Antigen binding molecules

Abstract

Disclosed herein are antigen binding molecules comprising a gamma c binding moiety and a moiety that binds to IL-21R [alpha]. 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 that is 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-21Ra and yc is necessary for efficient IL-21 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 IL-21Ra.

[0011] In some embodiments, the antigen binding molecule is an agonist of a yc-containing cytokine receptor.

[0012] In some embodiments, the antigen binding molecule is an antagonist of a yc-containing cytokine receptor.

[0013] In some embodiments, the antigen binding molecule is an agonist of a yc:IL-21Ra receptor.

[0014] In some embodiments, the antigen binding molecule is an antagonist of a yc:IL-21Ra receptor.

[0015] In some embodiments, the antigen binding molecule increases signaling mediated by a yc-containing cytokine receptor to which the antigen binding molecule binds.

[0016] In some embodiments, the antigen binding molecule increases signaling mediated by a yc:IL-21Ra receptor.

[0017] In some embodiments, the antigen binding molecule decreases signaling mediated by a yc-containing cytokine receptor to which the antigen binding molecule binds.

[0018] In some embodiments, the antigen binding molecule decreases signaling mediated by a yc:IL-21Ra receptor.

[0019] In some embodiments, the IL-21Ra binding moiety comprises a single domain antibody sequence, the IL-21Ra binding moiety comprising a single domain antibody sequence incorporating the CDRs:

[0020] (3DAS82) CDR1 having the amino acid sequence of SEQ ID NO: 378

[0021] CDR2 having the amino acid sequence of SEQ ID NO: 379

[0022] CDR3 having the amino acid sequence of SEQ ID NO: 380;

[0023] (2DAS6) CDR1 having the amino acid sequence of SEQ ID NO: 386

[0024] CDR2 having the amino acid sequence of SEQ ID NO: 387

[0025] CDR3 having the amino acid sequence of SEQ ID NO: 388; or (2DAS92) CDR1 having the amino acid sequence of SEQ ID NO: 394

[0026] CDR2 having the amino acid sequence of SEQ ID NO: 395

[0027] CDR3 having the amino acid sequence of SEQ ID NO: 396.

[0028] In some embodiments, the IL-21Ra binding moiety comprises or consists of an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 385, 393, or 401.

[0029] In some embodiments, the IL-21Ra binding moiety comprises a single domain antibody sequence incorporating the following FRs:

[0030] (3DAS82) FR1 having the amino acid sequence of SEQ ID NO: 381

[0031] FR2 having the amino acid sequence of SEQ ID NO: 382

[0032] FR3 having the amino acid sequence of SEQ ID NO: 383

[0033] FR4 having the amino acid sequence of SEQ ID NO: 384;

[0034] (2DAS6) FR1 having the amino acid sequence of SEQ ID NO: 389

[0035] FR2 having the amino acid sequence of SEQ ID NO: 390

[0036] FR3 having the amino acid sequence of SEQ ID NO: 391

[0037] FR4 having the amino acid sequence of SEQ ID NO: 392; or

[0038] (2DAS92) FR1 having the amino acid sequence of SEQ ID NO: 397

[0039] FR2 having the amino acid sequence of SEQ ID NO: 398

[0040] FR3 having the amino acid sequence of SEQ ID NO: 399

[0041] FR4 having the amino acid sequence of SEQ ID NO:400.

[0042] In some embodiments, the γc binding portion comprises a single domain antibody sequence incorporating the following CDRs:

[0043] CDR1 having the amino acid sequence of SEQ ID NO: 370

[0044] CDR2 having the amino acid sequence of SEQ ID NO: 371

[0045] A CDR3 having the amino acid sequence of SEQ ID NO: 372.

[0046] In some embodiments, the γc binding portion comprises, or consists of, an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:377.

[0047] In some embodiments, the γc binding portion comprises a single domain antibody sequence incorporating the following FRs:

[0048] FR1 having the amino acid sequence of SEQ ID NO: 373

[0049] FR2 having the amino acid sequence of SEQ ID NO: 374

[0050] FR3 having the amino acid sequence of SEQ ID NO: 375

[0051] FR4 having the amino acid sequence of SEQ ID NO:376.

[0052] In a second aspect, the present disclosure provides an antigen binding molecule, which is optionally isolated, that binds to IL-21Rα, wherein the antigen binding molecule comprises a single domain antibody sequence incorporating the following CDRs:

[0053] (3DAS82) CDR1 having the amino acid sequence of SEQ ID NO: 378

[0054] CDR2 having the amino acid sequence of SEQ ID NO:379

[0055] CDR3 having the amino acid sequence of SEQ ID NO:380;

[0056] (2DAS6) CDR1 having the amino acid sequence of SEQ ID NO:386

[0057] CDR2 having the amino acid sequence of SEQ ID NO:387

[0058] CDR3 having the amino acid sequence of SEQ ID NO:388; or

[0059] (2DAS92) CDR1 having the amino acid sequence of SEQ ID NO:394

[0060] CDR2 having the amino acid sequence of SEQ ID NO:395

[0061] CDR3 having the amino acid sequence of SEQ ID NO:396.

[0062] In some embodiments, the antigen binding molecule that binds to IL-21Ra comprises or consists of an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 385 (3DAS82), 393 (2DAS6), or 401 (2DAS92). In some embodiments, the antigen binding molecule comprises an amino acid sequence having at least one of: >70%, e.g., >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 385 (3DAS82), 393 (2DAS6), or 401 (2DAS92).

[0063] In some embodiments, the antigen binding molecule that binds to IL-21Ra comprises a single domain antibody sequence incorporating the following FRs:

[0064] (3DAS82) FR1 having the amino acid sequence of SEQ ID NO: 381

[0065] FR2 having the amino acid sequence of SEQ ID NO: 382

[0066] FR3 having the amino acid sequence of SEQ ID NO: 383

[0067] FR4 having the amino acid sequence of SEQ ID NO: 384;

[0068] (2DAS6) FR1 having the amino acid sequence of SEQ ID NO: 389

[0069] FR2 having the amino acid sequence of SEQ ID NO: 390

[0070] FR3 having the amino acid sequence of SEQ ID NO: 391

[0071] FR4 having the amino acid sequence of SEQ ID NO: 392; or

[0072] (2DAS92) FR1 having the amino acid sequence of SEQ ID NO: 397

[0073] FR2 having the amino acid sequence of SEQ ID NO: 398

[0074] FR3 having the amino acid sequence of SEQ ID NO: 399

[0075] FR4 having the amino acid sequence of SEQ ID NO: 400.

[0076] In some embodiments, the antigen binding molecule is a multispecific antigen binding molecule and further comprises an antigen binding moiety that binds to an antigen other than IL-21Ra. In some embodiments, the antigen other than IL-21Ra is yc.

[0077] In a third aspect, the present disclosure provides an antigen binding molecule, optionally isolated, that binds to yc, wherein the antigen binding molecule comprises a single domain antibody sequence incorporating the following CDRs:

[0078] CDR1 having the amino acid sequence of SEQ ID NO: 370

[0079] CDR2 having the amino acid sequence of SEQ ID NO: 371

[0080] CDR3 having the amino acid sequence of SEQ ID NO: 372.

[0081] In some embodiments, the antigen binding molecule that binds to yc comprises or consists of an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 377. In some embodiments, the antigen binding molecule comprises a single domain antibody sequence having at least one of: >70%, e.g., >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 377.

[0082] In some embodiments, the antigen binding molecule that binds to yc comprises a single domain antibody sequence incorporating the following FRs:

[0083] FR1 having the amino acid sequence of SEQ ID NO: 373

[0084] FR2 having the amino acid sequence of SEQ ID NO: 374

[0085] FR3 having the amino acid sequence of SEQ ID NO: 375

[0086] FR4 having the amino acid sequence of SEQ ID NO: 376.

[0087] In some embodiments, the antigen binding molecule that binds to yc is a multispecific antigen binding molecule, and wherein the antigen binding molecule further comprises an antigen binding moiety that binds to an antigen other than yc. In some embodiments, the antigen other than yc is IL-21Ra.

[0088] In some embodiments, the antigen binding molecule that binds to IL-21Ra or the antigen binding molecule that binds to yc is a bispecific antigen binding molecule.

[0089] In some embodiments of each aspect of the present disclosure, the antigen binding molecule further comprises: (iii) an antigen binding moiety that binds to a target antigen other than the yc-containing cytokine receptor polypeptide.

[0090] In some embodiments, the target antigen other than the yc-containing cytokine receptor polypeptide is a disease-associated antigen or an antigen expressed by an immune cell.

[0091] The present disclosure also provides a chimeric antigen receptor (CAR) comprising an antigen binding molecule according to the present disclosure.

[0092] 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.

[0093] The present disclosure also provides an expression vector or a plurality of expression vectors comprising a nucleic acid or a plurality of nucleic acids according to the present disclosure.

[0094] 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 a plurality of nucleic acids according to the present disclosure, or an expression vector or a plurality of expression vectors according to the present disclosure.

[0095] 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.

[0096] 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 a plurality of nucleic acids according to the present disclosure, an expression vector or a 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.

[0097] 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 a plurality of nucleic acids according to the present disclosure, an expression vector or a 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.

[0098] 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 a plurality of nucleic acids according to the present disclosure, an expression vector or a 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.

[0099] 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 a plurality of nucleic acids according to the present disclosure, an expression vector or a plurality of expression vectors according to the present disclosure, a cell according to the present disclosure, or a composition according to the present disclosure.

[0100] 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.

[0101] 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 cavity 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.

[0102] 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.

[0103] The present disclosure also provides a method for producing 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.

[0104] 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.

[0105] The present disclosure also provides a method for reducing the number / proportion of (e.g., depleting or increasing depletion 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 or a CAR according to the present disclosure.

[0106] The present disclosure also provides a method for reducing 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.

[0107] In some embodiments, the cell is an effector immune cell.

[0108] In some embodiments, the cell is a T cell or an NK cell.

[0109] In some embodiments, the yc-containing cytokine receptor is a yc:IL-21R alpha receptor.

[0110] The present disclosure also provides a method of promoting heteromultimerization of a polypeptide of a yc and yc-containing cytokine receptor complex (e.g., IL-21Ra) comprising contacting the polypeptide of a yc and yc-containing cytokine receptor complex (e.g., IL-21Ra) 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.

[0111] The present disclosure also provides a method of inhibiting heteromultimerization of a polypeptide of a yc and yc-containing cytokine receptor complex (e.g., IL-21Ra) comprising contacting the polypeptide of a yc and yc-containing cytokine receptor complex (e.g., IL-21Ra) 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.

[0112] The present disclosure includes combinations of the aspects and preferred features described, except where such a combination is clearly impermissible or clearly to be avoided. BRIEF DESCRIPTION OF DRAWINGS

[0113] Embodiments illustrating the principles of the application will now be discussed with reference to the accompanying drawings, in which:

[0114] Figure 1 . Dose-dependent STAT5 phosphorylation by agonist bispecific yc and IL-2Rβ binding antibodies. STAT5 phosphorylation was assessed in different leukocyte subpopulations. The percentage of phosphorylated STAT5 (pSTAT5) was measured after stimulation with different concentrations of bispecific yc and CD122 binding antibodies. High efficient stimulation of CD4+, CD8+ and NK cells was shown, but no stimulation of ISO controls.

[0115] Figure 2 . Agonist bispecific yc 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 yc and CD122 binding antibodies (Adk-1 or Adk-2). Treatment including agonist bispecific yc and CD122 binding antibodies resulted in a significant reduction of absolute tumor cell counts.

[0116] Figure 3 . Agonist bispecific yc and IL-2Rβ binding antibodies stimulate T cell proliferation in non-human primates in vivo. Expression of the proliferation marker Ki67 was used as a pharmacodynamic marker of immunostimulation in T cells. CD8 T cell proliferation occurred as early as 24 hours and persisted until 120 hours post-dose.

[0117] Figure 4Schematic of 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 QUANTI-Blue assay. Different steps highlighted in (A) agonist assay and (B) antagonist assay.

[0118] Figure 5 Bispecific yc and IL-21Ra binding antibodies modulate yc:IL-21Ra receptor 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.

[0119] Figure 6 Bispecific yc and IL-21Ra binding antibodies modulate yc:IL-21Ra receptor mediated signaling. Antagonist bispecific yc and IL-21Ra binding antibodies inhibit STAT5 phosphorylation. DETAILED DESCRIPTION

[0120] The present disclosure encompasses nucleotide and amino acid sequences of antigen binding molecules specific for common gamma chain (yc; CD132) and IL-21Ra (CD360). In some embodiments, the antigen binding molecules comprise a yc binding portion and a portion that binds to IL-21Ra (CD360).

[0121] In one aspect, the present disclosure describes the design of cytokine receptor agonists, wherein receptor activation is achieved by multispecific antigen binding molecules (e.g., bispecific antibodies or bifunctional proteins) that have anti-yc specificity and specificity for another polypeptide of a yc-containing cytokine receptor complex, by heterodimerization of the receptor components. The polypeptide of the yc-containing cytokine receptor complex other than yc can be IL-21Ra (CD360).

[0122] In another aspect, the present disclosure describes the design of cytokine receptor antagonists, wherein receptor activation is reduced by multispecific antigen binding molecules (e.g., bispecific antibodies or bifunctional proteins) that have anti-yc specificity and specificity for IL-21Ra (CD360), by inhibition of heterodimerization of the receptor components.

[0123] The antigen binding molecules of the present disclosure have beneficial properties that overcome the drawbacks and problems associated with therapeutic administration of cytokines or engineered cytokines (e.g., pegylated cytokines and antibody conjugated cytokines).

[0124] 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 lead to problems for patients such as: reduced efficacy, occurrence of anti-drug antibodies, activation of non-optimal signaling pathways, occurrence of adverse events and serious side effects such as vascular leakage syndrome (VLS).

[0125] Antigen binding molecules that bind to yc-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 reaction durability, and can be tailored to individual patients and specific diseases.

[0126] 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 signaling induction can be adjusted to achieve optimal downstream effects.

[0127] Common gamma chain (yc)

[0128] 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 is 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 in their entireties.

[0129] The canonical isoform of human yc (isoform 1) has the amino acid sequence set forth in SEQ ID NO: 437. The N-terminal 22 amino acids of SEQ ID NO: 437 constitute a signal peptide (SEQ ID NO: 503), thus 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) which 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.

[0130] 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 signaling transduction pathways. Signaling through yc family receptors promotes activation, proliferation, and survival of immune cells.

[0131] In this specification, “common gamma chain,” “common gamma,” “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 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 human yc.

[0132] 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.

[0133] A“fragment” generally refers to a portion of a reference protein. A“variant” generally refers to a protein having an amino acid sequence comprising one or more amino acid substitutions, insertions, deletions, or other modifications relative to the amino acid sequence of a reference protein, but retaining 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).

[0134] 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 an 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).

[0135] 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 an 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).

[0136] 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 an 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).

[0137] An isoform, fragment, variant, or homolog can optionally be a functional isoform, fragment, variant, or homolog, e.g., having a functional property / activity of a reference yc (e.g., human yc isoform 1), as determined by analysis in an assay suitable for the functional property or activity. For example, an isoform, fragment, variant, or homolog of yc can exhibit one or more of the following: association with one or more of IL-2R, IL-2R, IL-15R, IL-4R, IL-9R, IL-21R, or IL-7R, or binding to one or more of IL-2, IL-15, IL-4, IL-9, IL-21, or IL-7.

[0138] The minimum length of the fragment of yc can be one of 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, or 350 amino acids, and the maximum length can be one of 20, 30, 40, 50, 100, 150, 200, 250, 300, or 350 amino acids.

[0139] In some embodiments, 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.

[0140] 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.

[0141] Signaling through cytokine receptors comprising yc

[0142] There are many cytokines that signal through cytokine receptors that comprise yc, also referred to herein as yc-containing receptor complexes, for example, 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, for example, 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.

[0143] 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 chain family cytokines.

[0144] 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.

