Antigen binding molecules

Abstract

Disclosed herein are antigen-binding molecules comprising a gamma c-binding moiety and a moiety that binds to a polypeptide other than gamma c of a gamma c-containing cytokine receptor. Compositions comprising such antigen binding molecules and uses and methods of using the same are also disclosed.

Description

[0001] This application claims priority to US 63 / 437470 filed January 6, 2023, the contents and elements of which are incorporated herein by reference for all purposes. TECHNICAL FIELD

[0002] The present disclosure relates to the field of molecular biology and methods of medical treatment and prevention. In particular, the present disclosure relates to antigen binding molecules that bind to polypeptides of gamma c-containing cytokine receptors. BACKGROUND

[0003] Interleukins play an important role in maintaining T cell homeostasis and mediating appropriate immune responses. In particular, interleukins and related cytokines act as a means of communication for innate and adaptive immune cells as well as non-immune cells and tissues. As such, interleukins have a key role in the development, progression and control of cancer (Briukhovetska D. et al. Nat Rev Cancer 21, 481-499 (2021)).

[0004] The use of interleukins in therapy has shown great promise, but also has drawbacks and disappointing results.

[0005] IL-2 was the first interleukin approved for cancer treatment, although its use inevitably brings significant safety concerns. High doses of IL-2 required for effective treatment of certain diseases have high toxicity. The main side effects of this therapy include vascular leak syndrome (VLS), which leads to accumulation of fluid within blood vessels in organs such as the lungs and liver, with subsequent pulmonary oedema and liver damage. There is no treatment for VLS other than stopping therapy.

[0006] In addition, monotherapy with IL-15 and several engineered variants is ineffective (Waldman et al. (2020) Front Immunol. May 19; 11: 868).

[0007] The common cytokine receptor gamma chain (common gamma chain, gamma c or CD132) is a cytokine receptor polypeptide common to cytokine receptor complexes of at least six different interleukin receptors (i.e. receptors for IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21).

[0008] Cells expressing yc can form functional receptors for cytokine proteins and can transmit signals from one cell to another and direct cell differentiation programs. Heterodimerization of yc and other polypeptides is necessary and sufficient for efficient signal transduction through interactions of their cytoplasmic domains and subsequent kinase activation of multiple signaling pathways. For example, heterodimerization of IL-2Rβ and yc is necessary for efficient IL-2 signal transduction, and heterodimerization of IL-9R and yc is necessary for efficient IL-9 signal transduction.

[0009] Antigen binding molecules that bind to yc and IL-2Rβ are disclosed, for example, in WO 2017 / 021540 Al. SUMMARY

[0010] In one aspect, the present disclosure provides an antigen binding molecule, optionally isolated, comprising: (i) a yc binding moiety, and (ii) a moiety that binds to a polypeptide of a yc-containing cytokine receptor other than yc.

[0011] In some embodiments, the polypeptide of a yc-containing cytokine receptor other than yc is selected from the group consisting of: IL-2Rβ, IL-2Ra, IL-15Ra, IL-4Ra, IL-9Ra, IL-21Ra, and IL-7Ra.

[0012] In some embodiments, the polypeptide of a yc-containing cytokine receptor other than yc is IL-2Rβ.

[0013] In some embodiments, the polypeptide of a yc-containing cytokine receptor other than yc is not IL-2Rβ.

[0014] In some embodiments, the antigen binding molecule is an agonist of a yc-containing cytokine receptor.

[0015] In some embodiments, the antigen binding molecule is an antagonist of a yc-containing cytokine receptor.

[0016] In some embodiments, the antigen binding molecule is an agonist of a yc:IL-2Rβ receptor, a yc:IL-2Rβ:IL-2Ra receptor, and / or a yc:IL-2Rβ:IL-15Ra receptor.

[0017] In some embodiments, the antigen binding molecule is an antagonist of a yc:IL-2Rβ receptor, a yc:IL-2Rβ:IL-2Ra receptor, and / or a yc:IL-2Rβ:IL-15Ra receptor.

[0018] In some embodiments, the antigen binding molecule further comprises: (iii) an antigen binding moiety that binds to a target antigen other than a yc-containing cytokine receptor polypeptide.

[0019] In some embodiments, the target antigen other than a yc-containing cytokine receptor polypeptide is a disease-associated antigen or an antigen expressed by an immune cell.

[0020] The present disclosure also provides a chimeric antigen receptor (CAR) comprising an antigen binding molecule according to the present disclosure.

[0021] The present disclosure also provides a nucleic acid or plurality of nucleic acids, optionally isolated, encoding an antigen binding molecule according to the present disclosure or a CAR according to the present disclosure.

[0022] The present disclosure also provides an expression vector or plurality of expression vectors comprising a nucleic acid or plurality of nucleic acids according to the present disclosure.

[0023] The present disclosure also provides a cell comprising an antigen binding molecule according to the present disclosure, a CAR according to the present disclosure, a nucleic acid or plurality of nucleic acids according to the present disclosure, or an expression vector or plurality of expression vectors according to the present disclosure.

[0024] The present disclosure also provides a method comprising culturing a cell according to the present disclosure under conditions suitable for the cell to express an antigen binding molecule or a CAR according to the present disclosure.

[0025] The present disclosure also provides a composition comprising an antigen binding molecule according to the present disclosure, a CAR according to the present disclosure, a nucleic acid or plurality of nucleic acids according to the present disclosure, an expression vector or plurality of expression vectors according to the present disclosure, or a cell according to the present disclosure, and a pharmaceutically acceptable carrier, diluent, excipient, or adjuvant.

[0026] The present disclosure also provides an antigen binding molecule according to the present disclosure, a CAR according to the present disclosure, a nucleic acid or plurality of nucleic acids according to the present disclosure, an expression vector or plurality of expression vectors according to the present disclosure, a cell according to the present disclosure, or a composition according to the present disclosure, for use in a method of treatment or prevention.

[0027] The present disclosure also provides the use of an antigen binding molecule according to the present disclosure, a CAR according to the present disclosure, a nucleic acid or plurality of nucleic acids according to the present disclosure, an expression vector or plurality of expression vectors according to the present disclosure, a cell according to the present disclosure, or a composition according to the present disclosure, in the manufacture of a medicament for a method of treatment or prevention.

[0028] The present disclosure also provides a method of treatment or prevention comprising administering to a subject in need thereof a therapeutically or prophylactically effective amount of an antigen binding molecule according to the present disclosure, a CAR according to the present disclosure, a nucleic acid or plurality of nucleic acids according to the present disclosure, an expression vector or plurality of expression vectors according to the present disclosure, a cell according to the present disclosure, or a composition according to the present disclosure.

[0029] In some embodiments, the method of treatment or prevention is a method of treating or preventing a disease / disorder characterized by T cell dysfunction, cancer, infectious disease, or autoimmune disease.

[0030] In some embodiments, the cancer is selected from the group consisting of colon cancer, colon carcinoma, colorectal cancer, nasopharyngeal carcinoma, cervical cancer, oropharyngeal cancer, gastric cancer, hepatocellular carcinoma, head and neck cancer, head and neck squamous cell carcinoma (HNSCC), oral cancer, laryngeal cancer, prostate cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, bladder cancer, urothelial cancer, melanoma, advanced melanoma, renal cell carcinoma, ovarian cancer, or mesothelioma.

[0031] In some embodiments, the disease / disorder is characterized by lymphocytosis and / or inflammation.

[0032] The present disclosure also provides an in vitro complex, optionally isolated, comprising an antigen binding molecule according to the present disclosure or a CAR according to the present disclosure, bound to a polypeptide other than yc of a yc-containing cytokine receptor and yc.

[0033] The present disclosure also provides a method for generating or expanding a population of cells expressing a yc-containing cytokine receptor, the method comprising contacting a cell expressing a yc-containing cytokine receptor in vitro, in vivo, or ex vivo with an antigen binding molecule according to the present disclosure.

[0034] The present disclosure also provides a method for increasing proliferation, survival, and / or effector activity of a cell expressing a yc-containing cytokine receptor, the method comprising contacting a cell expressing a yc-containing cytokine receptor in vitro, in vivo, or ex vivo with an antigen binding molecule according to the present disclosure.

[0035] The present disclosure also provides a method for decreasing the number / proportion of (e.g., depleting or increasing depletion of) a cell expressing a yc-containing cytokine receptor, the method comprising contacting a cell expressing a yc-containing cytokine receptor in vitro, in vivo, or ex vivo with an antigen binding molecule according to the present disclosure or a CAR according to the present disclosure.

[0036] The present disclosure also provides a method for reducing the proliferation, survival and / or effector activity of a cell expressing a γc-containing cytokine receptor, the method comprising contacting the cell expressing the γc-containing cytokine receptor in vitro, in vivo, or ex vivo with an antigen binding molecule according to the present disclosure.

[0037] In some embodiments, the cell is an effector immune cell.

[0038] In some embodiments, the cell is a T cell or an NK cell.

[0039] In some embodiments, the γc-containing cytokine receptor is selected from the group consisting of: a γc:IL-2Rβ receptor, a γc:IL-2Rβ:IL-2Rα receptor, a γc:IL-2Rβ:IL-15Rα receptor, a γc:IL-4Rα receptor, a γc:IL-9Rα receptor, a γc:IL-21Rα receptor, and a γc:IL-7Rα receptor.

[0040] The present disclosure also provides a method of promoting heteromultimerization of a polypeptide of a γc and a γc-containing cytokine receptor complex, the method comprising contacting the polypeptide of the γc and the γc-containing cytokine receptor complex in vitro, in vivo, or ex vivo with an antigen binding molecule according to the present disclosure or a CAR according to the present disclosure.

[0041] The present disclosure also provides a method of inhibiting heteromultimerization of a polypeptide of a γc and a γc-containing cytokine receptor complex, the method comprising contacting the polypeptide of the γc and the γc-containing cytokine receptor complex in vitro, in vivo, or ex vivo with an antigen binding molecule or a CAR.

[0042] The present disclosure also provides a method of inhibiting heteromultimerization of a γc and an IL-2Rβ.

[0043] The present disclosure includes combinations of the described aspects and preferred features, except where such combinations are explicitly prohibited or should be clearly avoided. BRIEF DESCRIPTION OF DRAWINGS

[0044] Embodiments illustrating the principles of the application will now be discussed with reference to the accompanying drawings, in which:

[0045] Figure 1 . Dose-dependent STAT5 phosphorylation by agonist bispecific γc and IL-2Rβ binding antibodies. STAT5 phosphorylation was assessed in different leukocyte subsets. Percent of phosphorylated STAT5 (pSTAT5) was measured after stimulation with different concentrations of bispecific γc and CD122 binding antibodies. High efficient stimulation of CD4+, CD8+ and NK cells was shown, but no stimulation of ISO controls.

[0046] Figure 2Agonist bispecific γc and IL-2Rβ binding antibodies enhance tumor killing by EBV-specific T cells. Mice engrafted with EBV-BCL tumors were treated with EBV-specific T cells and Treg cells in the presence of IL-2 and agonist bispecific γc and CD122 binding antibodies (Adk-1 or Adk-2). Treatment involving agonist bispecific γc and CD122 binding antibodies resulted in a significant reduction in absolute tumor cell counts.

[0047] Figure 3 Agonist bispecific γc and IL-2Rβ binding antibodies stimulate T cell proliferation in vivo in non-human primates. Expression of the proliferation marker Ki67 was used as a pharmacodynamic marker of immune stimulation in T cells. CD8 T cell proliferation occurred as early as 24 hours and continued up to 120 hours post-dose.

[0048] Figure 4 Surface expression of cytokine receptors on commercially and in-house produced human embryonic kidney (HEK) 293 cytokine reporter cells.

[0049] Figure 5 Functional characterization of HEK-Blue IL-2Rβγ, HEK-Blue IL-7, HEK-Blue IL-9, HEK-Blue IL-21, HEK-Blue IL-15Rα / IL-2Rβγ, and HEK-Blue IL-4 / IL-7 cell lines by assessing STAT5 phosphorylation (pSTAT5) levels upon stimulation with the relevant cytokine.

[0050] Figures 6A-6B Schematic of the cell signaling assay. The assay involves stimulation of (HEK) 293 cytokine reporter cells with bispecific antibodies (or control antibodies) and assessment of pSTAT5 levels using the QUANTI-Blue assay. Different steps performed in (A) agonist assay and (B) antagonist assay are highlighted.

[0051] Figure 7 Antagonist bispecific γc and IL-2Rβ binding antibodies modulate IL-2Rβγ-mediated signaling and inhibit STAT5 phosphorylation.

[0052] Figures 8A-8B Antagonist bispecific γc and IL-2Rβ binding antibodies modulate IL-15Rα-mediated signaling and inhibit STAT5 phosphorylation.

[0053] Figures 9A-9B. Bispecific yc and IL-4Ra binding antibodies modulate IL-4R-mediated signaling. (A) Agonist bispecific yc and IL-4Ra binding antibodies increase STAT5 phosphorylation, (B) Antagonist bispecific yc and IL-4Ra binding antibodies inhibit STAT5 phosphorylation.

[0054] Figures 10A-10E . Bispecific yc and IL-7Ra binding antibodies modulate IL-7R-mediated signaling. (A-D) Agonist bispecific yc and IL-7Ra binding antibodies increase STAT5 phosphorylation, (B) Antagonist bispecific yc and IL-7Ra binding antibodies inhibit STAT5 phosphorylation.

[0055] Figures 11A-11D . Bispecific yc and IL-7Ra binding antibodies inhibit IL-4-mediated signaling. (A) Line graph showing STAT5 signaling levels following IL-4 stimulation after treatment of HEK-Blue IL-4 / IL-7 cells with 1 nM bispecific yc and IL-7Ra binding antibodies or monospecific control antibodies. (B) Line graph showing STAT5 signaling levels following IL-4 stimulation after treatment of HEK-Blue IL-4 / IL-7 cells with 30 nM bispecific yc and IL-7Ra binding antibodies or monospecific control antibodies. (C) Histogram showing STAT5 signaling levels following IL-4 (10 pM) stimulation after treatment of HEK-Blue IL-4 / IL-7 cells with 1 nM or 30 nM bispecific yc and IL-7Ra binding antibodies or monospecific control antibodies. (D) Histogram showing STAT5 signaling levels following IL-4 (100 pM) stimulation after treatment of HEK-Blue IL-4 / IL-7 cells with 1 nM or 30 nM bispecific yc and IL-7Ra binding antibodies or monospecific control antibodies.

[0056] Figures 12A-12D. Bispecific yc and IL-7Ra binding antibodies inhibit IL-21 mediated signaling. (A) Line graph showing STAT5 signaling levels following IL-21 stimulation after treatment of HEK-Blue IL-21 cells with 30 nM bispecific yc and IL-7Ra binding antibodies or monospecific control antibodies. (B) Line graph showing STAT5 signaling levels following IL-21 stimulation after treatment of HEK-Blue IL-21 cells with 100 nM bispecific yc and IL-7Ra binding antibodies or monospecific control antibodies. (C) Histogram showing STAT5 signaling levels following IL-21 (50 pM) stimulation after treatment of HEK-Blue IL-21 cells with 30 nM or 100 nM bispecific yc and IL-7Ra binding antibodies or monospecific control antibodies. (D) Histogram showing STAT5 signaling levels following IL-21 (100 pM) stimulation after treatment of HEK-Blue IL-21 cells with 30 nM or 100 nM bispecific yc and IL-7Ra binding antibodies or monospecific control antibodies.

[0057] Figures 13A-13B . Bispecific yc and IL-9Ra binding antibodies modulate IL-9R mediated signaling. (A) Agonist bispecific yc and IL-9Ra binding antibodies increase STAT5 phosphorylation, (B) Antagonist bispecific yc and IL-9Ra binding antibodies inhibit STAT5 phosphorylation.

[0058] Figure 14 A-14B. Bispecific yc and IL-21Ra binding antibodies modulate IL-21R mediated signaling. (A) Agonist bispecific yc and IL-21Ra binding antibodies increase STAT5 phosphorylation, (B) Additional agonist bispecific yc and IL-21Ra binding antibodies increase STAT5 phosphorylation.

[0059] Figure 15 . Bispecific yc and IL-21Ra binding antibodies modulate IL-21R mediated signaling. Antagonist bispecific yc and IL-21Ra binding antibodies inhibit STAT5 phosphorylation. DETAILED DESCRIPTION

[0060] The present disclosure encompasses nucleotide and amino acid sequences of antigen binding molecules specific for the common gamma chain (yc; CD132) and polypeptides other than yc of yc-containing cytokine receptor complexes. In some embodiments, the antigen binding molecules comprise a yc binding portion and a portion that binds to a polypeptide other than yc of a yc-containing cytokine receptor complex.

[0061] In one aspect, the present disclosure describes the design of cytokine receptor agonists, where receptor activation is achieved by multispecific antigen-binding molecules (e.g., bispecific antibodies or bifunctional proteins) that have anti-gamma c specificity and specificity for another polypeptide of a gamma c-containing cytokine receptor complex, effecting heterodimerization of the receptor components. The polypeptide of the gamma c-containing cytokine receptor complex other than gamma c can be one of IL-2R beta (CD122), IL-2R alpha (CD25), IL-15R alpha, IL-4R alpha (CD124), IL-9R alpha (CD129), IL-21R alpha (CD360), or IL-7R alpha (CD127).

[0062] In another aspect, the present disclosure describes the design of cytokine receptor antagonists, where receptor activation is reduced by multispecific antigen-binding molecules (e.g., bispecific antibodies or bifunctional proteins) that have anti-gamma c specificity and specificity for another polypeptide of a gamma c-containing cytokine receptor complex, effecting heterodimerization of the receptor components. The polypeptide of the gamma c-containing cytokine receptor complex other than gamma c can be one of IL-2R beta (CD122), IL-2R alpha (CD25), IL-15R alpha, IL-4R alpha (CD124), IL-9R alpha (CD129), IL-21R alpha (CD360), or IL-7R alpha (CD127).

[0063] The antigen-binding molecules of the present disclosure have beneficial properties that overcome the shortcomings and problems associated with therapeutic administration of cytokines or engineered cytokines (e.g., pegylated cytokines and antibody-coupled cytokines).

[0064] Administration of cytokines and engineered cytokines can have negative characteristics, such as: short half-life, need for toxic dosing levels, non-specific binding, and high levels of immunogenicity. These shortcomings can result in problems for patients, such as: reduced efficacy, occurrence of anti-drug antibodies, activation of non-optimal signaling pathways, occurrence of adverse events, and severe side effects (such as vascular leakage syndrome (VLS)).

[0065] Antigen-binding molecules that bind to gamma c-containing cytokine receptor complexes provide a technical advance over cytokine-based therapies. For example, the antigen-binding molecules of the present disclosure can be considered drug-like molecules and are more stable, have longer half-lives, increased persistence of response, and can be tailored to individual patients and specific diseases.

[0066] The use of the antigen binding molecules of the present disclosure have efficient cell signaling. Depending on the target cytokine receptor, the disease to be treated, and the status of the individual patient, the affinity of the binding and the level of signal induction can be adjusted to achieve the optimal downstream effects.

[0067] Common gamma chain (yc)

[0068] The human common gamma chain (yc; also known as CD132, IL-2RG, and CIDX) is a protein identified by UniProt P31785-1. The structure and function of yc are reviewed, for example, in Waickman et al., Cell Mol Life Sci. (2016) 73(2):253-269 and Leonard et al., Immunity (2019) 50(4):832-850, both of which are incorporated by reference herein in their entireties.

[0069] The canonical isoform of human yc (isoform 1) has the amino acid sequence set forth in SEQ ID NO: 437. The N-terminal 23 amino acids of SEQ ID NO: 437 constitute a signal peptide (SEQ ID NO: 503), so the mature form of human yc (i.e., after processing to remove the signal peptide) has the amino acid sequence set forth in SEQ ID NO: 438. Amino acids 23 to 262 of SEQ ID NO: 437 constitute the extracellular domain of yc (SEQ ID NO: 439), positions 263 to 283 form a single-pass transmembrane domain (SEQ ID NO: 518), and positions 284 to 369 form a cytoplasmic domain (SEQ ID NO: 519). The extracellular domain includes a fibronectin type III (FNIII) domain (set forth in SEQ ID NO: 547) that includes a WSXWS motif set forth in SEQ ID NO: 548. The WSXWS motif is conserved among type I cytokine receptor polypeptides, and the WSXWS motif of yc is believed to be important for conformational changes of the receptor.

[0070] All receptors of the yc receptor family include yc as a component polypeptide. Janus kinase 3 (JAK3) associates with yc, and upon activation of yc-containing cytokine receptors, JAK3 is phosphorylated and activated. The phosphorylated JAK3 then phosphorylates and activates downstream signaling proteins, such as STAT5, and also triggers signaling through the MAPK / ERK and PI3K / Akt signal transduction pathways. Signaling through yc family receptors promotes activation, proliferation, and survival of immune cells.

[0071] In the present specification, “common gamma chain,” “common gamma chain,” “yc,” or “CD132” refers to a common gamma chain from any species, including an isoform, fragment, variant, or homolog of yc from any species. In some embodiments, yc is a yc from a mammal (e.g., a therian, a placental, an epitherian, a preptotherian, an archontan, a primate (rhesus, cynomolgus, non-human primate, or human)). In some embodiments, yc is a human yc.

[0072] As used herein, an isoform, fragment, variant, or homolog of a given reference protein (e.g., yc) can be characterized by having at least 70% sequence identity, preferably one of >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99%, or 100% amino acid sequence identity to the amino acid sequence of the reference protein.

[0073] A “fragment” generally refers to a portion of a reference protein. A “variant” generally refers to a protein having an amino acid sequence that comprises one or more amino acid substitutions, insertions, deletions, or other modifications relative to the amino acid sequence of a reference protein, but retains a substantial degree of sequence identity (e.g., at least 60%) to the amino acid sequence of the reference protein. An “isoform” generally refers to a variant of a reference protein that is expressed by the same species as the reference protein. A “homolog” generally refers to a variant of a reference protein that is produced by a different species as compared to the species of the reference protein. Homologs include orthologs. For example, homologs of human yc include, e.g., mouse yc (UniProt P34902).

[0074] An isoform, fragment, variant, or homolog of a given reference protein can optionally be characterized by having at least 70% (preferably one of >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99%, or 100%) amino acid sequence identity to the amino acid sequence of the immature or mature (i.e., after processing to remove the signal peptide) form of a particular isoform of a related protein from a given species (e.g., human).

[0075] >96%, >97%, >98%, >99%, or 100%).

[0076] The isoform, fragment, variant, or homolog of yc according to the present disclosure can optionally be characterized by having at least 70% (preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) amino acid sequence identity to the amino acid sequence of an immature or mature yc isoform from a given species (e.g., human).

[0077] The isoform, fragment, variant, or homolog can optionally be a functional isoform, fragment, variant, or homolog, e.g., having functional properties / activities of a reference yc (e.g., human yc isoform 1), as determined by analysis by an assay suitable for the functional properties or activities. For example, the isoform, fragment, variant, or homolog of yc can exhibit one or more of the following: association with one or more of IL-2Rβ, IL-2Rα, IL-15Rα, IL-4Rα, IL-9Rα, IL-21Rα, or IL-7Rα, or binding to one or more of IL-2, IL-15, IL-4, IL-9, IL-21, or IL-7.

[0078] The fragment of yc can be one of a minimum length of 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, or 350 amino acids and a maximum length of one of 20, 30, 40, 50, 100, 150, 200, 250, 300, or 350 amino acids.

[0079] In some embodiments, the yc has at least 70% (preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) amino acid sequence identity to SEQ ID NO: 437 or 438.

[0080] In some embodiments, the fragment of yc comprises or consists of an amino acid sequence having at least 70% (preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) amino acid sequence identity to SEQ ID NO: 439.

[0081] Signaling through cytokine receptors comprising yc

[0082] There are many cytokines that signal through cytokine receptors that include yc, also referred to herein as yc-containing receptor complexes, e.g., IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. Such cytokines are considered to belong to the yc family of cytokines. The biology of yc family cytokines is reviewed, e.g., in Lin and Leonard, Cold Spring Harb Perspect Biol (2018) 10(9):a028449, Leonard et al., Immunity (2019) 50(4):832-850, and Pulliam et al., Immunol Lett. (2016) 169:61-72, both of which are incorporated by reference herein in their entireties.

[0083] Members of the common cytokine receptor yc chain family of cytokines signal through yc-containing receptor complexes. Such cytokines can be referred to herein as yc-associated cytokines. The yc subunit associates with different cytokine-specific receptor subunits to form unique heterodimeric receptors. Common yc chain family cytokines generally activate three major signaling pathways that promote cell survival and proliferation, the PI3K-Akt pathway, the RAS-MAPK pathway, and the JAK-STAT pathway. Differences in the expression patterns of the cytokines or their unique receptor components, as well as activation of different STAT proteins, can account for some of the different effects mediated by yc family cytokines.

[0084] Interleukin-2 (IL-2) is a cytokine that mediates its effects by binding to the IL-2 receptor expressed by lymphocytes. The main sources of IL-2 are activated CD4+ T cells and activated CD8+ T cells. Treatment with IL-2 is an approved immunotherapy for the treatment of cancer, acting by promoting the proliferation and activity of effector immune cells such as T cells and NK cells (see, e.g., Skorombolas and Frelinger, Expert Rev Clin Immunol. (2014) 10(2):207-217). However, high doses of IL-2, which are required for effective treatment of certain diseases, have high toxicity. IL-2 exerts its pleiotropic functions by binding to different combinations of receptor components (a chain (IL-2Ra), b chain (IL-2Rb), and yc) expressed on different cell types. The isolated IL-2Ra has been referred to as the “low-affinity” IL-2 receptor (binding affinity KD of about 10 nM) and does not participate in signal transduction. The complex of IL-2Rb and yc binds IL-2 with moderate affinity (KD of about 1 nM), although the affinity of IL-2Rb alone is very low (KD of about 100 nM), and the binding affinity of yc alone to IL-2 is almost undetectable. The complex of all three subunits IL-2Ra, IL-2Rb, and yc binds IL-2 with high affinity (KD of about 10 pM). The high-affinity a-b-yc IL-2R is usually present on CD4+ T regulatory cells (Tregs) as well as recently activated T cells. The moderate-affinity b-yc IL-2R is present at low levels on naive CD8+ cells but is prominent on antigen-experienced (memory) and memory phenotype (MP) CD8+ T cells as well as natural killer (NK) cells. Both MPCD8+ T cells and NK cells express very high levels of IL-2Rb and are readily responsive to IL-2. Engineered IL-2 molecules have been developed for therapy. Reasonably designed IL-2 variants (IL-2 muteins) have been developed to overcome some of the problems of IL-2 therapy (Khoryati et al. Sci Immunol. (2020) 5(50): eaba5264). In addition, pegylated IL-2 (PEG-IL-2) molecules have been developed for similar reasons (Zhang et al. Nat Biomed Eng. (2021) 5(11): 1288-1305). However, neither IL-2 muteins nor PEG-IL-2 have all the benefits that the antigen-binding molecules of the present disclosure have. For example, neither IL-2 muteins nor PEG-IL-2 are designed to specifically bind the optimal subset of IL-2R polypeptides (i.e., yc and IL-2Rb, but not IL-2Ra), they cannot be tuned in the same way as the antigen-binding molecules of the present disclosure, and they do not have the same low level of immunogenicity.

[0085] Interleukin-4 (IL-4) has multiple biological effects, including stimulating proliferation of activated B cells and T cells and differentiating B cells into plasma cells. It is a key regulator of humoral and adaptive immunity. IL-4 induces B cell class switching to IgE and upregulates MHC class II production. IL-4 reduces Th1 cell, macrophage, IFNy, and dendritic cell IL-12 production. The IL4 receptor is overexpressed by many epithelial cancers and can be a promising target for metastatic tumor therapy (Bankaitis et al. Clin Exp Metastasis. (2015) 32(8): 847-856). The cytokine-binding receptor chain for IL-4 is IL-4Ra. This receptor chain is widely expressed, with most cells carrying at least a small amount of the receptor chain. When IL-4 binds to IL-4Ra, the IL-4 / IL-4Ra complex will bind to a secondary receptor chain, yc or IL-13Ra1 (Junttila. Front Immunol. (2018) 9:888). Expression of these secondary chains varies among different cell types. In non-hematopoietic cells, yc expression is low or absent, while IL-13Ra1 is expressed in higher amounts in these cells. In contrast, lymphocytes express only low levels of IL-13Ra1 and relatively large amounts of yc. Finally, myeloid cells are in between non-hematopoietic cells and lymphocytes, as myeloid cells express both IL-13Ra1 and yc. IL-4 activates multiple signaling pathways. IL-4 activates JAK1 and JAK3 via the type I IL-4 receptor; however, IL-4 activates JAK1 and JAK2 or TYK2 (depending on cell type) via the type II IL-4 receptor (Keegan et al. Fac Rev. (2021) 10:71). Regardless of receptor type, IL-4 is associated with potent activation of STAT6, which docks on a critical phosphotyrosine on IL-4Ra. The type I IL-4 receptor (containing IL-4Ra and yc) also activates STAT5 signaling. In addition, IL-4 (via the type I IL-4 receptor) efficiently activates IRS2, so IL-4 subsequently activates various pathways, including Sos / Ras, PI3K / Akt, PKB / mTOR, or PKC.

[0086] Interleukin-7 (IL-7) is a molecule known for its growth-promoting effect on B-cell progenitors, plays a key role in health maintenance and disease prevention, and congenital IL-7 signaling deficiency leads to severe immunodeficiency. Elevated IL-7 levels are associated with poor prognosis in many cancers (Zarogoulidis et al., J Cancer. (2014) 5(9):765-773). IL-7 binds to a receptor composed of two chains, IL-7Ra and yc. yc is expressed by most hematopoietic cells, while IL-7Ra is almost exclusively expressed on lymphocytes. Upon binding to its receptor, IL-7 signals through two different pathways: JAK-STAT (Janus kinase signal transducer and activator of transcription) and PI3K / Akt, responsible for differentiation and survival, respectively (El Kassar and Gress, J Immunotoxicol. (2010) 7(1): 1-7). When IL-7 binds to IL-7Ra, it recruits yc, bringing together the intracellular domains of JAK1 and JAK3, respectively. Binding of IL-7 to the IL-7 receptor complex leads to downstream STAT1, STAT3, and STAT5 signaling. Under steady-state conditions, IL-7 signaling is mediated primarily through activation of signal transducer and activator of transcription 5 (STAT5). In contrast, under lymphopenic conditions, STAT1 expression is modulated, leading to IL-7-dependent STAT1 and STAT5 activation (Le Saout et al. JCI Insight. (2017) 2(22):e96228). Activation by IL-7 leads to phosphorylation of the Y449 residue on IL-7Ra (Jiang et al., Cytokine Growth Factor Rev. (2005) 16:513-533). The p85a subunit of PI3K binds directly to the phosphorylated Y449 via SH2 domains. This is followed by allosteric activation of the catalytic subunit P110. PI3K is recruited to the membrane, where it generates phosphatidylinositol PIP3 by phosphorylating PIP2. PIP3 activates downstream genes with plekstrin homology domains, such as PDK1 and Akt (Shiroki et al., J Immunol. (2007) 178: 1349-1356). Akt in turn phosphorylates genes that regulate cell metabolism, cell cycle progression, and survival, such as GSK3b, P27, and the death protein BAD.

[0087] Interleukin-9 (IL-9) is a cytokine that stimulates cell proliferation and prevents apoptosis. IL-9 is a pleiotropic cytokine that has been primarily studied under T helper 2 (TH2)-associated immunopathological conditions such as asthma and parasitic infections. A paradigm shift in the biology of IL-9 has occurred following the recent discovery of TH9 cells, a novel TH cell subset that secretes large amounts of IL-9. This has led to renewed interest in the cytokine, which has been neglected since its discovery as it was considered just another TH2 cytokine. Recent studies have shown that it has multiple cellular sources and is critically involved in the immune pathogenesis of inflammatory diseases and in the protection of immune tolerance (Chakraborty et al. Int J Mol Sci. (2019) 20(9): 2113). IL-9 acts through the interleukin-9 receptor complex, which comprises IL-9 receptor alpha (IL-9Ra) and yc. When IL-9 binds to the IL-9 receptor complex, it activates different signal transducer and activator of transcription (STAT) proteins, i.e. STAT1, STAT3, and STAT5, thereby linking the cytokine to various biological processes. IL-9 is a pleiotropic cytokine with direct and indirect effects on hematopoietic progenitor cells, lymphocytes, mast cells, and airway smooth muscle and epithelial cells (Lee et al. Pathology & Oncology Research (2020) 26:2017-2022). IL-9 also activates insulin receptor substrates (IRS) 1 and 2. Upon JAK-mediated phosphorylation, the IRS proteins interact with other SH2-containing signaling proteins such as the regulatory subunit of phosphatidylinositol-3 kinase (PI3K) p85, leading to the activation of the PI3K catalytic subunit pl lO. PI3K then activates downstream signaling molecules such as PI3K-dependent kinase (PDK) and Akt. Akt then phosphorylates BAD and protects cells by preventing Caspase-mediated apoptosis. IL-9 also activates the MAPK pathway in several cell lines of lymphoid and hematopoietic origin, but IL-9-mediated MAPK activation is weaker compared to other cytokines such as IL-3.

[0088] Interleukin-15 (IL-15) shares structural similarity with IL-2. Like IL-2, IL-15 binds to and signals through a complex composed of the IL-2 / IL-15 receptor beta chain (CD122) and gamma c (CD132). Interleukin 15 is considered a potent proinflammatory cytokine and has the ability to disrupt chromosomal stability and induce tumorigenesis (Zarogoulidis et al., J Cancer. (2014) 5(9):765-773). IL-15 signals using three different receptor chains in at least two different combinations and has effects on the immune system. Despite the lack of homology between the amino acid sequences of IL-15 and IL-2, the mature IL-15 protein binds to the IL-2Rβγ heterodimer, activates intracellular signals, leading to cell activation (Mishra et al., Clin Cancer Res. (2014) 20(8):2044-2050). The third component of the IL-15R complex is a unique alpha chain (IL-15Rα). In contrast to the IL-2Rα chain, which binds IL-2 with low affinity and only confers high affinity for IL-2 when noncovalently linked to the IL-2Rβγ complex, IL-15Rα is itself a high-affinity receptor for IL-15 (Giri et al., EMBO J. (1995) 14:3654-63).

[0089] Binding of IL-15 to the IL-2 / 15Rβγ heterodimer induces JAK1 activation, followed by phosphorylation of STAT3 via the beta chain and activation of JAK3 / STAT5 via the c gamma chain. Phosphorylated STAT3 and STAT5 proteins form heterodimers, which then translocate to the nucleus, where they activate the transcription of the anti-apoptotic protein bcl-2 and the proto-oncogenes c-myc, c-fos, and c-jun.

[0090] The Akt signaling mechanism utilizes the adaptor protein Shc, which binds to phosphotyrosine residues on IL-2 / 15Rβ leading to Grb2 activation and via the Shc→Grb2→Gab2→PI3K→Akt signaling pathway to AKT to increase cell proliferation, survival and / or effector activity (Gu et al. Mol Cell Biol. 2000; 20:7109-20.). In the third signaling pathway after IL-15 trans-presentation to IL-2 / 15Rβγ and Shc-mediated Grb2 activation, the latter binds to the guanine nucleotide exchange factor SOS to form a Grb2-SOS complex, which then activates the Ras-Raf pathway by promoting the removal of GDP from members of the Ras family of subunits, which in turn activates the mitogen-activated protein kinase (MAPK) pathway for cell proliferation (Adunyah et al. Biochem Biophys Res Commun. 1997; 232:754-8). Thus, IL-15-mediated Grb2 phosphorylation modulates both the PI3K and MAPK pathways. Overall, these signaling mechanisms induce the expression and activation of downstream effector molecules such as c-myc, c-fos, c-jun, Bcl-2 and NF-κΒ.

[0091] Interleukin-21 (IL-21) is a cytokine that has potent regulatory effects on immune system cells, including natural killer (NK) cells and cytotoxic T cells that can destroy virus-infected cells or cancer cells. The cytokine induces cell division / proliferation in its target cells. Several preclinical studies have shown that IL-21 has anti-tumor activity in different tumor models, the mechanism of which involves activation of NK and T or B cell responses (Croce et al. J Immunol Res. 2015; 2015:696578). IL-21 signals via a heterodimer of IL-21 receptor (IL-21R) and yc and utilizes JAK-STAT, MAPK and PI3K pathways.

[0092] IL-21 binding stabilizes the complex between IL-21R and yc, leading to the activation of JAK1 and JAK3, which allows the recruitment and phosphorylation of STAT proteins, mainly STAT3, but also STAT1 and STAT5. Binding of IL-21 to IL-21R can also activate the MAPK and PI3K signaling pathways. IL-21 induces the transcription of suppressor of cytokine signaling 1 (SOCS1) and SOCS3 proteins, which downregulate the JAK-STAT pathway.

