequence of SEQ ID NO: 90, SEQ ID NO: 91 , SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, or SEQ ID NO: 101 ;(b) a second polypeptide comprising an amino acid sequence of SEQ ID NO: 44;(c) a third polypeptide comprising an amino acid sequence of SEQ ID NO: 45; and a second antigen binding domain comprising:(d) a fourth polypeptide comprising an amino acid sequence of SEQ ID NO: 64;(e) a fifth polypeptide comprising an amino acid sequence of SEQ ID NO: 61 ;(f) a sixth polypeptide comprising an amino acid sequence of SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 66, or SEQ ID NO: 67; and(g) a seventh polypeptide comprising an amino acid sequence of SEQ ID NO: 62 or(II) a first antigen binding molecule comprising:(a) a first polypeptide comprising an amino acid sequence of SEQ ID NO: 90, SEQ ID NO: 91 , SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, or SEQ ID NO: 101 ;(b) a second polypeptide comprising an amino acid sequence of SEQ ID NO: 44;(c) a third polypeptide comprising an amino acid sequence of SEQ ID NO: 45; and a second antigen binding domain comprising:(d) a fourth polypeptide comprising an amino acid sequence of SEQ ID NO: 467;(e) a fifth polypeptide comprising an amino acid sequence of SEQ ID NO: 466;(f) a sixth polypeptide comprising an amino acid sequence of SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 66, or SEQ ID NO: 67; and(g) a seventh polypeptide comprising an amino acid sequence of SEQ ID NO: 62 or(III) a first antigen binding molecule comprising:(a) a first polypeptide comprising an amino acid sequence of SEQ ID NO: 91 ;(b) a second polypeptide comprising an amino acid sequence of SEQ ID NO: 44;(c) a third polypeptide comprising an amino acid sequence of SEQ ID NO: 45; a second antigen binding domain comprising:(d) a fourth polypeptide comprising an amino acid sequence of SEQ ID NO: 64;(e) a fifth polypeptide comprising an amino acid sequence of SEQ ID NO: 61 ;(f) a sixth polypeptide comprising an amino acid sequence of SEQ ID NO: 67; and(g) a seventh polypeptide comprising an amino acid sequence of SEQ ID NO: 62; or(IV) a first antigen binding molecule comprising:(a) a first polypeptide comprising an amino acid sequence of SEQ ID NO: 91 ;(b) a second polypeptide comprising an amino acid sequence of SEQ ID NO: 44;(c) a third polypeptide comprising an amino acid sequence of SEQ ID NO: 45; and a second antigen binding domain comprising:(d) a fourth polypeptide comprising an amino acid sequence of SEQ ID NO: 467;(e) a fifth polypeptide comprising an amino acid sequence of SEQ ID NO: 466;(f) a sixth polypeptide comprising an amino acid sequence of SEQ ID NO: 67; and(g) a seventh polypeptide comprising an amino acid sequence of SEQ ID NO: 62.

111. The cytokine receptor agonist according to any one of claims 1-78, wherein the IL2Ry binding domain, the first PD-1 binding domain, the Fc region, the second PD-1 binding domain, and the IL2Rp binding domain all form part of the same antigen binding molecule.

112. The cytokine receptor agonist according to claim 111 , wherein the cytokine receptor agonist comprises a hinge region comprising a first hinge peptide and a second hinge peptide, wherein(a) the first hinge peptide is fused at its C-terminus to the N-terminus of a CH2 domain of a first subunit of the Fc region and at its N-terminus to the C-terminus of (i) a CH1 domain or (ii) a CL domain of the first PD-1 binding domain and(b) the second hinge peptide is fused at its C-terminus to the N-terminus of a CH2 domain of a second Fc region subunit and fused at its N-terminus to the other of the C-terminus of (i) a CH1 domain or (ii) a CL domain of the first PD-1 binding domain.

113. The cytokine receptor agonist according to claim 112, wherein the first hinge peptide is fused at its N-terminus to the C-terminus of the CH1 domain of the first PD-1 binding domain and the second hinge peptide is fused at its N-terminus to the C-terminus of the CL domain of the first PD-1 binding domain.