[0145] IL-21 binding stabilizes the complex between IL-21Ra and yc, leading to activation of JAK1 and JAK3, which allows recruitment and phosphorylation of STAT proteins, primarily STAT3, but also STAT1 and STAT5. Binding of IL-21 to IL-21Ra can also activate MAPK and PI3K signaling pathways. IL-21 induces transcription of suppressor of cytokine signaling 1 (SOCS1) and SOCS3 proteins, which downregulate the JAK-STAT pathway.

[0146] In this specification, “yc-containing cytokine receptor-mediated signaling” 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.

[0147] yc-containing cytokine receptor-mediated signaling 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 constituent polypeptides / peptides. In some embodiments, the association includes electrostatic interactions (e.g., ionic bonds, hydrogen bonds) and / or van der Waals forces.

[0148] Gammac-containing cytokine receptor-mediated signaling can be mediated by a heteromultimeric polypeptide complex comprising one or more gammac polypeptides and additionally comprising one or more polypeptides of the gammac receptor family other than gammac (e.g., selected from IL-2R beta, IL-2R alpha, IL-15R alpha, IL-4R alpha, IL-9R alpha, IL-21R alpha, or IL-7R alpha). In some embodiments, gammac-containing cytokine receptor-mediated signaling can be mediated by a polypeptide complex that forms a receptor for a gammac family cytokine. For example, gammac-containing cytokine receptor-mediated signaling can be mediated by a polypeptide complex that forms a receptor for IL-2, IL-4, IL-7, IL-9, IL-15, or IL-21. In some embodiments, gammac-containing cytokine receptor-mediated signaling is mediated by a polypeptide complex that forms a receptor for IL-21.

[0149] In some embodiments, gammac-containing cytokine receptor-mediated signaling can be mediated by a polypeptide complex comprising gammac and IL-21R alpha (i.e., a gammac:IL-21R alpha complex). As explained above, gammac and IL-21R alpha interact to form the IL-21 receptor. This signaling can be referred to as gammac:IL-21R alpha-mediated signaling. In some embodiments, gammac-containing cytokine receptor-mediated signaling can be mediated by a polypeptide complex comprising IL-21, gammac, and IL-21R alpha (i.e., an IL-21:gammac:IL-21R alpha complex). This signaling can be referred to as IL-21:gammac:IL-21R alpha-mediated signaling (i.e., signaling mediated by binding of IL-21 to the IL-21 receptor).

[0150] The present disclosure relates to antigen binding molecules that selectively bind to more than one component of a gammac-containing cytokine receptor.

[0151] Disclosed herein are amino acid sequences of polypeptides of a gammac-containing cytokine receptor other than gammac and domains / fragments thereof (e.g., SEQ ID NOs 245-255 and 258-284).

[0152] In particular, the antigen binding molecules of the present disclosure are multispecific antigen binding molecules comprising (i) a gammac binding moiety, and (ii) a moiety that binds to IL-21R alpha. That is, the antigen binding molecules bind to (i) gammac, and (ii) IL-21R alpha.

[0153] Human IL-21Ra (also known as CD360) is the protein identified by UniProt Q9HBE5. The canonical isoform of human IL-21Ra has the amino acid sequence of SEQ ID NO: 273. The N-terminal 19 amino acids of SEQ ID NO: 273 constitute a signal peptide (SEQ ID NO: 278), thus the mature form of human IL-21Ra (i.e., after processing to remove the signal peptide) has the amino acid sequence set forth in SEQ ID NO: 279. Human IL-21Ra comprises an extracellular domain (SEQ ID NO: 274), a transmembrane domain (SEQ ID NO: 275), and a cytoplasmic domain (SEQ ID NO: 276).

[0154] In the present specification, “IL-21Ra” refers to IL-21Ra from any species, including isoforms, fragments, variants, or homologs from any species. In some embodiments, the IL-21Ra is 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, the IL-21Ra is human IL-21Ra.

[0155] Isoforms, fragments, variants, or homologs of IL-21Ra can exhibit association with yc or IL-21. Fragments of IL-21Ra 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.

[0156] In some embodiments, the IL-21Ra 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, the fragment of IL-21Ra 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.

[0157] Antigen binding molecules

[0158] The present disclosure provides antigen binding molecules that are capable of binding to (i.e., associating with) a yc-containing cytokine receptor and constituent polypeptides thereof.

[0159] In aspects of the disclosure, the antigen binding molecule comprises a yc-binding portion. In aspects of the disclosure, the antigen binding molecule comprises a portion 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 IL-21Ra.

[0160] In some embodiments, the antigen binding molecule comprises an IL-21Ra-binding portion.

[0161] In some embodiments, the antigen binding molecule comprises a yc-binding portion and an IL-21Ra-binding portion.

[0162] As used herein, “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 portions by which the antigen binding molecule binds to a target antigen.

[0163] An antigen binding portion can comprise or can be derived from an antibody (i.e., an immunoglobulin (Ig)) and antigen binding fragments thereof. As used herein, “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.

[0164] Single domain antibodies (sdAbs) - also referred to in the art in various ways as “single variable domains on heavy chain antibodies,” “VHHs,” “nanobodies,” and “heavy chain only antibodies (HcAbs)” - are described, for example, in Henry and MacKenzie, Front Immunol. (2018) 9:41 and Bever et al., Anal Bioanal Chem. (2016) 408(22):5985-6002, both of which are hereby incorporated by reference in their entireties.

[0165] Single domain antibodies are composed of a single monomeric antibody variable domain. The first single domain antibodies were engineered from heavy chain antibodies present in camelids, and cartilaginous fish also have heavy chain antibodies.

[0166] A single domain antibody according to the present disclosure generally comprises three complementarity determining regions, CDRs: CDR1, CDR2, and CDR3. Together, these three CDRs define the paratope of the molecule, which is the portion of the molecule through which it binds to a target antigen.

[0167] A single domain antibody also comprises framework regions (FRs) flanking each CDR, which provide a scaffold for the CDRs. From N-terminus to C-terminus, a single domain antibody comprises the following structure: N-terminus - [FR1] - [CDR1] - [FR2] - [CDR2] - [FR3] - [CDR3] - [FR4] - C-terminus.

[0168] Antigen binding moieties also include target antigen binding aptamers, such as nucleic acid aptamers (reviewed, e.g., in Zhou and Rossi, Nat Rev Drug Discov. (2017) 16(3): 181-202). In some embodiments, an 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) or VHH), affilin, armadillo repeat protein (ArmRP), OBody, or fibronectin— reviewed, e.g., 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).

[0169] In some embodiments, an antigen binding moiety comprises or consists of an antigen binding region of an antibody (e.g., an antigen binding fragment of an antibody). An 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. An antigen binding domain formed by a VH and a VL can also be referred to herein as an Fv region. An antigen binding moiety of an antigen binding molecule of the present disclosure can comprise a single variable domain (VHH) of a single domain antibody (sdAb) that binds to the relevant target antigen of the antigen binding moiety.

[0170] 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).

[0171] 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).

[0172] The 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), such as an IgG-like antigen binding molecule comprising two heavy chain polypeptides and two light chain polypeptides.

[0173] Antigen binding moieties of the present disclosure can be designed and made using sequences of monoclonal antibodies (mAbs) that are capable of binding to a given target antigen (e.g., HER3). Antigen binding regions of antibodies, such as single chain variable fragments (scFv), Fabs, and F(ab’)2 fragments, can also be used / provided. An “antigen binding region” is any fragment of an antibody that binds to the given antibody’s specific target.

[0174] 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. These six CDRs together define the paratope of the antibody, which is the part of the antibody that binds to the target antigen.

[0175] The VH region and the VL region comprise framework regions (FRs) flanking each CDR, which provide a scaffold for the CDRs. From N-terminal to C-terminal, the 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 the VL region comprises the following structure: N-terminal - [LC-FR1] - [LC-CDR1] - [LC-FR2] - [LC-CDR2] - [LC-FR3] - [LC-CDR3] - [LC-FR4] - C-terminal.

[0176] 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 (or VHH) 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.

[0177] 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).

[0178] 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 the 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).

[0179] 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.

[0180] 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).

[0181] 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.

[0182] 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.

[0183] 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 through fifth rows of the table below, the replacement amino acid of the substitution is another non-identical amino acid provided in the same row:

[0184]

[0185] 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.

[0186] 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:

[0187]

[0188] That is, in some embodiments, a nonpolar amino acid is substituted with another nonpolar amino acid that is not identical. In some embodiments, a polar amino acid is substituted with another polar amino acid that is not identical. In some embodiments, an acidic polar amino acid is substituted with another acidic polar amino acid that is not identical. In some embodiments, a basic polar amino acid is substituted with another basic polar amino acid that is not identical. In some embodiments, an uncharged amino acid is substituted with another uncharged amino acid that is not identical. In some embodiments, a positive amino acid is substituted with another positive amino acid that is not identical. In some embodiments, a negative amino acid is substituted with another negative amino acid that is not identical.

[0189] 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.

[0190] 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 or consists of a VH region and a VL region of an antibody that binds to yc, or a VHH sequence of a single domain 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 (described, e.g., in Floch et al., Hemasphere. (2022) 6(Suppl):694-695), TUGh4 (described, e.g., in Ishii et al., Int Immunol. (1994) 6(8): 1273-1277), and 3E12 (described, e.g., in He et al., Proc Natl Acad Sci U S A. (1995) 92(12):5689-5693). The single domain antibody (VHH) nb6 has also been shown to bind to yc (Yen et al., Cell. 2022; 185(8): 1414-1430). The present disclosure provides other single domain antibodies (VHH) that bind to yc (see, e.g., the clones listed in Table A2).

[0191] 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 the CDRs of an antibody that binds to IL-21Ra. In some embodiments, the antigen binding moiety comprises the FRs of an antibody that binds to IL-21Ra. In some embodiments, the antigen binding moiety comprises the CDRs and FRs of an antibody that binds to IL-21Ra. That is, in some embodiments, the antigen binding moiety comprises or consists of a VH region and a VL region of an antibody that binds to IL-21Ra, or a VHH sequence of a single domain 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, comprising a VH region and a VL region. Antibodies that bind to IL-21Ra include, for example, ATR-107 (described, e.g., in Hua et al., J Clin Pharmacol. (2014) 54(1): 14-22) and 4A9 (described, e.g., in Jin and Malek. J Leukoc Biol. (2006) 80(6): 1416-1423). The present disclosure provides other single domain antibodies (VHH) that bind to IL-21Ra (see, e.g., Tables A3 and B3).

[0192] 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., IL-21Ra). 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 decaspecific.

[0193] In some embodiments, the multispecific antigen binding molecules described herein exhibit at least monovalent binding to yc and also exhibit at least monovalent binding to a polypeptide other than yc of a yc-containing cytokine receptor (e.g., IL-21Ra). 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 scFv-KiH-Fc, CrossMab, and Duobody formats that exhibit monovalent binding to yc and also monovalent binding to IL-2Rß. Additionally, described herein are bispecific antigen binding molecules in the tandem VHH format that exhibit monovalent binding to yc and also monovalent binding to IL-21Ra.

[0194] 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 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 VHH-VHH, tandem scFv-VHH, triabody, triplehead, 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 Figure 2 . See also Brinkmann and Kontermann, MAbs. (2017) 9(2): 182-212 (incorporated by reference herein in its entirety), particularly Figure 2 . In some embodiments, the multispecific antigen-binding molecule is a simple bispecific antibody, such as a tandem scFv-scFv, tandem VHH-VHH, or tandem VHH-scFv antibody.

[0195] Tandem multispecific antigen-binding molecules (e.g., tandem VHHs) 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, and tandem VHH-scFv. In some embodiments, the multispecific antigen-binding molecule is provided in a tandem VHH format, such as tandem VHH-VHH.

[0196] 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-terminal to C-terminal), or VHH-linker-VHH. Thus, the different binding moieties can be combined in a variety of different ways. For example, two scFv molecules or two VHH molecules can be combined in a variety of different orientations. For example, a P2C4 scFv can be combined with a 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. Two VHH molecules (VHH1 and VHH2) can be combined at least in the following orientations (from N-terminal to C-terminal): (i) VHH1-linker-VHH2, and (ii) VHH2-linker-VHH1.

[0197] In some embodiments, the multispecific antigen-binding molecule comprises a linker between the binding moieties, e.g., a linker between a yc binding moiety and a moiety that binds to a polypeptide other than yc of a yc-containing cytokine receptor (e.g., an IL-21R a 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).

[0198] 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.

[0199] In some embodiments, the flexible linker is enriched 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, for example 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: 240) amino acid sequence. In some embodiments, the linker comprises or consists of the GGGGSGGGS (SEQ ID NO: 361) amino acid sequence. In some embodiments, the linker comprises or consists of the (G4S)3 (SEQ ID NO: 362) amino acid sequence. In some embodiments, the linker comprises or consists of the GGGSG (SEQ ID NO: 363) amino acid sequence.

[0200] 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.

[0201] In some embodiments, the linker is a rigid linker. In some embodiments, the rigid linker forms an alpha helical 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 herein by reference 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 amino acid sequence A(EAAAK)5A (SEQ ID NO: 360).

[0202] In some embodiments, the linker comprises the amino acid sequence EAAAK (SEQ ID NO: 364). In some embodiments, the linker comprises or consists of the amino acid sequence A(EAAAK)2A (SEQ ID NO: 365). In some embodiments, the linker comprises or consists of the amino acid sequence A(EAAAK)3A (SEQ ID NO: 366). In some embodiments, the linker comprises or consists of the amino acid sequence A(EAAAK)4A (SEQ ID NO: 367). In some embodiments, the linker comprises or consists of the amino acid sequence A(EAAAK)5A (SEQ ID NO: 360).

[0203] In some embodiments, the length of the linker is at least 3 amino acids. In some embodiments, the maximum length of the linker is 50 amino acids. In some embodiments, the minimum length of the linker is 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 some embodiments, the maximum length of a linker is 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.

[0204] 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.

[0205] 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 40 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.

[0206] 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.

[0207] 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.

[0208] In some embodiments, the length of 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.

[0209] In some embodiments, the length of the flexible linker is 3 to 12 amino acids. In some embodiments, the length of the short flexible linker is 3 to 10 amino acids. In some embodiments, the length of the short flexible linker is 3 to 8 amino acids. In some embodiments, the length of the short flexible linker is 3 to 6 amino acids.

[0210] In some embodiments, the length of the rigid linker is 10 to 44 amino acids. In some embodiments, the length of the long rigid linker is 12 to 44 amino acids. In some embodiments, the length of the long rigid linker is 17 to 44 amino acids. In some embodiments, the length of the long rigid linker is 22 to 44 amino acids. In some embodiments, the length of the long rigid linker is 27 to 44 amino acids.

[0211] 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.

[0212] Methods for producing multispecific antigen binding molecules include chemical cross-linking of antigen binding molecules or antibody fragments, for example with reducible disulfide bonds 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)2heterodimers.

[0213] Other methods for producing multispecific antigen-binding molecules include fusing antibody-producing hybridomas, for example, with polyethylene glycol to produce a cell hybrid hybridoma cell that is capable of secreting a bispecific antibody, for example, as described in Segal and Bast, (2001) Current Protocols in Immunology. Chapter 2:2.13.1-2.13.16.

[0214] The multispecific antigen-binding molecules according to the present disclosure can also be produced recombinantly, for example, by expression of nucleic acid constructs encoding the polypeptides of the antigen-binding molecules, 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.

[0215] 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 constructs can be expressed (e.g., in vitro) in a suitable host cell (e.g., a mammalian host cell) to produce recombinant bispecific antibodies, which can then be optionally purified.

[0216] In some embodiments, the yc-binding portion of the present disclosure comprises one or more polypeptides comprising a VH region containing heavy chain CDRs and a VL region containing light chain CDRs selected from the 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 A1 herein.

[0217] In some embodiments, the 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 the 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.

[0218] In some embodiments, the 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 the 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.

[0219] 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).

[0220] 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 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.

[0221] 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 for one of binding moieties B1-1 to B1-27 in column A of Table B1, 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.

[0222] 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 for one of binding moieties B1-1 to B1-27 in column A of Table B1, 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.

[0223] In some embodiments, the yc-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 B1, 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 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).

[0224] 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.

[0225] 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.

[0226] 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.

[0227] In some embodiments, the 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 shown 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 shown 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).