[0093] In the present specification, “signaling mediated by a yc-containing cytokine receptor” refers to signaling mediated by a multimeric receptor complex comprising yc (e.g., comprising yc and another member of the yc receptor family other than yc). “Signaling” refers to signal transduction and other cellular processes that control cellular activity.

[0094] Signaling mediated by a yc-containing cytokine receptor is signaling mediated by a yc-containing polypeptide complex (i.e., a polypeptide complex comprising one or more yc polypeptides and another member of the yc receptor family other than yc). A polypeptide complex according to the present disclosure can be characterized by non-covalent protein:protein interactions between the constituent polypeptides / peptides. In some embodiments, the association includes electrostatic interactions (e.g., ionic bonds, hydrogen bonds) and / or van der Waals forces.

[0095] Signaling mediated by a yc-containing cytokine receptor can be mediated by a heteromultimeric polypeptide complex comprising one or more yc polypeptides and additionally comprising one or more polypeptides of the yc receptor family other than yc (e.g., selected from IL-2R, IL-2R, IL-15R, IL-4R, IL-9R, IL-21R, or IL-7R ). In some embodiments, signaling mediated by a yc-containing cytokine receptor can be mediated by a polypeptide complex forming a yc family cytokine receptor. For example, signaling mediated by a yc-containing cytokine receptor can be mediated by a polypeptide complex forming a receptor for IL-2, IL-4, IL-7, IL-9, IL-15, or IL-21.

[0096] In some embodiments, signaling mediated by a yc-containing cytokine receptor can be mediated by a polypeptide complex comprising yc and another polypeptide of the yc receptor family (e.g., selected from IL-2R, IL-2R, IL-15R, IL-4R, IL-9R, IL-21R, or IL-7R ).

[0097] In some embodiments, signaling mediated by a yc-containing cytokine receptor can be mediated by a polypeptide complex comprising yc and IL-2R (i.e., a yc:IL-2R complex). As explained above, yc and IL-2R interact to form the intermediate affinity IL-2 receptor. This signaling can be referred to as yc:IL-2R -mediated signaling. In some embodiments, signaling mediated by a yc-containing cytokine receptor can be mediated by a polypeptide complex comprising IL-2, yc, and IL-2R (i.e., an IL-2:yc:IL-2R complex). This signaling can be referred to as IL-2:yc:IL-2R -mediated signaling (i.e., signaling mediated by binding of IL-2 to the intermediate affinity IL-2 receptor).

[0098] In some embodiments, the yc-containing cytokine receptor-mediated signaling can be mediated by a polypeptide complex comprising yc, IL-2Rβ, and IL-2Rα (i.e., a yc:IL-2Rβ:IL-2Rα complex). As explained above, yc, IL-2Rβ, and IL-2Rα interact to form a high affinity IL-2 receptor. This signaling can be referred to as yc:IL-2Rβ:IL-2Rα-mediated signaling. In some embodiments, the yc-containing cytokine receptor-mediated signaling can be mediated by a polypeptide complex comprising IL-2, yc, IL-2Rβ, and IL-2Rα (i.e., an IL-2:yc:IL-2Rβ:IL-2Rα complex). This signaling can be referred to as IL-2:yc:IL-2Rβ:IL-2Rα-mediated signaling (i.e., signaling mediated by binding of IL-2 to the high affinity IL-2 receptor).

[0099] In some embodiments, the yc-containing cytokine receptor-mediated signaling can be mediated by a polypeptide complex comprising yc, IL-2Rβ, and IL-15Rα (i.e., a yc:IL-2Rβ:IL-15Rα complex). As explained above, yc, IL-2Rβ, and IL-15Rα interact to form an IL-15 receptor. This signaling can be referred to as yc:IL-2Rβ:IL-15Rα-mediated signaling. In some embodiments, the yc-containing cytokine receptor-mediated signaling can be mediated by a polypeptide complex comprising IL-15, yc, IL-2Rβ, and IL-15Rα (i.e., an IL-15:yc:IL-2Rβ:IL-15Rα complex). This signaling can be referred to as IL-15:yc:IL-2Rβ:IL-15Rα-mediated signaling (i.e., signaling mediated by binding of IL-15 to the IL-15 receptor).

[0100] In some embodiments, the yc-containing cytokine receptor-mediated signaling can be mediated by a polypeptide complex comprising yc and IL-4Rα (i.e., a yc:IL-4Rα complex). As explained above, yc and IL-4Rα interact to form an IL-4 receptor. This signaling can be referred to as yc:IL-4Rα-mediated signaling. In some embodiments, the yc-containing cytokine receptor-mediated signaling can be mediated by a polypeptide complex comprising IL-4, yc, and IL-4Rα (i.e., an IL-4:yc:IL-4Rα complex). This signaling can be referred to as IL-4:yc:IL-4Rα-mediated signaling (i.e., signaling mediated by binding of IL-4 to the IL-4 receptor).

[0101] In some embodiments, the yc-containing cytokine receptor-mediated signaling can be mediated by a polypeptide complex comprising yc and IL-9Ra (i.e., yc:IL-9Ra complex). As explained above, yc and IL-9Ra interact to form the IL-9 receptor. This signaling can be referred to as yc:IL-9Ra-mediated signaling. In some embodiments, the yc-containing cytokine receptor-mediated signaling can be mediated by a polypeptide complex comprising IL-9, yc, and IL-9Ra (i.e., IL-9:yc:IL-9Ra complex). This signaling can be referred to as IL-9:yc:IL-9Ra-mediated signaling (i.e., signaling mediated by binding of IL-9 to the IL-9 receptor).

[0102] In some embodiments, the yc-containing cytokine receptor-mediated signaling can be mediated by a polypeptide complex comprising yc and IL-21Ra (i.e., yc:IL-21Ra complex). As explained above, yc and IL-21Ra interact to form the IL-21 receptor. This signaling can be referred to as yc:IL-21Ra-mediated signaling. In some embodiments, the yc-containing cytokine receptor-mediated signaling can be mediated by a polypeptide complex comprising IL-21, yc, and IL-21Ra (i.e., IL-21:yc:IL-21Ra complex). This signaling can be referred to as IL-21:yc:IL-21Ra-mediated signaling (i.e., signaling mediated by binding of IL-21 to the IL-21 receptor).

[0103] In some embodiments, the yc-containing cytokine receptor-mediated signaling can be mediated by a polypeptide complex comprising yc and IL-7Ra (i.e., yc:IL-7Ra complex). As explained above, yc and IL-7Ra interact to form the IL-7 receptor. This signaling can be referred to as yc:IL-7Ra-mediated signaling. In some embodiments, the yc-containing cytokine receptor-mediated signaling can be mediated by a polypeptide complex comprising IL-7, yc, and IL-7Ra (i.e., IL-7:yc:IL-7Ra complex). This signaling can be referred to as IL-7:yc:IL-7Ra-mediated signaling (i.e., signaling mediated by binding of IL-7 to the IL-7 receptor).

[0104] The present disclosure relates to antigen binding molecules that selectively bind to more than one component of a yc-containing cytokine receptor. Specifically, the antigen binding molecules of the present disclosure are multi-specific antigen binding molecules comprising (i) a yc-binding moiety, and (ii) a moiety that binds to one or more polypeptides of a yc-containing cytokine receptor other than yc (e.g., selected from IL-2Rβ, IL-2Ra, IL-15Ra, IL-4Ra, IL-9Ra, IL-21Ra, and IL-7Ra). That is, the antigen binding molecules bind to (i) yc and (ii) at least one of IL-2Rβ, IL-2Ra, IL-15Ra, IL-4Ra, IL-9Ra, IL-21Ra, or IL-7Ra.

[0105] Human IL-2Rβ (also known as CD122, IL15RB, and P70-75) is the protein identified by UniProt P14784. The canonical isoform of human IL-2Rβ (isoform 1) has the amino acid sequence set forth in SEQ ID NO: 434. The N-terminal 26 amino acids of SEQ ID NO: 434 constitute a signal peptide, thus the mature form of the human IL-2Rβ protein (i.e., after processing to remove the signal peptide) has the amino acid sequence set forth in SEQ ID NO: 435. Amino acids 27 to 240 of SEQ ID NO: 434 constitute the extracellular domain of IL-2Rβ, as set forth in SEQ ID NO: 436. Amino acids 241 to 265 of SEQ ID NO: 434 form a transmembrane domain (SEQ ID NO: 512), and positions 266 to 551 form a cytoplasmic domain (SEQ ID NO: 513).

[0106] In the present specification, “IL-2Rβ” or “CD122” refers to IL-2Rβ from any species, including isoforms, fragments, variants, or homologs of IL-2Rβ from any species. In some embodiments, the IL-2Rβ is IL-2Rβ from a mammal (e.g., a mammal of the order carnivora, a mammal of the order placentalia, a mammal of the order archonta, a mammal of the order primates, a mammal of the order chiroptera, a primate (rhesus macaque, cynomolgus macaque, non-human primate, or human)). In some embodiments, the IL-2Rβ is human IL-2Rβ.

[0107] The minimum length of a fragment of IL-2Rβ can be one of 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 400, 450, or 500 amino acids, and the maximum length can be one of 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 amino acids. The isoform, fragment, variant, or homolog of IL-2Rβ can exhibit association with one or more of yc, IL-2Ra, IL-15Ra, IL-2, or IL-15.

[0108] In some embodiments, the IL-2Rβ has at least 70% (preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) amino acid sequence identity to SEQ ID NO: 434 or 435. In some embodiments, the fragment of IL-2Rβ comprises or consists of an amino acid sequence having at least 70% (preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) amino acid sequence identity to SEQ ID NO: 436.

[0109] Human IL-2Ra (also known as CD25, IDDM10, IL2R, TCGFR, p55, and IMD41) is the protein identified by UniProt P01589. The canonical isoform of human IL-2Ra has the amino acid sequence set forth in SEQ ID NO: 507. The N-terminal 21 amino acids of SEQ ID NO: 507 constitute a signal peptide (SEQ ID NO: 514), so the mature form of human IL-2Ra (i.e., after processing to remove the signal peptide) has the amino acid sequence set forth in SEQ ID NO: 515. Human IL-2Ra comprises an extracellular domain (SEQ ID NO: 508), a transmembrane domain (SEQ ID NO: 509), and a cytoplasmic domain (SEQ ID NO: 510).

[0110] In the present specification, “IL-2Ra” refers to IL-2Ra from any species, including isoforms, fragments, variants, or homologs from any species. In some embodiments, the IL-2Ra is IL-2Ra from a mammal (e.g., a mammal of the order carnivora, a mammal of the order placentalia, a mammal of the order archonta, a mammal of the order primates, a mammal of the order chiroptera, a mammal of the order anthropoidea, a non-human primate, or a human). In some embodiments, the IL-2Ra is human IL-2Ra. Isoforms, fragments, variants, or homologs of IL-2Ra can exhibit association with one or more of yc or IL-2Rb or IL-2. Fragments of IL-2Ra can be a minimum of one of 10, 20, 30, 40, 50, 100, 150, or 200 amino acids and a maximum of one of 20, 30, 40, 50, 100, 150, 200, or 250 amino acids.

[0111] In some embodiments, the IL-2Ra comprises or consists of an amino acid sequence having at least 70% (preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) amino acid sequence identity to SEQ ID NO: 507 or 515. In some embodiments, the fragment of IL-2Ra comprises or consists of an amino acid sequence having at least 70% (preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) amino acid sequence identity to SEQ ID NO: 508.

[0112] Human IL-15Ra (also known as CD215) is the protein identified by UniProt Q13261. The canonical isoform of human IL-15Ra (Uniprot Q13261-1) has the amino acid sequence of SEQ ID NO: 511.

[0113] The N-terminal 30 amino acids of SEQ ID NO: 511 constitute a signal peptide (SEQ ID NO: 516), so the mature form of human IL-15Ra (i.e., after processing to remove the signal peptide) has the amino acid sequence set forth in SEQ ID NO: 517. Human IL-15Ra comprises an extracellular domain (SEQ ID NO: 520), a transmembrane domain (SEQ ID NO: 521), and a cytoplasmic domain (SEQ ID NO: 522).

[0114] In the present specification, “IL-15Ra” refers to IL-15Ra from any species, including isoforms, fragments, variants, or homologs from any species. In some embodiments, IL-15Ra is IL-15Ra from a mammal (e.g., a mammal of the order carnivora, a mammal of the order placentalia, a mammal of the order archonta, a mammal of the order primates, a mammal of the order chiroptera, a mammal of the order anthropoidea, a rhesus macaque, a cynomolgus macaque, a non-human primate, or a human). In some embodiments, IL-15Ra is human IL-15Ra. Isoforms, fragments, variants, or homologs of IL-15Ra can exhibit association with one or more of yc or IL-2Rb or IL-15. Fragments of IL-15Ra can be one of 10, 20, 30, 40, 50, 100, 150, or 200 amino acids in minimum length and one of 20, 30, 40, 50, 100, 150, 200, or 250 amino acids in maximum length.

[0115] In some embodiments, IL-15Ra comprises or consists of an amino acid sequence having at least 70% (preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) amino acid sequence identity to SEQ ID NO: 511 or 517. In some embodiments, a fragment of IL-15Ra comprises or consists of an amino acid sequence having at least 70% (preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) amino acid sequence identity to SEQ ID NO: 520.

[0116] Human IL-4Ra (also known as CD124, IL-4R) is the protein identified by UniProt P24394. The canonical isoform of human IL-4R (Uniprot P24394-1) has the amino acid sequence of SEQ ID NO: 523.

[0117] The N-terminal 25 amino acids of SEQ ID NO: 523 constitute a signal peptide (SEQ ID NO: 524), so the mature form of human IL-4Ra (i.e., after processing to remove the signal peptide) has the amino acid sequence set forth in SEQ ID NO: 525. Human IL-4Ra comprises an extracellular domain (SEQ ID NO: 526), a transmembrane domain (SEQ ID NO: 527), and a cytoplasmic domain (SEQ ID NO: 528).

[0118] In the present specification, “IL-4Ra” refers to IL-4Ra from any species, including isoforms, fragments, variants, or homologs from any species. In some embodiments, IL-4Ra is IL-4Ra from a mammal (e.g., a mammal of the order carnivora, a mammal of the order placentalia, a mammal of the order archonta, a mammal of the order primates, a mammal of the order chiroptera, a mammal of the order anthropoidea, a rhesus monkey, a cynomolgus monkey, a non-human primate, or a human). In some embodiments, IL-4Ra is human IL-4Ra.

[0119] Isoforms, fragments, variants, or homologs of IL-4R can exhibit association with yc or IL-4. Fragments of IL-4Ra can be at least one of 10, 20, 30, 40, 50, 100, 150, 200, 300, 400, 500, 600, 700, or 800 amino acids in length and at most one of 10, 20, 30, 40, 50, 100, 150, 200, 300, 400, 500, 600, 700, or 800 amino acids in length.

[0120] In some embodiments, IL-4Ra comprises or consists of an amino acid sequence having at least 70% (preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) amino acid sequence identity to SEQ ID NO: 523 or 525. In some embodiments, a fragment of IL-4Ra comprises or consists of an amino acid sequence having at least 70% (preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) amino acid sequence identity to SEQ ID NO: 526.

[0121] Human IL-9Ra (also known as IL-9R and CD129) is the protein identified by UniProt Q01113. The canonical isoform of human IL-9Ra (Uniprot Q01113-1) has the amino acid sequence of SEQ ID NO: 529.

[0122] The N-terminal 40 amino acids of SEQ ID NO: 529 constitute a signal peptide (SEQ ID NO: 530), so the mature form of human IL-9Ra (i.e., after processing to remove the signal peptide) has the amino acid sequence set forth in SEQ ID NO: 531. Human IL-9Ra comprises an extracellular domain (SEQ ID NO: 532), a transmembrane domain (SEQ ID NO: 533), and a cytoplasmic domain (SEQ ID NO: 534).

[0123] In this specification, "IL-9Rα" refers to IL-9Rα from any species, including isoforms, fragments, variants or homologs from any species. In some embodiments, IL-9Rα is IL-9Rα from a mammal (e.g., a therian mammal, a placental mammal, a eutherian, a prototherian, a chiropteran, a primate (rhesus monkey, cynomolgus monkey, non-human primate or human)). In some embodiments, IL-9Rα is human IL-9Rα.

[0124] An isoform, fragment, variant, or homolog of IL-9R can exhibit association with γc or IL-9. A fragment of IL-9Rα can have a minimum length of one of 10, 20, 30, 40, 50, 100, 150, 200, 300, 400, or 500 amino acids and a maximum length of one of 20, 30, 40, 50, 100, 150, 200, 300, 400, or 500 amino acids.

[0125] In some embodiments, IL-9Rα comprises or consists of an amino acid sequence having at least 70% (preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) amino acid sequence identity to SEQ ID NO: 529 or 531. In some embodiments, a fragment of IL-9Rα comprises or consists of an amino acid sequence having at least 70% (preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) amino acid sequence identity to SEQ ID NO: 532.

[0126] Human IL-21R α (also referred to as CD360) is the protein identified by UniProt Q9HBE5. The canonical isoform of human IL-21R α has the amino acid sequence of SEQ ID NO:535. SEQ ID NO:535 N-terminal 19 amino acids constitute signal peptide (SEQ ID NO:540), so the mature form of human IL-21R α (that is, after being processed to remove signal peptide) has the amino acid sequence shown in SEQ ID NO:541. Human IL-21R α includes extracellular domain (SEQ ID NO:536), transmembrane domain (SEQ ID NO:537) and cytoplasmic domain (SEQ ID NO:538).

[0127] In the present specification, “IL-21Rα” refers to IL-21Rα from any species, including isoforms, fragments, variants, or homologs from any species. In some embodiments, IL-21Rα is IL-21Rα from a mammal (e.g., a mammal of the order carnivora, a mammal of the order placentalia, a mammal of the order archonta, a mammal of the order primates, a mammal of the order chiroptera, a mammal of the order anthropoidea, a rhesus monkey, a cynomolgus monkey, a non-human primate, or a human). In some embodiments, IL-21Rα is human IL-21Rα.

[0128] Isoforms, fragments, variants, or homologs of IL-21Rα can exhibit association with yc or IL-21. Fragments of IL-21Rα can be at least one of 10, 20, 30, 40, 50, 100, 150, 200, 300, 400, or 500 amino acids in length and at most one of 20, 30, 40, 50, 100, 150, 200, 300, 400, or 500 amino acids in length.

[0129] In some embodiments, IL-21Rα comprises or consists of an amino acid sequence having at least 70% (preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) amino acid sequence identity to SEQ ID NO: 535 or 541. In some embodiments, a fragment of IL-21Rα comprises or consists of an amino acid sequence having at least 70% (preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) amino acid sequence identity to SEQ ID NO: 536.

[0130] Human IL-7Rα (also known as IL-7R, CD127) is the protein identified by UniProt P16871. The canonical isoform of human IL-7Rα (Uniprot P16871-1) has the amino acid sequence of SEQ ID NO: 539. The N-terminal 20 amino acids of SEQ ID NO: 539 constitute a signal peptide (SEQ ID NO: 542), so the mature form of human IL-7Rα (i.e., after processing to remove the signal peptide) has the amino acid sequence set forth in SEQ ID NO: 546.

[0131] The mature canonical isoform of human IL-7Rα comprises an extracellular domain (SEQ ID NO: 543), a transmembrane domain (SEQ ID NO: 544), and a cytoplasmic domain (SEQ ID NO: 545).

[0132] In the present specification, “IL-7Ra” refers to IL-7Ra from any species, including isoforms, fragments, variants, or homologs from any species. In some embodiments, the IL-7Ra is from a mammal (e.g., a theria mammal, a eutherian mammal, a euarchontoglires, a metatheria, a chiroptera, a primate (a rhesus macaque, a cynomolgus monkey, a non-human primate, or a human). In some embodiments, the IL-7Ra is a human IL-7Ra.

[0133] Isoforms, fragments, variants, or homologs of IL-7R can exhibit association with yc or IL-7. Fragments of IL-7Ra can be at least one of 10, 20, 30, 40, 50, 100, 150, 200, 300, 400, or 450 amino acids in length and at most one of 20, 30, 40, 50, 100, 150, 200, 300, 400, or 450 amino acids in length.

[0134] In some embodiments, the IL-7Ra comprises or consists of an amino acid sequence having at least 70% (preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) amino acid sequence identity to any one of SEQ ID NOs: 539 or 546. In some embodiments, the fragment of IL-7Ra comprises or consists of an amino acid sequence having at least 70% (preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) amino acid sequence identity to SEQ ID NO: 543.

[0135] Antigen binding molecules

[0136] The present disclosure provides antigen binding molecules capable of binding to (i.e., associating with) yc-containing cytokine receptors and constituent polypeptides thereof.

[0137] In aspects of the present disclosure, the antigen binding molecule comprises a yc-binding moiety. In aspects of the present disclosure, the antigen binding molecule comprises a moiety that binds to a polypeptide of a yc-containing cytokine receptor other than yc. The polypeptide of a yc-containing cytokine receptor other than yc can be selected from the group consisting of IL-2Rb, IL-2Ra, IL-15Ra, IL-4Ra, IL-9Ra, IL-21Ra, and IL-7Ra.

[0138] In some embodiments, the antigen binding molecule comprises an IL-2Rβ binding moiety. In some embodiments, the antigen binding molecule comprises an IL-2Rα binding moiety. In some embodiments, the antigen binding molecule comprises an IL-15Rα binding moiety. In some embodiments, the antigen binding molecule comprises an IL-4Rα binding moiety. In some embodiments, the antigen binding molecule comprises an IL-9Rα binding moiety. In some embodiments, the antigen binding molecule comprises an IL-21Rα binding moiety. In some embodiments, the antigen binding molecule comprises an IL-7Rα binding moiety.

[0139] In some embodiments, the antigen binding molecule comprises a yc binding moiety and an IL-2Rβ binding moiety. In some embodiments, the antigen binding molecule comprises a yc binding moiety and an IL-2Rα binding moiety. In some embodiments, the antigen binding molecule comprises a yc binding moiety and an IL-15Rα binding moiety. In some embodiments, the antigen binding molecule comprises a yc binding moiety and an IL-4Rα binding moiety. In some embodiments, the antigen binding molecule comprises a yc binding moiety and an IL-9Rα binding moiety. In some embodiments, the antigen binding molecule comprises a yc binding moiety and an IL-21Rα binding moiety. In some embodiments, the antigen binding molecule comprises a yc binding moiety and an IL-7Rα binding moiety.

[0140] In some embodiments, the antigen binding molecule does not comprise an IL-2Rβ binding moiety.

[0141] As used herein, an “antigen binding molecule” refers to a molecule that binds to a given target antigen. The antigen binding molecules of the present disclosure comprise one or more antigen binding moieties by which the antigen binding molecule binds to a target antigen.

[0142] An antigen binding moiety can comprise or can be derived from an antibody (i.e., an immunoglobulin (Ig)) and antigen binding fragments thereof. As used herein, an “antibody” includes monoclonal antibodies, polyclonal antibodies, monospecific and multispecific (e.g., bispecific, trispecific, etc.) antibodies, as well as antibody-derived antigen binding molecules such as scFv, scFab, diabodies, triabodies, scFv-Fc, minibodies, single domain antibodies (e.g., VhH), etc. Antigen binding fragments of antibodies include, for example, Fv, Fab, F(ab’)2, and F(ab’) fragments.

[0143] Antigen binding moieties also include target antigen binding aptamers, such as nucleic acid aptamers (e.g., reviewed in Zhou and Rossi, Nat Rev Drug Discov. (2017) 16(3): 181-202). In some embodiments, the antigen binding moiety comprises or consists of an antigen binding peptide / polypeptide, such as a peptide aptamer, thioredoxin, monobody, anticalin, Kunitz domain, avimer, knottin, fynomer, atrimer, DARPin, affimer, nanobody (i.e., single domain antibody (sdAb)), affilin, armadillo repeat protein (ArmRP), OBody, or fibronectin— e.g., reviewed in Reverdatto et al., Curr Top Med Chem. (2015) 15(12): 1082-1101, which is incorporated by reference herein in its entirety (see also, e.g., Boersma et al., J Biol Chem. (2011) 286:41273-85 and Emanuel et al., Mabs. (2011) 3:38-48).

[0144] In some embodiments, the antigen binding moiety comprises or consists of an antigen binding region of an antibody (e.g., an antigen binding fragment of an antibody). The antigen binding moiety of an antigen binding molecule of the present disclosure can comprise an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) of an antibody that binds to the relevant target antigen of the antigen binding moiety. The antigen binding domain formed by the VH and VL can also be referred to herein as an Fv region.

[0145] In some embodiments, the antigen binding moiety is or comprises an Fv of an antibody (e.g., provided as a scFv). In some embodiments, the antigen binding moiety is or comprises a Fab region of an antibody. In some embodiments, the antigen binding moiety is or comprises a full antibody (i.e., comprising a variable region and a constant region).

[0146] The antigen binding moiety can be or can comprise an antigen binding polypeptide or an antigen binding polypeptide complex. The antigen binding moiety can comprise more than one polypeptide, which together form the antigen binding moiety. The polypeptides can be covalently or non-covalently associated. In some embodiments, the polypeptides form part of a larger polypeptide comprising the polypeptides (e.g., in the case of a scFv comprising a VH and a VL, or in the case of a scFab comprising a VH-CH1 and a VL-CL).

[0147] An antigen binding moiety can refer to a non-covalent or covalent complex of more than one polypeptide (e.g., 2, 3, 4, 6, or 8 polypeptides), for example, an IgG-like antigen binding molecule comprising two heavy chain polypeptides and two light chain polypeptides.

[0148] Antigen binding moieties of the present disclosure can be designed and prepared using monoclonal antibody (mAb) sequences that are capable of binding to a given target antigen (e.g., IL-2Rβ). Antigen binding regions of antibodies, such as single-chain variable fragments (scFv), single-domain antibody fragments (VHH), Fab, and F(ab’)2 fragments, can also be used / provided. An “antigen binding region” is any fragment of an antibody that binds to a given antibody’s specific target.

[0149] Antibodies typically comprise six complementarity determining regions (CDRs); three in the heavy chain variable (VH) region: HC-CDR1, HC-CDR2, and HC-CDR3, and three in the light chain variable (VL) region: LC-CDR1, LC-CDR2, and LC-CDR3. Together, these six CDRs define the paratope of an antibody, which is the portion of the antibody that binds to a target antigen.

[0150] The VH and VL regions comprise framework regions (FRs) flanking each CDR, which provide a scaffold for the CDRs. From N-terminal to C-terminal, a VH region comprises the following structure: N-terminal - [HC-FR1] - [HC-CDR1] - [HC-FR2] - [HC-CDR2] - [HC-FR3] - [HC-CDR3] - [HC-FR4] - C-terminal; and a VL region comprises the following structure: N-terminal - [LC-FR1] - [LC-CDR1] - [LC-FR2] - [LC-CDR2] - [LC-FR3] - [LC-CDR3] - [LC-FR4] - C-terminal.

[0151] There are several different conventions for defining antibody CDRs and FRs, such as those described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991), Chothia et al., J. Mol. Biol. (1987) 196:901-917, and VBASE2 as described in Retter et al., Nucl. Acids Res. (2005) 33 (Suppl. 1):D671-D674. The CDRs and FRs of the VH and VL regions of the antibody clones described herein are defined according to the international IMGT (ImMunoGeneTics) information system (LeFranc et al., Nucleic Acids Res. (2015) 43 (Database issue):D413-22), which uses the IMGT V-DOMAIN numbering rules as described in Lefranc et al., Dev. Comp. Immunol. (2003) 27:55-77.

[0152] In some embodiments, the antigen binding moiety according to the present disclosure comprises or consists of an Fv portion that binds to its target antigen. In some embodiments, the VH and VL regions of the Fv portion are provided as a single polypeptide connected by a linker sequence, i.e., a single chain Fv (scFv).

[0153] The VL and light chain constant (CL) region of an antibody, together with the VH region and heavy chain constant 1 (CH1) region, make up a Fab region. In some embodiments, the antigen binding molecule comprises a Fab portion comprising VH, CH1, VL, and CL (e.g., CK or C ). In some embodiments, the Fab portion comprises a polypeptide comprising VH and CH1 (e.g., a VH-CH1 fusion polypeptide) and a polypeptide comprising VL and CL (e.g., a VL-CL fusion polypeptide). In some embodiments, the Fab portion comprises a polypeptide comprising VH and CL (e.g., a VH-CL fusion polypeptide) and a polypeptide comprising VL and CH1 (e.g., a VL-CH1 fusion polypeptide); that is, in some embodiments, the Fab portion is a CrossFab portion. In some embodiments, the VH, CH1, VL, and CL regions of the Fab portion or CrossFab portion are provided as a single polypeptide connected by a linker sequence, i.e., as a single chain Fab (scFab) or single chain CrossFab (scCrossFab).

[0154] In some embodiments, the antigen binding molecules described herein comprise, or consist of, a whole antibody that binds to its target antigen. As used herein, “whole antibody” refers to an antibody having a structure substantially similar to that of an immunoglobulin (Ig). Different classes of immunoglobulins and their structures are described, for example, in Schroeder and Cavacini. J Allergy Clin Immunol. (2010) 125(202):S41-S52, which is incorporated by reference herein in its entirety.

[0155] G-type immunoglobulins (i.e., IgG) are glycoproteins of about 150 kDa comprising two heavy chains and two light chains. From N- to C-terminus, the heavy chain comprises VH, followed by a heavy chain constant region comprising three constant domains (CH1, CH2, and CH3), and similarly, the light chain comprises VL, followed by CL. Depending on the heavy chain, immunoglobulins can be classified as IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgA (e.g., IgA1, IgA2), IgD, IgE, or IgM. The light chain can be kappa (K) or lambda (l).

[0156] In this document, “CH2 domain” refers to an amino acid sequence corresponding to the CH2 domain of an immunoglobulin (Ig). According to the EU numbering system (described in Edelman et al., Proc Natl Acad Sci USA. (1969) 63(1): 78-85), the CH2 domain is the region of the Ig formed by positions 231 to 340 of the immunoglobulin constant domain. “CH3 domain” refers to an amino acid sequence corresponding to the CH3 domain of an immunoglobulin (Ig). According to the EU numbering system, the CH3 domain is the region of the Ig formed by positions 341 to 447 of the immunoglobulin constant domain. “CH2-CH3 region” refers to an amino acid sequence corresponding to the CH2 and CH3 domains of an immunoglobulin (Ig). According to the EU numbering system, the CH2-CH3 region is the region of the Ig formed by positions 231 to 447 of the immunoglobulin constant domain.

[0157] In some embodiments described herein, one or more amino acids of an amino acid sequence referred to herein (e.g., an amino acid sequence of an antigen binding molecule, e.g., an amino acid sequence of a CDR or VH / VL region) are substituted with another amino acid. A substitution includes the substitution of an amino acid residue with a non-identical "replacement" amino acid residue. The replacement amino acid residue of a substitution according to the present disclosure can be a naturally occurring amino acid residue (i.e., encoded by the genetic code) that is non-identical to the amino acid residue at the relevant position of the equivalent, unsubstituted amino acid sequence, selected from the group consisting of: alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine (Cys), glutamine (Gln), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (lie), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), and valine (Val). In some embodiments, the replacement amino acid can be a non-naturally occurring amino acid residue, i.e., an amino acid residue other than those listed in the preceding sentence. Examples of non-naturally occurring amino acid residues include norleucine, ornithine, norvaline, homoserine, aminoisobutyric acid, and other amino acid residue analogs such as those described in Ellman et al., Meth. Enzym. (1991) 202:301-336.

[0158] In some embodiments, the substitution can be biochemically conservative. In some embodiments, where the amino acid to be substituted is provided in one of the first five rows of the table below, the replacement amino acid of the substitution is another non-identical amino acid provided in the same row:

[0159]

[0160] By way of example, in some embodiments where the substitution is of a Met residue, the replacement amino acid can be selected from the group consisting of Ala, Val, Leu, lie, Trp, Tyr, Phe, and norleucine.

[0161] In some embodiments, the replacement amino acid in a substitution can have the same side chain polarity as the amino acid residue it replaces. In some embodiments, the replacement amino acid in a substitution can have the same side chain charge (at pH 7.4) as the amino acid residue it replaces:

[0162]

[0163]

[0164] That is, in some embodiments, a nonpolar amino acid is substituted with another non-identical nonpolar amino acid. In some embodiments, a polar amino acid is substituted with another non-identical polar amino acid. In some embodiments, an acidic polar amino acid is substituted with another non-identical acidic polar amino acid. In some embodiments, a basic polar amino acid is substituted with another non-identical basic polar amino acid. In some embodiments, an uncharged amino acid is substituted with another non-identical uncharged amino acid. In some embodiments, a positive amino acid is substituted with another non-identical positive amino acid. In some embodiments, a negative amino acid is substituted with another non-identical negative amino acid.

[0165] In some embodiments, the substitution can be functionally conservative. That is, in some embodiments, the substitution can not affect (or can not substantially affect) one or more functional properties (e.g., target binding) of the antigen binding molecule comprising the substitution as compared to an equivalent non-substituted molecule.

[0166] In some embodiments, the antigen binding molecule comprises an antigen binding moiety that binds to yc. In some embodiments, the antigen binding moiety comprises a CDR of an antibody that binds to yc. In some embodiments, the antigen binding moiety comprises a FR of an antibody that binds to yc. In some embodiments, the antigen binding moiety comprises a CDR and a FR of an antibody that binds to yc. That is, in some embodiments, the antigen binding moiety comprises a VH region and a VL region of an antibody that binds to yc. In some embodiments, the antigen binding moiety comprises or consists of a Fv portion or a Fab portion of an antibody that binds to yc, comprising a VH region and a VL region. Antibodies that bind to yc include, for example, REGN7257 (e.g., described in Floch et al., Hemasphere. (2022) 6(Suppl):694-695), TUGh4 (e.g., described in Ishii et al., Int Immunol. (1994) 6(8): 1273-1277), and 3E12 (e.g., described in He et al., Proc Natl Acad Sci USA. (1995) 92(12):5689-5693). The VHH antibody nb6 has also been shown to bind to yc ((Yen et al., Cell. 2022; 185(8): 1414-1430).

[0167] In some embodiments, the antigen binding molecule comprises an antigen binding moiety that binds to IL-2Rβ. In some embodiments, the antigen binding moiety comprises a CDR of an antibody that binds to IL-2Rβ. In some embodiments, the antigen binding moiety comprises a FR of an antibody that binds to IL-2Rβ. In some embodiments, the antigen binding moiety comprises a CDR and a FR of an antibody that binds to IL-2Rβ. That is, in some embodiments, the antigen binding moiety comprises a VH region and a VL region of an antibody that binds to IL-2Rβ. In some embodiments, the antigen binding moiety comprises or consists of a Fv portion or a Fab portion of an antibody that binds to IL-2Rβ, including the VH region and the VL region. Antibodies that bind to IL-2Rβ include, for example, Mibeta1 (described in Tsudo et al. Proc Natl Acad Sci USA. (1989) 86(6): 1982-1986), TM-beta1 (described in Tanaka et al. J Immunol. (1991) 147(7): 2222-2228), and TU27 (described in Takeshita et al. J Exp Med. (1989) 169(4): 1323-1332).

[0168] In some embodiments, the antigen binding molecule comprises an antigen binding moiety that binds to IL-2Rα. In some embodiments, the antigen binding moiety comprises a CDR of an antibody that binds to IL-2Rα. In some embodiments, the antigen binding moiety comprises a FR of an antibody that binds to IL-2Rα. In some embodiments, the antigen binding moiety comprises a CDR and a FR of an antibody that binds to IL-2Rα. That is, in some embodiments, the antigen binding moiety comprises a VH region and a VL region of an antibody that binds to IL-2Rα. In some embodiments, the antigen binding moiety comprises or consists of a Fv portion or a Fab portion of an antibody that binds to IL-2Rα, including the VH region and the VL region. Antibodies that bind to IL-2Rα include, for example, daclizumab (described in Vincenti et al. N Engl J Med. (1998) 338(3): 161-165; DrugBank Accession Number DB00111), Basiliximab (described in Kapic et al. Med Arh. (2004) 58(6): 373-376; DrugBank Accession Number DB00074), and 7G7 / B6 (described in Rubin et al. Hybridoma (1985) 4(2): 91-102).