114. The cytokine receptor agonist according to claim 113 wherein the first hinge peptide consists of the sequence EPKSCDKTHTCPPCP (SEQ ID NO: 42).

115. The cytokine receptor agonist according to any one of claims 113-114, wherein the second hinge peptide is a truncated hinge peptide, optionally wherein the second hinge peptide consists of the sequence DKTHTCPPCP (SEQ ID NO: 43).

116. The cytokine receptor agonist according to any one of claims 111-115, wherein the N-terminus of the second PD-1 binding domain is fused to the C-terminus of the Fc region.

117. The cytokine receptor agonist according to claim 116, wherein the N-terminus of the VH of the second PD-1 binding domain is fused to the C-terminus of the CH3 domain of a first Fc region subunit.

118. The cytokine receptor agonist according to any one of claims 116-117, wherein the second PD-1 binding domain is fused to the Fc region subunit via a peptide linker.

119. The cytokine receptor agonist according to claim 118, wherein the peptide linker consists of 10 amino acids.

120. The cytokine receptor agonist according to claim 119, wherein the second PD-1 binding domain is fused to the Fc region subunit via a GGSGGGGSGG (SEQ ID NO: 34) peptide linker.121 . The cytokine receptor agonist according to any one of claims 111-120, wherein(i) the second PD-1 binding domain is a Fab molecule, optionally wherein the CH1 domain and CL domain of the second PD-1 binding domain comprise charged modifications; and / or the first PD-1 binding domain is a cross-Fab molecule; or(ii) the first PD-1 binding domain is a Fab molecule, optionally wherein the CH1 domain and CL domain of the first PD-1 binding domain comprise charged modifications; and / or the second PD-1 binding domain is a cross-Fab molecule.

122. The cytokine receptor agonist according to any one of claims 111-121 , wherein the cytokine receptor agonist comprises:(a) a first polypeptide comprising in order from N-terminus to C-terminus: a VL and CH1 of the first PD-1 binding domain, a first subunit of the Fc region, and a VH and CH1 of the second PD-1 binding domain;(b) a second polypeptide comprising in order from N-terminus to C-terminus: the IL2Ry binding domain, a VH and CL of the first PD-1 binding domain, and a second subunit of the Fc region; and(c) a third polypeptide comprising in order from N-terminus to C-terminus: the IL2Rp binding domain, and a VL and CL of the second PD-1 binding domain.

123. The cytokine receptor agonist according to claim 122, wherein the cytokine receptor agonist comprises:(a) a first polypeptide comprising in order from N-terminus to C-terminus: a VL and CH1 of the first PD-1 binding domain, a first hinge peptide, a first subunit of the Fc region, and a VH and CH1 of the second PD-1 binding domain;(b) a second polypeptide comprising in order from N-terminus to C-terminus: the IL2Ry binding domain, a VH and CL of the first PD-1 binding domain, a second hinge peptide, wherein the second hinge peptide is a truncated hinge peptide, and a second subunit of the Fc region; and(c) a third polypeptide comprising in order from N-terminus to C-terminus: the IL2Rp binding domain, and a VL and CL of the second PD-1 binding domain.

124. The cytokine receptor agonist according to claim 111-123, wherein(I) the first antigen binding molecule comprises:(a) a first polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 68,(b) a second polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111 , SEQ ID NO: 112, or SEQ ID NO: 113; and(c) a third polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 67; or(II) the first antigen binding molecule comprises:(a) a first polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 68,(b) a second polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 103; and(c) a third polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 67.

125. The cytokine receptor agonist according to claim 111-124, wherein(I) the cytokine receptor agonist comprises:(a) a first polypeptide comprising an amino acid sequence of SEQ ID NO: 68,(b) a second polypeptide comprising an amino acid sequence of SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 1 10, SEQ ID NO: 111 , SEQ ID NO: 112, or SEQ ID NO: 113; and(c) a third polypeptide comprising an amino acid sequence of SEQ ID NO: 67; or(II) the cytokine receptor agonist comprises:(a) a first polypeptide comprising an amino acid sequence of SEQ ID NO: 68,(b) a second polypeptide comprising an amino acid sequence of SEQ ID NO: 103; and(c) a third polypeptide comprising an amino acid sequence of SEQ ID NO: 67.

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