[0228] In some embodiments, the yc-binding moiety of the present disclosure comprises a single domain antibody sequence incorporating the CDRs of 2RGT38, as shown in Table A2 herein.

[0229] In some embodiments, the yc-binding moiety comprises a single domain antibody sequence incorporating a CDR1, a CDR2, and a CDR3, as shown in 2RGT38 in Table A2, optionally wherein 1 or 2 or 3 amino acids in CDR1, and / or 1 or 2 or 3 amino acids in CDR2, and / or 1 or 2 or 3 amino acids in CDR3 are substituted with another amino acid.

[0230] In some embodiments, the yc-binding moiety of the present disclosure comprises a single domain antibody sequence comprising the FRs of 2RGT38, as shown in Table B2 herein.

[0231] In some embodiments, the yc-binding moiety comprises a single domain antibody sequence comprising a FR1, a FR2, a FR3, and a FR4, as shown in one of the binding moieties in Table B2, optionally wherein 1 or 2 or 3 amino acids in the FR1, and / or 1 or 2 or 3 amino acids in the FR2, and / or 1 or 2 or 3 amino acids in the FR3, and / or 1 or 2 or 3 amino acids in the FR4 are substituted with another amino acid.

[0232] In some embodiments, the yc-binding moiety of the present disclosure comprises a single domain antibody sequence comprising the VHH sequence of 2RGT38, as shown in Table A2 herein.

[0233] In some embodiments, the yc-binding moiety comprises a single domain antibody sequence 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 the amino acid sequence shown in column A of Table A2. That is, in some embodiments, the yc-binding moiety comprises a single domain antibody sequence 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 SEQ ID NO: 377.

[0234] In some embodiments, the IL-21Ra-binding moiety of the present disclosure comprises a single domain antibody sequence incorporating the CDRs of a clone selected from the group consisting of 2DAS92, 3DAS82, and 2DAS6, as shown in Table A3 herein.

[0235] In some embodiments, the IL-21Ra-binding moiety comprises a single domain antibody sequence incorporating a CDR1, a CDR2, and a CDR3, as shown in one of the binding moieties in Table A3, optionally wherein 1 or 2 or 3 amino acids in the CDR1, and / or 1 or 2 or 3 amino acids in the CDR2, and / or 1 or 2 or 3 amino acids in the CDR3 are substituted with another amino acid.

[0236] In some embodiments, the IL-21Ra-binding moiety of the present disclosure comprises a single domain antibody sequence incorporating the FRs of a clone selected from the group consisting of 2DAS92, 3DAS82, and 2DAS6, as shown in Table B3 herein.

[0237] In some embodiments, the IL-21Rα binding portion comprises a single domain antibody sequence comprising FR1, FR2, FR3, and FR4 as shown in one of the binding portions in Table B3, optionally wherein one or two or three amino acids in FR1, and / or one or two or three amino acids in FR2, and / or one or two or three amino acids in FR3, and / or one or two or three amino acids in FR4 are substituted with another amino acid.

[0238] In some embodiments, an IL-21Rα binding portion of the present disclosure comprises a single domain antibody sequence comprising a cloned VHH sequence selected from: and , as shown in Table A3 herein.

[0239] In some embodiments, the IL-21Rα portion comprises a single domain antibody sequence having at least 70%, e.g., ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% amino acid sequence identity to the amino acid sequence shown in column A of Table A3.

[0240] In some embodiments, an antigen binding molecule according to the present disclosure comprises: (i) a γc binding portion according to the embodiments described herein, and (ii) an IL-21Rα binding portion according to the embodiments described herein.

[0241] 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 an 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) heavy chain constant sequence. 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).

[0242] It will be appreciated that, depending on the modification of the Fc region of the antigen binding molecules as described herein, further substitutions may be provided to the CH2 and / or CH3 regions.

[0243] 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.

[0244] In one aspect, the present disclosure provides an antigen binding molecule that binds to yc and comprises a single domain antibody sequence incorporating the CDRs of 2RGT38, as shown in Table A2 herein.

[0245] In some embodiments, the antigen binding molecules that bind to yc comprise a single domain antibody sequence incorporating a CDR1, a CDR2, and a CDR3, as shown in the binding portions in Table A2, optionally wherein 1 or 2 or 3 amino acids in CDR1, and / or 1 or 2 or 3 amino acids in CDR2, and / or 1 or 2 or 3 amino acids in CDR3 are substituted with another amino acid.

[0246] In some embodiments, the antigen binding molecules that bind to yc comprise a single domain antibody sequence comprising the FRs of 2RGT38, as shown in Table B2 herein.

[0247] In some embodiments, the antigen binding molecules comprise a single domain antibody sequence comprising a FR1, a FR2, a FR3, and a FR4, as shown in one of the clones in Table B2, optionally wherein 1 or 2 or 3 amino acids in FR1, and / or 1 or 2 or 3 amino acids in FR2, and / or 1 or 2 or 3 amino acids in FR3, and / or 1 or 2 or 3 amino acids in FR4 are substituted with another amino acid.

[0248] In some embodiments, the antigen binding molecules comprise or consist of a single domain antibody sequence comprising the CDRs and FRs of clone 2RGT38, as shown in Table A2 and Table B2.

[0249] In some embodiments, the antigen binding molecules comprise a single domain antibody sequence comprising the VHH sequence of 2RGT38, as shown in Table A2 herein.

[0250] In some embodiments, the antigen binding molecule comprises a single domain antibody sequence having at least 70%, e.g., >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99%, or 100% identity of an amino acid sequence to an amino acid sequence set forth in Column A of Table A2. That is, in some embodiments, the antigen binding molecule comprises a single domain antibody sequence having at least 70%, e.g., >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99%, or 100% identity of an amino acid sequence to SEQ ID NO:377.

[0251] In another aspect, the present application provides an antigen binding molecule that binds to IL-21Ra and comprises a single domain antibody sequence that incorporates CDRs selected from the clone of: 2DAS92, 3DAS82, and 2DAS6, as set forth in Table A3 herein.

[0252] In some embodiments, the antigen binding molecule that binds to IL-21Ra comprises a single domain antibody sequence that incorporates CDR1, CDR2, and CDR3, as set forth in one of the binding portions in Table A3, optionally wherein 1 or 2 or 3 amino acids in CDR1, and / or 1 or 2 or 3 amino acids in CDR2, and / or 1 or 2 or 3 amino acids in CDR3 are substituted with another amino acid.

[0253] In some embodiments, the antigen binding molecule that binds to IL-21Ra of the present disclosure comprises a single domain antibody sequence that comprises FRs selected from the clone of: 2DAS92, 3DAS82, and 2DAS6, as set forth in Table B3 herein.

[0254] In some embodiments, the antigen binding molecule that binds to IL-21Ra comprises a single domain antibody sequence that comprises FR1, FR2, FR3, and FR4, as set forth in one of the binding portions in Table B3, optionally wherein 1 or 2 or 3 amino acids in FR1, and / or 1 or 2 or 3 amino acids in FR2, and / or 1 or 2 or 3 amino acids in FR3, and / or 1 or 2 or 3 amino acids in FR4 are substituted with another amino acid.

[0255] In some embodiments, the antigen binding molecule comprises, or consists of, a single domain antibody sequence comprising CDRs and FRs selected from the following clones: 2DAS92, 3DAS82, and 2DAS6, as shown in Table A3 and Table B3.

[0256] In some embodiments, the antigen binding molecule that binds to IL-21Ra comprises a single domain antibody sequence comprising a VHH sequence selected from the following clones: 2DAS92, 3DAS82, and 2DAS6, as shown in Table A3 herein.

[0257] In some embodiments, the antigen binding molecule that binds to IL-21Ra comprises a single domain antibody sequence having at least 70%, e.g., > 80%, > 85%, > 90%, > 91%, > 92%, > 93%, > 94%, > 95%, > 96%, > 97%, > 98%, > 99%, or 100% identity to an amino acid sequence shown in Column A of Table A3.

[0258] It will be appreciated that antigen binding molecules comprising a yc binding moiety, and IL-21Ra binding moieties according to the present disclosure, can have a yc binding moiety comprising a single domain antibody sequence as defined above and / or an IL-21Ra binding moiety comprising a single domain antibody sequence as defined above. For example, in some embodiments, the yc binding moiety comprises a single domain antibody sequence having the CDRs of clone 2RGT38, and the IL-21Ra binding moiety comprises a single domain antibody sequence having the CDRs of clone 2DAS92. Exemplary combinations of yc binding moieties and IL-21Ra binding moieties in antigen binding molecules according to the present disclosure are shown in Table A4.

[0259] Fc region

[0260] In some embodiments, the antigen binding molecule of the present disclosure comprises an Fc region.

[0261] In some embodiments, the antigen binding molecule of the present disclosure does not comprise an Fc region.

[0262] 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.

[0263] 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.

[0264] 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).

[0265] 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.

[0266] 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.

[0267] 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).

[0268] 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.

[0269] 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.

[0270] 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.

[0271] 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.

[0272] 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.

[0273] 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.

[0274] 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.

[0275] 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.

[0276] 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.

[0277] 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.

[0278] 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.

[0279] 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.

[0280] 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.

[0281] 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.

[0282] 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.

[0283] 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.

[0284] 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.

[0285] 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.

[0286] 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.

[0287] 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.

[0288] In some embodiments, the Fc region comprises a modification corresponding to the substitution combination L234A / L235A / P329G.

[0289] In some embodiments, the Fc region comprises a modification corresponding to the substitution combination L234A / L235A / P329G / N297Q.

[0290] In some embodiments, the Fc region comprises a modification corresponding to the substitution combination L234A / L235E / G237A / A330S / P331S.

[0291] In some embodiments, the Fc region comprises a modification corresponding to the substitution S228P in, e.g., IgG4.

[0292] 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).

[0293] 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.

[0294] In some embodiments, the antigen binding molecule of the present disclosure comprises an Fc region comprising a pair of substitutions in the CH3 region of the Fc region according to one of the following formats 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.

[0295] 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.

[0296] 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.

[0297] 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).

[0298] 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.

[0299] 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.

[0300] 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.

[0301] 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.

[0302] 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 IgG1 CH3 and IgA CH3 are exchanged.

[0303] 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).

[0304] 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).

[0305] 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).

[0306] 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).

[0307] 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).

[0308] 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.

[0309] In some embodiments, the 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, the CH3 region comprising a knob modification comprises 366W. In some embodiments, the knob modification is or includes T366W.

[0310] 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 comprises 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 comprises Y407V, T366S, and L368A.

[0311] In some embodiments, the antigen binding molecules of the present disclosure comprise a CH3 region comprising 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.

[0312] 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.

[0313] Additional antigen binding moieties

[0314] In some embodiments, the antigen binding molecules comprise an additional antigen binding moiety. In some embodiments, the additional antigen binding moiety binds to a target antigen other than a polypeptide of a gamma c-containing cytokine receptor (e.g., a target antigen that is not gamma c or IL-21R alpha). That is, in some embodiments, the antigen binding molecules of the present disclosure comprise (i) a gamma c binding moiety, (ii) a moiety that binds to IL-21R alpha, and (iii) a moiety that binds to a target antigen (e.g., an antigen that is not a polypeptide of a gamma c-containing cytokine receptor).

[0315] It will be appreciated that the role of part (iii) is to localise the antigen binding molecule to a cell expressing its target. This can be used to direct the effects of parts (i) and (ii) of the antigen binding molecule to a cell expressing the target of part (iii). By way of illustration, in embodiments in which part (ii) is an IL-21Ra binding moiety and in which part (iii) is a CD8 binding moiety, the role of part (iii) is to target the yc:IL-21Ra receptor agonist / antagonist activity conferred by parts (i) and (ii) to CD8+ T cells.

[0316] Part (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 parts (i) and (ii) of the antigen binding molecule to such organs. By way of illustration, in embodiments in which part (ii) is an IL-21Ra binding moiety and in which part (iii) is a cancer cell antigen binding moiety, the role of part (iii) is to target the yc:IL-2Rβ receptor agonist / antagonist activity conferred by parts (i) and (ii) to cells expressing the yc:IL-21Ra receptor in the vicinity of cells expressing the cancer cell antigen.

[0317] Thus, it will be appreciated that part (iii) is used to target / localise the antigen binding molecule to a cell comprising / expressing the target antigen of part (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 part (iii).

[0318] The target of part (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.

[0319] In some embodiments, the target antigen is a disease-associated antigen or an antigen expressed by an immune cell.

[0320] A “disease-associated antigen” refers to an antigen whose presence is indicative of a given disease / disease state, or an antigen whose levels are 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 cause or pathology of a disease, or can be abnormally 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.

[0321] 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.

[0322] 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 an 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 severity of symptoms 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.

[0323] 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.

[0324] 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.

[0325] 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.

[0326] In some embodiments, the target antigen is selected from the group consisting of PD-1, 4-1BB, and CD8.

[0327] Chimeric antigen receptor (CAR)

[0328] 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.

[0329] 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.

[0330] 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.

[0331] 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.

[0332] 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.

[0333] 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.

[0334] 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.

[0335] 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.

[0336] Functional properties

[0337] 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:

[0338] binds to a polypeptide other than yc of a yc-containing cytokine receptor;

[0339] binds to a polypeptide other than yc of a yc-containing cytokine receptor;

[0340] binds to IL-21 Ra;

[0341] binds to a polypeptide other than yc of a yc and yc-containing cytokine receptor;

[0342] binds to yc and IL-21 Ra;

[0343] binds to a cell expressing yc;

[0344] binds to a cell expressing a polypeptide other than yc of a yc and yc-containing cytokine receptor;

[0345] binds to a cell expressing IL-21 Ra;

[0346] binds to a cell expressing yc and a polypeptide other than yc of a yc and yc-containing cytokine receptor;

[0347] binds to a cell expressing yc and IL-21 Ra;

[0348] binds to a cell expressing a receptor comprising yc and a polypeptide other than yc of a yc and yc-containing cytokine receptor;

[0349] binds to a cell expressing a yc:IL-21 Ra receptor;

[0350] increases multimerization of a polypeptide other than yc of a yc and yc-containing cytokine receptor (e.g., IL-21 Ra);

[0351] increases multimerization of yc and IL-21 Ra;

[0352] decreases multimerization of a polypeptide other than yc of a yc and yc-containing cytokine receptor (e.g., IL-21 Ra);

[0353] decreases multimerization of yc and IL-21 Ra;

[0354] increases signaling mediated by a yc-containing cytokine receptor (e.g., yc:IL-21 Ra receptor) bound by the antigen binding molecule;

[0355] increases signaling mediated by a yc:IL-21 Ra receptor;

[0356] decreases signaling mediated by a yc-containing cytokine receptor (e.g., yc:IL-21 Ra receptor) bound by the antigen binding molecule;

[0357] decreases signaling mediated by a yc:IL-21 Ra receptor;

[0358] increasing proliferation, survival, and / or effector activity of a cell expressing a gamma c-containing cytokine receptor (e.g., a gamma c:IL-21R alpha receptor) bound by the antigen binding molecule;

[0359] increasing proliferation, survival, and / or effector activity of a cell expressing a gamma c:IL-21R alpha receptor;

[0360] decreasing proliferation, survival, and / or effector activity of a cell expressing a gamma c-containing cytokine receptor (e.g., a gamma c:IL-21R alpha receptor) bound by the antigen binding molecule;

[0361] decreasing proliferation, survival, and / or effector activity of a cell expressing a gamma c:IL-21R alpha receptor;

[0362] decreasing expression of one or more immune cell exhaustion markers by a cell expressing a gamma c-containing cytokine receptor (e.g., a gamma c:IL-21R alpha receptor) bound by the antigen binding molecule;

[0363] decreasing expression of one or more immune cell exhaustion markers by a cell expressing a gamma c:IL-21R alpha receptor;

[0364] increasing expression of one or more immune cell exhaustion markers by a cell expressing a gamma c-containing cytokine receptor (e.g., a gamma c:IL-21R alpha receptor) bound by the antigen binding molecule;

[0365] increasing expression of one or more immune cell exhaustion markers by a cell expressing a gamma c:IL-21R alpha receptor;