[0169] In some embodiments, the antigen binding molecule comprises an antigen binding moiety that binds to IL-15Ra. In some embodiments, the antigen binding moiety comprises a CDR of an antibody that binds to IL-15Ra. In some embodiments, the antigen binding moiety comprises a FR of an antibody that binds to IL-15Ra. In some embodiments, the antigen binding moiety comprises a CDR and a FR of an antibody that binds to IL-15Ra. That is, in some embodiments, the antigen binding moiety comprises a VH region and a VL region of an antibody that binds to IL-15Ra. In some embodiments, the antigen binding moiety comprises or consists of a Fv portion or a Fab portion of an antibody that binds to IL-15Ra, including the VH region and the VL region. Antibodies that bind to IL-15Ra include, for example, JM7A4 (described in Dubois et al. Immunity (2002) 17(5):537-547).

[0170] In some embodiments, the antigen binding molecule comprises an antigen binding moiety that binds to IL-4Ra. In some embodiments, the antigen binding moiety comprises a CDR of an antibody that binds to IL-4Ra. In some embodiments, the antigen binding moiety comprises a FR of an antibody that binds to IL-4Ra. In some embodiments, the antigen binding moiety comprises a CDR and a FR of an antibody that binds to IL-4Ra. That is, in some embodiments, the antigen binding moiety comprises a VH region and a VL region of an antibody that binds to IL-4Ra. In some embodiments, the antigen binding moiety comprises or consists of a Fv portion or a Fab portion of an antibody that binds to IL-4Ra, including the VH region and the VL region. Antibodies that bind to IL-4Ra include, for example, Dupilumab (also known as SAR231893 and REGN668, described in Wenzel et al. N Engl J Med. (2013) 368(26):2455-2466; DrugBank Accession Number DB12159) and 4R34.1.1 (described in Kim et al., Sci Rep. (2019) 9(1):7772).

[0171] In some embodiments, the antigen binding molecule comprises an antigen binding moiety that binds to IL-9Ra. In some embodiments, the antigen binding moiety comprises a CDR of an antibody that binds to IL-9Ra. In some embodiments, the antigen binding moiety comprises a FR of an antibody that binds to IL-9Ra. In some embodiments, the antigen binding moiety comprises a CDR and a FR of an antibody that binds to IL-9Ra. That is, in some embodiments, the antigen binding moiety comprises a VH region and a VL region of an antibody that binds to IL-9Ra. In some embodiments, the antigen binding moiety comprises or consists of a Fv portion or a Fab portion of an antibody that binds to IL-9Ra, including the VH region and the VL region. Antibodies that bind to IL-9Ra include, for example, RZ-66 (described in Takatsuka et al., Nature Immunology. (2018) 19(9): 1025-1034) and AH9R7 (described in Smedt et al., J Immunol. (2000) 164(4): 1761-1767).

[0172] In some embodiments, the antigen binding molecule comprises an antigen binding moiety that binds to IL-21Ra. In some embodiments, the antigen binding moiety comprises a CDR of an antibody that binds to IL-21Ra. In some embodiments, the antigen binding moiety comprises a FR of an antibody that binds to IL-21Ra. In some embodiments, the antigen binding moiety comprises a CDR and a FR of an antibody that binds to IL-21Ra. That is, in some embodiments, the antigen binding moiety comprises a VH region and a VL region of an antibody that binds to IL-21Ra. In some embodiments, the antigen binding moiety comprises or consists of a Fv portion or a Fab portion of an antibody that binds to IL-21Ra, including the VH region and the VL region. Antibodies that bind to IL-21Ra include, for example, ATR-107 (described in Hua et al., J Clin Pharmacol. (2014) 54(1): 14-22) and 4A9 (described in Jin and Malek, J Leukoc Biol. (2006) 80(6): 1416-1423).

[0173] In some embodiments, the antigen binding molecule comprises an antigen binding moiety that binds to IL-7Ra. In some embodiments, the antigen binding moiety comprises a CDR of an antibody that binds to IL-7Ra. In some embodiments, the antigen binding moiety comprises a FR of an antibody that binds to IL-7Ra. In some embodiments, the antigen binding moiety comprises a CDR and a FR of an antibody that binds to IL-7Ra. That is, in some embodiments, the antigen binding moiety comprises a VH region and a VL region of an antibody that binds to IL-7Ra. In some embodiments, the antigen binding moiety comprises or consists of a Fv portion or a Fab portion of an antibody that binds to IL-7Ra, comprising a VH region and a VL region. Antibodies that bind to IL-7Ra include, for example, Lusvertikimab (OSE-127; e.g., described in Belarif et al., Nature Communications 9, 4483. 2018), RN168 (PF-06342674; e.g., described in Herold et al., JCI Insight. 2019 Dec 19;4(24):e126054), 4A10 (e.g., described in Hixon et al., Leukemia. 2020 Jan;34(l):35-49), GSK2618960 (e.g., described in Ellis et al., Br J Clin Pharmacol. (2019) 85(2):304-315), B12 (e.g., described in Akkapeddi et al., Leukemia. (2019) 33(9); 2155-2168), and A7R34 (e.g., described in Sudo et al., Proc Natl Acad Sci USA. (1993) 90(19):9125-9129).

[0174] The antigen binding molecules of the present disclosure are multispecific. By “multispecific” is meant that the antigen binding molecule binds to more than one target. Specifically, the antigen binding molecule binds to (i) yc and (ii) a polypeptide other than yc of a yc-containing cytokine receptor (e.g., selected from the group consisting of: IL-2R, IL-2R, IL-15R, IL-4R, IL-9R, IL-21R, and IL-7R ). It will be understood that the multispecific antigen binding molecules are at least bispecific. The term “bispecific” means that the antigen binding molecule binds to at least two different antigenic determinants. In some embodiments, the antigen binding molecule is bispecific, trispecific, tetraspecific, pentaspecific, hexaspecific, heptaspecific, octaspecific, nonaspecific, or deca-specific.

[0175] The multispecific antigen binding molecules described herein exhibit at least monovalent binding to yc and also exhibit at least monovalent binding to a polypeptide of the yc-containing cytokine receptor other than yc (e.g., selected from IL-2Rβ, IL-2Ra, IL-15Ra, IL-4Ra, IL-9Ra, IL-21Ra, and IL-7Ra). Binding valency refers to the number of binding sites for a given antigenic determinant in an antigen binding molecule. For example, described herein are bispecific antigen binding molecules in the formats of scFv-Kih-Fc, CrossMab, and Duobody, which exhibit monovalent binding to yc, and monovalent binding to IL-2Rβ.

[0176] The multispecific antigen binding molecules according to the present disclosure can be provided in any suitable format, such as those described in Kontermann, MAbs. (2012) 4(2): 182-197, which is incorporated by reference herein in its entirety. For example, the antigen binding molecules according to the present disclosure can be bispecific antibody conjugates (e.g., IgG2, F(ab’)2, or CovX-Body), bispecific IgG or IgG-like molecules (e.g., IgG, scFv4-Ig, IgG-scFv, scFv-IgG, DVD-Ig, IgG-sVD, sVD-IgG, 2 in 1-IgG, mAb 2 or tandem antibody common LC), asymmetric bispecific IgG or IgG-like molecules (e.g., kihIgG, kih IgG common LC, CrossMab, kih IgG-scFab, mAb-Fv, charge-pair antibody, or SEED-Body), small bispecific antibody molecules (e.g., diabody (Db), dsDb, DART, scDb, tandAb, tandem scFv (taFv), tandem dAb / VHH, tandem scFV-VHH, triabody, triple head, Fab-scFv, or F(ab’)2-scFv2), bispecific Fc and C H 3 fusion proteins (e.g., taFv-Fc, Di-diabody, scDb-C H 3, scFv-Fc-scFv, HCAb-VHH, scFv-kih-Fc, or scFv-kih-C H 3) or bispecific fusion proteins (e.g., scFv2-albumin, scDb-albumin, taFv-toxin, DNL-Fab3, DNL-Fab4-IgG, DNL-Fab4-IgG-cytokine2). See, in particular, Kontermann, MAbs. (2012) 4(2): 182-19, which is incorporated by reference herein in its entirety. Figure 2See also Brinkmann and Kontermann, MAbs. (2017) 9(2): 182-212 (incorporated by reference in its entirety), in particular Figure 2 In some embodiments, the multispecific antigen binding molecule is a simple bispecific antibody, such as a tandem scFV-scFv, a tandem VHH-VHH, or a tandem VHH-scFv antibody.

[0177] Tandem multispecific antigen binding molecules (e.g., tandem scFv) comprise two (or more) binding moieties (e.g., scFv and / or VHH moieties) and a linker. In some embodiments, the multispecific antigen binding molecule is provided in a tandem format, where the binding moieties are connected by a linker. Examples of tandem format antigen binding molecules include tandem scFV-scFv, tandem VHH-VHH, tandem scFv-VHH, and tandem VHH-scFv. In some embodiments, the multispecific antigen binding molecule is provided in a tandem scFv format, such as a tandem scFv-scFv. In some embodiments, the multispecific antigen binding molecule is provided in a tandem VHH format, such as a tandem VHH-VHH. In some embodiments, the multispecific antigen binding molecule is provided in a tandem VHH-scFV format. In some embodiments, the multispecific antigen binding molecule is provided in a tandem scFV-VHH format.

[0178] The binding moieties are connected by a linker, typically in the orientation: VH-VL-linker-VH-VL or VL-VH-linker-VL-VH (from N-terminus to C-terminus). Thus, the different binding moieties can be combined in a variety of different ways. For example, two scFv molecules can be combined in a variety of different orientations. For example, the P2C4 scFv can be combined with the P1A3 scFv in at least the following orientations: (i) P2C4 VL-P2C4 VH-linker-P1A3 VL-P1A3 VH, (ii) P1A3 VL-P1A3 VH-linker-P2C4 VL-P2C4 VH, (iii) P2C4 VH-P2C4 VL-linker-P1A3 VH-P1A3 VL, and (iv) P1A3 VH-P1A3 VL-linker-P2C4 VH-P2C4 VL.

[0179] In some embodiments, the multispecific antigen binding molecule comprises a linker between the binding moieties, e.g., between the yc binding moiety and the moiety that binds to a polypeptide other than yc of the yc-containing cytokine receptor (e.g., an IL-2Rß binding moiety). Such linkers are described by Brinkmann and Kontermann (MAbs. (2017) 9(2): 182-212), which is incorporated by reference herein in its entirety. In some embodiments, the linker is a linker described in Brinkmann and Kontermann (MAbs. (2017) 9(2): 182-212).

[0180] In some embodiments, the linker is an amino acid linker. In some embodiments, the linker is a flexible linker. In some embodiments, the linker is a rigid linker. In some embodiments, the linker is a short flexible linker. In some embodiments, the linker is a long rigid linker.

[0181] In some embodiments, the flexible linker is rich in small or polar amino acids, such as Gly and / or Ser, to provide flexibility and solubility. In some embodiments, the linker is a glycine-rich linker. In some embodiments, the linker is a serine-rich linker. In some embodiments, the linker is an amino acid linker in which at least 50% of the total amino acids are glycine amino acids, e.g., one of >55%, >60%, >65%, >70%, >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, or >99% of the total amino acids are glycine amino acids. In some embodiments, the linker comprises or consists of the GGGGS (SEQ ID NO: 502) amino acid sequence. In some embodiments, the linker comprises or consists of the GGGGSGGGS (SEQ ID NO: 720) amino acid sequence. In some embodiments, the linker comprises or consists of the (G3S)4 (SEQ ID NO: 721) amino acid sequence. In some embodiments, the linker comprises or consists of the GGGSG (SEQ ID NO: 722) amino acid sequence. In some embodiments, the linker comprises or consists of the NSGAAA (SEQ ID NO: 729) amino acid sequence.

[0182] In some embodiments, the linker sequence comprises at least one glycine residue and / or at least one serine residue. In some embodiments, the linker sequence comprises or consists of glycine and serine residues. In some embodiments, the linker sequence has the structure: (GxS)n or (GxS)nGm; wherein G = glycine, S = serine, x = 3 or 4, n = 2, 3, 4, 5, or 6, m = 0, 1, 2, or 3. In some embodiments, the linker sequence comprises one or more (e.g., 1, 2, 3, 4, 5, or 6) copies (e.g., in tandem) of the sequence motif G4S. In some embodiments, the linker sequence comprises or consists of (G4S)4 or (G4S)6. In some embodiments, the linker sequence is 1-2, 1-3, 1-4, 1-5, 1-10, 1-15, 1-20, 1-25, or 1-30 amino acids in length.

[0183] In some embodiments, the linker is a rigid linker. In some embodiments, the rigid linker forms an alpha helix structure between the binding moieties. Rigid linkers are discussed by Arai et al. (Protein Engineering, Design and Selection, 14(8), 2001, 529-532), which is incorporated by reference herein in its entirety. In some embodiments, the linker is a linker described in Arai et al. (Protein Engineering, Design and Selection, 14(8), 2001, 529-532). In some embodiments, the linker comprises or consists of the A(EAAAK)5A (SEQ ID NO: 719) amino acid sequence.

[0184] In some embodiments, the linker comprises the EAAAK (SEQ ID NO: 723) amino acid sequence. In some embodiments, the linker comprises or consists of the A(EAAAK)2A (SEQ ID NO: 724) amino acid sequence. In some embodiments, the linker comprises or consists of the A(EAAAK)3A (SEQ ID NO: 725) amino acid sequence. In some embodiments, the linker comprises or consists of the A(EAAAK)4A (SEQ ID NO: 726) amino acid sequence. In some embodiments, the linker comprises or consists of the A(EAAAK)5A (SEQ ID NO: 719) amino acid sequence.

[0185] In some embodiments, the linker is at least 3 amino acids in length. In some embodiments, the linker is at most 50 amino acids in length. In some embodiments, the linker is at least one of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or 49 amino acids in length. In some embodiments, the linker is at most one of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids in length.

[0186] In some embodiments, the linker is 3 to 50 amino acids, 4 to 50 amino acids, 5 to 50 amino acids, 6 to 50 amino acids, 7 to 50 amino acids, 8 to 50 amino acids, 9 to 50 amino acids, 10 to 50 amino acids, 11 to 50 amino acids, 12 to 50 amino acids, 13 to 50 amino acids, 14 to 50 amino acids, 15 to 50 amino acids, 16 to 50 amino acids, 17 to 50 amino acids, 18 to 50 amino acids, 19 to 50 amino acids, or 20 to 50 amino acids in length.

[0187] In some embodiments, the linker is 3 to 40 amino acids, 4 to 40 amino acids, 5 to 40 amino acids, 6 to 40 amino acids, 7 to 50 amino acids, 8 to 40 amino acids, 9 to 40 amino acids, 10 to 40 amino acids, 11 to 40 amino acids, 12 to 40 amino acids, 13 to 40 amino acids, 14 to 40 amino acids, 15 to 40 amino acids, 16 to 40 amino acids, 17 to 40 amino acids, 18 to 40 amino acids, 19 to 40 amino acids, or 20 to 40 amino acids in length.

[0188] In some embodiments, the linker is 3 to 30 amino acids, 4 to 30 amino acids, 5 to 30 amino acids, 6 to 30 amino acids, 7 to 30 amino acids, 8 to 30 amino acids, 9 to 30 amino acids, 10 to 30 amino acids, 11 to 30 amino acids, 12 to 30 amino acids, 13 to 30 amino acids, 14 to 30 amino acids, 15 to 30 amino acids, 16 to 30 amino acids, 17 to 30 amino acids, 18 to 30 amino acids, 19 to 30 amino acids, or 20 to 30 amino acids in length.

[0189] In some embodiments, the linker is 3 to 20 amino acids, 4 to 20 amino acids, 5 to 20 amino acids, 6 to 20 amino acids, 7 to 20 amino acids, 8 to 20 amino acids, 9 to 20 amino acids, 10 to 20 amino acids, 11 to 20 amino acids, 12 to 20 amino acids, 13 to 20 amino acids, 14 to 20 amino acids, 15 to 20 amino acids, 16 to 20 amino acids, 17 to 20 amino acids, 18 to 20 amino acids, or 19 to 20 amino acids in length.

[0190] In some embodiments, the linker is 3 to 4 amino acids, 3 to 5 amino acids, 3 to 6 amino acids, 3 to 7 amino acids, 3 to 8 amino acids, 3 to 9 amino acids, 3 to 10 amino acids, 3 to 11 amino acids, 3 to 12 amino acids, 3 to 13 amino acids, 3 to 14 amino acids, 3 to 15 amino acids, 3 to 16 amino acids, 3 to 17 amino acids, 3 to 18 amino acids, 3 to 19 amino acids, or 3 to 20 amino acids in length.

[0191] In some embodiments, the flexible linker is 3 to 12 amino acids in length. In some embodiments, the short flexible linker is 3 to 10 amino acids in length. In some embodiments, the short flexible linker is 3 to 8 amino acids in length. In some embodiments, the short flexible linker is 3 to 6 amino acids in length.

[0192] In some embodiments, the rigid linker is 10 to 44 amino acids in length. In some embodiments, the long rigid linker is 12 to 44 amino acids in length. In some embodiments, the long rigid linker is 17 to 44 amino acids in length. In some embodiments, the long rigid linker is 22 to 44 amino acids in length. In some embodiments, the long rigid linker is 27 to 44 amino acids in length.

[0193] The skilled person is readily able to design and prepare multispecific (e.g. bispecific) antigen binding molecules according to the present disclosure based on their common general knowledge and, for example, with reference to the publications mentioned herein. Such techniques are described, for example, in Brinkmann and Kontermann, MAbs. (2017) 9(2): 182-212 and Ma et al., Front Immunol. (2021) 12:626616, both of which are incorporated herein in their entirety by reference.

[0194] Methods for producing multispecific antigen binding molecules include chemical cross-linking of antigen binding molecules or antibody fragments, for example with reducible disulfide or non-reducible thioether linkages, for example as described in Segal and Bast, (2001) Current Protocols in Immunology. Chapter 2:2.13.1-2.13.16, which is incorporated herein in its entirety by reference. For example, N-succinimidyl-3-(-2-pyridyldithio)-propionate (SPDP) can be used to chemically cross-link Fab fragments, for example via the hinge region SH-groups, resulting in disulfide-linked bispecific F(ab)2 heterodimers.

[0195] Other methods for producing multispecific antigen binding molecules include fusing antibody-producing hybridomas, for example with polyethylene glycol, to produce cell hybridoma cells capable of secreting bispecific antibodies, for example as described in Segal and Bast, (2001) Current Protocols in Immunology. Chapter 2:2.13.1-2.13.16.

[0196] Multispecific antigen binding molecules according to the present disclosure can also be produced recombinantly, for example by expression of nucleic acid constructs encoding the antigen binding molecule polypeptides, for example as described in Hornig and Methods Mol Biol. (2012) 907:713-27 or French, Methods Mol Med. (2000) 40:333-339, the entire contents of both of which are incorporated herein by reference.

[0197] For example, DNA constructs encoding the light and heavy chain variable domains of two antigen binding fragments (i.e., the light and heavy chain variable domains of an antigen binding fragment capable of binding yc, and the light and heavy chain variable domains of an antigen binding fragment capable of binding to another target protein) can be prepared by molecular cloning techniques, including sequences encoding suitable linkers or dimerization domains between the antigen binding fragments. Thereafter, the recombinant bispecific antibodies can be produced by expressing (e.g., in vitro) the constructs in suitable host cells (e.g., mammalian host cells), and the expressed recombinant bispecific antibodies can then be optionally purified.

[0198] In some embodiments, a yc-binding moiety of the present disclosure comprises one or more polypeptides comprising a VH region comprising heavy chain CDRs and a VL region comprising light chain CDRs cloned from P1A3, P1A3_B3, P1A3_E8, P1A3_E9, P2B9, P1A3_B4, P1A3_FW2, P1A10, P1B6, P1C10, P1D7, P1E8, P2B2, P2B7, P2D11, P2F10, P2H4, P2D3, P1G4, P1B12, P1C7, P1A3_A, P1A3_Q, P1A3_AQ, P1A3_ANQ, P1A10_AQ, and P1A10_ANQ, as shown in Table A1 herein.

[0199] In some embodiments, a yc-binding moiety comprises a polypeptide comprising a VH region comprising a HC-CDR1, a HC-CDR2, and a HC-CDR3 as shown in one of binding moieties A1-1 to A1-27 in column A of Table A1, optionally wherein 1 or 2 or 3 amino acids in the HC-CDR1, and / or 1 or 2 or 3 amino acids in the HC-CDR2, and / or 1 or 2 or 3 amino acids in the HC-CDR3 are substituted with another amino acid.

[0200] In some embodiments, a yc-binding moiety comprises a polypeptide comprising a VL region comprising a LC-CDR1, a LC-CDR2, and a LC-CDR3 as shown in one of binding moieties A1-1 to A1-27 in column B of Table A1, optionally wherein 1 or 2 or 3 amino acids in the LC-CDR1, and / or 1 or 2 or 3 amino acids in the LC-CDR2, and / or 1 or 2 or 3 amino acids in the LC-CDR3 are substituted with another amino acid.

[0201] In some embodiments, the yc-binding moiety comprises one or more polypeptides comprising: (i) a VH region comprising a HC-CDR1, a HC-CDR2, and a HC-CDR3 as shown in Column A of Table A1, and (ii) a VL region comprising a LC-CDR1, a LC-CDR2, and a LC-CDR3 as shown in Column B of Table A1, wherein the sequences of Column A and Column B are selected from the same row of Table A1 (i.e., wherein the sequences of Column A and Column B have the same binding moiety selected from A1-1 to A1-27).

[0202] In some embodiments, the yc-binding moiety of the present disclosure comprises one or more polypeptides comprising a VH region comprising heavy chain FRs and a VL region comprising light chain FRs selected from the following clones: P1A3, P1A3_B3, P1A3_E8, P1A3_E9, P2B9, P1A3_B4, P1A3_FW2, P1A10, P1B6, P1C10, P1D7, P1E8, P2B2, P2B7, P2D11, P2F10, P2H4, P2D3, P1G4, P1B12, P1C7, P1A3_A, P1A3_Q, P1A3_AQ, P1A3_ANQ, P1A10_AQ, and P1A10_ANQ, as shown in Table B1 herein.

[0203] In some embodiments, the yc-binding moiety comprises a polypeptide comprising a VH region comprising a HC-FR1, a HC-FR2, a HC-FR3, and a HC-FR4 as shown in Column A of Table B1 for one of binding moieties B1-1 to B1-27, optionally wherein 1 or 2 or 3 amino acids in the HC-FR1, and / or 1 or 2 or 3 amino acids in the HC-FR2, and / or 1 or 2 or 3 amino acids in the HC-FR3, and / or 1 or 2 or 3 amino acids in the HC-FR4 are substituted with another amino acid.

[0204] In some embodiments, the yc-binding moiety comprises a polypeptide comprising a VH region comprising a HC-FR1, a HC-FR2, a HC-FR3, and a HC-FR4 as shown in Column A of Table B1 for one of binding moieties B1-1 to B1-27, optionally wherein 1 or 2 or 3 amino acids in the HC-FR1, and / or 1 or 2 or 3 amino acids in the HC-FR2, and / or 1 or 2 or 3 amino acids in the HC-FR3, and / or 1 or 2 or 3 amino acids in the HC-FR4 are substituted with another amino acid.

[0205] In some embodiments, the yc-binding moiety comprises one or more polypeptides comprising: (i) a VH region comprising HC-FR1, HC-FR2, HC-FR3, and HC-FR4 as shown in Column A of Table B1, and (ii) a VL region comprising LC-FR1, LC-FR2, LC-FR3, and LC-FR4 as shown in Column B of Table B1, wherein the sequences of Column A and Column B are selected from the same row of Table B1 (i.e., wherein the sequences of Column A and Column B have the same binding moiety selected from B1-1 to B1-27).

[0206] In some embodiments, the yc-binding moiety of the present disclosure comprises one or more polypeptides comprising a VH region and a VL region selected from the following clones: P1A3, P1A3_B3, P1A3_E8, P1A3_E9, P2B9, P1A3_B4, P1A3_FW2, P1A10, P1B6, P1C10, P1D7, P1E8, P2B2, P2B7, P2D11, P2F10, P2H4, P2D3, P1G4, P1B12, P1C7, P1A3_A, P1A3_Q, P1A3_AQ, P1A3_ANQ, P1A10_AQ, and P1A10_ANQ, as shown in Table C1 herein.

[0207] In some embodiments, the yc-binding moiety comprises a polypeptide comprising a VH region having at least 70% (e.g., one of >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99%, or 100%) amino acid sequence identity to an amino acid sequence shown in Column A of Table C1.

[0208] In some embodiments, the yc-binding moiety comprises a polypeptide comprising a VL region having at least 70% (e.g., one of >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99%, or 100%) amino acid sequence identity to an amino acid sequence shown in Column B of Table C1.

[0209] In some embodiments, a yc-binding moiety of the present disclosure comprises one or more polypeptides comprising a VH region having at least 70% (e.g., one of >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99%, or 100%) amino acid sequence identity to an amino acid sequence recited in Column A of Table C1 and a VL region having at least 70% (e.g., one of >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99%, or 100%) amino acid sequence identity to an amino acid sequence recited in Column B of Table C1, wherein the sequences of Column A and Column B are selected from the same row of Table C1 (i.e., wherein the sequences of Column A and Column B have the same binding moiety selected from C1-1 to C1-27).

[0210] In some embodiments, an IL-2Rβ-binding moiety of the present disclosure comprises one or more polypeptides comprising a VH region comprising heavy chain CDRs and a VL region comprising light chain CDRs selected from the following clones: P2C4, P2C4_A4, P2C4_A2, P2C4_B5, P2C4_C1, P2C4_C4, P2C4_C7, P2C4_D10, P2C4_E6, P2C4_E7, P2C4_F8, P2C4_C1D10, P2C4_FW2, P2H7, P2D12, P1G11, P2C4_A9, P2C4_B6, P2C4_E9, P2C4_B8, P2C4_A22, P2C4_C12, P2C4_E2, P2C4_E3, P2C4_E8, P2C4_F11, P2C4_G2, P2C4_G11, P2C4_H1, P2C4_H2, P2C4_H3, P1E7, P1A20, P1F3, P1D10, P1E1, P2A21, P2C9, P2C10, P2C11, P2E6, P2E11, P2F9, P2F10, P2C4FW2-AQ, P2C4FW2-ANQ, P2C4WT-AQ, and P2C4WT-ANQ, as shown in Table A2 herein.

[0211] In some embodiments, an IL-2Rβ-binding moiety comprises a polypeptide comprising a VH region comprising a HC-CDR1, a HC-CDR2, and a HC-CDR3, as shown in Column A of Table A2 for one of binding moieties A2-1 to A2-48, optionally wherein 1 or 2 or 3 amino acids in the HC-CDR1, and / or 1 or 2 or 3 amino acids in the HC-CDR2, and / or 1 or 2 or 3 amino acids in the HC-CDR3 are substituted with another amino acid.

[0212] In some embodiments, the IL-2Rβ binding moiety comprises a polypeptide comprising a VL region comprising a LC-CDR1, a LC-CDR2, and a LC-CDR3 as shown in one of binding moieties A2-1 to A2-48 in column B of Table A2, optionally wherein 1 or 2 or 3 amino acids in the LC-CDR1, and / or 1 or 2 or 3 amino acids in the LC-CDR2, and / or 1 or 2 or 3 amino acids in the LC-CDR3 are substituted with another amino acid.

[0213] In some embodiments, the IL-2Rβ binding moiety comprises one or more polypeptides comprising: (i) a VH region comprising a HC-CDR1, a HC-CDR2, and a HC-CDR3 as shown in column A of Table A2, and (ii) a VL region comprising a LC-CDR1, a LC-CDR2, and a LC-CDR3 as shown in column B of Table A2, wherein the sequences of column A and column B are selected from the same row of Table A2 (i.e., wherein the sequences of column A and column B have the same binding moiety selected from A2-1 to A2-48).

[0214] In some embodiments, the IL-2Rβ binding moiety of the present disclosure comprises one or more polypeptides comprising a VH region comprising heavy chain FRs and a VL region comprising light chain FRs selected from the clones: P2C4, P2C4_A4, P2C4_A2, P2C4_B5, P2C4_C1, P2C4_C4, P2C4_C7, P2C4_D10, P2C4_E6, P2C4_E7, P2C4_F8, P2C4_C1D10, P2C4_FW2, P2H7, P2D12, P1G11, P2C4_A9, P2C4_B6, P2C4_E9, P2C4_B8, P2C4_A22, P2C4_C12, P2C4_E2, P2C4_E3, P2C4_E8, P2C4_F11, P2C4_G2, P2C4_G11, P2C4_H1, P2C4_H2, P2C4_H3, P1E7, P1A20, P1F3, P1D10, P1E1, P2A21, P2C9, P2C10, P2C11, P2E6, P2E11, P2F9, P2F10, P2C4FW2-AQ, P2C4FW2-ANQ, P2C4WT-AQ, and P2C4WT-ANQ, as shown in Table B2 herein.

[0215] In some embodiments, the IL-2Rβ binding moiety comprises a polypeptide comprising a VH region comprising a HC-FR1, a HC-FR2, a HC-FR3, and a HC-FR4, as shown in one of binding moieties B2-1 to B2-48 in Column A of Table B2, optionally wherein 1 or 2 or 3 amino acids in the HC-FR1, and / or 1 or 2 or 3 amino acids in the HC-FR2, and / or 1 or 2 or 3 amino acids in the HC-FR3, and / or 1 or 2 or 3 amino acids in the HC-FR4 are substituted with another amino acid.

[0216] In some embodiments, the IL-2Rβ binding moiety comprises a polypeptide comprising a VH region comprising a HC-FR1, a HC-FR2, a HC-FR3, and a HC-FR4, as shown in one of binding moieties B2-1 to B2-48 in Column A of Table B2, optionally wherein 1 or 2 or 3 amino acids in the HC-FR1, and / or 1 or 2 or 3 amino acids in the HC-FR2, and / or 1 or 2 or 3 amino acids in the HC-FR3, and / or 1 or 2 or 3 amino acids in the HC-FR4 are substituted with another amino acid.

[0217] In some embodiments, the IL-2Rβ binding moiety comprises one or more polypeptides comprising: (i) a VH region comprising a HC-FR1, a HC-FR2, a HC-FR3, and a HC-FR4 as shown in Column A of Table B2, and (ii) a VL region comprising a LC-FR1, a LC-FR2, a LC-FR3, and a LC-FR4 as shown in Column B of Table B2, wherein the sequences of Column A and Column B are selected from the same row of Table B2 (i.e., wherein the sequences of Column A and Column B have the same binding moiety selected from B2-1 to B2-48).

[0218] In some embodiments, the IL-2Rβ binding moiety of the present disclosure comprises one or more polypeptides comprising a VH region and a VL region selected from the clones P2C4, P2C4_A4, P2C4_A2, P2C4_B5, P2C4_C1, P2C4_C4, P2C4_C7, P2C4_D10, P2C4_E6, P2C4_E7, P2C4_F8, P2C4_C1D10, P2C4_FW2, P2H7, P2D12, P1G11, P2C4_A9, P2C4_B6, P2C4_E9, P2C4_B8, P2C4_A22, P2C4_C12, P2C4_E2, P2C4_E3, P2C4_E8, P2C4_F11, P2C4_G2, P2C4_G11, P2C4_H1, P2C4_H2, P2C4_H3, P1E7, P1A20, P1F3, P1D10, P1E1, P2A21, P2C9, P2C10, P2C11, P2E6, P2E11, P2F9, P2F10, P2C4FW2-AQ, P2C4FW2-ANQ, P2C4WT-AQ, and P2C4WT-ANQ, as shown in Table C2 herein.

[0219] In some embodiments, the IL-2Rβ binding moiety comprises a polypeptide comprising a VH region having at least 70% (e.g., one of ≥ 80%, ≥ 85%, ≥ 90%, ≥ 91%, ≥ 92%, ≥ 93%, ≥ 94%, ≥ 95%, ≥ 96%, ≥ 97%, ≥ 98%, ≥ 99%, or 100%) amino acid sequence identity to an amino acid sequence shown in column A of Table C2.

[0220] In some embodiments, the IL-2Rβ binding moiety comprises a polypeptide comprising a VL region having at least 70% (e.g., one of ≥ 80%, ≥ 85%, ≥ 90%, ≥ 91%, ≥ 92%, ≥ 93%, ≥ 94%, ≥ 95%, ≥ 96%, ≥ 97%, ≥ 98%, ≥ 99%, or 100%) amino acid sequence identity to an amino acid sequence shown in column B of Table C2.

[0221] In some embodiments, the IL-2Rβ binding moiety of the present disclosure comprises one or more polypeptides comprising a VH region having at least 70% (e.g., one of >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99%, or 100%) amino acid sequence identity to an amino acid sequence recited in Column A of Table C1 and a VL region having at least 70% (e.g., one of >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99%, or 100%) amino acid sequence identity to an amino acid sequence recited in Column B of Table C1, wherein the sequences of Column A and Column B are selected from the same row of Table C2 (i.e., wherein the sequences of Column A and Column B have the same binding moiety selected from C2-1 to C2-48).

[0222] In some embodiments, an antigen binding molecule according to the present disclosure comprises: (i) a yc binding moiety according to the embodiments described herein, and (ii) a moiety that binds to one or more polypeptides of a yc-containing cytokine receptor other than yc (e.g., selected from IL-2Rβ, IL-2Rα, IL-15Rα, IL-4Rα, IL-9Rα, IL-21Rα, and IL-7Rα).

[0223] In some embodiments, an antigen binding molecule according to the present disclosure comprises: (i) a yc binding moiety according to the embodiments described herein, and (ii) an IL-2Rβ binding moiety according to the embodiments described herein.

[0224] In some embodiments, an antigen binding molecule according to the present disclosure is an antigen binding molecule according to the embodiments described in WO 2017 / 021540 Al, WO 2019 / 092181 Al, WO 2020 / 094834 Al, and WO 2020 / 094836 Al, all of which are incorporated by reference in their entireties.

[0225] In some embodiments, the antigen binding molecules of the present disclosure comprise one or more regions of an immunoglobulin heavy chain constant sequence (e.g., CH1, CH2, CH3, etc.). In some embodiments, the immunoglobulin heavy chain constant sequence is or is derived from a heavy chain constant sequence of IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgA (e.g., IgA1, IgA2), IgD, IgE, or IgM (e.g., human IgG (e.g., hIgG1, hIgG2, hIgG3, hIgG4), hIgA (e.g., hIgA1, hIgA2), hIgD, hIgE, or hIgM). In some embodiments, the immunoglobulin heavy chain constant sequence is or is derived from a heavy chain constant sequence of a human IgG1 allotype (e.g., G1m1, G1m2, G1m3, or G1m17).

[0226] It will be appreciated that further substitutions can be provided to the CH2 and / or CH3 regions in accordance with modifications to the Fc region of the antigen binding molecules as described herein.

[0227] In some embodiments, the antigen binding molecules of the present disclosure comprise one or more regions of an immunoglobulin light chain constant sequence. In some embodiments, the immunoglobulin light chain constant sequence is a human immunoglobulin kappa constant sequence (IGKC; CK). In some embodiments, the immunoglobulin light chain constant sequence is a human immunoglobulin lambda constant sequence (IGLC; CL), e.g., IGLC1, IGLC2, IGLC3, IGLC6, or IGLC7.

[0228] Fc region

[0229] In some embodiments, the antigen binding molecules of the present disclosure comprise an Fc region.

[0230] In some embodiments, the antigen binding molecules of the present disclosure do not comprise an Fc region.

[0231] As used herein, an “Fc region” refers to a polypeptide complex formed by the interaction of two polypeptides, each comprising a CH2-CH3 region of an immunoglobulin (Ig) heavy chain constant sequence.

[0232] In the present text, a "CH2 region" refers to an amino acid sequence corresponding to the CH2 region of an immunoglobulin (Ig). According to the EU numbering system described in Edelman et al., Proc Natl Acad Sci USA (1969) 63(1): 78-85, the CH2 region is the region of the Ig formed by positions 231 to 340 of the immunoglobulin constant region. A "CH3 region" refers to an amino acid sequence corresponding to the CH3 region of an immunoglobulin (Ig). According to the EU numbering system described in Edelman et al., Proc Natl Acad Sci USA. (1969) 63(1): 78-85, the CH3 region is the region of the Ig formed by positions 341 to 447 of the immunoglobulin constant region. A "CH2-CH3 region" refers to an amino acid sequence corresponding to the CH2 and CH3 regions of an immunoglobulin (Ig). According to the EU numbering system described in Edelman et al., Proc Natl Acad Sci USA. (1969) 63(1): 78-85, the CH2-CH3 region is the region of the Ig formed by positions 231 to 447 of the immunoglobulin constant region.