[0366] decreasing multimerization of a gamma c and a polypeptide of a gamma c-containing cytokine receptor other than gamma c (e.g., IL-21R alpha), decreasing signaling mediated by a gamma c-containing cytokine receptor (e.g., a gamma c:IL-21R alpha receptor) bound by the antigen binding molecule, and / or decreasing proliferation, survival, and / or effector activity of a cell expressing a gamma c-containing cytokine receptor (e.g., a gamma c:IL-21R alpha receptor) bound by the antigen binding molecule independent of Fc-mediated function;

[0367] decreasing multimerization of a gamma c and IL-21R alpha, decreasing signaling mediated by a gamma c:IL-21R alpha receptor independent of Fc-mediated function, and / or decreasing proliferation, survival, and / or effector activity of a cell expressing a gamma c:IL-21R alpha receptor;

[0368] increasing killing / depletion of, and / or decreasing the number / proportion of, a cell comprising / expressing one or more target antigens of a constituent antigen binding moiety of the antigen binding molecule (i.e., a gamma c, and / or a polypeptide of a gamma c-containing cytokine receptor other than gamma c (e.g., IL-21R alpha));

[0369] increased cell killing / elimination of, and / or decreased number / proportion of, cells comprising / expressing yc and / or IL-21Ra;

[0370] 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);

[0371] increased stability and / or half-life compared to IL-21;

[0372] increased upregulation of signaling mediated by yc:IL-21Ra receptor bound by the antigen binding molecule compared to a cytokine that binds to a yc-containing cytokine receptor;

[0373] increased upregulation of signaling mediated by yc:IL-21Ra receptor compared to IL-21;

[0374] decreased upregulation of signaling mediated by yc:IL-21Ra receptor bound by the antigen binding molecule compared to a cytokine that binds to a yc-containing cytokine receptor;

[0375] decreased upregulation of signaling mediated by yc:IL-21Ra receptor compared to IL-21;

[0376] increased upregulation of proliferation, survival, and / or effector activity of cells expressing yc:IL-21Ra receptor bound by the antigen binding molecule compared to a cytokine that binds to a yc-containing cytokine receptor;

[0377] decreased upregulation of signaling mediated by yc:IL-21Ra receptor compared to IL-21;

[0378] decreased upregulation of proliferation, survival, and / or effector activity of cells expressing yc:IL-21Ra receptor bound by the antigen binding molecule compared to a cytokine that binds to a yc-containing cytokine receptor;

[0379] decreased upregulation of proliferation, survival, and / or effector activity of cells expressing yc:IL-21Ra receptor compared to IL-21;

[0380] decreased downregulation of expression of one or more immune cell exhaustion markers by cells expressing yc:IL-21Ra receptor bound by the antigen binding molecule compared to a cytokine that binds to a yc-containing cytokine receptor;

[0381] compared to IL-21, the downregulation of expression of one or more immune cell exhaustion marker by cells expressing the yc:IL-21Ra receptor is increased;

[0382] compared to a cytokine that binds to a yc-containing cytokine receptor, the downregulation of expression of one or more immune cell exhaustion marker by cells expressing the yc-containing cytokine receptor (e.g. the yc:IL-21Ra receptor) to which the antigen binding molecule binds is reduced;

[0383] compared to IL-21, the downregulation of expression of one or more immune cell exhaustion marker by cells expressing the yc:IL-21Ra receptor is reduced; and / or

[0384] enhancing the anti-cancer activity of a cancer antigen-specific immune cell, e.g. in vivo.

[0385] It will be appreciated that a given antigen binding molecule can exhibit more than one of the properties listed in the preceding paragraphs. The properties listed in the preceding paragraphs for a given antigen binding molecule can be assessed 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 for antigen binding molecules having the desired functional properties.

[0386] 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 labelled with a detectable entity to facilitate its detection. The assay can comprise evaluating the listed properties following treatment of cells with a range of amounts / concentrations (e.g. a dilution series) of a given antigen binding molecule alone. It will be appreciated that the cells preferably express the target antigen of the antigen binding molecule.

[0387] 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 ”. By way of illustration, the EC 50 of a given antigen binding molecule for 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.

[0388] Depending on the property, the EC 50 may also be referred to as the “half maximal inhibitory concentration” or “IC 50The "IC50" is the concentration of a test agent that induces a 50% inhibition of the maximum level of a given property observed.

[0389] In the context of comparing functional properties of agents (e.g., in the context of comparing antigen binding molecules of the present disclosure to other polypeptides), comparisons are made at equivalent concentrations and / or amounts of the relevant agents.

[0390] The antigen binding molecules and antigen binding moieties described herein preferably exhibit specific binding to yc and / or IL-21Ra. 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 / moiety that specifically binds to a target molecule preferably binds the target with greater affinity and / or for a longer duration than it binds to other non-target molecules.

[0391] 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 that is detected in a given assay.

[0392] In some embodiments, the extent of binding of an antigen binding molecule / moiety to a non-target molecule is less than about 10% of the binding of the antigen binding molecule / moiety 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 an antigen binding molecule / moiety 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 / moiety to a non-target molecule. This can optionally be at least one of 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, or 2.0.

[0393] The binding affinity of an antigen binding molecule / moiety for its target is typically expressed as its dissociation constant (K D) are described. 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 radiolabeled antigen binding assays (RIA).

[0394] In some embodiments, the antigen binding molecules / portions described herein bind to yc and / or IL-21Rα with an affinity in the micromolar range, i.e., Kd D = 9.9 x 10 -4 M to 1 x 10 -6 M. In some embodiments, the antigen binding molecules / portions described herein bind to yc and / or IL-21Rα with an affinity in the sub-micromolar range, i.e., Kd D < 1 x 10 -6 M. In some embodiments, the antigen binding molecules / portions described herein bind to yc and / or IL-21Rα with an affinity in the nanomolar range, i.e., Kd D = 9.9 x 10 -7 M to 1 x 10 -9 M. In some embodiments, the antigen binding molecules / portions described herein bind to yc and / or IL-21Rα with an affinity in the sub-nanomolar range, i.e., Kd D < 1 x 10 -9 M. In some embodiments, the antigen binding molecules / portions described herein bind to yc and / or IL-21Rα with an affinity in the picomolar range, i.e., Kd D = 9.9 x 10 -10 M to 1 x 10 -12 M. In some embodiments, the antigen binding molecules / portions described herein bind to yc and / or IL-21Rα with an affinity in the sub-picomolar range, i.e., Kd D < 1 x 10 -12 M.

[0395] 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 and / or IL-21Ra, which is composed 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 and / or IL-21Ra, which is composed of a discontinuous amino acid sequence of the amino acid sequence.

[0396] 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-crystallography 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.

[0397] 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.

[0398] The antigen binding molecules and antigen binding moieties preferably bind to the extracellular domain of the target antigen. The extracellular domain of yc and IL-21Ra is as described above.

[0399] The antigen binding molecules can bind to a cell expressing yc and / or IL-21Ra.

[0400] 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 CD3d), 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.

[0401] 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.

[0402] The ability of an antigen binding molecule to bind to a given cell type, e.g., a cell expressing yc and / or IL-21Ra, can be analyzed by contacting the cell with the antigen binding molecule and detecting the antigen binding molecule bound to the cell, 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 and / or IL-21Ra can be analyzed by methods such as flow cytometry and immunofluorescence microscopy.

[0403] In some embodiments, the antigen binding molecule increases multimerization of yc and IL-21Ra. In some embodiments, the antigen binding molecule decreases multimerization of yc and IL-21Ra.

[0404] 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.

[0405] It will be appreciated that multimerization of the polypeptides of the yc and yc-containing cytokine receptors other than yc is heteromultimerization, as the constituent polypeptides of the multimer are not identical. Thus, the multimers formed by multimerization of the polypeptides of the yc and yc-containing cytokine receptors other than yc according to the present disclosure are heteromultimers, and not homomultimers.

[0406] The antigen binding molecules of the present disclosure can promote multimerization of yc and IL-21Ra by virtue of the antigen binding moieties of which they are composed binding to the respective polypeptides. Binding to yc and IL-21Ra brings the polypeptides in close physical proximity (e.g., within 50 angstroms, e.g., within 40, 30, 25, 20, 15, 10, or 5 angstroms), thereby facilitating their association.

[0407] 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 appropriately labeled interaction partners, e.g., as described in Ciruela, Curr Opin Biotechnol. (2008) 19(4):338-43. Other suitable techniques include protein fragment complementation systems, e.g., NanoLuc and NanoBiT, which systems are described, e.g., in Thirukkumaran et al., Front Chem. (2020) 7:938 and Dixon et al., ACS Chem Biol. (2016) 11(2):400-408.

[0408] An antigen binding molecule according to the present 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).

[0409] In some embodiments, an antigen binding molecule increases signaling mediated by a yc-containing cytokine receptor (e.g., a yc:IL-21Ra receptor). It will be understood that an antigen binding molecule increases signaling mediated by a yc-containing cytokine receptor to which the antigen binding molecule binds. That is, an antigen binding molecule increases signaling mediated by a yc-containing cytokine receptor comprising or consisting of a polypeptide complex comprising a polypeptide for which the antigen binding molecule comprises a binding moiety. To illustrate, in embodiments in which an antigen binding molecule comprises (i) a yc-binding moiety and (ii) an IL-21Ra-binding moiety, the antigen binding molecule can increase signaling through a yc-containing cytokine receptor comprising yc and IL-21Ra (e.g., a yc:IL-21Ra receptor).

[0410] Such antigen binding molecules can variously be described as “upregulating”, “inducing”, “enhancing”, “promoting”, “stimulating”, “triggering” or “potentiating” signalling 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 “agonist” or “activating” activity on the relevant yc-containing cytokine receptor.

[0411] In some embodiments, the antigen binding molecule reduces signalling mediated by a yc-containing cytokine receptor (e.g. a yc:IL-21Ra receptor). It will be understood that the antigen binding molecule reduces signalling mediated by a yc-containing cytokine receptor to which the antigen binding molecule binds. That is, the antigen binding molecule reduces signalling mediated by a yc-containing cytokine receptor comprising or consisting of a polypeptide complex comprising a polypeptide, for which the antigen binding molecule comprises a binding moiety. By way of illustration, in embodiments in which the antigen binding molecule comprises (i) a yc binding moiety and (ii) an IL-21Ra binding moiety, the antigen binding molecule can reduce signalling by a yc-containing cytokine receptor comprising yc and IL-21Ra (e.g. a yc:IL-21Ra receptor). Such antigen binding molecules can variously be described as “downregulating”, “preventing”, “diminishing”, “inhibiting”, “reducing”, “attenuating”, “blocking” or “decreasing” signalling 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 “antagonist” or “inhibitory” activity on the relevant yc-containing cytokine receptor.

[0412] Signalling mediated by a yc-containing cytokine receptor can be analysed using cells expressing the relevant receptor, e.g. using an assay for detecting and / or quantifying receptor-mediated signalling. Suitable assays include, for example, assays for detecting phosphorylation / activity / expression of factors that are phosphorylated / activated / expressed as a result of signalling through the yc-containing cytokine receptor.

[0413] Such assays can comprise contacting a cell expressing a given yc-containing cytokine receptor with an antigen binding molecule according to the present disclosure. By way of illustration, an assay for investigating the ability of an antigen binding molecule to increase yc:IL-21Ra-mediated signalling can comprise contacting a cell expressing a yc:IL-21Ra receptor with an antigen binding molecule comprising a yc binding moiety and an IL-21Ra binding moiety.

[0414] For example, yc-containing cytokine receptor-mediated signaling can be investigated 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).

[0415] In some embodiments, the antigen binding molecule increases JAK / STAT signaling mediated by the yc:IL-21Ra receptor. In some embodiments, the antigen binding molecule increases MAPK / ERK signaling mediated by the yc:IL-21Ra receptor. In some embodiments, the antigen binding molecule increases PI3K / Akt signaling mediated by the yc:IL-21Ra receptor.

[0416] 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.

[0417] 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.

[0418] In some embodiments, the antigen binding molecule decreases JAK / STAT signaling mediated by the yc:IL-21Ra receptor. In some embodiments, the antigen binding molecule decreases MAPK / ERK signaling mediated by the yc:IL-21Ra receptor. In some embodiments, the antigen binding molecule decreases PI3K / Akt signaling mediated by the yc:IL-21Ra receptor.

[0419] In some embodiments, the antigen binding molecule decreases phosphorylation of STAT1, STAT3, STAT5, and / or ERK. In some embodiments, the antigen binding molecule decreases activation of STAT1, STAT3, STAT5, and / or ERK. In some embodiments, the antigen binding molecule decreases STAT1, STAT3, STAT5, and / or ERK activity.

[0420] In some embodiments, the antigen binding molecule decreases phosphorylation of STAT5. In some embodiments, the antigen binding molecule decreases activation of STAT5. In some embodiments, the antigen binding molecule decreases STAT5 activity.

[0421] 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 studied 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 studied 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.

[0422] 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 studied using a reporter cell line that stably expresses 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 studied using a reporter cell line that expresses a secretible reporter gene that can be detected quantitatively from the supernatant and can be readily measured.

[0423] In some embodiments, the antigen binding molecule increases proliferation, survival, and / or effector activity of a cell expressing a yc-containing cytokine receptor (e.g., yc:IL-21Ra receptor) to which the antigen binding molecule binds. In some embodiments, the antigen binding molecule decreases proliferation, survival, and / or effector activity of a cell expressing a yc-containing cytokine receptor (e.g., yc:IL-21Ra 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 of the relevant yc-containing cytokine receptor.

[0424] The ability of an antigen binding molecule to increase / decrease proliferation of a cell expressing a given yc-containing cytokine receptor can be analyzed by contacting the cell with the antigen binding molecule and subsequently evaluating 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 detected, for example, by detecting changes in cell number over time, or by incorporating a label that is diluted out as the cell divides. 3In vitro analysis of H-thymidine or by CFSE dilution assay, e.g., as described in Fulcher and Wong, Immunol Cell Biol. (1999) 77(6):559-564, incorporated by reference in its entirety. Proliferating cells can also be identified by analysis of 5-ethynyl-2'-deoxyuridine (EdU) incorporation by appropriate assays, e.g., as described 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 evaluated, e.g., by labeling cells and monitoring cell number over time.

[0425] Effector activity can be evaluated by analyzing an indicator of such activity. For example, the ability of an antigen-binding molecule to increase / decrease effector activity of cells expressing a given yc-containing cytokine receptor can be analyzed by contacting the cells with the antigen-binding molecule and subsequently evaluating the cells for gene and / or protein expression of one or more effector molecules (i.e., after a period of time sufficient to observe an effect on gene and / or protein expression of such factors). Effector molecules include, e.g., 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, e.g., by detecting mRNA encoding the relevant molecule, e.g., by quantitative real-time PCR (qRT-PCR). Protein expression can be determined, e.g., by antibody-based methods, e.g., by Western blot, immunohistochemistry, immunocytochemistry, flow cytometry, or ELISA.

[0426] The ability of an antigen-binding molecule to increase / decrease effector activity of cells expressing a given yc-containing cytokine receptor can also be analyzed by contacting the cells with the antigen-binding molecule and subsequently evaluating the cells for their ability to kill target cells expressing the antigen (for which the cells expressing the yc-containing cytokine receptor comprise 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 investigated, e.g., using any of the methods reviewed in Zaritskaya et al., Expert Rev Vaccines (2011) 9(6):601-616, incorporated by reference in its entirety. Examples of in vitro assays for cytotoxicity / cell killing assays include release assays, such as 51Cr 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 by reference in its entirety).

[0427] Effector activity can also be analyzed in vivo, e.g., in a suitable non-human animal model of a given disease / condition. Effector activity can be inferred by evaluating therapeutic / prophylactic effects in a relevant model that correlate with relevant effector activity.

[0428] 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).

[0429] 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 (e.g., yc:IL-21Ra receptor) to which the antigen binding molecule binds. 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 (e.g., yc:IL-21Ra receptor) to which the antigen binding molecule binds. It will be appreciated that a reduction in the level of expression of one or more immune cell exhaustion markers is a cellular level functional consequence of increased / reduced signaling through the relevant yc-containing cytokine receptor.

[0430] 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, e.g., by flow cytometry.

[0431] In this context, a “reduction” or “increase” in the level of expression of one or more immune cell exhaustion markers 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 markers) (i.e., the same type of cells). In some embodiments, a “reduction” in the level of expression of one or more immune cell exhaustion markers 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 markers).

[0432] In some embodiments, an increase in the level of expression 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).

[0433] In some embodiments, the antigen binding molecules of the present disclosure achieve their functional effects via mechanisms that do not involve killing / elimination of cells comprising / expressing the one or more target antigens of their constituent antigen binding portions (i.e., yc and / or IL-21Rα) (e.g., Fc-mediated killing / elimination of such cells).

[0434] In some embodiments, the antigen binding molecules of the present disclosure are capable of reducing multimerization of yc and IL-21Rα, reducing signaling mediated by the yc-containing cytokine receptor to which the antigen binding molecule binds (e.g., yc:IL-21Rα receptor), and / or reducing proliferation, survival, and / or effector activity of cells expressing the yc-containing cytokine receptor to which the antigen binding molecule binds (e.g., yc:IL-21Rα receptor) by mechanisms that do 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.

[0435] The ability of an antigen binding molecule to reduce multimerization of yc and IL-21Rα, reduce signaling mediated by the yc-containing cytokine receptor to which the antigen binding molecule binds (e.g., yc:IL-21Rα receptor), and / or reduce proliferation, survival, and / or effector activity of cells expressing the yc-containing cytokine receptor to which the antigen binding molecule binds (e.g., yc:IL-21Rα receptor) by mechanisms that do not require / involves Fc-mediated function can be assessed, for example, by analyzing the ability of an antigen binding molecule provided in a form lacking a functional Fc region to achieve one or more of the specified 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 a form lacking an Fc region (e.g., a scFv, Fab, etc.).

[0436] In some embodiments, the antigen binding molecule reduces multimerization of yc and IL-21Ra, reduces signaling mediated by a yc-containing cytokine receptor (e.g., yc:IL-21Ra receptor) to which the antigen binding molecule binds, and / or reduces proliferation, survival, and / or effector activity of a cell expressing a yc-containing cytokine receptor (e.g., yc:IL-21Ra receptor) to which the antigen binding molecule binds, through a mechanism that does not involve ADCC, ADCP, and / or CDC.

[0437] In some embodiments, the antigen binding molecule reduces multimerization of yc and IL-21Ra, reduces signaling mediated by a yc-containing cytokine receptor (e.g., yc:IL-21Ra receptor) to which the antigen binding molecule binds, and / or reduces proliferation, survival, and / or effector activity of a cell expressing a yc-containing cytokine receptor (e.g., yc:IL-21Ra receptor) to which the antigen binding molecule binds, through a mechanism that does not require the antigen binding molecule to bind to an Fc receptor (e.g., does not require the antigen binding molecule to bind to one or more of FcyRI, FcyRIIa, FcyRIIb, FcyRIIc, FcyRIIIa, and FcyRIIIb; e.g., does not require the antigen binding molecule to bind to C1q, and / or does not require N297 glycosylation.

[0438] In some embodiments, the antigen binding molecule of the present disclosure No induces ADCC, ADCP, or CDC of a cell comprising / expressing one or more target antigens (i.e., yc and / or IL-21Ra) of which the antigen binding moiety is a component.

[0439] No induces (i.e., No The antigen binding molecule capable of inducing ADCC / ADCP / CDC does not substantially elicit 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)). By “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.

[0440] 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, for example, according to the method described in Yamashita et al., Scientific Reports (2016) 6: 19772 (this document is incorporated herein in its entirety by reference) or by a method as described in, for example, Jedema et al., Blood (2004) 103: 2677-82 (incorporated herein in its entirety by reference). 51 The ability and extent to which a given antigen binding molecule is capable of inducing ADCP can be analyzed, for example, according to the method described in Kamen et al., J Immunol (2017) 198 (1 Supplement) 157.17 (incorporated herein in its entirety by reference). The ability and extent to which a given antigen binding molecule is capable of inducing CDC can be analyzed, for example, using a Clq binding assay, for example, as described in Schlothauer et al., Protein Engineering, Design and Selection (2016), 29 (10): 457-466 (incorporated herein in its entirety by reference).

[0441] In some embodiments, the antigen binding molecules according to the present disclosure can increase (i.e., upregulate, enhance, potentiate) cell killing of cells comprising / expressing one or more target antigens of its constituent antigen binding moiety (i.e., yc and / or IL-21Ra). In some embodiments, an “increased” level of cell killing refers to a level of cell killing that is greater than 1-fold, for example, 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 killing of such cells).

[0442] In some embodiments, an antigen binding molecule according to the present disclosure is capable of reducing the number / proportion of cells comprising / expressing one or more target antigens of which it comprises an antigen binding moiety (i.e., yc and / or IL-21Ra). In some embodiments, the antigen binding molecule is capable of depleting / enhancing the depletion of such cells. In some embodiments, “reducing” the number / proportion of cells means the number / proportion of cells is less than 1-fold, e.g., one of 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 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).

[0443] An antigen binding molecule according to the present disclosure can comprise one or more moieties for enhancing the reduction of the number / proportion of cells comprising / expressing one or more target antigens of which it comprises an antigen binding moiety (i.e., yc and / or IL-21Ra). For example, an antigen binding molecule according to the present disclosure may, for example, comprise an Fc region and / or a drug moiety.

[0444] In some embodiments, an antigen binding molecule according to the present disclosure comprises an Fc region that is capable of enhancing / guiding one or more of ADCC, ADCP, CDC against cells comprising / expressing one or more target antigens of which it comprises an antigen binding moiety (i.e., yc and / or IL-21Ra), and / or enhancing the formation of a MAC on the cell or enhancing the cell degranulation of the cell.

[0445] In some embodiments, an antigen binding molecule according to the present disclosure comprises a drug moiety. The antigen binding molecule can be conjugated to the 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 cells comprising / expressing one or more target antigens of which it comprises an antigen binding moiety (i.e., yc and / or IL-21Ra). In some embodiments, the drug moiety is or comprises a chemotherapeutic agent.

[0446] In some embodiments, the 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).

[0447] In some embodiments, the antigen binding molecules according to the present disclosure are capable of potentiating / directing T cell-mediated cytolytic activity against cells comprising / expressing one or more target antigens of which the constituent antigen binding moieties are (i.e., yc and / or IL-21Ra).

[0448] The antigen binding molecules of the present disclosure possess novel and / or improved properties over yc family cytokines.

[0449] In some embodiments, the antigen binding molecules according to the present disclosure possess cytokine-like properties in binding and triggering yc-containing cytokine receptor-mediated signaling, but in addition also possess drug (in particular antibody)-like biophysical and pharmacokinetic properties.

[0450] In some embodiments, the antigen binding molecules exhibit increased stability and / or half-life as compared to IL-21.

[0451] As used herein, “stability” can refer to resistance to degradation, aggregation, and / or unfolding. A molecule having increased stability as compared to a reference molecule can exhibit decreased degradation / degradation propensity, decreased aggregation / aggregation propensity, and / or decreased unfolding / unfolding propensity as compared to the reference molecule.

[0452] 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.

[0453] Stability can be evaluated according to methods described, e.g., 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) material (i.e., aggregates), and / or low molecular weight (LMW) material (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; analysis of effective surface charge and diffusion interaction parameter (KD) via zeta potential and diffusion interaction parameter (KD) analysis; and analysis of intrinsic tryptophan fluorescence by fluorescence spectroscopy.

[0454] 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, for example, 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).

[0455] In some embodiments, a 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.

[0456] In some embodiments, the antigen binding molecules of the disclosure are more effective at increasing signaling mediated by a yc-containing cytokine receptor (e.g., yc:IL-21Ra receptor) bound by the antigen binding molecule compared to a yc-containing cytokine receptor binding cytokine. In some embodiments, the antigen binding molecules of the disclosure are less effective at increasing signaling mediated by a yc-containing cytokine receptor (e.g., yc:IL-21Ra receptor) bound by the antigen binding molecule compared to a yc-containing cytokine receptor binding cytokine.

[0457] In some embodiments, the antigen binding molecules of the present disclosure are more effective than a cytokine that binds a γc-containing cytokine receptor in upregulating proliferation, survival, and / or effector activity of a cell expressing a γc-containing cytokine receptor (e.g., a γc:IL-21Rα receptor) to which the antigen binding molecule binds. In some embodiments, the antigen binding molecules of the present disclosure are less effective than a cytokine that binds a γc-containing cytokine receptor in upregulating proliferation, survival, and / or effector activity of a cell expressing a γc-containing cytokine receptor (e.g., a γc:IL-21Rα) to which the antigen binding molecule binds. In some embodiments, the antigen binding molecules of the present disclosure are more effective than a cytokine that binds a γc-containing cytokine receptor in reducing expression of one or more immune cell exhaustion markers by a cell expressing a γc-containing cytokine receptor (e.g., a γc:IL-21Rα receptor) to which the antigen binding molecule binds. In some embodiments, the antigen binding molecules of the present disclosure are less effective than a cytokine that binds a γc-containing cytokine receptor in reducing expression of one or more immune cell exhaustion markers by a cell expressing a γc-containing cytokine receptor (e.g., a γc:IL-21Rα receptor) to which the antigen binding molecule binds.

[0458] For example, in embodiments in which the antigen binding molecule comprises (i) a γc-binding moiety and (ii) an IL-21Rα-binding moiety, in some embodiments, the antigen binding molecule can more effectively increase signaling mediated by a γc:IL-21Rα receptor than IL-21, and / or can be more effective than IL-21 in upregulating cell proliferation, survival, and / or effector activity of a cell expressing a γc:IL-21Rα receptor. Similarly, the antigen binding molecule can be more effective than IL-21 in downregulating expression of one or more immune cell exhaustion markers by a cell expressing a γc:IL-21Rα receptor.

[0459] In some embodiments, the antigen binding molecule increases signaling mediated by a γc:IL-21Rα receptor and / or increases cell proliferation, survival, and / or effector activity of a cell expressing a γc:IL-21Rα receptor, wherein the EC 50 is less than the EC 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. 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.

[0460] In some embodiments, the antigen binding molecule decreases expression of one or more immune cell exhaustion markers by cells expressing the yc:IL-21Ra receptor, wherein the IC 50 less than the IC50exhibited by IL-21 50 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, as determined in the same assay. In some embodiments, the antigen binding molecule decreases expression of one or more immune cell exhaustion markers by cells expressing the yc:IL-21Ra receptor, wherein the IC 50 greater than the IC50exhibited by IL-21 50e.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.

[0461] 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.

[0462] In some embodiments, administration of the antigen binding molecules according to the present disclosure can be associated with one or more of inhibiting the development / progression of a cancer, delaying / preventing the onset of a cancer, reducing / delaying / preventing tumor growth, reducing / delaying / preventing tissue invasion, reducing / delaying / preventing metastasis, reducing the severity of one or more symptoms of a cancer, reducing the number of cancer cells, reducing cancer burden, reducing tumor size / volume, and / or increasing the survival (e.g., progression-free survival or overall survival) of a subject having a cancer, e.g., as determined in an appropriate model, as compared to an appropriate control condition.

[0463] It will be appreciated that the properties recited in the preceding paragraph are evaluated after a period of time sufficient to observe an effect associated with administration of the antigen binding molecules.

[0464] Linkers and additional sequences

[0465] The antigen binding molecules and polypeptides of the present disclosure can additionally comprise additional amino acids or amino acid sequences.

[0466] 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.

[0467] 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 a scFv moiety linked to a CH2CH3 region.

[0468] Linker sequences are known to those of skill in the art, e.g., 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, a linker sequence can be a flexible linker sequence. A flexible linker sequence allows 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.

[0469] In some embodiments, a linker sequence comprises at least one glycine residue and / or at least one serine residue. In some embodiments, a linker sequence comprises or consists of glycine and serine residues. In some embodiments, a 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, a 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, a linker sequence comprises or consists of (G4S)4 or (G4S)6. In some embodiments, a 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.

[0470] In some embodiments, the linker comprises or consists of the amino acid sequence GGGGS (SEQ ID NO: 240). In some embodiments, the linker comprises or consists of the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 141). In some embodiments, the linker comprises the amino acid sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 239). In some embodiments, the linker comprises or consists of the amino acid sequence NSGAAA (SEQ ID NO: 369).

[0471] In some embodiments, the linker between the VH sequence and the VL sequence comprises or consists of the amino acid sequence GGGGS (SEQ ID NO: 240). In some embodiments, the linker between the VH sequence and the VL sequence comprises or consists of the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 141). In some embodiments, the scFV comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 141). In some embodiments, the linker between the VH sequence and the VL sequence comprises or consists of the amino acid sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 239). In some embodiments, the scFV comprises the amino acid sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 239).

[0472] 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 amino acid sequence GGGGS (SEQ ID NO: 240). In some embodiments, the linker sequence between the VHH molecules comprises or consists of the amino acid sequence GGGGS (SEQ ID NO: 240).

[0473] 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 amino acid sequence EAAAK (SEQ ID NO: 364). In some embodiments, the linker sequence between the VHH molecules comprises or consists of the amino acid sequence EAAAK (SEQ ID NO: 364).

[0474] 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: 360) amino acid sequence. In some embodiments, the linker sequence between scFv molecules comprises or consists of an A(EAAAK)5A (SEQ ID NO: 360) amino acid sequence. In some embodiments, the linker sequence between VHH molecules comprises or consists of an A(EAAAK)5A (SEQ ID NO: 360) 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: 360) amino acid sequence.

[0475] Antigen binding molecules and polypeptides of the present disclosure can comprise amino acid sequences to facilitate expression, folding, trafficking, processing, purification, or detection of the antigen binding molecules / polypeptides. 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.

[0476] 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).

[0477] 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 generally removed by cleavage and thus are not included in the mature antigen binding molecules / polypeptides.

[0478] 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).

[0479] Labels and conjugates

[0480] In some embodiments, the antigen binding molecule or polypeptide according to the present disclosure comprises a detectable moiety.

[0481] In some embodiments, the detectable moiety is a fluorescent, phosphorescent, luminescent, immunodetectable (e.g., epitope tag), radioactive, chemical, nucleic acid, or enzymatic label. The antigen binding molecule or polypeptide can be labeled covalently or non-covalently with the detectable moiety.

[0482] 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 113m , 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. Immunologically detectable labels include haptens, peptides / polypeptides, antibodies, receptors, and ligands, such as biotin, avidin, streptavidin, or digoxigenin. Nucleic acid labels include aptamers.

[0483] In some embodiments, the antigen binding molecule / polypeptide comprises an epitope tag, e.g., His (e.g., 6XHis), FLAG, c-Myc, StrepTag, hemagglutinin (Ha tag), 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.

[0484] In some embodiments, the antigen binding molecule comprises a Ha tag and a His tag, optionally at the C terminus.

[0485] In some embodiments, the antigen binding molecule comprises a linker, a Ha tag, and a His tag, optionally at the C terminus. In some embodiments, the antigen binding molecule comprises SEQ ID NO: 368 (AAAYPYDVPDYGSHHHHHH), optionally at the C terminus. For example, in some embodiments, the antigen binding molecule comprises the sequence set forth in any one of SEQ ID NOs: 402-406, and further comprises SEQ ID NO: 368 at the C terminus.

[0486] In some embodiments, the antigen binding molecule / polypeptide comprises a moiety having a detectable activity, e.g., an enzyme moiety. Enzyme 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.

[0487] 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.

[0488] 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.

[0489] Nucleic acids and vectors

[0490] 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.

[0491] 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.

[0492] 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.

[0493] 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.

[0494] 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.

[0495] 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.

[0496] 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.

[0497] 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.

[0498] 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.

[0499] 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.

[0500] 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.

[0501] 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.

[0502] In some embodiments, transcription of the nucleic acids encoding the constituent polypeptides of the antigen binding molecule is under the control of different promoters.

[0503] In some embodiments, the nucleic acid / multiple nucleic acids or the vector / multiple vectors is multicistronic (e.g., bicistronic, tricistronic, 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.