[0233] In some embodiments, the CH2 region, the CH3 region, and / or the CH2-CH3 region according to the present disclosure correspond to a CH2 region / CH3 region / CH2-CH3 region of an IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgA (e.g., IgA1, IgA2), IgD, IgE, or IgM. In some embodiments, the CH2 region, the CH3 region, and / or the CH2-CH3 region correspond to a CH2 region / CH3 region / CH2-CH3 region of a human IgG (e.g., hIgG1, hIgG2, hIgG3, hIgG4), hIgA (e.g., hIgA1, hIgA2), hIgD, hIgE, or hIgM. In some embodiments, the CH2 region, the CH3 region, and / or the CH2-CH3 region correspond to a CH2 region / CH3 region / CH2-CH3 region of a human IgG1 allotype (e.g., G1m1, G1m2, G1m3, or G1m17).

[0234] The Fc region provides for interactions with Fc receptors and other molecules of the immune system to elicit functional effects. Fc-mediated effector functions are reviewed, for example, in Jefferis et al., Immunol Rev (1998) 163:59-76 (incorporated by reference in its entirety), and are elicited by Fc-mediated recruitment and activation of immune cells (e.g., macrophages, dendritic cells, neutrophils, basophils, eosinophils, platelets, mast cells, NK cells, and T cells) through interactions between the Fc region and Fc receptors expressed by immune cells, by recruitment of components of the complement pathway and subsequent activation of the complement cascade through binding of the Fc region to complement protein Clq. Fc-mediated functions include Fc receptor binding, antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), formation of membrane attack complexes (MAC), cell degranulation, cytokine and / or chemokine production, and antigen processing and presentation.

[0235] Modifications of antibody Fc regions that affect Fc-mediated functions are known in the art, such as those described, for example, in Wang et al., Protein Cell (2018) 9(1):63-73, which is incorporated by reference in its entirety. Exemplary Fc region modifications known to affect antibody effector functions are summarized in Table 1 of Wang et al., Protein Cell (2018) 9(1):63-73. In some embodiments, an antigen binding molecule of the present disclosure comprises an Fc region comprising a modification to increase or decrease Fc-mediated functions compared to an antigen binding molecule comprising a corresponding unmodified Fc region.

[0236] Where an Fc region / CH2 / CH3 is described as comprising a modification that “corresponds to” a reference substitution, the equivalent substitution in the homologous Fc / CH2 / CH3 is contemplated. To illustrate, a L234A / L235A substitution in human IgGl (numbered according to the EU 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) corresponds to a L to A substitution at positions 117 and 118 of the mouse Ig gamma-2A chain C region (UniProtKB: P01863-1, vl).

[0237] Where an Fc region is described as comprising a modification, the modification can be present in one or both polypeptide chains that together form the Fc region.

[0238] In some embodiments, the antigen binding molecules of the present disclosure comprise an Fc region comprising modifications. In some embodiments, the antigen binding molecules of the present disclosure comprise an Fc region comprising modifications in one or more CH2 and / or CH3 regions.

[0239] In some embodiments, the Fc region comprises modifications to increase Fc-mediated function. In some embodiments, the Fc region comprises modifications to increase ADCC. In some embodiments, the Fc region comprises modifications to increase ADCP. In some embodiments, the Fc region comprises modifications to increase CDC. The antigen binding molecules comprising an Fc region comprising modifications to increase Fc-mediated function (e.g., ADCC, ADCP, CDC) induce increased levels of the relevant effector function compared to antigen binding molecules comprising corresponding unmodified Fc regions.

[0240] In some embodiments, the Fc region comprises modifications to increase binding to an Fc receptor. In some embodiments, the Fc region comprises modifications to increase binding to an Fcy receptor. In some embodiments, the Fc region comprises modifications to increase binding to one or more of FcyRI, FcyRIIa, FcyRIIb, FcyRIIc, FcyRIIIa, and FcyRIIIb. In some embodiments, the Fc region comprises modifications to increase binding to FcyRIIIa. In some embodiments, the Fc region comprises modifications to increase binding to FcyRIIa. In some embodiments, the Fc region comprises modifications to increase binding to FcyRIIb. In some embodiments, the Fc region comprises modifications to increase binding to FcRn. In some embodiments, the Fc region comprises modifications to increase binding to a complement protein. In some embodiments, the Fc region comprises modifications to increase binding to Clq. In some embodiments, the Fc region comprises modifications to promote hexamerization of the antigen binding molecule. In some embodiments, the Fc region comprises modifications to increase the half-life of the antigen binding molecule. In some embodiments, the Fc region comprises modifications to increase co-engagement.

[0241] In some embodiments, the Fc region comprises modifications corresponding to the substitution combination F243L / R292P / Y300L / V305I / P396L, as described in Stavenhagen et al. Cancer Res. (2007) 67: 8882-8890. In some embodiments, the Fc region comprises modifications corresponding to the substitution combination S239D / I332E or S239D / I332E / A330L, as described in Lazar et al., Proc Natl Acad Sci USA. (2006) 103: 4005-4010. In some embodiments, the Fc region comprises modifications corresponding to the substitution combination S298A / E333A / K334A, as described in Shields et al., J Biol Chem. (2001) 276: 6591-6604. In some embodiments, the Fc region comprises modifications corresponding to the substitution combination L234Y / L235Q / G236W / S239M / H268D / D270E / S298A to one heavy chain polypeptide and the substitution combination D270E / K326D / A330M / K334E to another heavy chain polypeptide, as described in Mimoto et al., MAbs. (2013) 5: 229-236. In some embodiments, the Fc region comprises modifications corresponding to the substitution combination G236A / S239D / I332E, as described in Richards et al., Mol Cancer Ther. (2008) 7: 2517-2527.

[0242] In some embodiments, the Fc region comprises modifications corresponding to the substitution combination K326W / E333S, as described in Idusogie et al. J Immunol. (2001) 166(4):2571-5. In some embodiments, the Fc region comprises modifications corresponding to the substitution combination S267E / H268F / S324T, as described in Moore et al. MAbs. (2010) 2(2): 181-9. In some embodiments, the Fc region comprises modifications corresponding to the substitution combination described in Natsume et al., Cancer Res. (2008) 68(10):3863-72. In some embodiments, the Fc region comprises modifications corresponding to the substitution combination E345R / E430G / S440Y, as described in Diebolder et al. Science (2014) 343(6176): 1260-3.

[0243] In some embodiments, the Fc region comprises modifications corresponding to the substitution combination M252Y / S254T / T256E, as described in Dall’Acqua et al. J Immunol. (2002) 169: 5171-5180.

[0244] These so-called "YTE" modifications at the CH2-CH3 interface of the Fc region have been shown to increase binding affinity for MHC class I neonatal Fc receptor (FcRn) at pH 6.0, which is localized within the acidic endosomes of endothelial and hematopoietic cells, which increases the efficient recycling of the administered mAb and half-life in plasma.

[0245] In some embodiments, the Fc region comprises modifications corresponding to the substitution combination M428L / N434S, as described in Zalevsky et al. Nat Biotechnol. (2010) 28: 157-159.

[0246] In some embodiments, the Fc region comprises modifications corresponding to the substitution combination S267E / L328F, as described in Chu et al., Mol Immunol. (2008) 45:3926-3933. In some embodiments, the Fc region comprises modifications corresponding to the substitution combination N325S / L328F, as described in Shang et al. Biol Chem. (2014) 289:15309-15318.

[0247] In some embodiments, the Fc region comprises modifications to reduce / prevent Fc-mediated function. In some embodiments, the Fc region comprises modifications to reduce / prevent ADCC. In some embodiments, the Fc region comprises modifications to reduce / prevent ADCP. In some embodiments, the Fc region comprises modifications to reduce / prevent CDC. An antigen binding molecule comprising an Fc region comprising modifications to reduce / prevent Fc-mediated function (e.g., ADCC, ADCP, CDC) induces a reduced level of an associated effector function compared to an antigen binding molecule comprising a corresponding unmodified Fc region.

[0248] In some embodiments, the Fc region comprises a modification to reduce / prevent binding to an Fc receptor. In some embodiments, the Fc region comprises a modification to reduce / prevent binding to an Fcy receptor. In some embodiments, the Fc region comprises a modification to reduce / prevent binding to one or more of FcyRI, FcyRIIa, FcyRIIb, FcyRIIc, FcyRIIIa, and FcyRIIIb. In some embodiments, the Fc region comprises a modification to reduce / prevent binding to FcyRIIIa. In some embodiments, the Fc region comprises a modification to reduce / prevent binding to FcyRIIa. In some embodiments, the Fc region comprises a modification to reduce / prevent binding to FcyRIIb. In some embodiments, the Fc region comprises a modification to reduce / prevent binding to a complement protein. In some embodiments, the Fc region comprises a modification to reduce / prevent binding to Clq. In some embodiments, the Fc region comprises a modification to reduce / prevent glycosylation of the amino acid residue corresponding to N297.

[0249] In some embodiments, the Fc region is unable to induce one or more Fc- mediated functions (i.e., lacks the ability to elicit the relevant Fc-mediated function). Thus, an antigen binding molecule comprising such an Fc region also lacks the ability to induce the relevant function. Such an antigen binding molecule can be described as being devoid of the relevant function.

[0250] In some embodiments, the Fc region is unable to induce ADCC. In some embodiments, the Fc region is unable to induce ADCP. In some embodiments, the Fc region is unable to induce CDC. In some embodiments, the Fc region is unable to induce ADCC and / or is unable to induce ADCP and / or is unable to induce CDC.

[0251] In some embodiments, the Fc region is unable to bind to an Fc receptor. In some embodiments, the Fc region is unable to bind to an Fcy receptor. In some embodiments, the Fc region is unable to bind to one or more of FcyRI, FcyRIIa, FcyRIIb, FcyRIIc, FcyRIIIa, and FcyRIIIb. In some embodiments, the Fc region is unable to bind to FcyRIIIa. In some embodiments, the Fc region is unable to bind to FcyRIIa. In some embodiments, the Fc region is unable to bind to FcyRIIb. In some embodiments, the Fc region is unable to bind to FcRn. In some embodiments, the Fc region is unable to bind to a complement protein. In some embodiments, the Fc region is unable to bind to Clq. In some embodiments, the Fc region is not glycosylated at the amino acid residue corresponding to N297.

[0252] In some embodiments, the Fc region comprises a modification corresponding to N297A or N297Q or N297G, as described in Leabman et al., MAbs. (2013) 5: 896-903. In some embodiments, the Fc region comprises a modification corresponding to L235E, as described in Alegre et al., J Immunol. (1992) 148: 3461-3468. In some embodiments, the Fc region comprises a modification corresponding to the substitution combination L234A / L235A or F234A / L235A, as described in Xu et al., Cell Immunol. (2000) 200: 16-26. In some embodiments, the Fc region comprises a modification corresponding to P329A or P329G, as described in Schlothauer et al., Protein Engineering, Design and Selection (2016), 29(10): 457-466. In some embodiments, the Fc region comprises a modification corresponding to the substitution combination L234A / L235A / P329G, as described in Lo et al. J. Biol. Chem (2017) 292(9): 3900-3908. In some embodiments, the Fc region comprises a modification corresponding to the substitution combination described in Rother et al., Nat Biotechnol. (2007) 25: 1256-1264. In some embodiments, the Fc region comprises a modification corresponding to the substitution combination S228P / L235E, as described in Newman et al., Clin. Immunol. (2001) 98: 164-174. In some embodiments, the Fc region comprises a modification corresponding to the substitution combination H268Q / V309L / A330S / P331S, as described in An et al., MAbs. (2009) 1: 572-579. In some embodiments, the Fc region comprises a modification corresponding to the substitution combination V234A / G237A / P238S / H268A / V309L / A330S / P331S, as described in Vafa et al., Methods. (2014) 65: 114-126. In some embodiments, the Fc region comprises a modification corresponding to the substitution combination L234A / L235E / G237A / A330S / P331S, as described in US 2015 / 0044231 Al.

[0253] The substitution combination“L234A / L235A” and corresponding substitutions such as, for example, F234A / L235A in human IgG4 are known to disrupt Fc binding to Fcy receptors and inhibit ADCC, ADCP, and also reduce Clq binding, thus reducing CDC (Schlothauer et al., Protein Engineering, Design and Selection (2016) 29(10): 457-466, incorporated herein by reference in its entirety). The substitutions“P329G” and“P329A” reduce Clq binding (and thus CDC). Substitution of“N297” with“A”,“G” or“Q” is known to abolish glycosylation, thus reducing Fc binding to Clq and Fcy receptors, and thus reducing CDC and ADCC. Lo et al. J. Biol. Chem (2017) 292(9): 3900-3908 (incorporated herein by reference in its entirety) reports that the substitution combination L234A / L235A / P329G abolishes complement binding and fixation and Fcy receptor-dependent, antibody-dependent, cell-mediated cytotoxicity in murine IgG2a and human IgGl.

[0254] The substitution combination L234A / L235E / G237A / A330S / P331S in IgGl Fc is disclosed in US2015 / 0044231 Al to abolish induction of phagocytosis, ADCC and CDC.

[0255] In some embodiments, the Fc region comprises a modification corresponding to the substitution S228P, as described in Silva et al., J Biol Chem. (2015) 290(9): 5462-5469. The substitution S228P in IgG4 Fc reduces Fab arm exchange, which can be undesirable.

[0256] In some embodiments, the Fc region comprises a modification corresponding to the substitution combination L234A / L235A. In some embodiments, the Fc region comprises a modification corresponding to the substitution P329G. In some embodiments, the Fc region comprises a modification corresponding to the substitution N297Q.

[0257] In some embodiments, the Fc region comprises a modification corresponding to the substitution combination L234A / L235A / P329G.

[0258] In some embodiments, the Fc region comprises a modification corresponding to the substitution combination L234A / L235A / P329G / N297Q.

[0259] In some embodiments, the Fc region comprises a modification corresponding to the substitution combination L234A / L235E / G237A / A330S / P331S.

[0260] In some embodiments, the Fc region comprises a modification corresponding to the substitution S228P in, e.g., IgG4.

[0261] In some embodiments, the Fc region comprises a CH2-CH3 region comprising an amino acid difference at one or more of the following positions relative to the amino acid sequence of the CH2-CH3 region of a reference Fc region: 234 or 235 (according to the EU numbering system). In some embodiments, the Fc region comprises a CH2-CH3 region comprising one or more of the following specific amino acid residues: A234 or A235 (according to the EU numbering system). In some embodiments, the Fc region comprises a CH2-CH3 region comprising A234 and A235. In some embodiments, the Fc region comprises a CH2-CH3 region comprising one or more of the following amino acid substitutions relative to the amino acid sequence of the CH2-CH3 region of a reference Fc region: L234A or L235A (according to the EU numbering system). In some embodiments, the Fc region comprises a CH2-CH3 region comprising the following amino acid substitutions relative to the amino acid sequence of the CH2-CH3 region of a reference Fc region: L234A and L235A (according to the EU numbering system).

[0262] In some embodiments, in particular embodiments wherein the antigen binding molecule is a multispecific, e.g., bispecific, antigen binding molecule, the antigen binding molecule comprises an Fc region comprising modifications in one or more of the CH2 and CH3 regions that promote association of the Fc region. The recombinant co-expression of the constituent polypeptides of the antigen binding molecule and the subsequent association results in several possible combinations. In order to improve the yield of the desired polypeptide combination in the antigen binding molecule in recombinant production, it is advantageous to introduce modifications in the Fc region that promote the association of the desired heavy chain polypeptide combination. The modifications can promote, e.g., hydrophobic and / or electrostatic interactions between the CH2 and / or CH3 regions of the different polypeptide chains. Suitable modifications are described, e.g., in Ha et al., Front Immnol. (2016) 7:394, which is incorporated by reference in its entirety.

[0263] In some embodiments, the antigen binding molecule of the present disclosure comprises an Fc region comprising a pair of substitutions according to one of the following formats in the CH3 region of the Fc region as shown in Table 1 of Ha et al., Front Immnol. (2016) 7:394: KiH, KiHs-s, HA-TF, ZW1, 7.8.60, DD-KK, EW-RVT, EW-RVTs-s, SEED, or A107.

[0264] In some embodiments, the multispecific (e.g., bispecific) antigen binding molecules of the present disclosure have an Fc region comprising a“knob-into-hole” or“KiH” modification, e.g., as described in, e.g., US 7,695,936 and Carter, J Immunol Meth. (2001) 248:7-15. In such embodiments, one CH3 region of the Fc region comprises a“knob” modification and the other CH3 region comprises a“hole” modification. The“knob” and“hole” modifications are located within the respective CH3 regions, such that the“knob” can be seated in the“hole” to promote heterodimerization (and inhibit homodimerization) of the polypeptides and / or to stabilize the heterodimer. The knob is constructed by substituting an amino acid with a large side chain (e.g., tyrosine or tryptophan) for an amino acid with a small side chain. The hole is created by substituting an amino acid with a small side chain (e.g., alanine or threonine) for an amino acid with a large side chain.

[0265] In some embodiments, one CH3 region of the Fc region of the antigen binding molecules of the present disclosure comprises the substitution T366W (numbering of positions in the Fc region herein is in accordance with the EU numbering system set forth in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991) and the other CH3 region of the Fc region comprises the substitution Y407V. In some embodiments, one CH3 region of the Fc region of the antigen binding molecules comprises the substitution T366W and the other CH3 region of the Fc region comprises the substitutions T366S and L368A. In some embodiments, one CH3 region of the Fc region of the antigen binding molecules comprises the substitution T366W and the other CH3 region of the Fc region comprises the substitutions Y407V, T366S and L368A.

[0266] In some embodiments, one CH3 region comprises the substitution S354C and the other CH3 region of the Fc region comprises the substitution Y349C. Introduction of these cysteine residues results in the formation of a disulfide bridge between the two CH3 regions of the Fc region, further stabilizing the heterodimer (Carter, J Immunol Methods (2001) 248:7-15).

[0267] In some embodiments, one CH3 region comprises substitutions K392D and K409D, and the other CH3 region of the Fc region comprises substitutions E356K and D399K. The “DDKK” knob-in-hole technology is described, e.g., in WO 2014 / 131694 Al, facilitating assembly of the heavy chains, providing complementary amino acid residues.

[0268] In some embodiments, the antigen binding molecules of the present disclosure comprise an Fc region modified as described in Labrijn et al., Proc Natl Acad Sci USA. (2013) 110(13):5145-50, referred to as the “Duobody” format. In some embodiments, one CH3 region comprises substitution K409R, and the other CH3 region of the Fc region comprises substitution K405L.

[0269] In some embodiments, the antigen binding molecules of the present disclosure comprise an Fc region modified as described in Strop et al., J Mol Biol. (2012) 420(3):204-19, the so-called “EEE-RRR” format. In some embodiments, one CH3 region comprises substitutions D221E, P228E, and L368E, and the other CH3 region of the Fc region comprises substitutions D221R, P228R, and K409R.

[0270] In some embodiments, the antigen binding molecules comprise an Fc region comprising the “EW-RVT” modifications described in Choi et al., Mol Cancer Ther. (2013) 12(12):2748-59. In some embodiments, one CH3 region comprises substitutions K360E and K409W, and the other CH3 region of the Fc region comprises substitutions Q347R, D399V, and F405T.

[0271] In some embodiments, the antigen binding molecules of the present disclosure comprise an Fc region comprising the “SEED” modification (as described in Davis et al., Protein Eng Des Sel. (2010) 23(4):195-202), in which the beta strand segments of human IgGl CH3 and IgA CH3 are exchanged.

[0272] In some embodiments, one CH3 region comprises substitutions S364H and F405A, and the other CH3 region of the Fc region comprises substitutions Y349T and T394F (see, e.g., Moore et al., MAbs (2011) 3(6):546-57).

[0273] In some embodiments, one CH3 region comprises substitutions T350V, L351Y, F405A, and Y407V, and the other CH3 region of the Fc region comprises substitutions T350V, T366L, K392L, and T394W (see, e.g., Von Kreudenstein et al., MAbs (2013) 5(5):646-54).

[0274] In some embodiments, one CH3 region comprises substitutions K360D, D399M, and Y407A, and the other CH3 region of the Fc region comprises substitutions E345R, Q347R, T366V, and K409V (see, e.g., Leaver-Fay et al., Structure (2016) 24(4):641-51).

[0275] In some embodiments, one CH3 region comprises substitutions K370E and K409W, and the other CH3 region of the Fc region comprises substitutions E357N, D399V, and F405T (see, e.g., Choi et al., PLoS One (2015) 10(12):e0145349).

[0276] In some embodiments, the antigen binding molecules of the present disclosure comprise an Fc region comprising modifications to increase stability (e.g., thermal stability and / or freeze-thaw stability). In some embodiments, the antigen binding molecules comprise modifications to one or more CH2 and CH3 regions to increase stability (e.g., thermal stability and / or freeze-thaw stability).

[0277] In some embodiments, the antigen binding molecules of the present disclosure comprise a CH3 region comprising a paired CH3 region “KiH” or “KiH S-S ” modification (e.g., within an Fc region, e.g., within a CH2-CH3 region forming an Fc region). Such paired CH3 regions can comprise a CH3 region comprising a knob modification and a CH3 region comprising a hole modification.

[0278] In some embodiments, a CH3 region comprising a knob modification comprises a tryptophan or tyrosine residue at position 366 (i.e., 366W or 366Y). In some embodiments, the knob modification is or includes T366W or T366Y. In some embodiments, a CH3 region comprising a knob modification comprises 366W. In some embodiments, the knob modification is or includes T366W.

[0279] In some embodiments, the CH3 region comprising a knob modification comprises 407V, 407A, 407S, or 407T; 366S, 366V, or 366A; and 368A, 368V, 368S, or 368T. In some embodiments, the knob modification is or includes Y407V, Y407A, Y407S, or Y407T; T366S, T366V, or T366A; and L368A, L368V, L368S, or L368T. In some embodiments, the CH3 region comprising a knob modification comprises 407V, 366S, and 368A. In some embodiments, the knob modification is or includes Y407V, T366S, and L368A.

[0280] In some embodiments, the antigen binding molecules of the present disclosure comprise a CH3 region that comprises a modification to form an interchain disulfide bond (i.e., between polypeptides comprising the CH2-CH3 region forming the Fc region). Such a modification can include the introduction of one or more cysteine residues into one or both CH3 regions of the constituent polypeptides of the polypeptide complex of the present disclosure. More specifically, such a modification can result in the CH3:CH3 interface formed between the CH3 regions of the polypeptides of the polypeptide complex of the present disclosure comprising a disulfide bond formed between cysteine residues (one from each polypeptide). In some embodiments, one CH3 region comprises 349C, and the other CH3 region comprises 354C. In some embodiments, one CH3 region comprises Y349C, and the other CH3 region comprises S354C.

[0281] In some embodiments, the CH3 region comprising a knob modification comprises 366W and S354C. In some embodiments, the CH3 region comprising a hole modification comprises Y407V, T366S, L368A, and Y349C.

[0282] Additional antigen binding moieties

[0283] In some embodiments, the antigen binding molecule comprises an additional antigen binding moiety. In some embodiments, the additional antigen binding moiety binds to a target antigen other than a yc-containing cytokine receptor polypeptide (e.g., a target antigen that is not yc, IL-2Rβ, IL-2Rα, IL-15Rα, IL-4Rα, IL-9Rα, IL-21Rα, or IL-7Rα). That is, in some embodiments, the antigen binding molecules of the present disclosure comprise (i) a yc binding moiety, (ii) a moiety that binds to one or more polypeptides of a yc-containing cytokine receptor other than yc (e.g., selected from IL-2Rβ, IL-2Rα, IL-15Rα, IL-4Rα, IL-9Rα, IL-21Rα, and IL-7Rα), and (iii) a moiety that binds to a target antigen (e.g., an antigen that is not a yc-containing cytokine receptor polypeptide).

[0284] It will be appreciated that the role of moiety (iii) is to localise the antigen binding molecule to a cell that expresses its target. This can be used to direct the effects of moieties (i) and (ii) of the antigen binding molecule to a cell that expresses the target of moiety (iii). By way of illustration, in embodiments in which moiety (ii) is an IL-2Rβ binding moiety and in which moiety (iii) is a CD8 binding moiety, the role of moiety (iii) is to target the yc:IL-2Rβ receptor agonist / antagonist activity conferred by moieties (i) and (ii) to CD8+ T cells.

[0285] Moiety (iii) can also be used to target the antigen binding molecule to an anatomical site / tissue / organ of interest. This can be used to direct the effects of moieties (i) and (ii) of the antigen binding molecule to such an organ. By way of illustration, in embodiments in which moiety (ii) is an IL-2Rβ binding moiety and in which moiety (iii) is a cancer cell antigen binding moiety, the role of moiety (iii) is to target the yc:IL-2Rβ receptor agonist / antagonist activity conferred by moieties (i) and (ii) to cells expressing the yc:IL-2Rβ receptor in the vicinity of cells expressing the cancer cell antigen.

[0286] Thus, it will be appreciated that moiety (iii) is used to target / localise the antigen binding molecule to a cell comprising / expressing the target antigen of moiety (iii), and / or to increase the local concentration of the antigen binding molecule in the vicinity of a cell comprising / expressing the target antigen of moiety (iii).

[0287] The target of moiety (iii) can be any target antigen. In some embodiments, the target antigen can be a peptide / polypeptide, glycoprotein, lipoprotein, glycan, glycolipid, lipid, or fragment thereof. The antigen is preferably expressed at the cell surface of a cell expressing the antigen.

[0288] In some embodiments, the target antigen is a disease-associated antigen or an antigen expressed by an immune cell.

[0289] A“disease-associated antigen” refers to an antigen whose presence is indicative of a given disease / disease state, or an antigen whose level is positively correlated with a given disease / disease state. A disease-associated antigen can be an antigen whose expression is correlated with the development, progression, or symptom severity of a given disease. A disease-associated antigen can be related to the etiology or pathology of a disease, or can be aberrantly expressed as a result of a disease. A disease-associated antigen can be an antigen of an infectious agent or pathogen, a cancer-associated antigen, or an autoimmune disease-associated antigen.

[0290] In some embodiments, the disease-associated antigen is an antigen of a pathogen. The pathogen can be prokaryotic (bacterial), eukaryotic (e.g., protozoan, helminth, fungal), viral, or prion. In some embodiments, the pathogen is an intracellular pathogen. In some embodiments, the pathogen is a virus, e.g., a virus as described above. In some embodiments, the pathogen is a bacterium.

[0291] In some embodiments, the target antigen is a cancer-associated antigen. A cancer-associated antigen is an antigen whose expression or overexpression is associated with cancer. In some embodiments, the cancer-associated antigen is a receptor molecule, e.g., a cell surface receptor. In some embodiments, the cancer-associated antigen is a cell signaling molecule, e.g., a cytokine, chemokine, interferon, interleukin, or lymphokine. In some embodiments, the cancer-associated antigen is a growth factor or hormone. In some embodiments, the cancer-associated antigen is a viral antigen. A cancer cell antigen can be aberrantly expressed by a cancer cell (e.g., a cancer cell antigen can be expressed with aberrant localization), or can be expressed with aberrant structure by a cancer cell. A cancer cell antigen can be capable of eliciting an immune response. In some embodiments, the antigen is expressed at the cell surface of a cancer cell (i.e., the cancer cell antigen is a cancer cell surface antigen). In some embodiments, the portion of the antigen bound by an antigen-binding molecule described herein is displayed on the outer surface of a cancer cell (i.e., is extracellular). A cancer cell antigen can be a cancer-associated antigen. In some embodiments, a cancer cell antigen is an antigen whose expression is correlated with the development, progression, or symptom severity of cancer. A cancer-associated antigen can be associated with the etiology or pathology of cancer, or can be aberrantly expressed as a result of cancer. In some embodiments, a cancer cell antigen is an antigen whose expression is upregulated (e.g., at the RNA and / or protein level) by a cancer cell, e.g., as compared to the expression level of a comparable non-cancer cell (e.g., a non-cancer cell derived from the same tissue / cell type). In some embodiments, a cancer-associated antigen can be preferentially expressed by a cancer cell, and not by a comparable non-cancer cell (e.g., a non-cancer cell derived from the same tissue / cell type). In some embodiments, a cancer-associated antigen can be a product of a mutated oncogene or a mutated tumor suppressor gene. In some embodiments, a cancer-associated antigen can be a product of an overexpressed cellular protein, a cancer antigen produced by an oncogenic virus, a carcinoembryonic antigen, or a cell surface glycolipid or glycoprotein.

[0292] Cancer-associated antigens are reviewed by Zarour HM, DeLeo A, Finn OJ, et al. Categories of Tumor Antigens. In: Kufe DW, Pollock RE, Weichselbaum RR, et al, editors. Holland-Frei Cancer Medicine. 6th ed. Hamilton (ON): BC Decker; 2003. Cancer-associated antigens include carcinoembryonic antigens: CEA, immature laminin receptor, TAG-72; oncoviral antigens such as HPV E6 and E7; overexpressed proteins: BING-4, calcium-activated chloride channel 2, cyclin-B1, 9D7, Ep-CAM, EphA3, HER2 / neu, telomerase, mesothelin, SAP-1, survivin; cancer-testis antigens: BAGE, CAGE, GAGE, MAGE, SAGE, XAGE, CT9, CT10, NY-ESO-1, PRAME, SSX-2; lineage-restricted antigens: MART1, GplOO, tyrosinase, TRP-1 / 2, MC1R, prostate-specific antigen; mutated antigens: beta-catenin, BRCA1 / 2, CDK4, CML66, fibronectin, MART-2, p53, Ras, TGF-beta RII; post-translationally altered antigens: MUC1; idiotype antigens: Ig, TCR. Other cancer cell antigens include heat shock protein 70 (HSP70), heat shock protein 90 (HSP90), glucose-regulated protein 78 (GRP78), vimentin, nucleolin, fetal pancreatic acinar protein (FAPP), placental alkaline phosphatase-like protein 2 (ALPPL-2), siglec-5, stress-induced phosphoprotein 1 (STIP1), protein tyrosine kinase 7 (PTK7), and cyclophilin B. In some embodiments, the cancer cell antigen is a cancer cell antigen described in Zhao and Cao, Front Immunol. (2019) 10:2250, which is incorporated by reference in its entirety.

[0293] In some embodiments, the target antigen is an immune cell surface molecule. An immune cell surface molecule is any molecule expressed in or at the cell membrane of an immune cell. In some embodiments, the portion of the immune cell surface molecule bound by the antigen-binding moiety is located on the outer surface of the immune cell (i.e., extracellularly). The immune cell surface molecule can be expressed at the cell surface of any immune cell. In some embodiments, the immune cell can be a cell of hematopoietic origin, such as a neutrophil, eosinophil, basophil, dendritic cell, lymphocyte, or monocyte. The lymphocyte can be, for example, a T cell, B cell, natural killer (NK) cell, NKT cell, or innate lymphoid cell (ILC), or a precursor thereof (e.g., a thymocyte or pro-B cell). The immune cell can express a CD3 polypeptide (e.g., CD3 gamma, CD3 epsilon, CD3 zeta, or CD3 delta), a TCR polypeptide (TCR alpha or TCR beta), CD27, CD28, CD4, or CD8. In some embodiments, the immune cell is a T cell, such as a CD3+ T cell. In some embodiments, the T cell is a CD3+, CD4+ T cell. In some embodiments, the T cell is a CD3+, CD8+ T cell. In some embodiments, the T cell is a T helper cell (TH cell). In some embodiments, the T cell is a cytotoxic T cell (e.g., a cytotoxic T lymphocyte (CTL)). In some embodiments, the immune cell is a T cell or NK cell.

[0294] In some embodiments, the immune cell surface molecule can be a CD3-TCR complex polypeptide, such as TCR alpha, TCR beta, TCR gamma, TCR delta, TRAC, TRBC1, TRBC2, TRGC1, TRGC2, TRDC, CD3 epsilon, CD3 delta, CD3 gamma, CD3 zeta, or CD3 eta. In some embodiments, the immune cell surface molecule is CD3, CD8, CD4, or CD28. In some embodiments, the immune cell surface molecule is a checkpoint molecule (e.g., PD-1, CTLA-4, LAG-3, TIM-3, VISTA, TIGIT, or BTLA) or a ligand thereof. In some embodiments, the immune cell surface molecule is a costimulatory molecule (e.g., CD28, OX40, 4-1BB, ICOS, or CD27) or a ligand thereof.

[0295] In some embodiments, the target antigen is selected from the group consisting of PD-1, 4-1BB, and CD8.

[0296] Chimeric antigen receptor (CAR)

[0297] The present disclosure also provides chimeric antigen receptors (CARs). CARs are recombinant receptors that provide antigen binding and T cell activation functions. CAR structure and engineering are reviewed, for example, in Dotti et al., Immunol Rev (2014) 257(1): 107-126, incorporated by reference in its entirety. CARs comprise an antigen binding region linked to a cell membrane anchoring region and a signaling region. An optional hinge region can provide separation between the antigen binding region and the cell membrane anchoring region, and can act as a flexible linker.

[0298] The antigen binding domain of a CAR according to the present disclosure comprises or consists of an antigen binding molecule as described herein. Thus, a CAR according to the present disclosure comprises an antigen binding molecule as described herein.

[0299] It will be appreciated that an antigen binding molecule according to the present disclosure forms or is comprised in the antigen binding domain of a CAR. Thus, in some embodiments, an antigen binding molecule of the present disclosure is comprised in a CAR.

[0300] It will also be appreciated that an antigen binding molecule according to the present disclosure can be a CAR. A CAR having an antigen binding domain comprising or consisting of an antigen binding molecule of the present disclosure is an antigen binding molecule. The antigen binding domain of a CAR of the present disclosure can be provided in any suitable form, for example, a scFv, a scFab, and the like.

[0301] A cell membrane anchoring region is located between the antigen binding region and the signaling region of a CAR, providing anchoring of the CAR to the cell membrane of a cell expressing the CAR, with the antigen binding region located in the extracellular space and the signaling region located inside the cell. In some embodiments, a CAR comprises a cell membrane anchoring region comprising or consisting of an amino acid sequence comprising, consisting of, or derived from the transmembrane region amino acid sequence of one of CD3-zeta, CD4, CD8, or CD28. As used herein, a region “derived from” a reference amino acid sequence comprises an amino acid sequence having at least 60% (e.g., one of at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the reference sequence.

[0302] The signaling region of a CAR allows for activation of a T cell. The CAR signaling region can comprise an amino acid sequence of an intracellular domain of CD3-zeta, which provides an immunoreceptor tyrosine-based activation motif (ITAM) for phosphorylation and activation of a T cell expressing the CAR. Signaling regions comprising sequences of other ITAM-containing proteins, such as FcyRI, have also been used for CARs (Haynes et al., J Immunol. (2001) 166(1): 182-187). The signaling region of a CAR can also comprise a costimulatory sequence derived from a costimulatory molecule’s signaling region to facilitate activation of a T cell expressing the CAR upon binding to a target protein. Suitable costimulatory molecules include CD28, OX40, 4-1BB, ICOS, and CD27. In some cases, a CAR is engineered to provide costimulation of different intracellular signaling pathways. For example, CD28- associated costimulation preferentially activates the phosphoinositide-3 kinase (PI3K) pathway, whereas 4-1BB-mediated signaling is through TNF receptor-associated factor (TRAF) adaptor proteins. Thus, the signaling region of a CAR sometimes contains costimulatory sequences derived from the signaling region of more than one costimulatory molecule. In some embodiments, a CAR of the present disclosure comprises one or more costimulatory sequences comprising or consisting of an amino acid sequence comprising, consisting of, or derived from an amino acid sequence of an intracellular domain of one or more of CD28, OX40, 4-1BB, ICOS, and CD27.

[0303] An optional hinge region can provide separation between the antigen binding domain and the transmembrane domain, and can act as a flexible linker. The hinge region can be derived from IgGl. In some embodiments, a CAR of the present disclosure comprises a hinge region comprising or consisting of an amino acid sequence comprising, consisting of, or derived from an amino acid sequence of a hinge region of IgGl.

[0304] Also provided is a cell comprising a CAR according to the present disclosure. A CAR according to the present disclosure can be used to generate an immune cell expressing the CAR, e.g., a CAR-T or CAR-NK cell. Engineering a CAR into an immune cell can be performed during in vitro culture.