[0504] Production of antigen binding molecules and polypeptides

[0505] Antigen binding molecules and polypeptides according to the present disclosure can be prepared according to methods known to the skilled person for producing polypeptides.

[0506] 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.

[0507] 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.

[0508] 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.

[0509] 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 described above.

[0510] 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.

[0511] 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.

[0512] 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 content 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).

[0513] Bioreactors include 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. Bioreactors monitor and control 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.

[0514] 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.

[0515] 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.

[0516] 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.

[0517] 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.

[0518] Cells comprising / expressing antigen binding molecules and polypeptides

[0519] The present disclosure also provides a cell comprising or expressing an antigen binding molecule or polypeptide according to the present 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 present disclosure.

[0520] It will be understood that where reference is made herein to a cell in the singular (i.e. “a”, “an”, “the”), a plurality of / population of such cells is also contemplated.

[0521] The cell can be a eukaryotic cell, e.g. a mammalian cell. The mammal can be a primate (rhesus macaque, 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).

[0522] 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 (herein 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.

[0523] The present disclosure also provides a method for producing a cell comprising a nucleic acid or vector according to the present disclosure, the method comprising introducing a nucleic acid, a plurality of nucleic acids, a vector, or a plurality of vectors according to the present disclosure into the cell. In some embodiments, introducing an isolated nucleic acid or vector according to the present disclosure into the cell comprises transformation, transfection, electroporation, or transduction (e.g. retroviral transduction).

[0524] The present disclosure also provides a method for producing a cell expressing / comprising an antigen binding molecule or polypeptide according to the present disclosure, the method comprising introducing a nucleic acid, a plurality of nucleic acids, a vector, or a plurality of vectors according to the present 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.

[0525] The present disclosure also provides a cell obtained or obtainable by a method according to the present disclosure.

[0526] Compositions

[0527] The present disclosure also provides compositions comprising the antigen binding molecules, polypeptides, nucleic acids, expression vectors, and cells described herein.

[0528] 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.

[0529] 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).

[0530] 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.

[0531] 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.

[0532] 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.

[0533] 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.

[0534] 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.

[0535] 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.

[0536] Therapeutic and prophylactic applications

[0537] The antigen binding molecules, CARs, nucleic acids, expression vectors, cells, and compositions described herein are useful in therapeutic and prophylactic methods.

[0538] 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.

[0539] 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).

[0540] “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 stage chronic stage.

[0541] 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 derives a therapeutic or prophylactic benefit from modulation of signalling mediated by the yc:IL-21Ra receptor, and / or from manipulation of the number / proportion of cells expressing the yc:IL-21Ra receptor.

[0542] 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 / disorders that would derive therapeutic or prophylactic benefit from an increase 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-21Ra-binding moiety, the antigen binding molecule can be used to treat / prevent diseases / disorders that would derive therapeutic or prophylactic benefit from an increase in signaling mediated by the yc:IL-21Ra receptor.

[0543] 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 / disorders that would derive therapeutic or prophylactic benefit from a decrease 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-21Ra-binding moiety, the antigen binding molecule can be used to treat / prevent diseases / disorders that would derive therapeutic or prophylactic benefit from a decrease in signaling mediated by the yc:IL-21Ra receptor, and / or to treat / prevent diseases / disorders that would derive therapeutic or prophylactic benefit from a decrease in the number / proportion of cells comprising / expressing the yc:IL-21Ra receptor.

[0544] The articles of the present disclosure can also be used to treat / prevent diseases / disorders that would derive therapeutic or prophylactic benefit from a functional consequence of an increase in the level of signaling mediated by the yc-containing cytokine receptor (e.g., the yc:IL-21Ra receptor). For example, the articles of the present disclosure can be used to treat / prevent diseases / disorders that would derive therapeutic or prophylactic benefit from an increase in the proliferation and / or population expansion of cells expressing the yc-containing cytokine receptor (e.g., the yc:IL-21Ra receptor), and from an increase in the 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 diseases / disorders that would derive therapeutic or prophylactic benefit from an increase in the number / proportion and / or activity of immune cells (e.g., effector immune cells (e.g., effector T cells and / or NK cells)).

[0545] The articles of the present disclosure can also be used to treat / prevent diseases / conditions that would derive therapeutic or prophylactic benefit from reduced functional consequences of reduced levels of signaling mediated by a yc-containing cytokine receptor (e.g., yc:IL-21Ra receptor), and / or from reduced functional consequences of reduced numbers / proportions of cells comprising / expressing a yc-containing cytokine receptor (e.g., yc:IL-21Ra). For example, the articles of the present disclosure can be used to treat / prevent diseases / conditions that would derive therapeutic or prophylactic benefit from reduced proliferation and / or population expansion of cells expressing a yc-containing cytokine receptor (e.g., yc:IL-21Ra receptor), and from reduced survival and / or numbers / proportions and / or activity of such cells. In particular, the articles of the present disclosure can be used to treat / prevent diseases / conditions that would derive therapeutic or prophylactic benefit from reduced numbers / proportions and / or activity of immune cells (e.g., effector immune cells (e.g., effector T cells and / or NK cells)).

[0546] The diseases / conditions to be treated / prevented in accordance with the present disclosure can be diseases / conditions 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: reduced levels of signaling mediated by IL-21, reduced levels of signaling mediated by yc:IL-21Ra receptor, reduced levels of numbers / proportions / activity of cells expressing a yc-containing cytokine receptor (e.g., yc:IL-21Ra receptor), reduced levels of numbers / proportions / activity of lymphocytes, reduced levels of numbers / proportions / activity of effector immune cells, reduced levels of numbers / proportions / activity of T cells (e.g., effector T cells), and / or reduced levels of numbers / proportions / activity of NK cells.

[0547] Thus, in some embodiments, the diseases / conditions to be treated / prevented in accordance with the present disclosure are diseases / conditions characterized by one or more of the following: reduced levels of signaling mediated by IL-21, reduced levels of signaling mediated by a yc-containing cytokine receptor (e.g., yc:IL-21Ra receptor), reduced levels of numbers / proportions / activity of cells expressing a yc-containing cytokine receptor (e.g., yc:IL-21Ra receptor), reduced levels of numbers / proportions / activity of lymphocytes, reduced levels of numbers / proportions / activity of effector immune cells, reduced levels of numbers / proportions / activity of T cells (e.g., effector T cells), and / or reduced levels of numbers / proportions / activity of NK cells.

[0548] The disease / condition to be treated / prevented according to the present disclosure can be one 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 signalling mediated by IL-21, an increase in the level of signalling mediated by a yc-containing cytokine receptor (e.g. yc:IL-21Ra receptor), an increase in the number / proportion / activity level of cells expressing a yc-containing cytokine receptor (e.g. yc:IL-21Ra 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.

[0549] Thus, in some embodiments, the disease / condition to be treated / prevented according to the present disclosure is one that is characterised by one or more of: an increase in the level of signalling mediated by IL-21, an increase in the level of signalling mediated by a yc-containing cytokine receptor (e.g. yc:IL-21Ra receptor), an increase in the number / proportion / activity level of cells expressing a yc-containing cytokine receptor (e.g. yc:IL-21Ra 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.

[0550] In the preceding four paragraphs, the “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.

[0551] According to various aspects of the disclosure, there is provided a method which is to, or comprises (e.g. in the context of treating / preventing a disease / condition described herein):

[0552] increasing multimerisation of yc and IL-21Ra;

[0553] decreasing multimerisation of yc and IL-21Ra;

[0554] increasing signalling mediated by a yc-containing cytokine receptor (e.g. yc:IL-21Ra receptor) bound by the antigen binding molecule;

[0555] decreasing signalling mediated by a yc-containing cytokine receptor (e.g. yc:IL-21Ra receptor) bound by the antigen binding molecule;

[0556] increasing signaling mediated by IL-21 ;

[0557] decreasing signaling mediated by IL-21 ;

[0558] increasing proliferation, survival and / or effector activity of cells expressing a gamma c-containing cytokine receptor (e.g., gamma c:IL-21 Ra receptor) bound by the antigen binding molecule;

[0559] decreasing proliferation, survival and / or effector activity of cells expressing a gamma c-containing cytokine receptor (e.g., gamma c:IL-21 Ra receptor) bound by the antigen binding molecule;

[0560] decreasing expression of one or more immune cell exhaustion markers by cells expressing a gamma c-containing cytokine receptor (e.g., gamma c:IL-21 Ra receptor) bound by the antigen binding molecule;

[0561] increasing expression of one or more immune cell exhaustion markers by cells expressing a gamma c-containing cytokine receptor (e.g., gamma c:IL-21 Ra receptor) bound by the antigen binding molecule;

[0562] increasing killing / depletion of cells comprising / expressing one or more target antigens (i.e., gamma c and / or IL-21 Ra) of a constituent antigen binding moiety of the antigen binding molecule, and / or decreasing the number / proportion of such cells;

[0563] and / or

[0564] enhancing anti-cancer activity of cancer antigen-specific immune cells.

[0565] 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.

[0566] 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)

[0567] increasing the level of multimerization of gamma c IL-21 Ra;

[0568] decreasing the level of multimerization of gamma c and the polypeptide other than gamma c (e.g., IL-21 Ra) of the gamma c-containing cytokine receptor;

[0569] increasing the level of signaling mediated by the gamma c-containing cytokine receptor (e.g., gamma c:IL-21 Ra receptor) bound by the antigen binding molecule;

[0570] reducing the level of signaling mediated by a yc-containing cytokine receptor (e.g., yc:IL-21Ra receptor) bound by the antigen binding molecule;

[0571] increasing the level of proliferation, survival, and / or effector activity of a cell expressing a yc-containing cytokine receptor (e.g., yc:IL-21Ra receptor) bound by the antigen binding molecule;

[0572] reducing the level of proliferation, survival, and / or effector activity of a cell expressing a yc-containing cytokine receptor (e.g., yc:IL-21Ra receptor) bound by the antigen binding molecule;

[0573] reducing the level of expression of one or more immune cell exhaustion markers of a cell expressing a yc-containing cytokine receptor (e.g., yc:IL-21Ra receptor) bound by the antigen binding molecule;

[0574] increasing the level of expression of one or more immune cell exhaustion markers of a cell expressing a yc-containing cytokine receptor (e.g., yc:IL-21Ra receptor) bound by the antigen binding molecule;

[0575] increasing killing / depletion of cells comprising / expressing one or more target antigens (i.e., yc and / or IL-21Ra) of which the constituent antigen binding moiety of the antigen binding molecule is specific, and / or reducing the number / proportion of such cells;

[0576] and / or

[0577] anti-cancer activity of the cancer antigen-specific immune cells is enhanced.

[0578] 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 routine in the art.

[0579] 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.

[0580] Lymphocytopenia can be defined as a total lymphocyte count <1000 / mcL (1 x 10 9 / L) in adults or <3000 / mcL (<3 x 10 9Diseases / conditions characterized by lymphopenia include, for example: T-lymphocyte lymphopenia, B-lymphocyte lymphopenia, NK-lymphocyte lymphopenia, 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).

[0581] 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.

[0582] 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.

[0583] 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.

[0584] A dysfunctional T-cell can be characterized by one or more of the following (i.e., as compared to a normal, non-dysfunctional T-cell): 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).

[0585] 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.

[0586] 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.

[0587] The cancer according to the present disclosure can be any unwanted cell proliferation (or any disease that manifests itself as 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.

[0588] 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.

[0589] 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.

[0590] 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.

[0591] 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.

[0592] 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).

[0593] 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).

[0594] 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.

[0595] 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 infection.

[0596] 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). IL-21 R-mediated signalling is required to maintain polyfunctional T cells during chronic viral infection (Frohlich A. et al. Science. (2009) 324:5934).

[0597] In some embodiments, the disease / condition to be treated / prevented in accordance with the present disclosure is a chronic infection, optionally a bacterial, viral, fungal or parasitic infection. In some embodiments, the disease / condition to be treated / prevented in accordance with the present disclosure is a chronic viral infection.

[0598] 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.

[0599] Examples of viral infections that can be treated include infection by 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 papilloma virus (HPV).

[0600] Examples of fungal infections that can be treated include infection by Alternaria sp, Aspergillus sp, Candida sp and Histoplasma sp. The fungal infection can be fungal sepsis or histoplasmosis.

[0601] Examples of parasitic infections that can be treated include infection by a Plasmodium species (e.g. P. falciparum, P. yoelli, P. ovale, P. vivax or P. chabaudi chabaudi). The parasitic infection can be a disease such as malaria, leishmaniasis or toxoplasmosis.

[0602] In some embodiments, the disease / condition to be treated / prevented according to 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.

[0603] Autoimmune diseases develop when self-reactive B lymphocytes (autoantibodies) and T lymphocytes cause pathological and / or functional damage to organs / tissues containing target self-antigens. Signaling mediated by yc-containing cytokine receptors plays an important role in the initiation, development, maintenance, and progression of autoimmune diseases. Excessive cytokine signaling can lead to inflammation, autoimmunity, and cancer. The yc family cytokines (IL-7, IL-2, IL-4, IL-9, IL-15, and / or IL-21) bind to yc-containing cytokine receptors and activate three major signaling pathways that promote cell survival and proliferation, namely the PI3K-Akt pathway, the RAS-MAPK pathway, and the JAK-STAT pathway. These pathways are often upregulated in patients with autoimmune diseases, and thus there is a beneficial effect associated with inhibiting / reducing yc family cytokine signaling (i.e., inhibiting / reducing signaling mediated by yc-containing cytokine receptors). Autoimmune diseases are associated with increased cytokine signaling (e.g., signaling mediated by yc-containing cytokine receptors). Treatment with the antigen-binding molecules of the present disclosure can reduce cytokine signaling and have a positive impact on patients with autoimmune diseases. Modulating the signaling of cytokines that play a key role in the initiation and / or effector phase of autoimmune attacks is a strategy that has shown success. For example, there are several drugs on the market that block TNFa (etanercept, infliximab, adalimumab) for the treatment of autoimmune diseases. For example, Etanercept, infliximab, adalimumab) are used to treat autoimmune diseases.

[0604] Interleukin-21 (IL-21) (produced mainly by follicular helper T (Tfh) and T helper 17 (Th17) cells) has been shown to play an important role in the immune system, for example, by promoting the proliferation and development of Tfh and Th17 cells, balancing helper T cell subsets, inducing B cell production and differentiation into plasma cells, and enhancing the production of immunoglobulins (Long D. et al. Journal of Autoimmunity, 2019, 99: 1-14). These effects are mediated mainly by the activation of JAK / STAT, MAPK, and PI3K pathways. Some IL-21 target genes, such as B lymphocyte-induced maturation protein-1 (Blimp-1), suppressor of cytokine signaling (SOCS), CXCR5, and Bcl-6, play an important role in the immune response. Thus, IL-21 / IL-21R-mediated signaling is implicated in autoimmune diseases. For example, IL-21 levels are increased in the peripheral blood and tissues of patients with systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), type 1 diabetes (T1D), immune thrombocytopenia (ITP), primary Sjogren’s syndrome (pSS), autoimmune thyroid disease (AITD), and psoriasis (Long D. et al. Journal of Autoimmunity, 2019, 99: 1-14).

[0605] In some embodiments, the autoimmune disease is selected from the group consisting of inflammatory bowel disease (IBD), Crohn’s disease, syndrome, lupus, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), multiple sclerosis (MS), alopecia areata, psoriasis, psoriatic arthritis, myasthenia gravis, sarcoidosis, type 1 diabetes, ulcerative colitis, Addison’s disease, Grave’s disease, immune thrombocytopenia (ITP), autoimmune thyroid disease (AITD), Hashimoto’s thyroiditis, autoimmune vasculitis, pernicious anemia, graft-versus-host disease (GVHD), vitiligo, and / or celiac disease.

[0606] The articles of the present disclosure can also be used in conjunction with methods for treating / preventing diseases / conditions, including adoptive cell transfer (ACT), in particular ACT of immune cells (e.g., effector immune cells, such as T cells and / or NK cells).

[0607] 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 before being 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 generally 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 the subject. Adoptive transfer can be performed with the aim of introducing a cell or population of cells into a subject, and / or increasing the frequency of a cell or population of cells in a subject.