[0305] Functional properties

[0306] An antigen binding molecule described herein can be characterized by features relating to certain functional properties. In some embodiments, an antigen binding molecule described herein can have one or more of the following properties:

[0307] binds to a polypeptide other than yc of a yc-containing cytokine receptor;

[0308] binds to a polypeptide other than yc of a yc-containing cytokine receptor;

[0309] binds to IL-2Rβ;

[0310] binds to IL-2Rα;

[0311] binds to IL-15Rα;

[0312] binds to IL-4Rα;

[0313] binds to IL-9Rα;

[0314] binds to IL-21Rα;

[0315] binds to IL-7Rα;

[0316] binds to a polypeptide other than γc of a γc-containing cytokine receptor;

[0317] binds to γc and IL-2Rβ;

[0318] binds to γc and IL-2Rα;

[0319] binds to γc and IL-15Rα;

[0320] binds to γc and IL-4Rα;

[0321] binds to γc and IL-9Rα;

[0322] binds to γc and IL-21Rα;

[0323] binds to γc and IL-7Rα;

[0324] binds to a cell expressing γc;

[0325] binds to a cell expressing a polypeptide other than γc of a γc-containing cytokine receptor; binds to a cell expressing IL-2Rβ;

[0326] binds to a cell expressing IL-2Rα;

[0327] binds to a cell expressing IL-15Rα;

[0328] binds to a cell expressing IL-4Rα;

[0329] binds to a cell expressing IL-9Rα;

[0330] binds to a cell expressing IL-21Rα;

[0331] binds to a cell expressing IL-7Rα;

[0332] binds to a cell expressing a polypeptide other than γc of a γc-containing cytokine receptor;

[0333] binds to a cell expressing yc and IL-2Rβ;

[0334] binds to a cell expressing yc and IL-2Rα;

[0335] binds to a cell expressing yc and IL-15Rα;

[0336] binds to a cell expressing yc and IL-4Rα;

[0337] binds to a cell expressing yc and IL-9Rα;

[0338] binds to a cell expressing yc and IL-21Rα;

[0339] binds to a cell expressing yc and IL-7Rα;

[0340] binds to a cell expressing a receptor comprising yc and a polypeptide other than yc of a yc-containing cytokine receptor;

[0341] binds to a cell expressing a yc:IL-2Rβ receptor;

[0342] binds to a cell expressing a yc:IL-2Rβ:IL-2Rα receptor;

[0343] binds to a cell expressing a yc:IL-2Rβ:IL-15Rα receptor;

[0344] binds to a cell expressing a yc:IL-4Rα receptor;

[0345] binds to a cell expressing a yc:IL-9Rα receptor;

[0346] binds to a cell expressing a yc:IL-21Rα receptor;

[0347] binds to a cell expressing a yc:IL-7Rα receptor;

[0348] increases multimerization of yc and a polypeptide other than yc of a yc-containing cytokine receptor (e.g., selected from IL-2Rβ, IL-2Rα, IL-15Rα, IL-4Rα, IL-9Rα, IL-21Rα, and IL-7Rα);

[0349] decreases multimerization of yc and a polypeptide other than yc of a yc-containing cytokine receptor (e.g., selected from IL-2Rβ, IL-2Rα, IL-15Rα, IL-4Rα, IL-9Rα, IL-21Rα, and IL-7Rα);

[0350] increasing signaling mediated by a yc-containing cytokine receptor bound by the antigen binding molecule (e.g., selected from yc:IL-2R beta receptor, yc:IL-2R beta:IL-2R alpha, yc:IL-2R beta:IL-15R alpha, yc:IL-4R alpha receptor, yc:IL-9R alpha receptor, yc:IL-21R alpha receptor, and yc:IL-7R alpha receptor);

[0351] decreasing signaling mediated by a yc-containing cytokine receptor bound by the antigen binding molecule (e.g., selected from yc:IL-2R beta receptor, yc:IL-2R beta:IL-2R alpha, yc:IL-2R beta:IL-15R alpha, yc:IL-4R alpha receptor, yc:IL-9R alpha receptor, yc:IL-21R alpha receptor, and yc:IL-7R alpha receptor);

[0352] increasing signaling mediated by IL-2, IL-4, IL-7, IL-9, IL-15, and / or IL-21;

[0353] decreasing signaling mediated by IL-2, IL-4, IL-7, IL-9, IL-15, and / or IL-21;

[0354] increasing signaling mediated by IL-2;

[0355] increasing signaling mediated by IL-4;

[0356] increasing signaling mediated by IL-7;

[0357] increasing signaling mediated by IL-9;

[0358] increasing signaling mediated by IL-15;

[0359] increasing signaling mediated by IL-21;

[0360] increasing signaling mediated by IL-2 and IL-15;

[0361] decreasing signaling mediated by IL-2;

[0362] decreasing signaling mediated by IL-4;

[0363] decreasing signaling mediated by IL-7;

[0364] decreasing signaling mediated by IL-9;

[0365] decreasing signaling mediated by IL-15;

[0366] decreasing signaling mediated by IL-21;

[0367] decrease signaling mediated by IL-2 and IL-15;

[0368] increase proliferation, survival, and / or effector activity of a cell expressing a yc-containing cytokine receptor (e.g., selected from the group consisting of yc:IL-2Rß receptor, yc:IL-2Rß:IL-2Ra, yc:IL-2Rß:IL-15Ra, yc:IL-4Ra receptor, yc:IL-9Ra receptor, yc:IL-21Ra receptor, and yc:IL-7Ra receptor) bound by the antigen binding molecule;

[0369] decrease proliferation, survival, and / or effector activity of a cell expressing a yc-containing cytokine receptor (e.g., selected from the group consisting of yc:IL-2Rß receptor, yc:IL-2Rß:IL-2Ra, yc:IL-2Rß:IL-15Ra, yc:IL-4Ra receptor, yc:IL-9Ra receptor, yc:IL-21Ra receptor, and yc:IL-7Ra receptor) bound by the antigen binding molecule;

[0370] decrease expression of one or more immune cell exhaustion markers by a cell expressing a yc-containing cytokine receptor (e.g., selected from the group consisting of yc:IL-2Rß receptor, yc:IL-2Rß:IL-2Ra, yc:IL-2Rß:IL-15Ra, yc:IL-4Ra receptor, yc:IL-9Ra receptor, yc:IL-21Ra receptor, and yc:IL-7Ra receptor) bound by the antigen binding molecule;

[0371] increase expression of one or more immune cell exhaustion markers by a cell expressing a yc-containing cytokine receptor (e.g., selected from the group consisting of yc:IL-2Rß receptor, yc:IL-2Rß:IL-2Ra, yc:IL-2Rß:IL-15Ra, yc:IL-4Ra receptor, yc:IL-9Ra receptor, yc:IL-21Ra receptor, and yc:IL-7Ra receptor) bound by the antigen binding molecule;

[0372] receptor, and / or reduce proliferation, survival, and / or effector activity of cells expressing a gamma c-containing cytokine receptor (e.g., selected from the group consisting of a gamma c:IL-2R beta receptor, a gamma c:IL-2R beta:IL-2R alpha, a gamma c:IL-2R beta:IL-15R alpha, a gamma c:IL-4R alpha receptor, a gamma c:IL-9R alpha receptor, a gamma c:IL-21R alpha receptor, and a gamma c:IL-7R alpha receptor) bound by the antigen binding molecule;

[0373] increase cell killing / elimination of, and / or reduce the number / proportion of, cells comprising / expressing one or more target antigens of which a constituent antigen binding moiety of the antigen binding molecule is specific (i.e., a polypeptide other than gamma c of a gamma c and / or gamma c-containing cytokine receptor (e.g., selected from the group consisting of IL-2R beta, IL-2R alpha, IL-15R alpha, IL-4R alpha, IL-9R alpha, IL-21R alpha, and IL-7R alpha));

[0374] increased stability and / or half-life as compared to one or more gamma c family cytokines (e.g., IL-2, IL-15, IL-4, IL-9, IL-21, and / or IL-7);

[0375] increased upregulation of signaling mediated by a gamma c-containing cytokine receptor (e.g., selected from the group consisting of a gamma c:IL-2R beta receptor, a gamma c:IL-2R beta:IL-2R alpha,

[0376] a gamma c:IL-2R beta:IL-15R alpha, a gamma c:IL-4R alpha receptor, a gamma c:IL-9R alpha receptor, a gamma c:IL-21R alpha receptor, and a gamma c:IL-7R alpha receptor) bound by the antigen binding molecule as compared to a cytokine binding to a gamma c-containing cytokine receptor;

[0377] increased upregulation of signaling mediated by a gamma c-containing cytokine receptor (e.g., selected from the group consisting of a gamma c:IL-2R beta receptor, a gamma c:IL-2R beta:IL-2R alpha,

[0378] upregulation of signaling mediated by a yc:IL-2Rβ receptor, a yc:IL-2Rβ:IL-2Rα receptor, a yc:IL-2Rβ:IL-15Rα receptor, a yc:IL-4Rα receptor, a yc:IL-9Rα receptor, a yc:IL-21Rα receptor, and a yc:IL-7Rα receptor;

[0379] upregulation of proliferation, survival, and / or effector activity of a cell expressing a yc-containing cytokine receptor bound by an antigen binding molecule (e.g., selected from a yc:IL-2Rβ receptor, a yc:IL-2Rβ:IL-2Rα receptor, a yc:IL-2Rβ:IL-15Rα receptor, a yc:IL-4Rα receptor, a yc:IL-9Rα receptor, a yc:IL-21Rα receptor, and a yc:IL-7Rα receptor) compared to a cytokine that binds to a yc-containing cytokine receptor;

[0380] downregulation of proliferation, survival, and / or effector activity of a cell expressing a yc-containing cytokine receptor bound by an antigen binding molecule (e.g., selected from a yc:IL-2Rβ receptor, a yc:IL-2Rβ:IL-2Rα receptor, a yc:IL-2Rβ:IL-15Rα receptor, a yc:IL-4Rα receptor, a yc:IL-9Rα receptor, a yc:IL-21Rα receptor, and a yc:IL-7Rα receptor) compared to a cytokine that binds to a yc-containing cytokine receptor;

[0381] upregulation of expression of one or more immune cell exhaustion markers of a cell expressing a yc-containing cytokine receptor bound by an antigen binding molecule (e.g., selected from a yc:IL-2Rβ receptor, a yc:IL-2Rβ:IL-2Rα receptor, a yc:IL-2Rβ:IL-15Rα receptor, a yc:IL-4Rα receptor, a yc:IL-9Rα receptor, a yc:IL-21Rα receptor, and a yc:IL-7Rα receptor) compared to a cytokine that binds to a yc-containing cytokine receptor;

[0382] downregulation of expression of one or more immune cell exhaustion markers of a cell expressing a yc-containing cytokine receptor bound by an antigen binding molecule (e.g., selected from a yc:IL-2Rβ receptor, a yc:IL-2Rβ:IL-2Rα receptor, a yc:IL-2Rβ:IL-15Rα receptor, a yc:IL-4Rα receptor, a yc:IL-9Rα receptor, a yc:IL-21Rα receptor, and a yc:IL-7Rα receptor) compared to a cytokine that binds to a yc-containing cytokine receptor; and / or

[0383] enhancing anti-cancer activity of a cancer antigen-specific immune cell, e.g., in vivo.

[0384] It will be appreciated that a given antigen binding molecule can exhibit more than one of the properties listed in the preceding paragraph. The properties listed in the preceding paragraph for a given antigen binding molecule can be evaluated using a suitable assay. For example, the assay can be, e.g., an in vitro assay, optionally a cell-based assay or a cell-free assay. In some embodiments, the assay can be, e.g., an in vivo assay, i.e., performed in a non-human animal. In some embodiments, the assay can be, e.g., an ex vivo assay, i.e., performed using cells / tissues / organs obtained from a subject. Such assays can be used to screen antigen binding molecules having a desired functional property.

[0385] Where the assay is a cell-based assay, the assay can comprise treating cells with a given antigen binding molecule to determine whether the antigen binding molecule exhibits one or more of the listed properties. The assay can employ a substance labeled with a detectable entity to facilitate its detection. The assay can comprise evaluating the listed property after treating the cells with a range of amounts / concentrations (e.g., a dilution series) of the given antigen binding molecule alone. It will be appreciated that the cells preferably express the target antigen of the antigen binding molecule.

[0386] Analysis of such assay results can comprise determining the concentration at which 50% of the maximum level of the relevant activity is achieved. The concentration at which 50% of the maximum level of the relevant activity is achieved for a given agent can be referred to as the “half maximal effective concentration” of the agent with respect to the relevant activity, and can also be referred to as the “EC 50 ”. To illustrate, the EC 50 for a given antigen binding molecule binding to human yc can be the concentration of the antigen binding molecule at which binding to human yc is at 50% of the maximum level.

[0387] Depending on the property, the EC 50 may also be referred to as the “half maximal inhibitory concentration” or “IC 50 ”, which is the concentration of an agent at which 50% of the maximum level of inhibition of a given property is observed.

[0388] Where functional properties of agents are being compared (e.g., where antigen binding molecules of the disclosure are compared to other polypeptides), the comparison is made at equivalent concentrations and / or amounts of the relevant agents.

[0389] The antigen binding molecules and antigen binding moieties described herein preferably exhibit specific binding to yc, IL-2Rβ, IL-2Rα, IL-15Rα, IL-4Rα, IL-9Rα, IL-21Rα, and / or IL-7Rα. As used herein, “specific binding” refers to binding that is selective for an antigen, and can be distinguished from non-specific binding to non-target antigens. An antigen binding molecule / moieties that specifically binds to a target molecule preferably binds the target with greater affinity and / or for a longer duration than binding to other, non-target molecules.

[0390] The ability of a given polypeptide to specifically bind to a given molecule can be determined by analysis according to methods known in the art, such as by ELISA, surface plasmon resonance (SPR; see, e.g., Hearty et al., Methods Mol Biol. (2012) 907:411-442), bio-layer interferometry (BLI; see, e.g., Lad et al., J Biomol Screen (2015) 20(4):498-507), flow cytometry, or by radio-labeled antigen binding assays (RIA), enzyme-linked immunosorbent assays. Through such analysis, binding to a given molecule can be measured and quantified. In some embodiments, binding can be a reaction detected in a given assay.

[0391] In some embodiments, the extent of binding of an antigen binding molecule / portion to a non-target molecule is less than about 10% of the binding of the antigen binding molecule / portion to a target molecule, as measured, e.g., by ELISA, SPR, BLI, or RIA. Alternatively, binding specificity can be reflected in terms of binding affinity, where the antigen binding molecule / portion binds to a target molecule with a K D at least 0.1 orders of magnitude (i.e., 0.1 x 10 n , where n is an integer representing orders of magnitude) greater than the dissociation constant (K D ) of the antigen binding molecule / portion to a non-target molecule. This can optionally be one of at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, or 2.0.

[0392] The binding affinity of an antigen binding molecule / portion for its target is typically described by its dissociation constant (K D ). Binding affinity can be measured by methods known in the art, such as by ELISA, surface plasmon resonance (SPR; see, e.g., Hearty et al., Methods Mol Biol (2012) 907:411-442; or Rich et al., Anal Biochem. (2008) 373(1):112-20), bio-layer interferometry (see, e.g., Lad et al., J Biomol Screen (2015) 20(4):498-507; or Concepcion et al., Comb Chem High Throughput Screen. (2009) 12(8):791-800), microscale thermophoresis (MST) analysis (see, e.g., Jerabek-Willemsen et al., Assay Drug Dev Technol. (2011) 9(4):342-353), or by radio-labeled antigen binding assays (RIA).

[0393] In some embodiments, the antigen binding molecules / portions described herein bind to γc, IL-2Rβ, IL-2Rα, IL-15Rα, IL-4Rα, IL-9Rα, IL-21Rα and / or IL-7Rα with an affinity in the micromolar range, i.e., K D =9.9x 10 -4 to 1x 10 -6 In some embodiments, the antigen binding molecules / portions described herein bind to γc, IL-2Rβ, IL-2Rα, IL-15Rα, IL-4Rα, IL-9Rα, IL-21Rα and / or IL-7Rα with submicromolar affinity, i.e., K D <1x 10 -6 In some embodiments, the antigen binding molecules / portions described herein bind to γc, IL-2Rβ, IL-2Rα, IL-15Rα, IL-4Rα, IL-9Rα, IL-21Rα and / or IL-7Rα with an affinity in the nanomolar range, i.e., K D =9.9x 10 -7 to 1x 10 -9 In some embodiments, the antigen binding molecules / portions described herein bind to γc, IL-2Rβ, IL-2Rα, IL-15Rα, IL-4Rα, IL-9Rα, IL-21Rα and / or IL-7Rα with sub-nanomolar affinity, i.e., K D <1x 10 -9 In some embodiments, the antigen binding molecules / portions described herein bind to γc, IL-2Rβ, IL-2Rα, IL-15Rα, IL-4Rα, IL-9Rα, IL-21Rα and / or IL-7Rα with an affinity in the picomolar range, i.e., K D =9.9x 10 -10 to 1x 10 -12 In some embodiments, the antigen binding molecules / portions described herein bind to γc, IL-2Rβ, IL-2Rα, IL-15Rα, IL-4Rα, IL-9Rα, IL-21Rα and / or IL-7Rα with sub-picomolar affinity, i.e., K D <1x 10 -12 M.

[0394] The antigen binding molecules and antigen binding moieties of the present disclosure can bind to a particular region of interest of their target antigen. For example, they can bind to a linear epitope of yc, IL-2Rβ, IL-2Rα, IL-15Rα, IL-4Rα, IL-9Rα, IL-21Rα, and / or IL-7Rα, which consists of a contiguous amino acid sequence (i.e., the amino acid primary sequence). In some embodiments, they can bind to a conformational epitope of yc, IL-2Rβ, IL-2Rα, IL-15Rα, IL-4Rα, IL-9Rα, IL-21Rα, and / or IL-7Rα, which consists of a discontinuous amino acid sequence of an amino acid sequence.

[0395] The region of a given target molecule to which an antigen binding molecule binds can be determined by one of skill in the art using various methods well known in the art, including X-ray co-crystallographic analysis of antibody-antigen complexes, peptide scanning, mutagenesis mapping, hydrogen-deuterium exchange analysis by mass spectrometry, phage display, competitive ELISA, and proteolysis-based “protection” methods. Such methods are described, for example, in Gershoni et al., BioDrugs (2007) 21(3): 145-156, which is incorporated by reference herein in its entirety.

[0396] When the target antigen is expressed at the surface of a cell (i.e., in or at the cell membrane), the antigen binding molecules and antigen binding moieties preferably bind to the target antigen in a region accessible to the antigen binding molecules (i.e., extracellular antigen binding molecules). In some embodiments, the antigen binding molecules and antigen binding moieties are capable of binding to the target antigen when the target antigen is expressed at the surface of a cell.

[0397] The antigen binding molecules and antigen binding moieties preferably bind to an extracellular domain of the target antigen. The extracellular domains of yc, IL-2Rβ, IL-2Rα, IL-15Rα, IL-4Rα, IL-9Rα, IL-21Rα, and IL-7Rα are described above.

[0398] The antigen binding molecules can bind to a cell expressing yc, IL-2Rβ, IL-2Rα, IL-15Rα, IL-4Rα, IL-9Rα, IL-21Rα, and / or IL-7Rα.

[0399] Such cells include immune cells, e.g., effector immune cells. The immune cells can be cells of hematopoietic origin, e.g., neutrophils, eosinophils, basophils, dendritic cells, lymphocytes, or monocytes. The lymphocytes can be, e.g., T cells, B cells, NK cells, NKT cells, or innate lymphoid cells (ILCs), or precursors thereof (e.g., thymocytes or pre-B cells). The immune cells can express a CD3 polypeptide (e.g., CD3y, CD3s, CD3zeta, or CD35), a TCR polypeptide (TCRa or TCRp), CD27, CD28, CD4, or CD8. In some embodiments, the immune cells are T cells, e.g., CD3+ T cells. In some embodiments, the T cells are CD3+, CD4+ T cells. In some embodiments, the T cells are CD3+, CD8+ T cells. In some embodiments, the T cells are T helper cells (TH cells). In some embodiments, the T cells are cytotoxic T cells (e.g., cytotoxic T lymphocytes (CTLs)). In some embodiments, the immune cells are T cells or NK cells.

[0400] An “effector immune cell” can be an immune cell that exhibits effector function. The effector immune cell can be a CD8+ T cell, a CD8+ cytotoxic T lymphocyte (CD8+ CTL), a CD4+ T cell, a CD4+ T helper cell, an NK cell, an IFNy-producing cell, a memory T cell, a central memory T cell, an antigen experienced T cell, or a CD45RO+ T cell. The effector immune cell can be characterized by one or more of the following properties: granzyme B expression, IFNy expression, CD107a expression, IL-2 expression, TNFa expression, perforin expression, granulysin expression, and / or FAS ligand (FASL) expression. In some embodiments, the effector immune cell according to the present disclosure is a granzyme B-expressing cell.

[0401] The ability of an antigen binding molecule to bind to a given cell type (e.g., a cell expressing one or more particular molecules, e.g., selected from yc, IL-2R, IL-2R, IL-15R, IL-4R, IL-9R, IL-21R, and / or IL-7R ) can be analyzed by contacting the cells with the antigen binding molecule and detecting antigen binding molecule bound to the cells, e.g., after a washing step to remove unbound antigen binding molecule. The ability of an antigen binding molecule to bind to a cell expressing yc, IL-2R, IL-2R, IL-15R, IL-4R, IL-9R, IL-21R, and / or IL-7R can be analyzed by methods such as flow cytometry and immunofluorescence microscopy.

[0402] In some embodiments, the antigen binding molecule increases multimerization of the polypeptide other than yc of the yc and yc-containing cytokine receptors (e.g., selected from the group consisting of IL-2R, IL-2R, IL-15R, IL-4R, IL-9R, IL-21R, and IL-7R ). In some embodiments, the antigen binding molecule decreases multimerization of the polypeptide other than yc of the yc and yc-containing cytokine receptors (e.g., selected from the group consisting of IL-2R, IL-2R, IL-15R, IL-4R, IL-9R, IL-21R, and IL-7R ).

[0403] As used herein, “multimerization” refers to the formation of a multimeric polypeptide complex (i.e., formed by non-covalent protein:protein interactions, as described above). A multimer comprises two or more polypeptides, and can be, for example, a dimer, a trimer, a tetramer, a pentamer, a hexamer, a heptamer, an octamer, a nonamer, or a decamer. Thus, multimerization can be dimerization, trimerization, tetramerization, etc.

[0404] It will be appreciated that multimerization of the polypeptide other than yc of the yc and yc-containing cytokine receptors is heteromultimerization, as the constituent polypeptides of the multimer are not identical. Thus, the multimers formed by multimerization of the polypeptide other than yc of the yc and yc-containing cytokine receptors according to the present disclosure are heteromultimers, and not homomultimers.

[0405] The antigen binding molecules of the present disclosure can promote multimerization of the polypeptide other than yc of the yc and yc-containing cytokine receptors by virtue of the antigen binding moieties of which they are composed binding to the respective polypeptides. Binding to the polypeptide other than yc of the yc and yc-containing cytokine receptors brings the polypeptides into close physical proximity (e.g., within 50 angstroms, e.g., within 40, 30, 25, 20, 15, 10, or 5 angstroms), thereby facilitating their association.

[0406] The ability of an antigen binding molecule to increase / promote or decrease / inhibit association between two polypeptides can be analyzed using techniques known to the skilled person. For example, cells expressing the relevant polypeptides can be contacted with a given test antigen binding molecule in vitro, and then the association of the relevant polypeptides can be analyzed. Suitable techniques employed in the analysis include, for example, resonance energy transfer techniques such as fluorescence resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET), using appropriate labeled interaction partners, for example as described in Ciruela, Curr Opin Biotechnol. (2008) 19(4):338-43. Other suitable techniques include protein fragment complementation systems, for example NanoLuc and NanoBiT, which are described, for example, in Thirukkumaran et al., Front Chem. (2020) 7:938 and Dixon et al., ACS Chem Biol. (2016) 11(2):400-408.

[0407] An antigen binding molecule according to the disclosure can increase or decrease the level of multimerization relative to the level observed in the absence of the antigen binding molecule or in the presence of an appropriate control antigen binding molecule (e.g., an antigen binding molecule known not to affect multimerization of the relevant polypeptide). In some embodiments, an “increased” level of multimerization refers to a level of multimerization that is greater than 1-fold, e.g., one of ≥1.01-fold, ≥1.02-fold, ≥1.03-fold, ≥1.04-fold, ≥1.05-fold, ≥1.1-fold, ≥1.2-fold, ≥1.3-fold, ≥1.4-fold, ≥1.5-fold, ≥1.6-fold, ≥1.7-fold, ≥1.8-fold, ≥1.9-fold, ≥2-fold, ≥3-fold, ≥4-fold, ≥5-fold, ≥6-fold, ≥7-fold, ≥8-fold, ≥9-fold, or ≥10-fold, of the level observed in the absence of the antigen binding molecule or in the presence of an appropriate control antigen binding molecule (e.g., an antigen binding molecule known not to affect multimerization of the relevant polypeptide). In some embodiments, a “decreased” level of multimerization refers to a level of multimerization that is less than 1-fold, e.g., one of ≤0.99-fold, ≤0.95-fold, ≤0.9-fold, ≤0.85-fold, ≤0.8-fold, ≤0.75-fold, ≤0.7-fold, ≤0.65-fold, ≤0.6-fold, ≤0.55-fold, ≤0.5-fold, ≤0.45-fold, ≤0.4-fold, ≤0.35-fold, ≤0.3-fold, ≤0.25-fold, ≤0.2-fold, ≤0.15-fold, ≤0.1-fold, ≤0.05-fold, or ≤0.01-fold, of the level observed in the absence of the antigen binding molecule or in the presence of an appropriate control antigen binding molecule (e.g., an antigen binding molecule known not to affect multimerization of the relevant polypeptide).

[0408] In some embodiments, the antigen binding molecule increases signaling mediated by a yc-containing cytokine receptor (e.g., selected from the group consisting of yc:IL-2Rß receptor, yc:IL-2Rß:IL-2Ra, yc:IL-2Rß:IL-15Ra, yc:IL-4Ra receptor, yc:IL-9Ra receptor, yc:IL-21Ra receptor, and yc:IL-7Ra receptor). It will be understood that the antigen binding molecule increases signaling mediated by a yc-containing cytokine receptor to which the antigen binding molecule binds. That is, the antigen binding molecule increases signaling mediated by a yc-containing cytokine receptor that comprises or consists of a polypeptide complex comprising a polypeptide, for which the antigen binding molecule comprises a binding moiety. To illustrate, in embodiments in which the antigen binding molecule comprises (i) a yc-binding moiety and (ii) an IL-2Rß-binding moiety, the antigen binding molecule can increase signaling through a yc-containing cytokine receptor comprising yc and IL-2Rß (e.g., a yc:IL-2Rß receptor).

[0409] Such antigen binding molecules can variously be described as “upregulating,” “inducing,” “enhancing,” “promoting,” “stimulating,” “triggering,” or “potentiating” signaling mediated by the relevant yc-containing cytokine receptor. They can also be referred to as “agonists” of the relevant yc-containing cytokine receptor, or as having “agonistic” or “activating” activity on the relevant yc-containing cytokine receptor.

[0410] In some embodiments, the antigen binding molecule reduces signaling mediated by a yc-containing cytokine receptor (e.g., selected from the group consisting of yc:IL-2Rß receptor, yc:IL-2Rß:IL-2Ra, yc:IL-2Rß:IL-15Ra, yc:IL-4Ra receptor, yc:IL-9Ra receptor, yc:IL-21Ra receptor, and yc:IL-7Ra receptor). It will be understood that the antigen binding molecule reduces signaling mediated by a yc-containing cytokine receptor to which the antigen binding molecule binds. That is, the antigen binding molecule reduces signaling mediated by a yc-containing cytokine receptor that comprises or consists of a polypeptide complex comprising a polypeptide, for which the antigen binding molecule comprises a binding moiety. To illustrate, in embodiments in which the antigen binding molecule comprises (i) a yc binding moiety and (ii) an IL-2Rß binding moiety, the antigen binding molecule can reduce signaling through a yc-containing cytokine receptor comprising yc and IL-2Rß (e.g., a yc:IL-2Rß receptor). Such antigen binding molecules can be variously described as “down-regulating,” “preventing,” “reducing,” “inhibiting,” “decreasing,” “attenuating,” “blocking,” or “reducing” signaling mediated by the relevant yc-containing cytokine receptor. They can also be referred to as “antagonists” of the relevant yc-containing cytokine receptor, or as having “antagonistic” or “inhibitory” activity toward the relevant yc-containing cytokine receptor.

[0411] In some embodiments, the antigen binding molecule inhibits IL-2 signaling. In some embodiments, the antigen binding molecule inhibits IL-4 signaling. In some embodiments, the antigen binding molecule inhibits IL-7 signaling. In some embodiments, the antigen binding molecule inhibits IL-9 signaling. In some embodiments, the antigen binding molecule inhibits IL-15 signaling. In some embodiments, the antigen binding molecule inhibits IL-21 signaling. In some embodiments, the antigen binding molecule inhibits IL-2 signaling and IL-15 signaling.

[0412] In some embodiments, the antigen binding molecule inhibits IL-2-mediated signaling. In some embodiments, the antigen binding molecule inhibits IL-4-mediated signaling. In some embodiments, the antigen binding molecule inhibits IL-7-mediated signaling. In some embodiments, the antigen binding molecule inhibits IL-9-mediated signaling. In some embodiments, the antigen binding molecule inhibits IL-15-mediated signaling. In some embodiments, the antigen binding molecule inhibits IL-21-mediated signaling. In some embodiments, the antigen binding molecule inhibits IL-2-mediated signaling and IL-15-mediated signaling.

[0413] Cytokine signaling can be analyzed using cells expressing the relevant cytokine receptor, e.g., the relevant cytokine receptor for assaying IL-7 signaling would be the yc:IL-7Ra receptor. Suitable assays include, e.g., assays for detecting phosphorylation / activity / expression of factors that are phosphorylated / activated / expressed as a result of signaling through yc-containing cytokine receptors.

[0414] For example, cytokine signaling can be investigated by assessing phosphorylation of one or more signaling molecules of a signal transduction pathway (e.g., JAK / STAT, MAPK / ERK, or PI3K / Akt pathway) triggered by signaling through the relevant yc-containing cytokine receptor. For example, yc-containing cytokine receptor-mediated signaling levels can be analyzed by detecting and / or quantifying phosphorylation levels of STAT1, STAT3, STAT5, and / or ERK (e.g., STAT5 and / or ERK).

[0415] Cytokine signaling levels can also be assessed by analyzing one or more correlates of signaling through the relevant receptor. For example, signaling can be investigated by detecting and / or quantifying expression or activity of a factor whose expression / activity is up- or down-regulated as a result of the particular cytokine signaling. In some embodiments, cytokine signaling can be investigated by detecting and / or quantifying expression of a factor whose expression is up-regulated as a result of cytokine signaling (e.g., IL-2, IL-4, IL-7, IL-9, IL-15, and / or IL-21 signaling).

[0416] Signaling levels can also be analyzed using reporter-based methods. For example, cytokine signaling can be investigated using a reporter cell line stably expressing a luciferase reporter whose expression is driven by signaling of signaling mediated through the relevant receptor.

[0417] yc-containing cytokine receptor-mediated signaling can be analyzed using cells expressing the relevant receptor, e.g., using assays for detecting and / or quantifying receptor-mediated signaling. Suitable assays include, e.g., assays for detecting phosphorylation / activity / expression of factors that are phosphorylated / activated / expressed as a result of signaling through yc-containing cytokine receptors.

[0418] Such assays can include contacting cells expressing a given yc-containing cytokine receptor with an antigen binding molecule according to the present disclosure. For example, an assay for studying the ability of an antigen binding molecule to increase yc:IL-2R signaling can include contacting cells expressing a yc:IL-2R receptor with an antigen binding molecule comprising a yc-binding moiety and an IL-2R beta-binding moiety.

[0419] For example, yc-containing cytokine receptor-mediated signaling can be studied by assessing phosphorylation of one or more signal transduction molecules of a signal transduction pathway (e.g., JAK / STAT, MAPK / ERK, or PI3K / Akt pathway) triggered by signaling through the relevant yc-containing cytokine receptor. For example, yc-containing cytokine receptor-mediated signaling levels can be analyzed by detecting and / or quantifying the phosphorylation levels of STAT1, STAT3, STAT5, and / or ERK (e.g., STAT5 and / or ERK).

[0420] In some embodiments, the antigen binding molecule increases JAK / STAT signaling mediated by a yc-containing cytokine receptor (e.g., selected from the group consisting of yc:IL-2R beta receptor, yc:IL-2R beta:IL-2R alpha, yc:IL-2R beta:IL-15R alpha, yc:IL-4R alpha receptor, yc:IL-9R alpha receptor, yc:IL-21R alpha receptor, and yc:IL-7R alpha receptor). In some embodiments, the antigen binding molecule increases MAPK / ERK signaling mediated by a yc-containing cytokine receptor (e.g., selected from the group consisting of yc:IL-2R beta receptor, yc:IL-2R beta:IL-2R alpha, yc:IL-2R beta:IL-15R alpha, yc:IL-4R alpha receptor, yc:IL-9R alpha receptor, yc:IL-21R alpha receptor, and yc:IL-7R alpha receptor). In some embodiments, the antigen binding molecule increases PI3K / Akt signaling mediated by a yc-containing cytokine receptor (e.g., selected from the group consisting of yc:IL-2R beta receptor, yc:IL-2R beta:IL-2R alpha, yc:IL-2R beta:IL-15R alpha, yc:IL-4R alpha receptor, yc:IL-9R alpha receptor, yc:IL-21R alpha receptor, and yc:IL-7R alpha receptor).

[0421] In some embodiments, the antigen binding molecule increases phosphorylation of STAT1, STAT3, STAT5, and / or ERK. In some embodiments, the antigen binding molecule increases activation of STAT1, STAT3, STAT5, and / or ERK. In some embodiments, the antigen binding molecule increases STAT1, STAT3, STAT5, and / or ERK activity.

[0422] In some embodiments, the antigen binding molecule increases phosphorylation of STAT5. In some embodiments, the antigen binding molecule increases activation of STAT5. In some embodiments, the antigen binding molecule increases STAT5 activity.

[0423] In some embodiments, the antigen binding molecule reduces JAK / STAT signaling mediated by a yc-containing cytokine receptor (e.g., selected from yc:IL-2Rß receptor, yc:IL-2Rß:IL-2Ra, yc:IL-2Rß:IL-15Ra, yc:IL-4Ra receptor, yc:IL-9Ra receptor, yc:IL-21Ra receptor, and yc:IL-7Ra receptor). In some embodiments, the antigen binding molecule reduces MAPK / ERK signaling mediated by a yc-containing cytokine receptor (e.g., selected from yc:IL-2Rß receptor, yc:IL-2Rß:IL-2Ra, yc:IL-2Rß:IL-15Ra, yc:IL-4Ra receptor, yc:IL-9Ra receptor, yc:IL-21Ra receptor, and yc:IL-7Ra receptor). In some embodiments, the antigen binding molecule reduces PI3K / Akt signaling mediated by a yc-containing cytokine receptor (e.g., selected from yc:IL-2Rß receptor, yc:IL-2Rß:IL-2Ra, yc:IL-2Rß:IL-15Ra, yc:IL-4Ra receptor, yc:IL-9Ra receptor, yc:IL-21Ra receptor, and yc:IL-7Ra receptor).

[0424] In some embodiments, the antigen binding molecule reduces phosphorylation of STAT1, STAT3, STAT5, and / or ERK. In some embodiments, the antigen binding molecule reduces activation of STAT1, STAT3, STAT5, and / or ERK. In some embodiments, the antigen binding molecule reduces STAT1, STAT3, STAT5, and / or ERK activity.

[0425] In some embodiments, the antigen binding molecule reduces phosphorylation of STAT5. In some embodiments, the antigen binding molecule reduces activation of STAT5. In some embodiments, the antigen binding molecule reduces STAT5 activity.

[0426] The level of signaling mediated by a given yc-containing cytokine receptor can also be evaluated by analyzing one or more relevant indicators of signaling through the relevant receptor. For example, yc-containing cytokine receptor-mediated signaling can be investigated by detecting and / or quantifying the expression or activity of a factor whose expression / activity is up- or down-regulated as a result of signaling through the relevant receptor. In some embodiments, yc-containing cytokine receptor-mediated signaling can be investigated by detecting and / or quantifying the expression of a factor whose expression is up-regulated as a result of yc-containing cytokine receptor-mediated signaling.

[0427] The level of signaling mediated by a given yc-containing cytokine receptor can also be analyzed using a reporter gene-based approach. For example, yc-containing cytokine receptor-mediated signaling can be investigated using a reporter cell line stably expressing a luciferase reporter gene whose expression is driven by signaling of signaling mediated through the relevant receptor. Additionally, yc-containing cytokine receptor-mediated signaling can be investigated using a reporter cell line expressing a secretible reporter gene that can be quantitatively detected from the supernatant and can be readily measured (e.g., the cytokine reporter cells described and utilized in Examples 3-6).

[0428] In some embodiments, the antigen binding molecule increases the proliferation, survival, and / or effector activity of a cell expressing a yc-containing cytokine receptor (e.g., selected from the group consisting of yc:IL-2Rβ receptor, yc:IL-2Rβ:IL-2Rα, yc:IL-2Rβ:IL-15Rα, yc:IL-4Rα receptor, yc:IL-9Rα receptor, yc:IL-21Rα receptor, and yc:IL-7Rα receptor) to which the antigen binding molecule binds. In some embodiments, the antigen binding molecule decreases the proliferation, survival, and / or effector activity of a cell expressing a yc-containing cytokine receptor (e.g., selected from the group consisting of yc:IL-2Rβ receptor, yc:IL-2Rβ:IL-2Rα, yc:IL-2Rβ:IL-15Rα, yc:IL-4Rα receptor, yc:IL-9Rα receptor, yc:IL-21Rα receptor, and yc:IL-7Rα receptor) to which the antigen binding molecule binds. It will be understood that the increase / decrease in proliferation, survival, and / or effector activity is a result of increased / decreased cellular level function by signaling through the relevant yc-containing cytokine receptor.

[0429] The ability of an antigen binding molecule to increase / decrease the ability of a cell expressing a given yc-containing cytokine receptor to proliferate can be analyzed by contacting the cell with the antigen binding molecule and subsequently assessing the proliferation of the cell (i.e., after a period of time sufficient to observe an effect on cell proliferation / survival). Cell proliferation can be assessed, for example, by detecting changes in cell number over time, or by incorporating a thymidine analog, such as BrdU, into the DNA of proliferating cells. The ability of an antigen binding molecule to increase / decrease the ability of a cell expressing a given yc-containing cytokine receptor to survive can be assessed, for example, by labeling the cell and monitoring cell number over time. 3 In vitro assays for H-thymidine or by CFSE dilution assays, for example as described in Fulcher and Wong, Immunol Cell Biol. (1999) 77(6):559-564, incorporated herein by reference in its entirety, can be used. Proliferating cells can also be identified by analysis of incorporation of 5-ethynyl-2'-deoxyuridine (EdU) by an appropriate assay, as described, for example, in Buck et al., Biotechniques. (2008) 44(7):927-9 and Sali and Mitchison, PNAS USA. (2008) 105(7):2415-2420. Cell survival can be assessed, for example, by labeling the cell and monitoring cell number over time.

[0430] Effector activity can be assessed by analyzing an indicator of such activity. For example, the ability of an antigen binding molecule to increase / decrease the effector activity of a cell expressing a given yc-containing cytokine receptor can be analyzed by contacting the cell with the antigen binding molecule and subsequently assessing the gene and / or protein expression of one or more effector molecules by the cell (i.e., after a period of time sufficient to observe an effect on the gene and / or protein expression of such factors). Effector molecules include, for example, granzyme B, IFNy, CD 107a, IL-2, TNFa, perforin, granulysin, and FAS ligand (FASL). Gene and / or protein expression of such effector molecules can be determined by any suitable means. Gene expression can be determined, for example, by detecting mRNA encoding the relevant molecule, for example, by quantitative real-time PCR (qRT-PCR). Protein expression can be determined, for example, by antibody-based methods, for example, by Western blot, immunohistochemistry, immunocytochemistry, flow cytometry, or ELISA.

[0431] The ability of an antigen binding molecule to increase / decrease the effector activity of a cell expressing a given yc-containing cytokine receptor can also be analyzed by contacting the cell with the antigen binding molecule and subsequently evaluating the ability of the cell to kill a target cell expressing the antigen (for which the cell expressing the yc-containing cytokine receptor comprises a specific receptor (e.g., a TCR or CAR)) (i.e., after a period of time sufficient to observe an effect on cell killing). Cell killing can be studied, for example, using any of the methods reviewed in Zaritskaya et al., Expert Rev Vaccines (2011) 9(6):601-616, incorporated herein by reference in its entirety. Examples of in vitro assays for cytotoxicity / cell killing assays include release assays, such as 51 Cr release assays, lactate dehydrogenase (LDH) release assays, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) release assays, and calcein acetoxy methyl (calcein-AM) release assays. These assays measure cell killing based on detection of factors released by lysed cells. Cell killing of a given test cell type by a given effector immune cell type can be analyzed, for example, by co-culturing the test cells with the effector immune cells and measuring the number / proportion of live / dead (e.g., lysed) test cells after a suitable period of time. Other suitable assays include the xCELLigence real-time cytolytic in vitro potency assay described in Cerignoli et al., PLoS One. (2018) 13(3):e0193498, incorporated herein by reference in its entirety.

[0432] Effector activity can also be analyzed in vivo, for example, in a suitable non-human animal model of a given disease / condition. Effector activity can be inferred by evaluating the therapeutic / prophylactic effect in a relevant model associated with a relevant effector activity.

[0433] In the context of the present disclosure, an "increase" or "decrease" in a level of signaling / proliferation / survival / effector activity is relative to the level of the relevant property exhibited in the absence of the antigen binding molecule or in the presence of an appropriate control antigen binding molecule (e.g., an antigen binding molecule known not to affect signaling mediated by the relevant yc-containing cytokine receptor / an antigen binding molecule known not to affect proliferation / survival / effector activity of a cell expressing the relevant yc-containing cytokine receptor), i.e., the same type of cell. In some embodiments, an "increased" level of signaling / proliferation / survival / effector activity refers to a level of signaling / proliferation / survival / effector activity that is greater than 1-fold, e.g., one of >1.01-fold, >1.02-fold, >1.03-fold, >1.04-fold, >1.05-fold, >1.1-fold, >1.2-fold, >1.3-fold, >1.4-fold, >1.5-fold, >1.6-fold, >1.7-fold, >1.8-fold, >1.9-fold, >2-fold, >3-fold, >4-fold, >5-fold, >6-fold, >7-fold, >8-fold, >9-fold, or >10-fold, of the level of signaling / proliferation / survival / effector activity observed in the absence of the antigen binding molecule or in the presence of an appropriate control antigen binding molecule (e.g., an antigen binding molecule known not to affect signaling mediated by the relevant yc-containing cytokine receptor / an antigen binding molecule known not to affect proliferation / survival / effector activity of a cell expressing the relevant yc-containing cytokine receptor). In some embodiments, a "decreased" level of signaling / proliferation / survival / effector activity refers to a level of signaling / proliferation / survival / effector activity that is less than 1-fold, e.g., one of <0.99-fold, <0.95-fold, <0.9-fold, <0.85-fold, <0.8-fold, <0.75-fold, <0.7-fold, <0.65-fold, <0.6-fold, <0.55-fold, <0.5-fold, <0.45-fold, <0.4-fold, <0.35-fold, <0.3-fold, <0.25-fold, <0.2-fold, <0.15-fold, <0.1-fold, <0.05-fold, or <0.01-fold, of the level of signaling / proliferation / survival / effector activity observed in the absence of the antigen binding molecule or in the presence of an appropriate control antigen binding molecule (e.g., an antigen binding molecule known not to affect signaling mediated by the relevant yc-containing cytokine receptor / an antigen binding molecule known not to affect proliferation / survival / effector activity of a cell expressing the relevant yc-containing cytokine receptor).

[0434] In some embodiments, the antigen binding molecule reduces expression of one or more immune cell exhaustion markers by cells expressing a yc-containing cytokine receptor to which the antigen binding molecule binds (e.g., selected from the group consisting of yc:IL-2Rß receptor, yc:IL-2Rß:IL-2Ra, yc:IL-2Rß:IL-15Ra, yc:IL-4Ra receptor, yc:IL-9Ra receptor, yc:IL-21Ra receptor, and yc:IL-7Ra receptor). In some embodiments, the antigen binding molecule increases expression of one or more immune cell exhaustion markers by cells expressing a yc-containing cytokine receptor to which the antigen binding molecule binds (e.g., selected from the group consisting of yc:IL-2Rß receptor, yc:IL-2Rß:IL-2Ra, yc:IL-2Rß:IL-15Ra, yc:IL-4Ra receptor, yc:IL-9Ra receptor, yc:IL-21Ra receptor, and yc:IL-7Ra receptor). It will be appreciated that a reduction in the level of expression of one or more immune cell exhaustion markers is a result of increased / reduced cell level function of signaling through the relevant yc-containing cytokine receptor.

[0435] For example, the ability of an antigen binding molecule to reduce / increase expression of one or more immune cell exhaustion markers by cells expressing a given yc-containing cytokine receptor can be analyzed by contacting the cells with the antigen binding molecule and subsequently evaluating the gene and / or protein expression of the one or more immune cell exhaustion markers by the cells (i.e., after a period of time sufficient to observe an effect on the gene and / or protein expression of such factors). Immune cell exhaustion markers include, for example, immune checkpoint molecules (e.g., PD-1, CTLA-4, LAG-3, TIM-3, VISTA, TIGIT, and BTLA), CD160, and CD244. In some embodiments, cell surface expression of one or more immune cell exhaustion markers can be evaluated, for example, by flow cytometry.

[0436] In the context of the present disclosure, a "reduction" or "increase" in the expression level of one or more immune cell exhaustion marker is relative to the level exhibited in the absence of the antigen binding molecule or in the presence of an appropriate control antigen binding molecule (e.g., an antigen binding molecule known not to affect expression of the one or more immune cell exhaustion marker) (i.e., the same type of cell). In some embodiments, a "reduction" in the expression level of one or more immune cell exhaustion marker refers to a level that is less than 1-fold, e.g., e.g., ≤ 0.99-fold, ≤ 0.95-fold, ≤ 0.9-fold, ≤ 0.85-fold, ≤ 0.8-fold, ≤ 0.75-fold, ≤ 0.7-fold, ≤ 0.65-fold, ≤ 0.6-fold, ≤ 0.55-fold, ≤ 0.5-fold, ≤ 0.45-fold, ≤ 0.4-fold, ≤ 0.35-fold, ≤ 0.3-fold, ≤ 0.25-fold, ≤ 0.2-fold, ≤ 0.15-fold, ≤ 0.1-fold, ≤ 0.05-fold, or ≤ 0.01-fold of the level observed in the absence of the antigen binding molecule or in the presence of an appropriate control antigen binding molecule (e.g., an antigen binding molecule known not to affect expression of the one or more immune cell exhaustion marker).

[0437] In some embodiments, an "increase" in the expression level of one or more immune cell exhaustion marker refers to a level that is greater than 1-fold, e.g., one of ≥ 1.01-fold, ≥ 1.02-fold, ≥ 1.03-fold, ≥ 1.04-fold, ≥ 1.05-fold, ≥ 1.1-fold, ≥ 1.2-fold, ≥ 1.3-fold, ≥ 1.4-fold, ≥ 1.5-fold, ≥ 1.6-fold, ≥ 1.7-fold, ≥ 1.8-fold, ≥ 1.9-fold, ≥ 2-fold, ≥ 3-fold, ≥ 4-fold, ≥ 5-fold, ≥ 6-fold, ≥ 7-fold, ≥ 8-fold, ≥ 9-fold, or ≥ 10-fold of the level observed in the absence of the antigen binding molecule or in the presence of an appropriate control antigen binding molecule (e.g., an antigen binding molecule known not to affect expression of the one or more immune cell exhaustion marker).

[0438] In some embodiments, the antigen binding molecules of the present disclosure achieve their functional effects via a mechanism that does not involve killing / depleting cells that comprise / express the one or more target antigens of their constituent antigen binding moieties (i.e., polypeptides other than yc of yc- and / or yc-containing cytokine receptor (e.g., selected from IL-2R, IL-2R, IL-15R, IL-4R, IL-9R, IL-21R, and IL-7R ) of the cell.

[0439] In some embodiments, the antigen binding molecules of the present disclosure are capable of reducing multimerization of a polypeptide other than yc (e.g., selected from the group consisting of IL-2Rβ, IL-2Ra, IL-15Ra, IL-4Ra, IL-9Ra, IL-21Ra, and IL-7Ra) of a yc and yc-containing cytokine receptor, reducing signaling mediated by a yc-containing cytokine receptor (e.g., selected from the group consisting of yc:IL-2Rβ receptor, yc:IL-2Rβ:IL-2Ra, yc:IL-2Rβ:IL-15Ra, yc:IL-4Ra receptor, yc:IL-9Ra receptor, yc:IL-21Ra receptor, and yc:IL-7Ra receptor) bound by the antigen binding molecule, and / or reducing proliferation, survival, and / or effector activity of a cell expressing a yc-containing cytokine receptor (e.g., selected from the group consisting of yc:IL-2Rβ receptor, yc:IL-2Rβ:IL-2Ra, yc:IL-2Rβ:IL-15Ra, yc:IL-4Ra receptor, yc:IL-9Ra receptor, yc:IL-21Ra receptor, and yc:IL-7Ra receptor) bound by the antigen binding molecule by a mechanism that does not require or involve Fc-mediated function (i.e., independent of Fc-mediated function). That is, in some embodiments, the antigen binding molecules are capable of achieving one or more of the effects listed in the preceding sentence in a manner that does not depend on the Fc region.

[0440] The ability of an antigen binding molecule to reduce multimerization of a polypeptide other than yc of a yc and yc-containing cytokine receptor (e.g., selected from the group consisting of IL-2Rβ, IL-2Rα, IL-15Rα, IL-4Rα, IL-9Rα, IL-21Rα, and IL-7Rα), reduce signaling mediated by a yc-containing cytokine receptor (e.g., selected from the group consisting of yc:IL-2Rβ receptor, yc:IL-2Rβ:IL-2Rα, yc:IL-2Rβ:IL-15Rα, yc:IL-4Rα receptor, yc:IL-9Rα receptor, yc:IL-21Rα receptor, and yc:IL-7Rα receptor) bound by the antigen binding molecule, and / or reduce proliferation, survival, and / or effector activity of a cell expressing a yc-containing cytokine receptor (e.g., selected from the group consisting of yc:IL-2Rβ receptor, yc:IL-2Rβ:IL-2Rα, yc:IL-2Rβ:IL-15Rα, yc:IL-4Rα receptor, yc:IL-9Rα receptor, yc:IL-21Rα receptor, and yc:IL-7Rα receptor) bound by the antigen binding molecule through a mechanism that does not require / involves Fc-mediated function can be evaluated, for example, by analyzing the ability of an antigen binding molecule provided in the form lacking a functional Fc region to achieve one or more of the particular effects. For example, the relevant functional properties can be investigated using an antigen binding molecule comprising a“silent” Fc region (e.g., comprising L234A, L235A, and P329G substitutions) or using an antigen binding molecule provided in the form lacking an Fc region (e.g., scFv, Fab, etc.).

[0441] In some embodiments, the antigen binding molecule reduces multimerization of a polypeptide other than yc of a yc and yc-containing cytokine receptor (e.g., selected from the group consisting of IL-2Rβ, IL-2Rα, IL-15Rα, IL-4Rα, IL-9Rα, IL-21Rα, and IL-7Rα), reduces signaling mediated by a yc-containing cytokine receptor (e.g., selected from the group consisting of yc:IL-2Rβ receptor, yc:IL-2Rβ:IL-2Rα, yc:IL-2Rβ:IL-15Rα, yc:IL-4Rα receptor, yc:IL-9Rα receptor, yc:IL-21Rα receptor, and yc:IL-7Rα receptor) bound by the antigen binding molecule, and / or reduces proliferation, survival, and / or effector activity of a cell expressing a yc-containing cytokine receptor (e.g., selected from the group consisting of yc:IL-2Rβ receptor, yc:IL-2Rβ:IL-2Rα, yc:IL-2Rβ:IL-15Rα, yc:IL-4Rα receptor, yc:IL-9Rα receptor, yc:IL-21Rα receptor, and yc:IL-7Rα receptor) bound by the antigen binding molecule through a mechanism that does not involve ADCC, ADCP, and / or CDC.

[0442] In some embodiments, the antigen binding molecule reduces multimerization of a polypeptide other than yc of a yc-containing cytokine receptor (e.g., selected from the group consisting of IL-2Rβ, IL-2Rα, IL-15Rα, IL-4Rα, IL-9Rα, IL-21Rα, and IL-7Rα), reduces signaling mediated by a yc-containing cytokine receptor (e.g., selected from the group consisting of yc:IL-2Rβ receptor, yc:IL-2Rβ:IL-2Rα, yc:IL-2Rβ:IL-15Rα, yc:IL-4Rα receptor, yc:IL-9Rα receptor, yc:IL-21Rα receptor, and yc:IL-7Rα receptor) bound by the antigen binding molecule, and / or reduces proliferation, survival, and / or effector activity of a cell expressing a yc-containing cytokine receptor (e.g., selected from the group consisting of yc:IL-2Rβ receptor, yc:IL-2Rβ:IL-2Rα, yc:IL-2Rβ:IL-15Rα, yc:IL-4Rα receptor, yc:IL-9Rα receptor, yc:IL-21Rα receptor, and yc:IL-7Rα receptor) bound by the antigen binding molecule, by a mechanism that does not require binding of the antigen binding molecule to an Fc receptor (e.g., does not require binding of the antigen binding molecule to one or more of FcyRI, FcyRIIa, FcyRIIb, FcyRIIc, FcyRIIIa, and FcyRIIIb; e.g., does not require binding of the antigen binding molecule to C1q and / or does not require N297 glycosylation.

[0443] In some embodiments, the antigen binding molecule of the present disclosure does not induce ADCC, ADCP, or CDC of a cell comprising / expressing one or more target antigens of a constituent antigen binding moiety thereof (i.e., yc and / or a polypeptide other than yc of a yc-containing cytokine receptor (e.g., selected from the group consisting of IL-2Rβ, IL-2Rα, IL-15Rα, IL-4Rα, IL-9Rα, IL-21Rα, and IL-7Rα)).

[0444] An antigen-binding molecule that does not induce (i.e., is not capable of inducing) ADCC / ADCP / CDC elicits substantially no ADCC / ADCP / CDC activity against the relevant cell type, e.g., as determined by analyzing the relevant activity in an appropriate assay. By “substantially no ADCC / ADCP / CDC activity” is meant that the level of ADCC / ADCP / CDC is not significantly greater than that of an appropriate negative control molecule in a given assay (e.g., an antigen-binding molecule lacking an Fc region or an antigen-binding molecule comprising a “silent” Fc region (e.g., as described in Schlothauer et al., Protein Engineering, Design and Selection (2016), 29(10): 457-466, which is incorporated by reference above)). The “substantially no activity” can be a level of relevant activity that is <5-fold, e.g., <4-fold, <3-fold, <2.5-fold, <2-fold, or <1.5-fold of the level of activity of an appropriate negative control molecule determined in a given assay.

[0445] The ability and extent to which a given antigen-binding molecule is capable of inducing ADCC of a given target cell type can be analyzed, e.g., according to the method described in Yamashita et al., Scientific Reports (2016) 6: 19772 (which is incorporated by reference in its entirety), or by a method such as described in, e.g., Jedema et al., Blood (2004) 103:2677-82 (which is incorporated by reference in its entirety). 51 The ability and extent to which a given antigen-binding molecule is capable of inducing ADCP can be analyzed, e.g., according to the method described in Kamen et al., J Immunol (2017) 198 (1 Supplement) 157.17 (which is incorporated by reference in its entirety). The ability and extent to which a given antigen-binding molecule is capable of inducing CDC can be analyzed, e.g., using a Clq binding assay, e.g., as described in Schlothauer et al., Protein Engineering, Design and Selection (2016), 29(10): 457-466 (which is incorporated by reference in its entirety).

[0446] In some embodiments, the antigen binding molecules according to the present disclosure can increase (i.e., upregulate, enhance, augment) cell killing of cells comprising / expressing one or more target antigens of which the constituent antigen binding moieties are comprised (i.e., yc and / or a polypeptide of a yc-containing cytokine receptor other than yc (e.g., selected from IL-2Rb, IL-2Ra, IL-15Ra, IL-4Ra, IL-9Ra, IL-21Ra, and IL-7Ra)). In some embodiments, an “increased” level of cell killing refers to a level of cell killing that is greater than 1-fold, e.g., one of >1.01-fold, >1.02-fold, >1.03-fold, >1.04-fold, >1.05-fold, >1.1-fold, >1.2-fold, >1.3-fold, >1.4-fold, >1.5-fold, >1.6-fold, >1.7-fold, >1.8-fold, >1.9-fold, >2-fold, >3-fold, >4-fold, >5-fold, >6-fold, >7-fold, >8-fold, >9-fold, or >10-fold, of the level of cell killing observed in the absence of the antigen binding molecule or in the presence of an appropriate control antigen binding molecule (e.g., an antigen binding molecule known not to affect such cell killing).

[0447] In some embodiments, the antigen binding molecules according to the present disclosure can reduce the number / proportion of cells comprising / expressing one or more target antigens of which the constituent antigen binding moieties are comprised (i.e., yc and / or a polypeptide of a yc-containing cytokine receptor other than yc (e.g., selected from IL-2Rb, IL-2Ra, IL-15Ra, IL-4Ra, IL-9Ra, IL-21Ra, and IL-7Ra)). In some embodiments, the antigen binding molecules can deplete / enhance depletion of such cells. In some embodiments, a “reduced” number / proportion of cells refers to a number / proportion of cells that is less than 1-fold, e.g., one of <0.99-fold, <0.95-fold, <0.9-fold, <0.85-fold, <0.8-fold, <0.75-fold, <0.7-fold, <0.65-fold, <0.6-fold, <0.55-fold, <0.5-fold, <0.45-fold, <0.4-fold, <0.35-fold, <0.3-fold, <0.25-fold, <0.2-fold, <0.15-fold, <0.1-fold, <0.05-fold, or <0.01-fold, of the number / proportion observed in the absence of the antigen binding molecule or in the presence of an appropriate control antigen binding molecule (e.g., an antigen binding molecule known not to affect the number / proportion of such cells).

[0448] Antigen binding molecules according to the present disclosure can comprise one or more moieties for potentiating the reduction in the number / proportion of cells comprising / expressing one or more target antigens of which the constituent antigen binding moieties of the antigen binding molecules comprise (i.e., yc and / or a polypeptide other than yc of a yc-containing cytokine receptor (e.g., selected from IL-2Rb, IL-2Ra, IL-15Ra, IL-4Ra, IL-9Ra, IL-21Ra, and IL-7Ra)). For example, antigen binding molecules according to the present disclosure may, for example, comprise an Fc region and / or a drug moiety.

[0449] In some embodiments, antigen binding molecules according to the present disclosure comprise an Fc region that is capable of potentiating / guiding one or more of ADCC, ADCP, CDC against a cell comprising / expressing one or more target antigens of which the constituent antigen binding moieties of the antigen binding molecules comprise (i.e., yc and / or a polypeptide other than yc of a yc-containing cytokine receptor (e.g., selected from IL-2Rb, IL-2Ra, IL-15Ra, IL-4Ra, IL-9Ra, IL-21Ra, and IL-7Ra)), and / or potentiating the formation of a MAC on the cell or potentiating cell degranulation of the cell.

[0450] In some embodiments, antigen binding molecules according to the present disclosure comprise a drug moiety. The antigen binding molecules can be conjugated to a drug moiety. Antibody drug conjugates are reviewed, for example, in Parslow et al., Biomedicines. 2016 Sep; 4(3): 14 (herein incorporated by reference in its entirety). In some embodiments, the drug moiety is or comprises a cytotoxic agent, such that the antigen binding molecule exhibits cytotoxicity against a cell comprising / expressing one or more target antigens of which the constituent antigen binding moieties of the antigen binding molecules comprise (i.e., yc and / or a polypeptide other than yc of a yc-containing cytokine receptor (e.g., selected from IL-2Rb, IL-2Ra, IL-15Ra, IL-4Ra, IL-9Ra, IL-21Ra, and IL-7Ra)). In some embodiments, the drug moiety is or comprises a chemotherapeutic agent.

[0451] In some embodiments, antigen binding molecules according to the present disclosure comprise an immune cell engaging moiety. In some embodiments, the antigen binding molecules comprise a CD3 polypeptide binding moiety (e.g., an antigen binding domain capable of binding to a CD3 polypeptide).

[0452] In some embodiments, the antigen binding molecules according to the present disclosure are capable of enhancing / directing T cell-mediated cytotoxic activity against cells comprising / expressing one or more target antigens of the constituent antigen binding moieties thereof (i.e., yc and / or polypeptides of the yc-containing cytokine receptors other than yc (e.g., selected from the group consisting of IL-2Rb, IL-2Ra, IL-15Ra, IL-4Ra, IL-9Ra, IL-21Ra, and IL-7Ra)).

[0453] The antigen binding molecules of the present disclosure have novel and / or improved properties over yc family cytokines.

[0454] In some embodiments, the antigen binding molecules according to the present disclosure have cytokine-like properties in binding and triggering yc-containing cytokine receptor-mediated signaling, but in addition also have drug (in particular antibody) like biophysical and pharmacokinetic properties.

[0455] In some embodiments, the antigen binding molecules exhibit increased stability and / or half-life compared to one or more yc family cytokines (e.g., IL-2, IL-15, IL-4, IL-9, IL-21, and / or IL-7). By way of illustration, an antigen binding molecule comprising (i) a yc binding moiety and (ii) an IL-2Rb binding moiety preferably exhibits increased stability and / or half-life compared to IL-2.

[0456] As used herein, "stability" can refer to resistance to degradation, aggregation, and / or unfolding. A molecule having increased stability compared to a reference molecule can exhibit decreased degradation / degradation propensity, decreased aggregation / aggregation propensity, and / or decreased unfolding / unfolding propensity compared to the reference molecule.

[0457] Degradation / aggregation can be determined by detecting and optionally quantifying degraded / aggregated / unfolded material, e.g., in a sample containing the relevant molecule. Stability can be evaluated according to methods well known in the art of molecular biology. Such methods can involve evaluating the antigen binding molecule to determine the level of degradation (fragmentation), aggregation, unfolding, and / or the proportion of degraded / aggregated / unfolded / monomeric material.

[0458] Stability can be evaluated according to, e.g., the methods described in Thiagarajan et al., mAbs. (2016) 8(6): 1088-1097, which is incorporated by reference herein in its entirety. Such methods include analysis by size exclusion chromatography (SEC) to detect correctly assembled molecules (termed monomers), high molecular weight (HMW) species (i.e., aggregates), and / or low molecular weight (LMW) species (i.e., fragments). Other methods include analysis of melting onset temperature (Tonset), thermal unfolding temperature (Tm), and apparent enthalpy associated with the unfolding transition by differential scanning calorimetry (DSC); analysis of effective surface charge via zeta potential and diffusion interaction parameter (KD); and analysis of intrinsic tryptophan fluorescence by fluorescence spectroscopy.

[0459] As used herein, “half-life” refers to the time required for the concentration of a given molecule to fall to half of its initial value. The half-life can be in plasma (plasma half-life) or in serum (serum half-life). The half-life of a given molecule can be evaluated by monitoring the levels of the molecule over time under particular conditions. Half-life can be assessed, e.g., by the method of Viera and Rajewsky, Eur J Immunol. (1988) 18(2):313-6, or the method of Souders et al., MAbs. (2015) 7(5):912-921.

[0460] In some embodiments, stability / half-life that is “increased” relative to a given reference molecule (e.g., a given yc family cytokine) can be a stability / half-life that is greater than 1-fold (e.g., one of ≥ 1.01-fold, ≥ 1.02-fold, ≥ 1.03-fold, ≥ 1.04-fold, ≥ 1.05-fold, ≥ 1.1-fold, ≥ 1.2-fold, ≥ 1.3-fold, ≥ 1.4-fold, ≥ 1.5-fold, ≥ 1.6-fold, ≥ 1.7-fold, ≥ 1.8-fold, ≥ 1.9-fold, ≥ 2-fold, ≥ 3-fold, ≥ 4-fold, ≥ 5-fold, ≥ 6-fold, ≥ 7-fold, ≥ 8-fold, ≥ 9-fold, or ≥ 10-fold) of the stability / half-life of the reference molecule.

[0461] In some embodiments, the antigen binding molecules of the present disclosure are more potent at increasing signaling mediated by the gamma c-containing cytokine receptor (e.g., selected from the group consisting of gamma c:IL-2R beta receptor, gamma c:IL-2R beta:IL-2R alpha, gamma c:IL-2R beta:IL-15R alpha, gamma c:IL-4R alpha receptor, gamma c:IL-9R alpha receptor, gamma c:IL-21R alpha receptor, and gamma c:IL-7R alpha receptor) bound by the antigen binding molecule compared to a cytokine that binds the gamma c-containing cytokine receptor. In some embodiments, the antigen binding molecules of the present disclosure are less potent at increasing signaling mediated by the gamma c-containing cytokine receptor (e.g., selected from the group consisting of gamma c:IL-2R beta receptor, gamma c:IL-2R beta:IL-2R alpha, gamma c:IL-2R beta:IL-15R alpha, gamma c:IL-4R alpha receptor, gamma c:IL-9R alpha receptor, gamma c:IL-21R alpha receptor, and gamma c:IL-7R alpha receptor) bound by the antigen binding molecule compared to a cytokine that binds the gamma c-containing cytokine receptor.

[0462] In some embodiments, the antigen binding molecules of the present disclosure are more effective than a cytokine that binds a yc-containing cytokine receptor in upregulating proliferation, survival, and / or effector activity of a cell expressing a yc-containing cytokine receptor to which the antigen binding molecule binds (e.g., selected from the group consisting of yc:IL-2Rß receptor, yc:IL-2Rß:IL-2Ra, yc:IL-2Rß:IL-15Ra, yc:IL-4Ra receptor, yc:IL-9Ra receptor, yc:IL-21Ra receptor, and yc:IL-7Ra receptor). In some embodiments, the antigen binding molecules of the present disclosure are less effective than a cytokine that binds a yc-containing cytokine receptor in upregulating proliferation, survival, and / or effector activity of a cell expressing a yc-containing cytokine receptor to which the antigen binding molecule binds (e.g., selected from the group consisting of yc:IL-2Rß receptor, yc:IL-2Rß:IL-2Ra, yc:IL-2Rß:IL-15Ra, yc:IL-4Ra receptor, yc:IL-9Ra receptor, yc:IL-21Ra receptor, and yc:IL-7Ra receptor). In some embodiments, the antigen binding molecules of the present disclosure are more effective than a cytokine that binds a yc-containing cytokine receptor in reducing expression of one or more immune cell exhaustion markers of a cell expressing a yc-containing cytokine receptor to which the antigen binding molecule binds (e.g., selected from the group consisting of yc:IL-2Rß receptor, yc:IL-2Rß:IL-2Ra, yc:IL-2Rß:IL-15Ra, yc:IL-4Ra receptor, yc:IL-9Ra receptor, yc:IL-21Ra receptor, and yc:IL-7Ra receptor). In some embodiments, the antigen binding molecules of the present disclosure are less effective than a cytokine that binds a yc-containing cytokine receptor in reducing expression of one or more immune cell exhaustion markers of a cell expressing a yc-containing cytokine receptor to which the antigen binding molecule binds (e.g., selected from the group consisting of yc:IL-2Rß receptor, yc:IL-2Rß:IL-2Ra, yc:IL-2Rß:IL-15Ra, yc:IL-4Ra receptor, yc:IL-9Ra receptor, yc:IL-21Ra receptor, and yc:IL-7Ra receptor).

[0463] By way of illustration, in embodiments in which the antigen binding molecule comprises (i) a yc-binding moiety and (ii) an IL-2Rß-binding moiety, in some embodiments, the antigen binding molecule can be more effective than IL-2 in increasing signaling mediated by the yc:IL-2Rß receptor, and / or can be more effective than IL-2 in upregulating cell proliferation, survival, and / or effector activity of a cell expressing the yc:IL-2Rß receptor. Similarly, the antigen binding molecule can be more effective than IL-2 in downregulating expression of one or more immune cell exhaustion markers of a cell expressing the yc:IL-2Rß receptor.

[0464] In some embodiments, the antigen binding molecule increases signaling mediated by a yc-containing cytokine receptor (e.g., selected from the group consisting of yc:IL-2Rβ receptor, yc:IL-2Rβ:IL-2Rα, yc:IL-2Rβ:IL-15Rα, yc:IL-4Rα receptor, yc:IL-9Rα receptor, yc:IL-21Rα receptor, and yc:IL-7Rα receptor), and / or increases cell proliferation, survival, and / or effector activity of a cell expressing a yc-containing cytokine receptor (e.g., selected from the group consisting of yc:IL-2Rβ receptor, yc:IL-2Rβ:IL-2Rα, yc:IL-2Rβ:IL-15Rα, yc:IL-4Rα receptor, yc:IL-9Rα receptor, yc:IL-21Rα receptor, and yc:IL-7Rα receptor) to which the antigen binding molecule binds, wherein EC 50 is less than the EC for the relevant activity exhibited by a cytokine that binds to the relevant yc-containing cytokine receptor 50 , e.g., <0.99-fold, <0.95-fold, <0.9-fold, <0.85-fold, <0.8-fold, <0.75-fold, <0.7-fold, <0.65-fold, <0.6-fold, <0.55-fold, <0.5-fold, <0.45-fold, <0.4-fold, <0.35-fold, <0.3-fold, <0.25-fold, <0.2-fold, <0.15-fold, <0.1-fold, <0.05-fold, or <0.01-fold, as determined in the same assay. In some embodiments, the antigen binding molecule increases signaling mediated by a yc-containing cytokine receptor (e.g., selected from the group consisting of yc:IL-2Rβ receptor, yc:IL-2Rβ:IL-2Rα, yc:IL-2Rβ:IL-15Rα, yc:IL-4Rα receptor, yc:IL-9Rα receptor, yc:IL-21Rα receptor, and yc:IL-7Rα receptor), and / or increases cell proliferation, survival, and / or effector activity of a cell expressing a yc-containing cytokine receptor (e.g., selected from the group consisting of yc:IL-2Rβ receptor, yc:IL-2Rβ:IL-2Rα, yc:IL-2Rβ:IL-15Rα, yc:IL-4Rα receptor, yc:IL-9Rα receptor, yc:IL-21Rα receptor, and yc:IL-7Rα receptor) to which the antigen binding molecule binds, wherein EC 50 is greater than the EC for the relevant activity exhibited by a cytokine that binds to the relevant yc-containing cytokine receptor 501-fold, e.g., one of >1.01-fold, >1.02-fold, >1.03-fold, >1.04-fold, >1.05-fold, >1.1-fold, >1.2-fold, >1.3-fold, >1.4-fold, >1.5-fold, >1.6-fold, >1.7-fold, >1.8-fold, >1.9-fold, >2-fold, >3-fold, >4-fold, >5-fold, >6-fold, >7-fold, >8-fold, >9-fold, or >10-fold, as determined in the same assay.

[0465] In some embodiments, the antigen binding molecule reduces expression of one or more immune cell exhaustion markers by cells expressing a yc-containing cytokine receptor to which the antigen binding molecule binds (e.g., selected from the group consisting of yc:IL-2Rß receptor, yc:IL-2Rß:IL-2Ra, yc:IL-2Rß:IL-15Ra, yc:IL-4Ra receptor, yc:IL-9Ra receptor, yc:IL-21Ra receptor, and yc:IL-7Ra receptor), wherein the IC 50 less than the IC50exhibited by the relevant cytokine binding to the relevant yc-containing cytokine receptor 50 1-fold, e.g., one of >1.01-fold, >1.02-fold, >1.03-fold, >1.04-fold, >1.05-fold, >1.1-fold, >1.2-fold, >1.3-fold, >1.4-fold, >1.5-fold, >1.6-fold, >1.7-fold, >1.8-fold, >1.9-fold, >2-fold, >3-fold, >4-fold, >5-fold, >6-fold, >7-fold, >8-fold, >9-fold, or >10-fold, as determined in the same assay. 50 greater than the IC50exhibited by the relevant cytokine binding to the relevant yc-containing cytokine receptor 50 1-fold, e.g., one of >1.01-fold, >1.02-fold, >1.03-fold, >1.04-fold, >1.05-fold, >1.1-fold, >1.2-fold, >1.3-fold, >1.4-fold, >1.5-fold, >1.6-fold, >1.7-fold, >1.8-fold, >1.9-fold, >2-fold, >3-fold, >4-fold, >5-fold, >6-fold, >7-fold, >8-fold, >9-fold, or >10-fold, as determined in the same assay.

[0466] In some embodiments, the antigen binding molecules of the present disclosure promote anti-cancer and / or anti-infective activity in vivo, e.g., in an appropriate non-human animal model. In some embodiments, administration of the antigen binding molecules is associated with a reduction in the number of cancer cells in vivo, e.g., as compared to an appropriate control condition. In some embodiments, administration of the antigen binding molecules is associated with an increase in cancer cell killing in vivo, e.g., as compared to an appropriate control condition. In some embodiments, administration of the antigen binding molecules is associated with a reduction in pathogen burden in vivo, e.g., as compared to an appropriate control condition. In some embodiments, administration of the antigen binding molecules is associated with a reduction in the number of pathogen cells and / or the number of cells infected with a pathogen in vivo, e.g., as compared to an appropriate control condition.