[0608] 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.

[0609] 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 that are 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.

[0610] 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., produced 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.

[0611] 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., yc:IL-21Ra receptor).

[0612] The present disclosure provides a method of treating or preventing a disease or condition in a subject, the method comprising:

[0613] (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;

[0614] (ii) administering the generated / expanded population of immune cells to the subject.

[0615] In some embodiments, the adoptively transferred are autologous cells. In some embodiments, the adoptively transferred are allogeneic cells.

[0616] 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); generating 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 present disclosure; collecting the population of immune cells generated or expanded by culturing (i.e., in vitro / ex vivo) in the presence of an antigen binding molecule according to the present disclosure; mixing the population of immune cells generated or expanded by culturing (i.e., in vitro / ex vivo) in the presence of an antigen binding molecule according to the present disclosure with an adjuvant, diluent, or carrier; and / or administering the population of immune cells generated or expanded by culturing (i.e., in vitro / ex vivo) in the presence of an antigen binding molecule according to the present disclosure or a composition comprising such cells to the subject.

[0617] 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 present disclosure.

[0618] The skilled person will be able to determine appropriate reagents and procedures for generating / expanding a population of immune cells for adoptive transfer, and appropriate reagents and procedures for the adoptive transfer of such populations, for example with reference to Chia WK et al., Molecular Therapy (2014) 22(1): 132-139; Kalos and June Immunity (2013) 39(1): 49-60; and Cobbold et al., J Exp Med. (2005) 202:379-386.

[0619] In some aspects and embodiments, the disease / condition to be treated / prevented according to the present disclosure can be a disease characterized by an increased number / proportion and / or activity of cells expressing a yc-containing cytokine receptor (e.g., yc:IL-21Ra receptor).

[0620] In some aspects and embodiments, the disease / condition to be treated / prevented according to the present disclosure can be lymphocytosis, or a disease / condition characterized by lymphocytosis. Lymphocytosis can be defined as a total lymphocyte count > 3000 / mcL (1 x 10 9 / L) in adults or a total lymphocyte count > 9000 / mcL (<3 x 10 9 / L) in children < 2 years of age.

[0621] As the articles of the present disclosure can be used to reduce / decrease the number / proportion of cells comprising / expressing a yc-containing cytokine receptor (e.g., yc:IL-21Ra receptor) in a subject, it will be appreciated that they can be used to treat / prevent a disease characterized by an increased number / proportion and / or activity of cells expressing a yc-containing cytokine receptor (e.g., yc:IL-21Ra receptor). As explained herein, cells comprising / expressing a yc-containing cytokine receptor (e.g., yc:IL-21Ra receptor) include immune cells, e.g., effector immune cells (e.g., effector T cells and / or NK cells).

[0622] Diseases / conditions characterized by lymphocytosis include, for example: lymphoproliferative diseases / conditions, cancers, leukemias (e.g., chronic lymphocytic leukemia, acute lymphoblastic leukemia, acute lymphocytic leukemia), lymphomas, infectious diseases, EBV infection infectious mononucleosis, hepatitis (e.g., hepatitis A, hepatitis B, hepatitis C), CMV infection, HIV / AIDS, syphilis, pertussis, toxoplasmosis, Chagas disease, tuberculosis, brucellosis, hypothyroidism, autoimmune diseases, and rheumatoid arthritis.

[0623] In some embodiments, the disease / condition characterized by lymphocytosis is selected from the group consisting of: infectious diseases, EBV infection infectious mononucleosis, hepatitis (e.g., hepatitis A, hepatitis B, hepatitis C), CMV infection, HIV / AIDS, syphilis, pertussis, toxoplasmosis, Chagas disease, tuberculosis, brucellosis, hypothyroidism, autoimmune diseases, and rheumatoid arthritis.

[0624] Immune cell responses in inflammatory diseases or diseases characterized by inflammation can be driven by IL-21, and signaling mediated by yc:IL-21Ra-containing receptors.

[0625] In some embodiments, the disease / condition to be treated according to the present disclosure is a disease / condition characterized by inflammation. Diseases / conditions characterized by inflammation include, but are not limited to:

[0626] Diseases / conditions affecting the respiratory system, such as rhinosinusitis, rhinitis, pharyngitis, laryngitis, tracheitis, bronchitis, bronchiolitis, pneumonitis, pleuritis, and mediastinitis;

[0627] Diseases / conditions affecting the accessory digestive organs, such as hepatitis, ascending cholangitis, cholecystitis, pancreatitis, and peritonitis;

[0628] Diseases / conditions affecting the cardiovascular system, such as carditis, endocarditis, myocarditis, pericarditis, vasculitis, arteritis, phlebitis, and capillaritis;

[0629] Diseases / conditions affecting the urinary system, such as nephritis, glomerulonephritis, pyelonephritis, ureteritis, cystitis, and urethritis;

[0630] Diseases / conditions affecting the nervous system, such as encephalitis, myelitis, meningitis, arachnoiditis, and neuritis;

[0631] Diseases / conditions affecting the musculoskeletal system, such as arthritis, dermatomyositis, soft tissue, myositis, synovitis / tenosynovitis, bursitis, enthesitis, fasciitis, bursitis, epicondylitis, tendonitis, panniculitis, osteochondritis, osteitis / osteomyelitis, spondylitis, periostitis, and chondritis;

[0632] Diseases / conditions affecting the mouth and throat, such as stomatitis, gingivitis, gingivostomatitis, periodontitis, glossitis, tonsillitis, sialoadenitis, parotitis, cheilitis, pulpitis, and gnathitis;

[0633] Diseases / conditions affecting the gastrointestinal system, such as esophagitis, gastritis, gastroenteritis, enteritis, colitis, enterocolitis, duodenitis, ileitis, cecitis, appendicitis, proctitis, and Peutz-Jeghers syndrome;

[0634] Diseases / conditions affecting the skin, such as dermatitis, folliculitis, cellulitis, and hidradenitis;

[0635] Diseases / conditions affecting the eye, such as lacrimalitis, scleritis, episcleritis, keratitis, retinitis, chorioretinitis, blepharitis, conjunctivitis, and uveitis;

[0636] Diseases / conditions affecting the ear, such as otitis externa, otitis media, labyrinthitis, and mastoiditis;

[0637] Diseases / conditions of the reproductive system, such as oophoritis, salpingitis, endometritis, endometriosis, parametritis, cervicitis, vaginitis, vulvitis, mastitis, orchitis, epididymitis, prostatitis, seminal vesiculitis, balanitis, foreskin inflammation, balano-foreskin inflammation, chorioamnionitis, umbilical cord inflammation, and omphalitis;

[0638] Diseases / conditions of the endocrine system, such as insulitis, hypophysitis, thyroiditis, parathyroiditis, and adrenalitis;

[0639] Diseases / conditions of the lymphatic system, such as lymphangitis and lymphadenitis;

[0640] Cancers, including inflammation-induced and inflammation-associated cancers, such as lung cancer (e.g., lung adenocarcinoma, lung squamous cell carcinoma), prostate cancer, hematological malignancies (e.g., multiple myeloma), pancreatic cancer, cervical cancer, gastric cancer, esophageal cancer, head and neck cancer, colorectal cancer, colon cancer, liver cancer (e.g., hepatocellular carcinoma), and biliary tract cancer.

[0641] Inflammation and its role in health and disease are reviewed, for example, in Chen et al., Oncotarget (2018) 9(6):7204-7218, which is incorporated by reference herein in its entirety. Inflammation refers to the body’s response to cellular / tissue damage, which is characterized by edema, erythema (redness), heat, pain, and loss of function (stiffness and immobility), which are caused by local immune, vascular, and inflammatory cell responses to infection or injury. Injury can be caused by, for example, physical (e.g., mechanical) or chemical injury, trauma, infection, cancer, or an overactive / abnormal immune response (e.g., autoimmune disease). Inflammation forms part of the innate immune response, plays an important physiological role in wound healing and infection control, and helps restore tissue homeostasis.

[0642] However, many diseases are associated with an overactive inflammatory response (i.e., excessive inflammation and / or abnormally activated inflammation) and / or chronic (long-term) inflammation. In this document, excessive and / or chronic inflammation can be referred to as “pathological inflammation”. Pathological inflammation can refer to inflammation that is associated with (i.e., positively contributes to) the pathology of a disease.

[0643] The inflammation to be treated / prevented according to the present disclosure can be of any tissue / organ of the body. In some embodiments, the inflammation is of the lung (e.g., bronchiole, alveolus), airway (e.g., nasal cavity, oral cavity, pharynx, larynx, trachea, bronchus), heart, kidney, liver, skeletal muscle, blood vessel, eye, skin, pancreas, intestine, small intestine, large intestine, colon, joint, brain, or bone marrow. The inflammation can also occur in multiple tissues / organs simultaneously.

[0644] In some embodiments, inflammation can be of an organ or tissue of the respiratory system, such as the lungs (e.g., bronchioles, alveoli) or airways (e.g., nasal cavity, oral cavity, pharynx, larynx, trachea, bronchi). In some embodiments, inflammation can be of an organ or tissue of the cardiovascular system, such as the heart or blood vessels. In some embodiments, inflammation can be of an organ or tissue of the gastrointestinal system, such as the liver, intestines, small intestine, large intestine, colon, or pancreas. In some embodiments, inflammation can be of the eyes. In some embodiments, inflammation can be of the skin. In some embodiments, inflammation can be of the nervous system, such as the brain. In some embodiments, inflammation can be of the bone marrow. In some embodiments, inflammation can be of the joints. In some embodiments, inflammation can be of an organ or tissue of the urinary system, such as the kidney. In some embodiments, inflammation can be of an organ or tissue of the musculoskeletal system, such as muscle tissue. In some embodiments, inflammation can be of an organ or tissue of one or more organ systems.

[0645] In some embodiments, the inflammatory disease (or disease associated with inflammation) is characterized by lymphocytosis.

[0646] In some embodiments, the disease / disorder characterized by inflammation is selected from the group consisting of: chronic inflammatory disease, arthritis, rheumatoid arthritis, juvenile arthritis, systemic juvenile idiopathic arthritis, syndrome, lupus, systemic lupus erythematosus, pancreatitis, thyroiditis, periodontitis, dermatitis, dermatitis, atopic dermatitis, psoriasis, Hermansky-Pudlak syndrome, Grave's disease, diabetes, type 1 diabetes, type 2 diabetes, gestational-related hyperglycemia, multiple sclerosis, giant cell arteritis, Takayasu's arteritis, cardiovascular disease, atherosclerosis, atrial fibrillation, ventricular fibrillation, hypertrophic cardiomyopathy, dilated cardiomyopathy, myocarditis, heart failure with preserved ejection fraction, heart failure with reduced ejection fraction, Marfan syndrome, systemic sclerosis, scarring, scleroderma, Alzheimer's disease, hippocampal atrophy, lung disease, asthma, chronic obstructive pulmonary disease, pulmonary fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, hepatitis, cirrhosis, hepatotoxicity, acetaminophen-induced hepatotoxicity, alcoholic liver disease, pancreatitis, steatosis, nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, cholestasis, primary biliary cholangitis, primary sclerosing cholangitis, inflammatory bowel disease, Crohn's disease, colitis, ulcerative colitis, Addison's disease, Grave's disease, Hashimoto's thyroiditis, autoimmune vasculitis, pernicious anemia, celiac disease, endometriosis, stroke, nephropathy, kidney injury, acute kidney injury, nephrotoxicity, glomerulonephritis, chronic kidney disease, Alport syndrome, adult-onset Still's disease, Castleman disease, cytokine release syndrome, retinal fibrosis, age-related macular degeneration, wet age-related macular degeneration, COVID-19, Peutz-Jeghers syndrome, cancer, hematological malignancy, leukemia, plasmacytoma, Hodgkin's lymphoma, lung cancer, colorectal cancer, intestinal cancer, urological cancer, cancer of the vulva, endometrial cancer, ovarian cancer, prostate cancer, pancreatic cancer, bone cancer, glioblastoma, breast cancer, stomach cancer, kidney cancer, metastatic renal cell carcinoma, prostate cancer, skin cancer, liver cancer, hepatocellular carcinoma, frailty, age-related body fat accumulation, sarcopenia, age-related hyperlipidemia, age-related hypertriglyceridemia, age-related hypercholesterolemia, age-related hepatic steatosis, age-related nonalcoholic fatty liver disease (NAFLD), age-related nonalcoholic fatty liver (NAFL), age-related nonalcoholic steatohepatitis (NASH), age-related cardiovascular disease, age-related hypertension, age-related nephropathy, and age-related dermatopathy.

[0647] Administration of the articles of the present disclosure is preferably in a "therapeutically effective" or "prophylactically effective" amount, which is sufficient to show therapeutic or prophylactic benefit to the subject. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of the disease / condition and the particular article administered. Prescription of treatment (e.g. decisions on dosage etc) is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disease / disorder to be treated, the condition of the individual subject, the site of delivery, the method of administration and other factors known to medical practitioners. Examples of the techniques and protocols mentioned above can be found in Remington "The Science and Practice of Pharmacy" (Ed. A. Adejare), 23rdedition (2020), Academic Press.

[0648] It will be appreciated that in embodiments in which an antigen binding molecule according to the present disclosure or a composition comprising such an antigen binding molecule is administered to a subject, it is preferably administered in an amount / quantity sufficient to cause: (i) an increase or decrease in the multimerization level of a polypeptide other than yc of a yc-containing cytokine receptor, (ii) an increase or decrease in the level of signalling mediated by a yc-containing cytokine receptor (e.g. yc:IL-21Ra receptor) bound by the antigen binding molecule, (iii) an increase or decrease in the level of proliferation, survival and / or effector activity of a cell expressing a yc-containing cytokine receptor, (iv) an increase or decrease in the level of expression of one or more immune cell exhaustion markers by a cell expressing a yc-containing cytokine receptor, (v) an increase in cell killing / elimination of a cell comprising / expressing one or more target antigens of the constituent antigen binding moieties of the antigen binding molecule (i.e. yc and / or IL-21Ra), and / or a decrease in the number / proportion of such a cell, and / or (vi) an increase in anti-cancer activity of a cancer antigen-specific immune cell.

[0649] Administration of the articles of the present disclosure can be local, parenteral, systemic, intracavitary, intravenous, intraarterial, intramuscular, intrathecal, intraocular, intravitreal, intracorneal, subretinal, suprachoroidal, subcutaneous, intradermal, intrathecal, oral, nasal, or transdermal. Administration can be by injection or infusion. Where the articles of the present disclosure are for use in the treatment of cancer, administration can be intratumoral.

[0650] In some aspects and embodiments according to the disclosure, the articles of the disclosure can be delivered targeted, i.e., where the concentration of the relevant agent in the subject is increased in some parts of the body relative to other parts of the body. In some embodiments, the method comprises intravenous, intra-arterial, intramuscular, or subcutaneous administration, where the relevant article is formulated in a targeted agent delivery system. Suitable targeted delivery systems include, for example, nanoparticles, liposomes, micelles, beads, polymers, metal particles, dendrimers, antibodies, aptamers, nanotubes, or micron-sized silica rods. Such systems can comprise magnetic elements to direct the agent to a desired organ or tissue. Those of skill in the art will appreciate suitable nanocarriers and delivery systems.

[0651] In some cases, the articles of the disclosure are formulated for targeted delivery to specific cells, tissues, organs, and / or tumors.

[0652] Administration of the articles of the disclosure can be alone, or in combination with other treatments, either simultaneously or sequentially, depending on the disease / condition to be treated. The antigen binding molecules, cells, or compositions described herein and another prophylactic / therapeutic agent can be administered simultaneously or sequentially.

[0653] In some embodiments, the method comprises an additional therapeutic or prophylactic intervention. In some embodiments, the additional therapeutic or prophylactic intervention is selected from chemotherapy, immunotherapy, radiotherapy, surgery, vaccination, and / or hormone therapy. In some embodiments, the additional therapeutic or prophylactic intervention comprises leukapheresis. In some embodiments, the additional therapeutic or prophylactic intervention comprises stem cell transplantation.