[0467] In some embodiments, administration of the antigen binding molecules according to the present disclosure can be associated with one or more of: inhibition of cancer development / progression, delay / prevention of cancer onset, reduction / delay / prevention of tumor growth, reduction / delay / prevention of tissue invasion, reduction / delay / prevention of metastasis, reduction in the severity of one or more symptoms of cancer, reduction in the number of cancer cells, reduction in cancer burden, reduction in tumor size / volume, and / or increase in the survival of a subject having cancer (e.g., progression-free survival or overall survival), e.g., as determined in an appropriate model, as compared to an appropriate control condition.

[0468] It will be appreciated that the properties recited in the preceding paragraphs are evaluated after a period of time sufficient to observe an effect associated with administration of the antigen binding molecules.

[0469] Linkers and additional sequences

[0470] The antigen binding molecules and polypeptides of the present disclosure can additionally comprise additional amino acids or amino acid sequences.

[0471] The antigen binding molecules and polypeptides of the present disclosure can comprise one or more linker sequences between amino acid sequences, e.g., between amino acid sequences forming domains / regions as described herein.

[0472] In some embodiments, a linker sequence is positioned between a VH sequence and a VL sequence, providing a linkage between the VH and VL (e.g., in a scFv molecule). In some embodiments, a linker sequence is positioned between antigen binding moieties of an antigen binding molecule of the present disclosure, e.g., in an antigen binding molecule comprising a polypeptide comprising a tandem scFv-scFv. In some embodiments, a linker sequence is positioned between an antigen binding moiety / its components and a CH2CH3 region, e.g., in an antigen binding molecule comprising a polypeptide comprising an scFv moiety linked to a CH2CH3 region.

[0473] Linker sequences are known to those of skill in the art, for example, described in Chen et al., Adv Drug Deliv Rev. (2013) 65(10): 1357-1369, which is incorporated by reference herein in its entirety. In some embodiments, the linker sequence can be a flexible linker sequence. Flexible linker sequences allow for relative movement of the amino acid sequences connected by the linker sequence. Flexible linkers are known to those of skill in the art, several flexible linkers are identified in Chen et al., Adv Drug Deliv Rev. (2013) 65(10): 1357-1369. Flexible linker sequences typically comprise a high proportion of glycine and / or serine residues.

[0474] In some embodiments, the linker sequence comprises at least one glycine residue and / or at least one serine residue. In some embodiments, the linker sequence comprises or consists of glycine and serine residues. In some embodiments, the linker sequence has the structure: (GxS)n or (GxS)nGm; where G = glycine, S = serine, x = 3 or 4, n = 2, 3, 4, 5, or 6, and m = 0, 1, 2, or 3. In some embodiments, the linker sequence comprises one or more (e.g., 1, 2, 3, 4, 5, or 6) copies (e.g., in tandem) of the sequence motif G4S. In some embodiments, the linker sequence comprises or consists of (G4S)4 or (G4S)6. In some embodiments, the linker sequence is 1-2, 1-3, 1-4, 1-5, 1-10, 1-15, 1-20, 1-25, or 1-30 amino acids in length.

[0475] In some embodiments, the linker comprises or consists of the amino acid sequence GGGGS (SEQ ID NO: 502). In some embodiments, the linker comprises or consists of the amino acid sequence GGGGSGGGS (SEQ ID NO: 720). In some embodiments, the linker comprises the amino acid sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 501). In some embodiments, the linker comprises or consists of the amino acid sequence NSGAAA (SEQ ID NO: 729).

[0476] In some embodiments, the linker between the VH sequence and the VL sequence comprises or consists of the GGGGS (SEQ ID NO:502) amino acid sequence. In some embodiments, the linker between the VH sequence and the VL sequence comprises or consists of the GGGGSG (SEQ ID NO:718) amino acid sequence. In some embodiments, the linker between the VH sequence and the VL sequence comprises or consists of the GGGGSGGGGSGGGGSG (SEQ ID NO:333) amino acid sequence. In some embodiments, the scFV comprises the GGGGSGGGGSGGGGSG (SEQ ID NO:333) amino acid sequence.

[0477] In some embodiments, the linker sequence between the antigen binding moieties comprises or consists of a flexible linker. In some embodiments, the linker sequence between the antigen binding moieties comprises or consists of the GGGGS (SEQ ID NO:502) amino acid sequence. In some embodiments, the linker sequence between the scFv molecules comprises or consists of the GGGGS (SEQ ID NO:502) amino acid sequence. In some embodiments, the linker sequence between the VHH molecules comprises or consists of the GGGGS (SEQ ID NO:502) amino acid sequence. In some embodiments, the linker sequence between the scFv molecules and the VHH molecules comprises or consists of the GGGGS (SEQ ID NO:502) amino acid sequence.

[0478] In some embodiments, the linker sequence between the antigen binding moieties comprises or consists of a rigid linker. In some embodiments, the linker sequence between the antigen binding moieties comprises or consists of the EAAAK (SEQ ID NO:723) amino acid sequence. In some embodiments, the linker sequence between the scFv molecules comprises or consists of the EAAAK (SEQ ID NO:723) amino acid sequence. In some embodiments, the linker sequence between the VHH molecules comprises or consists of the EAAAK (SEQ ID NO:723) amino acid sequence. In some embodiments, the linker sequence between the scFv molecules and the VHH molecules comprises or consists of the EAAAK (SEQ ID NO:723) amino acid sequence.

[0479] In some embodiments, the linker sequence between antigen binding moieties comprises or consists of a rigid linker. In some embodiments, the linker sequence between antigen binding moieties comprises or consists of an A(EAAAK)5A (SEQ ID NO: 719) amino acid sequence. In some embodiments, the linker sequence between scFv molecules comprises or consists of an A(EAAAK)5A (SEQ ID NO: 719) amino acid sequence. In some embodiments, the linker sequence between VHH molecules comprises or consists of an A(EAAAK)5A (SEQ ID NO: 719) amino acid sequence. In some embodiments, the linker sequence between scFv molecules and VHH molecules comprises or consists of an A(EAAAK)5A (SEQ ID NO: 719) amino acid sequence.

[0480] Antigen binding molecules and polypeptides of the present disclosure can comprise an amino acid sequence to facilitate expression, folding, trafficking, processing, purification, or detection of the antigen binding molecule / polypeptide. For example, antigen binding molecules and polypeptides of the present disclosure can additionally comprise an amino acid sequence that forms a detectable moiety, e.g., as described below.

[0481] Antigen binding molecules and polypeptides of the present disclosure can additionally comprise a signal peptide (also known as a leader sequence or signal sequence). Signal peptides generally consist of a sequence of 5-30 hydrophobic amino acids that form a single alpha helix. Secreted proteins and proteins expressed at the cell surface often comprise a signal peptide. Signal peptides are well known for many proteins and are recorded in databases such as GenBank, UniProt, and Ensembl, and / or can be identified / predicted, e.g., using amino acid sequence analysis tools such as SignalP (Petersen et al., Nature Methods (2011) 8:785-786) or Signal-BLAST (Frank and Sippl, Bioinformatics (2008) 24:2172-2176).

[0482] Signal peptides can be present at the N-terminus of an antigen binding molecule / polypeptide and can be present in newly synthesized antigen binding molecules / polypeptides. Signal peptides provide efficient trafficking of antigen binding molecules / polypeptides. Signal peptides are typically removed by cleavage and thus are not included in the mature antigen binding molecule / polypeptide.

[0483] Signal peptides are known for many proteins and documented in databases such as GenBank, UniProt, Swiss-Prot, TrEMBL, Protein Information Resource, Protein Data Bank, Ensembl and InterPro, and / or can be identified / predicted, e.g., using amino acid sequence analysis tools such as SignalP (Petersen et al., Nature Methods (2011) 8:785-786) or Signal-BLAST (Frank and Sippl, Bioinformatics (2008) 24:2172-2176).

[0484] Labels and conjugates

[0485] In some embodiments, the antigen binding molecule or polypeptide according to the present disclosure comprises a detectable moiety.

[0486] In some embodiments, the detectable moiety is a fluorescent label, a phosphorescent label, a luminescent label, an immunodetectable label (e.g., an epitope tag), a radioactive label, a chemical, nucleic acid, or enzymatic label. The antigen binding molecule or polypeptide can be labeled covalently or non-covalently with the detectable moiety.

[0487] Fluorescent labels include, for example, fluorescein, rhodamine, allophycocyanin, eosin and NDB, green fluorescent protein (GFP), chelates of rare earths such as europium (Eu), terbium (Tb) and samarium (Sm), tetramethylrhodamine, Texas Red, 4-methylumbelliferone, 7-amino-4-methylcoumarin, Cy3 and Cy5. Radioactive labels include radioisotopes such as hydrogen 3 , sulfur 35 , carbon 14 , phosphorus 32 , iodine 123 , iodine 125 , iodine 126 , iodine 131 , iodine 133 , bromine 77 , technetium 99m , indium 111 , indium 113 m, gallium 67 , gallium 68 , ruthenium 95 , ruthenium 97 , ruthenium 103 , ruthenium 105 , mercury 207 , mercury 203 , rhenium 99m, rhenium 101 , rhenium 105 , scandium 47 , tellurium121m tellurium 122m tellurium 125m thulium 165 thulium 167 thulium 168 copper 67 fluorine 18 yttrium 90 palladium 100 bismuth 217 and antimony 211 Luminescent labels include radioluminescent, chemiluminescent (e.g., acridinium ester, luminol, isoluminol), and bioluminescent labels. Immunodetectable labels include haptens, peptides / polypeptides, antibodies, receptors, and ligands, such as biotin, avidin, streptavidin, or digoxigenin. Nucleic acid labels include aptamers.

[0488] In some embodiments, the antigen binding molecule / polypeptide comprises an epitope tag, e.g., His (e.g., 6XHis), FLAG, c-Myc, StrepTag, hemagglutinin, E, calmodulin binding protein (CBP), glutathione-s-transferase (GST), maltose binding protein (MBP), thioredoxin, S-peptide, T7 peptide, SH2 domain, avidin, streptavidin, and hapten (e.g., biotin, digoxigenin, dinitrophenol), optionally at the N or C terminus of the antigen binding molecule / polypeptide.

[0489] In some embodiments, the antigen binding molecule / polypeptide comprises a moiety with a detectable activity, e.g., an enzymatic moiety. Enzymatic moieties include, e.g., luciferase, glucose oxidase, galactosidase (e.g., beta-galactosidase), glucuronidase, phosphatase (e.g., alkaline phosphatase), peroxidase (e.g., horseradish peroxidase), and cholinesterase.

[0490] In some embodiments, the antigen binding molecule or polypeptide of the present disclosure comprises a chemical moiety. In some embodiments, the antigen binding molecule / polypeptide of the present disclosure is conjugated to a chemical moiety.

[0491] The chemical moiety can be a moiety for providing a therapeutic effect, i.e. a drug moiety. The drug moiety can be a small molecule (e.g. an organic compound of low molecular weight (<1000 Daltons, typically between about 300-700 Daltons)). Drug moieties are described, for example, in Parslow et al., Biomedicines. (2016) 4(3): 14 (incorporated by reference in its entirety). In some embodiments, the drug moiety can be or comprise a cytotoxic agent. In some embodiments, the drug moiety can be or comprise a chemotherapeutic agent. Drug moieties include, for example, calicheamicin, DM1, DM4, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), SN-38, doxorubicin, duocarmycin, D6.5, and PBD.

[0492] Nucleic acids and vectors

[0493] The present disclosure provides a nucleic acid or plurality of nucleic acids encoding an antigen binding molecule or polypeptide according to the present disclosure. In some embodiments, the nucleic acid comprises and / or consists of DNA and / or RNA.

[0494] An antigen binding molecule or polypeptide according to the present disclosure can be produced intracellularly by translation of RNA encoding the polypeptide. An antigen binding molecule or polypeptide according to the present disclosure can be produced intracellularly by transcription from a nucleic acid encoding the polypeptide followed by translation of the transcribed RNA.

[0495] In some embodiments, the nucleic acid can be or can comprise / contain in a vector or plurality of vectors. As used herein, a “vector” is a nucleic acid molecule used as a vehicle to transfer exogenous nucleic acid into a cell.

[0496] Accordingly, the present disclosure also provides a vector or plurality of vectors comprising a nucleic acid or plurality of nucleic acids according to the present disclosure. The vector can facilitate delivery of the nucleic acid encoding a polypeptide according to the present disclosure to a cell. The vector can be an expression vector comprising elements required for expression of a polypeptide according to the present disclosure. The vector can comprise elements that facilitate integration of the nucleic acid into the genomic DNA of a cell into which the vector is introduced.

[0497] Nucleic acids and vectors according to the present disclosure can be provided in purified or isolated form, i.e. from other nucleic acids or naturally occurring biological material.

[0498] A vector can be a vector for expressing a nucleic acid in a cell (i.e., an expression vector). Such a vector can include a promoter sequence operably linked to a nucleotide sequence encoding an antigen binding molecule or polypeptide according to the present disclosure. A vector can also include a stop codon (i.e., 3’ to the nucleotide sequence encoding the polypeptide in the nucleotide sequence of the vector) and an expression enhancer. Any suitable vector, promoter, enhancer, and stop codon known in the art can be used to express a peptide or polypeptide from a vector of the present disclosure.

[0499] The term“operably linked” can include instances in which a nucleic acid encoding a polypeptide according to the present disclosure and a regulatory nucleic acid sequence (e.g., a promoter and / or an enhancer) are covalently linked in such a way that expression of the nucleic acid encoding the polypeptide is placed under the influence or control of the regulatory nucleic acid sequence (thereby forming an expression cassette). Thus, a regulatory sequence is operably linked to a selected nucleic acid sequence if the regulatory sequence is capable of affecting the transcription of the selected nucleic acid sequence. The resulting transcript can then be translated into the desired polypeptide.

[0500] Vectors contemplated in connection with the present disclosure include DNA vectors, RNA vectors, plasmids (e.g., conjugative plasmids (e.g., F plasmids), non-conjugative plasmids, R plasmids, col plasmids, episomes), viral vectors (e.g., retroviral vectors, such as gamma retroviral vectors (e.g., murine leukemia virus (MLV)-derived vectors, such as SFG vectors), lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, vaccinia viral vectors, and herpes viral vectors), transposition-based vectors, and artificial chromosomes (e.g., yeast artificial chromosomes), such as described in Maus et al., Annu Rev Immunol. (2014) 32:189-225 and Morgan and Boyerinas, Biomedicines (2016) 4:9, each of which is incorporated by reference herein in its entirety. In some embodiments, a vector according to the present disclosure is a lentiviral vector.

[0501] In some embodiments, a vector can be a eukaryotic vector, i.e., a vector in a eukaryotic cell that includes elements required for expression of a protein from the vector. In some embodiments, a vector can be a mammalian vector, e.g., including a cytomegalovirus (CMV) or SV40 promoter to drive protein expression.

[0502] The constituent polypeptides of an antigen binding molecule according to the present disclosure can be encoded by different nucleic acids in a plurality of nucleic acids or different vectors in a plurality of vectors.

[0503] In some embodiments, the nucleic acid / multiple nucleic acids or the vector / multiple vectors comprises a nucleic acid encoding an internal ribosome entry site (IRES). In some embodiments, the IRES is located between the nucleotide sequences encoding the constituent polypeptides of the antigen binding molecule according to the present disclosure. In some embodiments, the nucleic acid / multiple nucleic acids or the vector / multiple vectors comprises nucleic acid that allows for the separate translation of two or more polypeptides from the same RNA transcript.

[0504] In some embodiments, the constituent polypeptides of the antigen binding molecule according to the present disclosure are encoded by nucleotide sequences provided in the same reading frame. In some embodiments, the nucleic acid / multiple nucleic acids or the vector / multiple vectors encode a fusion protein of the constituent polypeptides of the antigen binding molecule. In some embodiments, the fusion protein encoded by the nucleic acid / multiple nucleic acids or the vector / multiple vectors comprises a cleavage site (e.g., a cleavage site as described herein) between the amino acid sequences of the constituent polypeptides of the antigen binding molecule.

[0505] In some embodiments, transcription of the nucleic acids encoding the constituent polypeptides of the antigen binding molecule is under the control of different promoters.

[0506] In some embodiments, the nucleic acid / multiple nucleic acids or the vector / multiple vectors is / are multi-cistronic (e.g., bi-cistronic, tri-cistronic, etc.). That is, in some embodiments, the nucleic acid / multiple nucleic acids or the vector / multiple vectors comprises multiple nucleotide sequences encoding polypeptides. In some embodiments, the nucleic acids encoding the constituent polypeptides of the antigen binding molecule according to the present disclosure are provided in different cistrons.

[0507] Production of antigen binding molecules and polypeptides

[0508] Antigen binding molecules and polypeptides according to the present disclosure can be prepared according to methods known to the skilled person for producing polypeptides.

[0509] Antigen binding molecules and polypeptides can be prepared by chemical synthesis, e.g., liquid or solid phase synthesis. For example, peptides / polypeptides can be synthesized using methods described in, e.g., Chandrudu et al., Molecules (2013) 18: 4373-4388, which is incorporated by reference in its entirety.

[0510] Alternatively, the antigen binding molecules and polypeptides can be produced by recombinant expression. Molecular biology techniques suitable for recombinantly producing polypeptides are well known in the art, such as those described in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed.), Cold Spring Harbor Press, 2012 and Nat Methods. (2008) 5(2): 135-146, both of which are incorporated by reference herein in their entireties. Methods for recombinantly producing antigen binding molecules are also described in Frenzel et al., Front Immunol. (2013) 4:217 and Kunert and Reinhart, Appl Microbiol Biotechnol. (2016) 100:3451-3461, both of which are hereby incorporated by reference in their entireties.

[0511] In some cases, the antigen binding molecules of the present disclosure are composed of more than one polypeptide chain. In such cases, production of the antigen binding molecules can include transcription and translation of more than one polypeptide, and subsequent association of the polypeptide chains to form the antigen binding molecule.

[0512] For recombinant production according to the present disclosure, any cell suitable for expressing a polypeptide can be used. The cell can be a prokaryote or a eukaryote. In some embodiments, the cell is a prokaryotic cell, such as a cell of an archaea or a bacterium. In some embodiments, the bacterium can be a gram-negative bacterium, such as a bacterium of the Enterobacteriaceae family, for example Escherichia coli. In some embodiments, the cell is a eukaryotic cell, such as a yeast cell, a plant cell, an insect cell, or a mammalian cell, for example a cell as described above.

[0513] In some cases, the cell is not a prokaryotic cell, as some prokaryotic cells do not allow for the same folding or post-translational modifications as eukaryotic cells. Additionally, very high expression levels can be possible in eukaryotes, and proteins can be more easily purified from eukaryotes using appropriate tags. A particular plasmid can also be utilized that enhances protein secretion into the culture medium.

[0514] In some embodiments, the polypeptides can be prepared by cell-free protein synthesis (CFPS), for example according to the system described in Zemella et al. Chembiochem (2015) 16(17): 2420-2431, which is hereby incorporated by reference in its entirety.

[0515] Production can involve culturing or fermenting eukaryotic cells modified to express a polypeptide of interest. Culturing or fermenting can be performed in a bioreactor with appropriate supply of nutrients, air / oxygen, and / or growth factors. Secreted polypeptides can be collected by separating the cells from the culture medium / fermentation broth, extracting the protein components and isolating individual proteins. Culturing, fermentation, and isolation techniques are well known to those of skill in the art, for example, as described in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed.; incorporated by reference above).

[0516] A bioreactor includes one or more vessels in which cells can be cultured. Culturing in a bioreactor can be performed continuously, with reactants continuously flowing into the reactor and cultured cells continuously flowing out of the reactor. Alternatively, culturing can be performed in batches. A bioreactor monitors and controls environmental conditions, such as pH, oxygen, flow rate into and out of the vessel, and agitation within the vessel, so that optimal conditions are provided for the cells to be cultured.

[0517] After culturing cells expressing a polypeptide, the polypeptide of interest can be isolated. Any suitable method known in the art for isolating proteins from cells can be used. To isolate the polypeptide, it can be necessary to separate the cells from the nutrient medium. If the polypeptide is secreted from the cells, the cells can be separated from the medium containing the secreted polypeptide of interest by centrifugation. If the polypeptide of interest is aggregated within the cells, protein isolation can include centrifugation to separate the cells from the cell culture medium, treatment of the cell pellet with a lysis buffer, and cell disruption, for example, by sonication, rapid freeze-thaw, or osmotic lysis.

[0518] The polypeptide of interest can then be expected to be isolated from the supernatant or medium, which can contain other protein and non-protein components. One common method for isolating protein components from supernatant or medium is by precipitation. Proteins of different solubility precipitate at different concentrations of a precipitant, such as ammonium sulfate. For example, at low concentrations of precipitant, water-soluble proteins are extracted. Thus, by adding different increasing concentrations of precipitant, proteins of different solubility can be distinguished. Dialysis can be used subsequently to remove ammonium sulfate from the isolated proteins.

[0519] Other methods for distinguishing different proteins are known in the art, such as ion exchange chromatography and size chromatography. These can be used as an alternative to precipitation, or can be performed after precipitation.

[0520] Once the polypeptide of interest has been isolated from the culture, it can be desirable or necessary to concentrate the polypeptide. Many methods for concentrating proteins are known in the art, such as ultrafiltration or lyophilization.

[0521] Cells comprising / expressing antigen binding molecules and polypeptides

[0522] The disclosure also provides a cell comprising or expressing an antigen binding molecule or polypeptide according to the disclosure. Also provided is a cell comprising or expressing a nucleic acid, a plurality of nucleic acids, a vector, or a plurality of vectors according to the disclosure.

[0523] It will be understood that where reference is made herein to a cell in the singular (i.e. “a / an / the”) such cell(s) in the plural / population is also contemplated.

[0524] The cell can be a eukaryotic cell, e.g. a mammalian cell. The mammal can be a primate (rhesus monkey, cynomolgus monkey, non-human primate, or human) or a non-human mammal (e.g. a rabbit, a guinea pig, a rat, a mouse, or other rodent (including any animal of the order Rodentia), a cat, a dog, a pig, a sheep, a goat, a cow (including a bovine, e.g. a dairy cow, or any animal of the genus Bos), a horse (including any animal of the family Equidae), a donkey, and a non-human primate).

[0525] In some embodiments, the cell is or is derived from a cell type that is typically used to express polypeptides for human therapy. Exemplary cells are described, e.g., in Kunert and Reinhart, Appl Microbiol Biotechnol. (2016) 100:3451-3461 (incorporated by reference in its entirety), including, e.g., CHO, HEK 293, PER.C6, NS0, and BHK cells. In preferred embodiments, the cell is or is derived from a CHO cell.

[0526] The disclosure also provides a method for producing a cell comprising a nucleic acid or vector according to the disclosure, the method comprising introducing a nucleic acid, a plurality of nucleic acids, a vector, or a plurality of vectors according to the disclosure into the cell. In some embodiments, introducing an isolated nucleic acid or vector according to the disclosure into the cell comprises transformation, transfection, electroporation, or transduction (e.g. retroviral transduction).

[0527] The disclosure also provides a method for producing a cell expressing / comprising an antigen binding molecule or polypeptide according to the disclosure, the method comprising introducing a nucleic acid, a plurality of nucleic acids, a vector, or a plurality of vectors according to the disclosure into the cell. In some embodiments, the method additionally comprises culturing the cell under conditions suitable for the cell to express the nucleic acid or vector. In some embodiments, the method is performed in vitro.

[0528] The disclosure also provides a cell obtained or obtainable by a method according to the disclosure.

[0529] Compositions

[0530] The present disclosure also provides compositions comprising the antigen binding molecules, polypeptides, nucleic acids, expression vectors, and cells described herein.

[0531] The antigen binding molecules, polypeptides, nucleic acids, expression vectors, and cells described herein can be formulated into a pharmaceutical composition or drug for clinical use and can comprise a pharmaceutically acceptable carrier, diluent, excipient, or adjuvant. Accordingly, the present disclosure also provides a pharmaceutical composition / drug comprising the antigen binding molecules, polypeptides, nucleic acids / multiple nucleic acids, expression vectors / multiple expression vectors, or cells described herein.

[0532] The compositions of the present disclosure can comprise one or more pharmaceutically acceptable carriers (e.g., liposomes, micelles, microspheres, nanoparticles), diluents / excipients (e.g., starch, cellulose, cellulose derivatives, polyols, dextrose, maltodextrin, magnesium stearate), adjuvants, fillers, buffers, preservatives (e.g., vitamin A, vitamin E, vitamin C, retinyl palmitate, selenium, cysteine, methionine, citric acid, sodium citrate, methyl paraben, propyl paraben), antioxidants (e.g., vitamin A, vitamin E, vitamin C, retinyl palmitate, selenium), lubricants (e.g., magnesium stearate, talc, silicon dioxide, stearic acid, vegetable fat), binders (e.g., sucrose, lactose, starch, cellulose, gelatin, polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), xylitol, sorbitol, mannitol), stabilizers, solubilizers, surfactants (e.g., wetting agents), masking agents, or coloring agents (such as titanium dioxide).

[0533] As used herein, the term “pharmaceutically acceptable” pertains to compounds, ingredients, materials, compositions, dosage forms, etc., which are “safe” for use with humans and animals, that is, they pose no significant risk of toxicity, irritation, allergic response, or other problem, commensurate with a reasonable benefit / risk ratio, as defined in the art. Each carrier, diluent, excipient, adjuvant, filler, buffer, preservative, antioxidant, lubricant, binder, stabilizer, solubilizer, surfactant, masking agent, coloring agent, flavoring agent, or sweetening agent utilized in the compositions of the present disclosure must also be “acceptable” in the sense of being compatible with the other ingredients of the formulation. Suitable carriers, diluents, excipients, adjuvants, fillers, buffers, preservatives, antioxidants, lubricants, binders, stabilizers, solubilizers, surfactants, masking agents, coloring agents, flavoring agents, or sweetening agents can be found in standard pharmaceutical texts, e.g., Remington’s “The Science and Practice of Pharmacy” (A. Adejare, ed.), 23rdedition (2020), Academic Press.

[0534] The compositions can be formulated for topical, parenteral, systemic, intracavitary, intravenous, intraarterial, intramuscular, intrathecal, intraocular, intracorneal, intratumoral, subcutaneous, intradermal, intrathecal, oral, or transdermal routes of administration. In some embodiments, the pharmaceutical compositions / drugs can be formulated for administration by injection or infusion, or by ingestion.

[0535] Suitable formulations can include the relevant article in a sterile or isotonic medium. The drugs and pharmaceutical compositions can be formulated in fluid form, including gel form. Fluid formulations can be formulated for administration to selected areas of the human or animal body by injection or infusion, for example, through a catheter.

[0536] In some embodiments, the compositions are formulated for injection or infusion, for example, into a blood vessel, a tissue / organ of interest, or a tumor.

[0537] The present disclosure also provides methods for producing a pharmaceutically useful composition and a drug. Such methods can include one or more steps selected from the following: producing an antigen binding molecule, polypeptide, nucleic acid (or plurality of nucleic acids), expression vector (or plurality of expression vectors), or cell described herein; isolating an antigen binding molecule, polypeptide, nucleic acid (or plurality of nucleic acids), expression vector (or plurality of expression vectors), or cell described herein; and / or mixing an antigen binding molecule, polypeptide, nucleic acid (or plurality of nucleic acids), expression vector (or plurality of expression vectors), or cell described herein with a pharmaceutically acceptable carrier, adjuvant, excipient, or diluent.

[0538] For example, another aspect of the present disclosure relates to a method of formulating or producing a drug or pharmaceutical composition for treating a disease / disorder (e.g., a disease / disorder described herein), the method comprising formulating the pharmaceutical composition or drug by mixing an antigen binding molecule, polypeptide, nucleic acid (or plurality of nucleic acids), expression vector (or plurality of expression vectors), or cell described herein with a pharmaceutically acceptable carrier, adjuvant, excipient, or diluent.

[0539] Therapeutic and prophylactic applications

[0540] The antigen binding molecules, CARs, nucleic acids, expression vectors, cells, and compositions described herein are useful in therapeutic and prophylactic methods.

[0541] The present disclosure provides an antigen binding molecule, polypeptide, nucleic acid (or plurality of nucleic acids), expression vector (or plurality of expression vectors), cell, or composition described herein for use in a method of medical treatment or prevention. Also provided is an antigen binding molecule, polypeptide, nucleic acid (or plurality of nucleic acids), expression vector (or plurality of expression vectors), cell, or composition described herein for use in a method of treating or preventing a disease or disorder described herein. Also provided is the use of an antigen binding molecule, polypeptide, nucleic acid (or plurality of nucleic acids), expression vector (or plurality of expression vectors), cell, or composition described herein in the manufacture of a medicament for treating or preventing a disease or disorder described herein. Also provided is a method of treating or preventing a disease or disorder described herein, the method comprising administering to a subject a therapeutically or prophylactically effective amount of an antigen binding molecule, polypeptide, nucleic acid (or plurality of nucleic acids), expression vector (or plurality of expression vectors), cell, or composition described herein.

[0542] The method can be effective to reduce the development or progression of the disease / disorder, to alleviate symptoms of the disease / disorder, or to reduce the pathology of the disease / disorder. The method can be effective to prevent the progression of the disease / disorder, for example to prevent the disease / disorder from worsening or to slow the rate of development of the disease / disorder. In some embodiments, the method can result in an improvement in the disease / disorder, for example a reduction in symptoms of the disease / disorder or some other relevant indicator of severity / activity of the disease / disorder. In some embodiments, the method can prevent the development of the disease / disorder at a later stage (e.g. a chronic stage or metastasis).

[0543] “Treatment” can for example reduce the development or progression of the disease / disorder, alleviate symptoms of the disease / disorder, or reduce the pathology of the disease / disorder. Treatment or alleviation of the disease / disorder can be effective to prevent the progression of the disease / disorder, for example to prevent the disease / disorder from worsening or to slow the rate of development. In some embodiments, treatment or alleviation can result in an improvement in the disease / disorder, for example a reduction in symptoms of the disease / disorder or some other relevant indicator of severity / activity of the disease / disorder. Prevention of the disease / disorder can refer to preventing the disease / disorder from worsening or preventing the development of the disease / disorder, for example preventing the development of early stage disease / disorder to a later chronic stage.

[0544] It will be appreciated that the articles of the disclosure (i.e., the antigen binding molecules, CARs, nucleic acids, expression vectors, cells, and compositions described herein) can be used to treat / prevent any disease / disorder that would derive a therapeutic or prophylactic benefit from modulating signaling mediated by a gamma c-containing cytokine receptor (e.g., selected from the group consisting of gamma c:IL-2R beta receptor, gamma c:IL-2R beta:IL-2R alpha, gamma c:IL-2R beta:IL-15R alpha, gamma c:IL-4R alpha receptor, gamma c:IL-9R alpha receptor, gamma c:IL-21R alpha receptor, and gamma c:IL-7R alpha receptor), and / or from manipulating the number / proportion of cells expressing a gamma c-containing cytokine receptor (e.g., selected from the group consisting of gamma c:IL-2R beta receptor, gamma c:IL-2R beta:IL-2R alpha, gamma c:IL-2R beta:IL-15R alpha, gamma c:IL-4R alpha receptor, gamma c:IL-9R alpha receptor, gamma c:IL-21R alpha receptor, and gamma c:IL-7R alpha receptor).

[0545] In particular aspects and embodiments, it will be appreciated that the antigen binding molecules of the disclosure can be used to treat / prevent a disease / disorder that would derive a therapeutic or prophylactic benefit from an increase in the level of signaling mediated by a gamma c-containing cytokine receptor to which the antigen binding molecule binds. By way of illustration, in embodiments in which the antigen binding molecule comprises (i) a gamma c-binding moiety and (ii) an IL-2R beta-binding moiety, the antigen binding molecule can be used to treat / prevent a disease / disorder that would derive a therapeutic or prophylactic benefit from an increase in signaling mediated by a gamma c:IL-2R beta receptor.

[0546] In some embodiments, the antigen binding molecule can be used to treat / prevent a disease / disorder that would derive a therapeutic or prophylactic benefit from an increase in signaling mediated by a gamma c:IL-2R beta receptor.

[0547] In some embodiments, the antigen binding molecule can be used to treat / prevent a disease / disorder that would derive a therapeutic or prophylactic benefit from an increase in signaling mediated by a gamma c:IL-2R beta:IL-15R alpha receptor.

[0548] In particular aspects and embodiments, it will be appreciated that the antigen binding molecules of the present disclosure can be used to treat / prevent diseases / conditions that would derive therapeutic or prophylactic benefit from a reduction in the level of signaling mediated by the yc-containing cytokine receptor bound by the antigen binding molecule. By way of illustration, in embodiments in which the antigen binding molecule comprises (i) a yc-binding moiety and (ii) an IL-2Rβ-binding moiety, the antigen binding molecule can be used to treat / prevent diseases / conditions that would derive therapeutic or prophylactic benefit from a reduction in signaling mediated by the yc:IL-2Rβ receptor, and / or to treat / prevent diseases / conditions that would derive therapeutic or prophylactic benefit from a reduction in the number / proportion of cells comprising / expressing the yc:IL-2Rβ receptor.

[0549] In some embodiments, the antigen binding molecules can be used to treat / prevent diseases / conditions that would derive therapeutic or prophylactic benefit from a reduction in signaling mediated by the yc:IL-2Rβ receptor. In some embodiments, the antigen binding molecules can be used to treat / prevent diseases / conditions that would derive therapeutic or prophylactic benefit from a reduction in signaling mediated by the yc:IL-2Rβ:IL-15Rα receptor. In some embodiments, the antigen binding molecules can be used to treat / prevent diseases / conditions that would derive therapeutic or prophylactic benefit from a reduction in signaling mediated by both the yc:IL-2Rβ receptor and the yc:IL-2Rβ:IL-15Rα receptor.

[0550] The articles of manufacture of the present disclosure can also be used to treat / prevent diseases / conditions that would derive therapeutic or preventative benefit from the functional consequence of increased levels of signaling mediated by a γc-containing cytokine receptor (e.g., selected from the group consisting of γc:IL-2Rβ receptor, γc:IL-2Rβ:IL-2Rα, γc:IL-2Rβ:IL-15Rα, γc:IL-4Rα receptor, γc:IL-9Rα receptor, γc:IL-21Rα receptor, and γc:IL-7Rα receptor). For example, the articles of manufacture of the present disclosure can be used to treat / prevent diseases / disorders that would benefit from an increase in the proliferation and / or population expansion of cells expressing a γc-containing cytokine receptor (e.g., selected from the group consisting of γc:IL-2Rβ receptor, γc:IL-2Rβ:IL-2Rα, γc:IL-2Rβ:IL-15Rα, γc:IL-4Rα receptor, γc:IL-9Rα receptor, γc:IL-21Rα receptor, and γc:IL-7Rα receptor), as well as from an increase in the survival and / or increase in the number / ratio and / or activity of such cells. Specifically, the articles of manufacture of the present disclosure can be used to treat / prevent diseases / disorders that would benefit from an increase in the number / ratio and / or activity of immune cells (e.g., effector immune cells (e.g., effector T cells and / or NK cells)).

[0551] The articles of the present disclosure can also be used to treat / prevent any disease / condition that would derive therapeutic or prophylactic benefit from a reduced functional outcome of signaling levels mediated by a yc-containing cytokine receptor (e.g., selected from the group consisting of yc:IL-2Rß receptor, yc:IL-2Rß:IL-2Ra, yc:IL-2Rß:IL-15Ra, yc:IL-4Ra receptor, yc:IL-9Ra receptor, yc:IL-21Ra receptor, and yc:IL-7Ra receptor), and / or from a reduced functional outcome of a number / proportion of cells comprising / expressing a yc-containing cytokine receptor (e.g., selected from the group consisting of yc:IL-2Rß receptor, yc:IL-2Rß:IL-2Ra, yc:IL-2Rß:IL-15Ra, yc:IL-4Ra receptor, yc:IL-9Ra receptor, yc:IL-21Ra receptor, and yc:IL-7Ra receptor). For example, the articles of the present disclosure can be used to treat / prevent a disease / condition that would derive therapeutic or prophylactic benefit from a reduction in proliferation and / or population expansion of cells expressing a yc-containing cytokine receptor (e.g., selected from the group consisting of yc:IL-2Rß receptor, yc:IL-2Rß:IL-2Ra, yc:IL-2Rß:IL-15Ra, yc:IL-4Ra receptor, yc:IL-9Ra receptor, yc:IL-21Ra receptor, and yc:IL-7Ra receptor), and from a reduction in survival and / or number / proportion and / or activity of such cells. In particular, the articles of the present disclosure can be used to treat / prevent a disease / condition that would derive therapeutic or prophylactic benefit from a reduction in number / proportion and / or activity of immune cells (e.g., effector immune cells (e.g., effector T cells and / or NK cells)).

[0552] The disease / condition to be treated / prevented according to the present disclosure can be a disease / condition in which one or more of the following is correlated with the onset, development or progression of the disease / condition and / or the severity of one or more symptoms of the disease / condition, or is a risk factor for the onset, development or progression of the disease / condition: a decrease in the level of signaling mediated by a yc family cytokine (e.g., selected from IL-2, IL-15, IL-4, IL-9, IL-21 and IL-7), a decrease in the level of signaling mediated by a yc-containing cytokine receptor (e.g., selected from yc:IL-2R beta receptor, yc:IL-2R beta:IL-2R alpha, yc:IL-2R beta:IL-15R alpha, yc:IL-4R alpha receptor, yc:IL-9R alpha receptor, yc:IL-21R alpha receptor and yc:IL-7R alpha receptor), a decrease in the level of number / proportion / activity of cells expressing a yc-containing cytokine receptor (e.g., selected from yc:IL-2R beta receptor, yc:IL-2R beta:IL-2R alpha, yc:IL-2R beta:IL-15R alpha, yc:IL-4R alpha receptor, yc:IL-9R alpha receptor, yc:IL-21R alpha receptor and yc:IL-7R alpha receptor), a decrease in the level of number / proportion / activity of lymphocytes, a decrease in the level of number / proportion / activity of effector immune cells, a decrease in the level of number / proportion / activity of T cells (e.g., effector T cells), and / or a decrease in the level of number / proportion / activity of NK cells.

[0553] Thus, in some embodiments, the disease / condition to be treated / prevented according to the present disclosure is a disease / condition characterized by one or more of a decrease in the level of signaling mediated by a yc family cytokine (e.g., selected from IL-2, IL-15, IL-4, IL-9, IL-21 and IL-7), a decrease in the level of signaling mediated by a yc-containing cytokine receptor (e.g., selected from yc:IL-2R beta receptor, yc:IL-2R beta:IL-2R alpha, yc:IL-2R beta:IL-15R alpha, yc:IL-4R alpha receptor, yc:IL-9R alpha receptor, yc:IL-21R alpha receptor and yc:IL-7R alpha receptor), a decrease in the level of number / proportion / activity of cells expressing a yc-containing cytokine receptor (e.g., selected from yc:IL-2R beta receptor, yc:IL-2R beta:IL-2R alpha, yc:IL-2R beta:IL-15R alpha, yc:IL-4R alpha receptor, yc:IL-9R alpha receptor, yc:IL-21R alpha receptor and yc:IL-7R alpha receptor), a decrease in the level of number / proportion / activity of lymphocytes, a decrease in the level of number / proportion / activity of effector immune cells, a decrease in the level of number / proportion / activity of T cells (e.g., effector T cells), and / or a decrease in the level of number / proportion / activity of NK cells.

[0554] The disease / condition to be treated / prevented according to the present disclosure can be a disease / condition in which one or more of the following is correlated with the onset, development, or progression of the disease / condition and / or the severity of one or more symptoms of the disease / condition, or is a risk factor for the onset, development, or progression of the disease / condition: an increase in the level of signaling mediated by a yc family cytokine (e.g., selected from the group consisting of IL-2, IL-15, IL-4, IL-9, IL-21, and IL-7), an increase in the level of signaling mediated by a yc-containing cytokine receptor (e.g., selected from the group consisting of yc:IL-2Rß receptor, yc:IL-2Rß:IL-2Ra, yc:IL-2Rß:IL-15Ra, yc:IL-4Ra receptor, yc:IL-9Ra receptor, yc:IL-21Ra receptor, and yc:IL-7Ra receptor), an increase in the number / proportion / activity level of cells expressing a yc-containing cytokine receptor (e.g., selected from the group consisting of yc:IL-2Rß receptor, yc:IL-2Rß:IL-2Ra, yc:IL-2Rß:IL-15Ra, yc:IL-4Ra receptor, yc:IL-9Ra receptor, yc:IL-21Ra receptor, and yc:IL-7Ra receptor), an increase in the number / proportion / activity level of lymphocytes, an increase in the number / proportion / activity level of effector immune cells, an increase in the number / proportion / activity level of T cells (e.g., effector T cells), and / or an increase in the number / proportion / activity level of NK cells. Thus, in some embodiments, the disease / condition to be treated / prevented according to the present disclosure is a disease / condition characterized by one or more of an increase in the level of signaling mediated by a yc family cytokine (e.g., selected from the group consisting of IL-2, IL-15, IL-4, IL-9, IL-21, and IL-7), an increase in the level of signaling mediated by a yc-containing cytokine receptor (e.g., selected from the group consisting of yc:IL-2Rß receptor, yc:IL-2Rß:IL-2Ra, yc:IL-2Rß:IL-15Ra, yc:IL-4Ra receptor, yc:IL-9Ra receptor, yc:IL-21Ra receptor, and yc:IL-7Ra receptor), an increase in the number / proportion / activity level of cells expressing a yc-containing cytokine receptor (e.g., selected from the group consisting of yc:IL-2Rß receptor, yc:IL-2Rß:IL-2Ra, yc:IL-2Rß:IL-15Ra, yc:IL-4Ra receptor, yc:IL-9Ra receptor, yc:IL-21Ra receptor, and yc:IL-7Ra receptor), an increase in the number / proportion / activity level of lymphocytes, an increase in the number / proportion / activity level of effector immune cells, an increase in the number / proportion / activity level of T cells (e.g., effector T cells), and / or an increase in the number / proportion / activity level of NK cells.

[0555] In some embodiments, the antigen binding molecules can be used to treat / prevent diseases / conditions that would benefit from an increase in signaling mediated by IL-2. In some embodiments, the antigen binding molecules can be used to treat / prevent diseases / conditions that would benefit from an increase in signaling mediated by IL-15. In some embodiments, the antigen binding molecules can be used to treat / prevent diseases / conditions that would benefit from an increase in signaling mediated by IL-2 and IL-15.

[0556] In some embodiments, the antigen binding molecules can be used to treat / prevent diseases / conditions that would benefit from an increase in signaling mediated by IL-2. In some embodiments, the antigen binding molecules can be used to treat / prevent diseases / conditions that would benefit from an increase in signaling mediated by IL-15. In some embodiments, the antigen binding molecules can be used to treat / prevent diseases / conditions that would benefit from an increase in signaling mediated by IL-2 and IL-15.

[0557] In some embodiments, the antigen binding molecules can be used to treat / prevent diseases / conditions that would benefit from a decrease in signaling mediated by IL-2. In some embodiments, the antigen binding molecules can be used to treat / prevent diseases / conditions that would benefit from a decrease in signaling mediated by IL-15. In some embodiments, the antigen binding molecules can be used to treat / prevent diseases / conditions that would benefit from a decrease in signaling mediated by IL-2 and IL-15.

[0558] In some embodiments, the antigen binding molecules can be used to treat / prevent diseases / conditions that would benefit from a decrease in signaling mediated by IL-2. In some embodiments, the antigen binding molecules can be used to treat / prevent diseases / conditions that would benefit from a decrease in signaling mediated by IL-15. In some embodiments, the antigen binding molecules can be used to treat / prevent diseases / conditions that would benefit from a decrease in signaling mediated by IL-2 and IL-15.

[0559] In the foregoing paragraphs, “decrease / increase” can be relative to levels observed in a healthy, disease-free state, e.g., as determined in healthy control subjects and / or equivalent, disease-free tissue.

[0560] According to various aspects of the present disclosure, methods are provided that are to or include one or more of (e.g., in the context of treating / preventing a disease / condition described herein):

[0561] increasing multimerization of a polypeptide other than yc of yc- and yc-containing cytokine receptors (e.g., selected from IL-2R, IL-2R, IL-15R, IL-4R, IL-9R, IL-21R, and IL-7R );

[0562] decreasing multimerization of a polypeptide other than yc of yc- and yc-containing cytokine receptors (e.g., selected from IL-2R, IL-2R, IL-15R, IL-4R, IL-9R, IL-21R, and IL-7R );

[0563] increasing signaling mediated by yc-containing cytokine receptors (e.g., selected from yc:IL-2R receptors, yc:IL-2R :IL-2R, yc:IL-2R :IL-15R, yc:IL-4R receptors, yc:IL-9R receptors, yc:IL-21R receptors, and yc:IL-7R receptors) bound by the antigen binding molecule;

[0564] decreasing signaling mediated by yc-containing cytokine receptors (e.g., selected from yc:IL-2R receptors, yc:IL-2R :IL-2R, yc:IL-2R :IL-15R, yc:IL-4R receptors, yc:IL-9R receptors, yc:IL-21R receptors, and yc:IL-7R receptors) bound by the antigen binding molecule;

[0565] increasing signaling mediated by IL-2, IL-4, IL-7, IL-9, IL-15, and / or IL-21;

[0566] decreasing signaling mediated by IL-2, IL-4, IL-7, IL-9, IL-15, and / or IL-21;

[0567] increasing signaling mediated by IL-2 and IL-15;

[0568] decreasing signaling mediated by IL-2 and IL-15;

[0569] increasing proliferation, survival, and / or effector activity of cells expressing a yc-containing cytokine receptor bound by the antigen binding molecule (e.g., selected from the group consisting of yc:IL-2Rβ receptor, yc:IL-2Rβ:IL-2Rα, yc:IL-2Rβ:IL-15Rα, yc:IL-4Rα receptor, yc:IL-9Rα receptor, yc:IL-21Rα receptor, and yc:IL-7Rα receptor);

[0570] decreasing proliferation, survival, and / or effector activity of cells expressing a yc-containing cytokine receptor bound by the antigen binding molecule (e.g., selected from the group consisting of yc:IL-2Rβ receptor, yc:IL-2Rβ:IL-2Rα, yc:IL-2Rβ:IL-15Rα, yc:IL-4Rα receptor, yc:IL-9Rα receptor, yc:IL-21Rα receptor, and yc:IL-7Rα receptor);

[0571] decreasing expression of one or more immune cell exhaustion markers by cells expressing a yc-containing cytokine receptor bound by the antigen binding molecule (e.g., selected from the group consisting of yc:IL-2Rβ receptor, yc:IL-2Rβ:IL-2Rα, yc:IL-2Rβ:IL-15Rα, yc:IL-4Rα receptor, yc:IL-9Rα receptor, yc:IL-21Rα receptor, and yc:IL-7Rα receptor);

[0572] increasing expression of one or more immune cell exhaustion markers by cells expressing a yc-containing cytokine receptor bound by the antigen binding molecule (e.g., selected from the group consisting of yc:IL-2Rβ receptor, yc:IL-2Rβ:IL-2Rα, yc:IL-2Rβ:IL-15Rα, yc:IL-4Rα receptor, yc:IL-9Rα receptor, yc:IL-21Rα receptor, and yc:IL-7Rα receptor);

[0573] increasing cell killing / depletion of cells comprising / expressing one or more target antigens of the constituent antigen binding moieties of the antigen binding molecule (i.e., polypeptides other than yc of the yc and / or yc-containing cytokine receptor (e.g., selected from the group consisting of IL-2Rβ, IL-2Rα, IL-15Rα, IL-4Rα, IL-9Rα, IL-21Rα, and IL-7Rα)), and / or decreasing the number / proportion of such cells;

[0574] and / or

[0575] enhancing anti-cancer activity of cancer antigen-specific immune cells.

[0576] Also provided are agents according to the disclosure for use in such methods, and use of agents according to the disclosure in the manufacture of a pharmaceutical composition or medicament for use in such methods. It will be understood that the methods can comprise administering to the subject an antigen binding molecule, nucleic acid, expression vector, cell or composition described herein.

[0577] Similarly, following a therapeutic or prophylactic intervention according to the disclosure, one or more of the following can be observed in the subject (e.g., compared to levels prior to the intervention):

[0578] Increased multimerization of a polypeptide other than yc of yc and yc-containing cytokine receptors (e.g., selected from IL-2Rβ, IL-2Rα, IL-15Rα, IL-4Rα, IL-9Rα, IL-21Rα, and IL-7Rα);

[0579] Decreased multimerization of a polypeptide other than yc of yc and yc-containing cytokine receptors (e.g., selected from IL-2Rβ, IL-2Rα, IL-15Rα, IL-4Rα, IL-9Rα, IL-21Rα, and IL-7Rα);

[0580] Increased levels of signaling mediated by yc-containing cytokine receptors (e.g., selected from yc:IL-2Rβ receptor, yc:IL-2Rβ:IL-2Rα, yc:IL-2Rβ:IL-15Rα, yc:IL-4Rα receptor, yc:IL-9Rα receptor, yc:IL-21Rα receptor, and yc:IL-7Rα receptor) bound by the antigen binding molecule;

[0581] Decreased levels of signaling mediated by yc-containing cytokine receptors (e.g., selected from yc:IL-2Rβ receptor, yc:IL-2Rβ:IL-2Rα, yc:IL-2Rβ:IL-15Rα, yc:IL-4Rα receptor, yc:IL-9Rα receptor, yc:IL-21Rα receptor, and yc:IL-7Rα receptor) bound by the antigen binding molecule;

[0582] Increased levels of proliferation, survival, and / or effector activity of cells expressing yc-containing cytokine receptors (e.g., selected from yc:IL-2Rβ receptor, yc:IL-2Rβ:IL-2Rα, yc:IL-2Rβ:IL-15Rα, yc:IL-4Rα receptor, yc:IL-9Rα receptor, yc:IL-21Rα receptor, and yc:IL-7Rα receptor) bound by the antigen binding molecule;

[0583] a decrease in the level of proliferation, survival, and / or effector activity of cells expressing a yc-containing cytokine receptor to which the antigen binding molecule binds (e.g., selected from the group consisting of yc:IL-2Rß receptor, yc:IL-2Rß:IL-2Ra, yc:IL-2Rß:IL-15Ra, yc:IL-4Ra receptor, yc:IL-9Ra receptor, yc:IL-21Ra receptor, and yc:IL-7Ra receptor);

[0584] a decrease in the level of expression of one or more immune cell exhaustion markers of cells expressing a yc-containing cytokine receptor to which the antigen binding molecule binds (e.g., selected from the group consisting of yc:IL-2Rß receptor, yc:IL-2Rß:IL-2Ra, yc:IL-2Rß:IL-15Ra, yc:IL-4Ra receptor, yc:IL-9Ra receptor, yc:IL-21Ra receptor, and yc:IL-7Ra receptor);

[0585] an increase in the level of expression of one or more immune cell exhaustion markers of cells expressing a yc-containing cytokine receptor to which the antigen binding molecule binds (e.g., selected from the group consisting of yc:IL-2Rß receptor, yc:IL-2Rß:IL-2Ra, yc:IL-2Rß:IL-15Ra, yc:IL-4Ra receptor, yc:IL-9Ra receptor, yc:IL-21Ra receptor, and yc:IL-7Ra receptor);

[0586] an increase in cell killing / depletion of cells comprising / expressing one or more target antigens of which the constituent antigen binding moiety of the antigen binding molecule is specific (i.e., polypeptides other than yc of yc- and / or yc-containing cytokine receptors (e.g., selected from the group consisting of IL-2Rß, IL-2Ra, IL-15Ra, IL-4Ra, IL-9Ra, IL-21Ra, and IL-7Ra)), and / or a decrease in the number / proportion of such cells;

[0587] and / or

[0588] an increase in the anti-cancer activity of cancer antigen-specific immune cells.

[0589] In some embodiments, the therapeutic / prophylactic intervention according to the present disclosure can be described as being “associated with” one or more of the effects described in the preceding paragraphs. The skilled person is readily able to assess such properties using techniques that are routine practice in the art.

[0590] In some aspects and embodiments, the disease / condition to be treated / prevented according to the present disclosure can be lymphocytopenia, or a disease / condition characterized by lymphocytopenia.

[0591] Lymphocytopenia can be defined as a total lymphocyte count <1000 / mcL (1 x 10 9<3000 / mcL (<3 x 10 9 Diseases / conditions characterized by lymphopenia include, for example: T-lymphocyte deficiency, B-lymphocyte deficiency, NK-lymphocyte deficiency, idiopathic CD4+ lymphopenia, human immunodeficiency virus infection and acquired immunodeficiency syndrome (HIV / AIDS), COVID-19, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), myasthenia gravis, sarcoidosis, multiple sclerosis (MS), chemotherapy-related lymphopenia, severe combined immunodeficiency (SCID), Omenn syndrome, Wiskott-Aldrich syndrome, and chondro-dysplasia-hair hypoplasia (CHH).

[0592] In some embodiments, the disease / condition to be treated / prevented according to the present disclosure is a disease / condition characterized by T-cell dysfunction, cancer, infection, or an autoimmune disease / disorder.

[0593] As used herein, “T-cell dysfunction” refers to a state in which normal T-cell function is reduced / decreased / impaired / abnormal, resulting in a subject having T-cell dysfunction exhibiting an insufficient or inappropriate T-cell mediated immune response. T-cell dysfunction is reviewed, for example, in Xia et al., Front Immunol. (2019) 10: 1719 and Gao et al., Front Immunol. (2022) Sec. Autoimmune and Autoinflammatory Disorders, both of which are incorporated by reference herein in their entireties.

[0594] In some embodiments, T-cell dysfunction can be associated with T-cell anergy. T-cell anergy is caused by suboptimal T-cell stimulation. In some embodiments, T-cell dysfunction can be associated with T-cell exhaustion. T-cell exhaustion is caused by persistent T-cell stimulation and / or overstimulation.

[0595] Dysfunctional T-cells can be characterized by one or more of the following (i.e., as compared to normal, non-dysfunctional T-cells): reduced proliferative capacity, reduced effector function, reduced expression of one or more effector molecules (e.g., selected from granzyme B, IFNy, CD107a, IL-2, TNFa, perforin, granulysin, and FASL), reduced cytotoxicity (e.g., against cells expressing MHC:peptide complexes for which the T-cell expresses a particular receptor), and / or increased expression of one or more T-cell exhaustion markers (e.g., selected from PD-1, CTLA-4, LAG-3, TIM-3, VISTA, TIGIT, BTLA, CD160, and CD244).

[0596] In some embodiments, the disease / condition characterized by T cell dysfunction can be a cancer, an infectious disease (e.g., a chronic infection), or an autoimmune disease.

[0597] In some embodiments, the disease / condition to be treated / prevented according to the present disclosure is a cancer. As the articles of the present disclosure can be used to increase / enhance / upregulate an anti-cancer immune response, particularly a cell-mediated anti-cancer immune response, in a subject, it will be appreciated that they can be used to treat / prevent essentially all cancers.

[0598] The cancer according to the present disclosure can be any unwanted cell proliferation (or any disease that manifests itself as an unwanted cell proliferation), a neoplasm, or a tumor. The cancer can be benign or malignant. The cancer can be primary or secondary (e.g., metastatic). The neoplasm or tumor can be any abnormal growth or proliferation of cells, and can be located in (and / or derived from cells of) any organ / tissue.

[0599] The cancer can be derived from cells of, for example, the adrenal gland, adrenal medulla, anus, appendix, bladder, blood, bone, bone marrow, brain, breast, cecum, central nervous system (including or excluding the brain), cerebellum, cervix, colon, duodenum, endometrium, epithelial cells (e.g., kidney epithelial cells), gall bladder, esophagus, glial cells, heart, ileum, jejunum, kidney, lacrimal gland, larynx, liver, lung, lymph, lymph node, lymphoblast, maxilla, mediastinum, mesentery, myometrium, nasopharynx, omentum, oral cavity, ovary, pancreas, parotid gland, peripheral nervous system, peritoneum, pleura, prostate, salivary gland, sigmoid colon, skin, small intestine, soft tissue, spleen, stomach, testicle, thymus, thyroid, tongue, tonsil, trachea, uterus, vulva, and / or white blood cells.

[0600] The cancer can be or can include one or more tumors. The cancer can be a glioma, medulloblastoma, meningioma, neurofibroma, ependymoma, schwannoma, neurofibrosarcoma, astrocytoma and oligodendroglioma, melanoma, mesothelioma, myeloma, lymphoma, non-Hodgkin’s lymphoma (NHL), Hodgkin’s lymphoma, cutaneous T-cell lymphoma (CTCL), leukemia, chronic myelogenous leukemia (CML), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), myelodysplastic syndrome (MDS), hepatoma, squamous cell carcinoma, prostate cancer, breast cancer, lung cancer, NSCLC, colon cancer, ovarian cancer, pancreatic cancer, thymic carcinoma, hematological cancer, or sarcoma.

[0601] In some embodiments, the cancer according to the present disclosure is selected from the group consisting of: solid tumor, breast cancer, breast carcinoma, ductal carcinoma, gastric cancer, gastric carcinoma, gastric adenocarcinoma, colorectal cancer, colorectal carcinoma, colorectal adenocarcinoma, head and neck cancer, head and neck squamous cell carcinoma (SCCHN), lung cancer, non-small cell lung cancer, lung adenocarcinoma, squamous cell lung cancer, ovarian cancer, ovarian carcinoma, ovarian serous adenocarcinoma, renal cancer, renal cell carcinoma, renal clear cell carcinoma, renal cell adenocarcinoma, renal papillary cell carcinoma, pancreatic cancer, pancreatic adenocarcinoma, pancreatic ductal adenocarcinoma, cervical cancer, cervical squamous cell carcinoma, skin cancer, melanoma, esophageal cancer, esophageal adenocarcinoma, liver cancer, hepatocellular carcinoma, cholangiocarcinoma, uterine cancer, uterine corpus endometrial carcinoma, thyroid cancer, thyroid carcinoma, pheochromocytoma, paraganglioma, bladder cancer, bladder urothelial carcinoma, prostate cancer, prostate adenocarcinoma, sarcoma, and thymoma.

[0602] In some embodiments, the cancer to be treated can be colon cancer, colon cancer, colorectal cancer, nasopharyngeal cancer, cervical cancer, oropharyngeal cancer, gastric cancer, hepatocellular carcinoma, head and neck cancer, head and neck squamous cell carcinoma (HNSCC), oral cancer, laryngeal cancer, prostate cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, bladder cancer, urothelial cancer, melanoma, advanced melanoma, renal cell carcinoma, ovarian cancer, or mesothelioma.

[0603] In some embodiments, the cancer according to the present disclosure is selected from the group consisting of: gastric cancer (e.g., gastric carcinoma, gastric adenocarcinoma, gastrointestinal adenocarcinoma), head and neck cancer (e.g., gastrointestinal), breast cancer, ovarian cancer (e.g., ovarian carcinoma), lung cancer (e.g., NSCLC, lung adenocarcinoma, squamous lung cell carcinoma), melanoma, prostate cancer, oral cancer (e.g., oropharyngeal cancer), renal cancer (e.g., renal cell carcinoma), and colorectal cancer (e.g., colorectal carcinoma), esophageal cancer, pancreatic cancer, solid cancer, and liquid cancer (e.g., hematological cancer).

[0604] In some embodiments, the cancer to be treated / prevented is a primary cancer. In some embodiments, the cancer to be treated / prevented is a secondary cancer (i.e., metastatic).

[0605] The treatment can aim to one or more of: delay / prevent the onset / progression of a cancer symptom, reduce the severity of a cancer symptom, reduce the survival / growth / invasion / metastasis of cancer cells, reduce the number of cancer cells and / or increase the survival of the subject.

[0606] In some embodiments, the disease / condition to be treated / prevented in accordance with the present disclosure is an infectious disease. As the articles of the present disclosure can be used to increase / enhance / upregulate the immune response, particularly the cell-mediated immune response, of a subject, it will be appreciated that they can be used to treat / prevent essentially any disease caused by an infection.

[0607] The infectious disease can be caused by and / or characterised by, for example, a bacterial, viral, fungal or parasitic infection. In some embodiments, it can be particularly desirable to treat chronic / persistent infections and / or diseases caused by and / or characterised by chronic / persistent infections, for example, where such infections are associated with T cell dysfunction, for example T cell exhaustion. It is well known that T cell exhaustion is a state of T cell dysfunction that occurs during many chronic infections, including viral, bacterial and parasitic infections, as well as in cancer (Wherry, Nature Immunology (2011) 12(6):492-499).

[0608] Examples of bacterial infections that can be treated include infection by Bacillus spp., Bordetella pertussis, Clostridium spp., Corynebacterium spp., Vibrio chloerae, Staphylococcus spp., Streptococcus spp., Escherichia, Klebsiella, Proteus, Yersinia, Erwina, Salmonella (e.g. Salmonella typhi), Listeria sp, Helicobacter pylori, mycobacteria (e.g. Mycobacterium tuberculosis) and Pseudomonas aeruginosa. For example, the bacterial infection can be sepsis or tuberculosis.

[0609] Examples of viral infections that can be treated include infection with influenza virus, measles virus, hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), lymphocytic choriomeningitis virus (LCMV), herpes simplex virus, or human papillomavirus (HPV).

[0610] Examples of fungal infections that can be treated include infection with Alternaria sp, Aspergillus sp, Candida sp, and Histoplasma sp. The fungal infection can be fungal sepsis or histoplasmosis.

[0611] Examples of parasitic infections that can be treated include infection with a Plasmodium species (e.g., P. falciparum, P. yoelii, P. ovale, P. vivax, or P. chabaudi chabaudi). The parasitic infection can be a disease such as malaria, leishmaniasis, or toxoplasmosis.

[0612] In some embodiments, the disease / condition to be treated / prevented in accordance with the present disclosure is an autoimmune disease. Lymphopenia and / or T cell dysfunction is a feature of autoimmune diseases, such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), myasthenia gravis, and sarcoidosis, and will benefit from treatment with the articles of the present disclosure.

[0613] The articles of the present disclosure can also be used in conjunction with methods for treating / preventing a disease / condition, including adoptive cell transfer (ACT), particularly ACT of immune cells (e.g., effector immune cells, such as T cells and / or NK cells).

[0614] Adoptive cell transfer generally refers to the process of obtaining cells (e.g., immune cells) from a subject, typically by drawing a blood sample from which the cells are isolated. The cells are then typically modified and / or expanded, and then administered to the same subject (in the case of autologous / self cell adoptive transfer) or a different subject (in the case of allogeneic cell adoptive transfer). The treatment is typically aimed at providing the subject with a population of cells having certain desirable characteristics, or increasing the frequency of such cells having such characteristics in that subject. Adoptive transfer can be performed with the aim of introducing cells or cell populations into a subject, and / or increasing the frequency of cells or cell populations in a subject.

[0615] Adoptive transfer of immune cells is described, for example, in Kalos and June Immunity (2013) 39(1): 49-60 and Davis et al. Cancer J. (2015) 21(6): 486-491, both of which are incorporated by reference in their entirety. The skilled person is able to determine appropriate reagents and procedures for adoptive transfer of cells according to the present disclosure, for example with reference to Dai et al., J Nat Cancer Inst. (2016) 108(7): djv439, which is incorporated by reference in its entirety.

[0616] Adoptive cell transfer can comprise allogeneic transplantation or autologous transplantation. As used herein, “allogeneic transplantation” refers to the transplantation of cells, tissues or organs that are not genetically identical to the recipient subject into the recipient subject. The cells, tissues or organs can be from or can be derived from cells, tissues or organs of a donor subject that are not genetically identical to the recipient subject. Allogeneic transplantation is distinct from autologous transplantation, which refers to the transplantation of cells, tissues or organs from / derived from a donor subject that is genetically identical to the recipient subject (i.e. autologous material). It will be appreciated that adoptive transfer of allogeneic immune cells is a form of allogeneic transplantation and adoptive transfer of autologous immune cells is a form of autologous transplantation.

[0617] The articles of the present disclosure can be used for the generation / expansion of immune cell populations in vitro or ex vivo, which can then be administered to a subject. In particular, the present disclosure contemplates the treatment / prevention of a disease / condition (e.g. a disease / condition described herein) by adoptive transfer of immune cells generated (e.g. generated or expanded) according to the methods described herein. That is, the adoptively transferred immune cells can be generated / expanded by culturing in vitro or ex vivo in the presence of an antigen binding molecule according to the present disclosure.

[0618] The immune cells can be immune cells as described above. It will be appreciated that the immune cells comprise a yc-containing cytokine receptor (e.g. selected from yc:IL-2Rß receptor, yc:IL-2Rß:IL-2Ra, yc:IL-2Rß:IL-15Ra, yc:IL-4Ra receptor, yc:IL-9Ra receptor, yc:IL-21Ra receptor and yc:IL-7Ra receptor).

[0619] The present disclosure provides a method of treating or preventing a disease or condition in a subject, the method comprising:

[0620] (i) generating or expanding a population of immune cells by culturing in the presence of an antigen binding molecule according to the present disclosure, and;

[0621] (ii) administering the generated / expanded population of immune cells to the subject.

[0622] In some embodiments, the adoptively transferred is an autologous cell. In some embodiments, the adoptively transferred is an allogeneic cell.

[0623] In some embodiments, the method can comprise one or more of: obtaining an immune cell-containing sample (e.g., a blood sample) from the subject; isolating / purifying immune cells (e.g., PBMCs) from the immune cell-containing sample (e.g., blood sample); producing or expanding a population of immune cells by culturing (i.e., in vitro / ex vivo) in the presence of an antigen binding molecule according to the disclosure; collecting a population of immune cells produced or expanded by culturing (i.e., in vitro / ex vivo) in the presence of an antigen binding molecule according to the disclosure; mixing a population of immune cells produced or expanded by culturing (i.e., in vitro / ex vivo) in the presence of an antigen binding molecule according to the disclosure with an adjuvant, diluent, or carrier; and / or administering a population of immune cells produced or expanded by culturing (i.e., in vitro / ex vivo) in the presence of an antigen binding molecule according to the disclosure or a composition comprising such cells to the subject.

[0624] In some embodiments, the method can additionally comprise administering to the subject a therapeutically or prophylactically effective amount of an antigen binding molecule according to the disclosure.

[0625] The skilled person will be able to determine appropriate reagents and procedures for producing / expanding a population of immune cells for adoptive transfer, and appropriate reagents and procedures for adopti...

Claims

1. An antigen binding molecule, which is optionally isolated, comprising: (i) a γc binding moiety, and (ii) A portion that binds to a polypeptide other than γc of a γc-containing cytokine receptor. 2 . The antigen-binding molecule according to claim 1 , wherein the polypeptide other than γc of the γc-containing cytokine receptor is selected from the group consisting of IL-2Rβ, IL-2Rα, IL-15Rα, IL-4Rα, IL-9Rα, IL-21Rα, and IL-7Rα. The antigen-binding molecule according to claim 1 , wherein the polypeptide other than γc of the γc-containing cytokine receptor is IL-2Rβ. The antigen-binding molecule according to any one of claims 1 to 3, wherein the antigen-binding molecule is an antagonist of a γc-containing cytokine receptor or an agonist of a γc-containing cytokine receptor. The antigen-binding molecule according to any one of claims 1 to 4, wherein the antigen-binding molecule is an antagonist of γc:IL-2Rβ receptor, γc:IL-2Rβ:IL-2Rα receptor and / or γc:IL-2Rβ:IL-15Rα receptor.

6. The antigen-binding molecule according to any one of claims 1 to 5, wherein the antigen-binding molecule further comprises: (iii) an antigen-binding portion that binds to a target antigen other than a γc-containing cytokine receptor polypeptide. The antigen-binding molecule according to claim 6 , wherein the target antigen other than the γc-containing cytokine receptor polypeptide is a disease-associated antigen or an antigen expressed by immune cells.

8. A chimeric antigen receptor (CAR) comprising the antigen-binding molecule according to any one of claims 1 to 7.

9. A nucleic acid or nucleic acids, optionally isolated, encoding the antigen binding molecule according to any one of claims 1 to 7 or the CAR according to claim 8.

10. An expression vector or a plurality of expression vectors comprising the nucleic acid or nucleic acids according to claim 9.

11. A cell comprising the antigen binding molecule according to any one of claims 1 to 7, the CAR according to claim 8, the nucleic acid or nucleic acids according to claim 9, or the expression vector or expression vectors according to claim 10.

12. A method comprising culturing the cell according to claim 11 under conditions suitable for the cell to express an antigen binding molecule or CAR.

13. A composition comprising the antigen binding molecule according to any one of claims 1 to 7, the CAR according to claim 8, the nucleic acid or multiple nucleic acids according to claim 9, the expression vector or multiple expression vectors according to claim 10, or the cell according to claim 11, and a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.

14. The antigen binding molecule according to any one of claims 1 to 7, the CAR according to claim 8, the nucleic acid or nucleic acids according to claim 9, the expression vector or expression vectors according to claim 10, the cell according to claim 11, or the composition according to claim 13, for use in a method of treatment or prevention.

15. Use of the antigen binding molecule according to any one of claims 1 to 7, the CAR according to claim 8, the nucleic acid or multiple nucleic acids according to claim 9, the expression vector or multiple expression vectors according to claim 10, the cell according to claim 11, or the composition according to claim 13 in the preparation of a medicament for a method of treatment or prevention.

16. A method of treatment or prevention, comprising administering to a subject in need thereof a therapeutically or prophylactically effective amount of an antigen binding molecule according to any one of claims 1 to 7, a CAR according to claim 8, a nucleic acid or multiple nucleic acids according to claim 9, an expression vector or multiple expression vectors according to claim 10, a cell according to claim 11, or a composition according to claim 13.

17. The antigen binding molecule for use, the nucleic acid or nucleic acids, the expression vector or expression vectors, the cell or composition for use according to claim 14, the use according to claim 15, or the method according to claim 16, wherein the method of treatment or prevention is a method of treating or preventing a disease / disorder characterized by T cell dysfunction, cancer, an infectious disease or an autoimmune disease.

18. The antigen binding molecule for use, the nucleic acid or nucleic acids, the expression vector or expression vectors, the cell or composition, the use or method according to claim 17, wherein the cancer is selected from the group consisting of colon cancer, colorectal cancer, nasopharyngeal cancer, cervical cancer, oropharyngeal cancer, gastric cancer, hepatocellular carcinoma, head and neck cancer, head and neck squamous cell carcinoma (HNSCC), oral cancer, laryngeal cancer, prostate cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, bladder cancer, urothelial carcinoma, melanoma, advanced melanoma, renal cell carcinoma, ovarian cancer or mesothelioma.

19. The antigen binding molecule, nucleic acid or nucleic acids, expression vector or expression vectors, cell or composition for use according to claim 14, 17 or 18, the use according to claim 15, 17 or 18, or the method according to claim 16, 17 or 18, wherein the disease / disorder is characterized by lymphocytosis and / or inflammation.

20. An in vitro complex, which is optionally isolated, comprising the antigen binding molecule according to any one of claims 1 to 7, the CAR according to claim 8, bound to γc and a polypeptide other than γc of a γc-containing cytokine receptor. 21 . A method for producing or expanding a cell population expressing a γc-containing cytokine receptor, comprising contacting cells expressing a γc-containing cytokine receptor with an antigen-binding molecule according to any one of claims 1 to 7 in vitro, in vivo or ex vivo.

22. A method for increasing the proliferation, survival and / or effector activity of cells expressing a γc-containing cytokine receptor, comprising contacting the cells expressing a γc-containing cytokine receptor with an antigen-binding molecule according to any one of claims 1 to 7 in vitro, in vivo or ex vivo.

23. A method for reducing the number / ratio of cells expressing a cytokine receptor containing γc, comprising contacting cells expressing a cytokine receptor containing γc in vitro, in vivo or ex vivo with an antigen binding molecule according to any one of claims 1 to 7 or a cell comprising a CAR according to claim 8.

24. A method for reducing the proliferation, survival and / or effector activity of cells expressing a γc-containing cytokine receptor, comprising contacting the cells expressing a γc-containing cytokine receptor with an antigen-binding molecule according to any one of claims 1 to 7 in vitro, in vivo or ex vivo.

25. The method of any one of claims 21 to 24, wherein the cell is an effector immune cell.

26. The method of any one of claims 21 to 25, wherein the cell is a T cell or a NK cell.

27. The method of any one of claims 21 to 26, wherein the γc-containing cytokine receptor is selected from the group consisting of: γc:IL-2Rβ receptor, γc:IL-2Rβ:IL-2Rα receptor, γc:IL-2Rβ:IL-15Rα receptor, γc:IL-4Rα receptor, γc:IL-9Rα receptor, γc:IL-21Rα receptor, and γc:IL-7Rα receptor.

28. A method for promoting heterologous multimerization of γc and a polypeptide of a cytokine receptor complex containing γc, comprising contacting γc and a polypeptide of a cytokine receptor complex containing γc with an antigen binding molecule according to any one of claims 1 to 7 or a CAR according to claim 8 in vitro, in vivo or ex vivo.

29. A method for inhibiting heterologous multimerization of γc and polypeptides of a cytokine receptor complex containing γc, comprising contacting γc and polypeptides of a cytokine receptor complex containing γc with an antigen binding molecule according to any one of claims 1 to 7 or a CAR according to claim 8 in vitro, in vivo or ex vivo.

30. The method for inhibiting heteromultimerization of γc and a polypeptide of a cytokine receptor complex containing γc according to claim 28, wherein the polypeptide of a cytokine receptor complex containing γc is IL-2Rβ.

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