[0654] Simultaneous administration means that the antigen binding molecules, polypeptides, nucleic acids (or multiple nucleic acids), expression vectors (or multiple expression vectors), cells, or compositions and therapeutic agents are administered together, e.g., as a pharmaceutical composition containing both agents (combination preparation), or immediately after each other, and optionally via the same route of administration, e.g., to the same artery, vein, or other blood vessel. Sequential administration means that one antigen binding molecule / composition or therapeutic agent is administered followed by separate administration of the other agent after a given time interval. While this is the case in some embodiments, it is not required that both agents be administered by the same route. The time interval can be any time interval.

[0655] In some embodiments, the treatment of cancer also includes chemotherapy and / or radiation therapy. Chemotherapy and radiation therapy refer to the treatment of cancer with a drug or ionizing radiation (e.g., radiation therapy using X-rays or gamma rays), respectively. The drug can be a chemical entity, such as a small molecule drug, an antibiotic, a DNA intercalator, a protein inhibitor (e.g., a kinase inhibitor), or a biologic (e.g., an antibody, antibody fragment, aptamer, nucleic acid (e.g., DNA, RNA), peptide, polypeptide, or protein). The drug can be formulated as a pharmaceutical composition or medicament. The formulation can include one or more drugs (e.g., one or more active agents) and one or more pharmaceutically acceptable diluents, excipients, or carriers.

[0656] Chemotherapy can involve the administration of more than one drug. The drugs can be administered alone or in combination with other treatments, either simultaneously or sequentially, depending on the condition to be treated. Chemotherapy can be administered by one or more routes of administration, such as parenterally, intravenously, orally, subcutaneously, intradermally, or intratumorally. Chemotherapy can be administered according to a treatment regimen. A treatment regimen can be a predetermined schedule, plan, protocol, or time schedule of administration of chemotherapy, which can be prepared by an attending physician or medical practitioner and can be customized to the patient in need of treatment. A treatment regimen can indicate one or more of the following: the type of chemotherapy administered to the patient; the dosage of each drug or radiation; the time interval between administrations; the length of each treatment; the number and nature of any treatment holidays, if any; and the like. For co-treatment, a single treatment regimen can be provided, which indicates the manner of administration of each drug.

[0657] Multiple doses 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 can be provided. One or more or each dose can be accompanied by the simultaneous or sequential administration of another therapeutic agent.

[0658] The multiple doses can be separated by a predetermined time interval, which can be selected to be one of 1, 2, 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, or 31 days, or 1, 2, 3, 4, 5, or 6 months. For example, a dose can be given once every 7, 14, 21, or 28 days (plus or minus 3, 2, or 1 day).

[0659] Methods of modulating cells expressing cytokine receptors comprising yc

[0660] The antigen binding molecules of the disclosure can be used in methods involving causing cytokine receptor containing g c-mediated signaling in cells expressing cytokine receptor containing g c and functional consequences thereof. Such methods include methods for or comprising: generating and / or expanding a population of cells expressing cytokine receptor containing g c; increasing cytokine receptor containing g c-mediated signaling in cells expressing cytokine receptor containing g c; and / or increasing proliferation, survival and / or effector activity of cells expressing cytokine receptor containing g c.

[0661] The antigen binding molecules of the disclosure can also be used in methods involving inhibiting / preventing cytokine receptor containing g c-mediated signaling in cells expressing cytokine receptor containing g c and / or killing / depleting such cells and functional consequences thereof. Such methods include methods for or comprising: reducing the number / proportion of cells expressing cytokine receptor containing g c, or killing / depleting cells expressing cytokine receptor containing g c; reducing cytokine receptor containing g c-mediated signaling in cells expressing cytokine receptor containing g c; and / or reducing proliferation, survival and / or effector activity of cells expressing cytokine receptor containing g c.

[0662] Such methods typically comprise contacting a cell expressing a cytokine receptor containing g c (e.g. a g c:IL-21R a receptor) with an antigen binding molecule according to the disclosure.

[0663] It will be appreciated that the antigen binding molecule preferably comprises an antigen binding moiety specific for a cytokine receptor containing g c polypeptide of a cell-expressed cytokine receptor containing g c. By way of illustration, in embodiments in which the antigen binding molecule comprises (i) a g c binding moiety and (ii) an IL-21R a binding moiety, the cell expresses a g c:IL-21R a receptor.

[0664] The method can be performed in vitro or ex vivo and can comprise contacting a cell expressing a cytokine receptor containing g c in vitro or ex vivo with an antigen binding molecule according to the disclosure. Such methods can be particularly useful for generating / expanding a population of cells for subsequent administration to a subject, i.e. in interventions to treat / prevent disease by adoptive cell transfer (ACT).

[0665] In some embodiments, the method can be performed in vivo and can comprise administering an antigen binding molecule according to the disclosure to a subject. In such cases, the cell expressing a cytokine receptor containing g c can be contacted in vivo with the antigen binding molecule, i.e. within the subject to which the antigen binding molecule is administered.

[0666] It will be appreciated that antigen binding molecules of the disclosure can be used in essentially any method, with the cytokine bound by the cytokine receptor containing yc to which the antigen binding molecule binds also being used in these methods. By way of illustration, antigen binding molecules comprising (i) a yc binding portion and (ii) an IL-21 Ra binding portion can be used in methods in which IL-21 is useful.

[0667] Detection methods

[0668] Antigen binding molecules described herein can be used in methods involving detection of a polypeptide other than yc (e.g. IL-21 Ra) of a yc and / or cytokine receptor containing yc. Antigen binding molecules can also be used in methods involving detection of a cell expressing a polypeptide other than yc (e.g. IL-21 Ra) of a yc and / or cytokine receptor containing yc. Antigen binding molecules can also be used in methods involving detection of a cell expressing a cytokine receptor containing yc (e.g. yc:IL-21 Ra receptor). Such methods can be in vitro or in vivo methods. Such methods can involve detection of a binding complex of an antigen binding molecule and yc and / or IL-21 Ra; and / or a cell expressing yc and / or IL-21 Ra; and / or a cell expressing a yc:IL-21 Ra receptor.

[0669] Thus, there is provided a method comprising contacting a sample containing or suspected of containing yc and / or IL-21 Ra; and / or a cell expressing yc and / or IL-21 Ra; and / or a cell expressing a yc:IL-21 Ra receptor with an antigen binding molecule according to the disclosure, and detecting the formation of a complex of the antigen binding molecule yc and / or IL-21 Ra; and / or a cell expressing yc and / or IL-21 Ra; and / or a cell expressing a yc:IL-21 Ra receptor.

[0670] Suitable method formats are well known in the art, including immunoassays such as sandwich assays, e.g. ELISA. The method can involve labelling the antigen binding molecule or the target or both with a detectable moiety (e.g. as described above). In some embodiments, the detectable moiety is a fluorescent label, a luminescent label, an immunodetectable label or a radioactive label. In some embodiments, the detectable moiety can be selected from: a radionucleotide, a positron emitting radionuclide (e.g. for positron emission tomography (PET)), an MRI contrast agent or a fluorescent label. In vitro or in vivo analysis can involve analysis by positron emission tomography (PET), magnetic resonance imaging (MRI) or fluorescence imaging, e.g. by detecting the appropriately labelled substance.

[0671] Such methods can provide a methodological basis for the diagnosis and / or prognostic evaluation of a disease or condition. Such methods can be performed in vitro on a patient sample, or after processing of a patient sample. Once a sample has been collected, the patient need not be present for the in vitro method to be performed, and thus the method can be one that is not practiced on the human or animal body.

[0672] In some embodiments, the method can comprise detecting or quantifying, for example, yc and / or IL-21 Ra in a patient sample; and / or cells expressing yc and / or IL-21 Ra; and / or cells expressing yc:IL-21 Ra receptors. Where the method comprises quantifying a molecule / complex, the method can further comprise comparing the determined amount to a standard or reference value as part of a diagnostic or prognostic evaluation. Other diagnostic / prognostic tests can be used in conjunction with those described herein to improve the accuracy of the diagnosis or prognosis, or to confirm results obtainable by use of the tests described herein.

[0673] The sample can be taken from any tissue or body fluid. The sample can comprise or can be derived from: an amount of blood; an amount of serum derived from the blood of an individual, which can comprise the fluid portion of blood obtained after removal of fibrin clot and blood cells; a tissue sample or biopsy; pleural fluid; cerebrospinal fluid (CSF); or cells isolated from the individual. In some embodiments, the sample can be obtained from or derived from a tissue affected by the disease / condition (e.g., a tissue exhibiting symptoms of the disease or involved in the pathogenesis of the disease / condition).

[0674] Subjects

[0675] A subject according to various aspects of the disclosure can be any animal or human. Therapeutic and prophylactic applications can be in humans or animals (veterinary use).

[0676] A subject to which an article of the disclosure is to be administered (e.g., according to a therapeutic or prophylactic intervention) can be a subject in need of such an intervention. The subject is preferably a mammal, more preferably a human. The subject can be a non-human mammal, but more preferably is a human. The subject can be male or female. The subject can be a patient.

[0677] The subject can have (e.g., can have been diagnosed with) a disease or condition described herein, can be suspected of having such a disease / condition, or can be at risk of developing / contracting such a disease / condition. In embodiments according to the disclosure, the subject can be selected for treatment according to a method based on the profile of one or more markers of such a disease / condition.

[0678] Kits

[0679] The disclosure also provides kits of parts.

[0680] In some embodiments, the kits can have at least one container with a predetermined 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.

[0681] In some embodiments, the kits can include materials for producing 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 some embodiments, the kits of parts can include materials for formulating an antigen binding molecule, polypeptide, nucleic acid (or plurality of nucleic acids), expression vector (or plurality of expression vectors), cell, or composition described herein into a pharmaceutical composition / drug (e.g., in a composition further comprising a pharmaceutically acceptable carrier, diluent, excipient, or adjuvant).

[0682] The kits can provide an antigen binding molecule, polypeptide, nucleic acid (or plurality of nucleic acids), expression vector (or plurality of expression vectors), cell, or composition along with instructions for administering to a patient for the treatment of a particular disease / condition (e.g., a disease / condition described herein).

[0683] In some embodiments, the kits can further include at least one container with a predetermined amount of another therapeutic agent (e.g., as described herein). In such embodiments, the kits can further include a second drug or pharmaceutical composition, such that the two drugs or pharmaceutical compositions can be administered simultaneously or separately, such that they provide combined therapy for a particular disease / condition.

[0684] The kits can also include reagents, buffers, and / or standards required to perform the methods according to the present disclosure. The kits according to the present disclosure can include instructions for use, e.g., in the form of a pamphlet or brochure. The instructions can include protocols for carrying out any one or more of the methods described herein.

[0685] Sequence identity

[0686] As used herein, "sequence identity" refers to the percentage of nucleotide / amino acid residues in a subject sequence that are identical with the nucleotide / amino acid residues in a reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Pairwise and multiple sequence alignments for the purpose of determining percent sequence identity between two or more amino acid or nucleic acid sequences can be achieved in a variety of ways known to one of skill in the art, for example, using publicly available computer software such as Clustal Omega (https: / / www.ebi.ac.uk / Tools / msa / clustalo / ), ClustalW (https: / / www.ebi.ac.uk / Tools / psa / clustalw / ), and the like. J. Bioinformatics (2005) 21 :951-960), T-coffee (Notredame et al. J Mol Biol. (2000) 302:205-217), Kalign (Lassmann and Sonnhammer, BMC Bioinformatics. (2005) 6(298)) and MAFFT (Katoh and Standley Molecular Biology and Evolution (2013) 30(4):772-780) software. When using such software, preferably the default parameters are used, such as gap penalty and extension penalty.

[0687] Sequence

[0688]

[0689]

[0690]

[0691]

[0692]

[0693]

[0694]

[0695]

[0696]

[0697]

[0698]

[0699]

[0700]

[0701]

[0702]

[0703]

[0704]

[0705]

[0706]

[0707]

[0708]

[0709]

[0710]

[0711]

[0712]

[0713]

[0714]

[0715]

[0716]

[0717]

[0718]

[0719]

[0720]

[0721]

[0722]

[0723]

[0724]

[0725]

[0726]

[0727]

[0728]

[0729]

[0730]

[0731]

[0732]

[0733]

[0734]

[0735]

[0736]

[0737]

[0738]

[0739]

[0740]

[0741]

[0742]

[0743]

[0744]

[0745]

[0746]

[0747]

[0748]

[0749]

[0750]

[0751]

[0752]

[0753] Table Al - gamma chain binding moieties

[0754]

[0755]

[0756] Table Bl - gamma chain binding moieties

[0757]

[0758]

[0759] Table Cl - gamma chain binding moieties

[0760]

[0761]

[0762] Table A2 - gamma chain binding moieties

[0763]

[0764] Table B2 - gamma chain binding moieties

[0765]

[0766] Table A3 - IL-21 Ra binding moieties

[0767]

[0768] Table B3 - IL-21 Ra binding moieties

[0769]

[0770]

[0771] ***

[0772] The present disclosure includes combinations of the described aspects and preferred features, except where such combinations are explicitly not permitted or should be explicitly avoided.

[0773] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0774] Aspects and embodiments of the present disclosure will now be described, by way of example, with reference to the accompanying drawings. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned herein are hereby incorporated by reference.

[0775] Throughout the specification and claims the word "comprise" and variations thereof such as "comprising" and "comprises" is not intended to exclude other integers or steps than those listed. Throughout the specification and claims the word "comprise" and variations thereof such as "comprising" and "comprises" is not intended to exclude other integers or steps than those listed.

[0776] As used herein, an amino acid sequence or region of a polypeptide that "corresponds" to a specified reference amino acid sequence or region of a polypeptide has 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 amino acid sequence of the amino acid sequence / polypeptide / region. An amino acid sequence / region / position of a polypeptide / amino acid sequence that "corresponds" to a specified reference amino acid sequence / region / position of a polypeptide / amino acid sequence can be identified by performing a sequence alignment of the subject sequence to the reference sequence, e.g., using sequence alignment software such as Clustal Omega (https: / / www.ebi.ac.uk / Tools / psa / clustalo / ) (Sievers et al., 2014, Bioinformatics 30, 1268-1270). J. 2005, Bioinformatics 21, 951-960).

[0777] It must be noted that, as used herein, the singular form "a", "an", and "the" include plural references unless the context clearly dictates otherwise. Ranges can be expressed herein as from "about" one particular value, and / or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations by use of the antecedent "about", it will be understood that the particular value forms another embodiment.

[0778] Where nucleic acid sequences are disclosed herein, the reverse complements are also expressly contemplated.

[0779] The methods described herein can preferably be performed in vitro. The term "in vitro" is intended to encompass procedures performed with cells in culture, while the term "in vivo" is intended to encompass procedures performed with / upon intact multicellular organisms.

[0780] EMBODIMENTS

[0781] Example 1 : Production and characterization of bispecific yc and CD122 binding antibodies in WO 2017 / 021540 Al, WO 2019 / 092181 Al, WO 2020 / 094834 Al and WO 2020 / 094836 Al

[0782] Generation and characterization of bispecific gamma c and CD122 binding antibody agonists of intermediate affinity gamma c / IL-2R beta receptors are described, e.g., in WO 2017 / 021540 Al, WO 2019 / 092181 Al, WO 2020 / 094834 Al, and WO 2020 / 094836 Al, which are incorporated by reference herein in their entireties.

[0783] 1.1 WO 2017 / 021540 Al

[0784] Example 1 of WO 2017 / 021540 Al discloses the identification of yc binding clones and CD122 binding clones from a human antibody phage display library via in vitro selection. Example 2 describes the generation of bispecific molecules comprising a yc binding arm and a CD122 binding arm (i.e., bispecific anti-yc, anti-CD122 antibodies). The bispecific anti-yc, anti-CD122 antibodies employed the following combinations of yc and CD122 binding clones:

[0785] Example 3 and Figure 21A demonstrate that bispecific antibodies comprising a yc binding arm and / or a CD122 binding arm bind to cells expressing yc or CD122, as determined by flow cytometry analysis. Example 4 and Figure 20 demonstrate that bispecific molecules comprising various different combinations of a yc binding arm and a CD122 binding arm bind to recombinant yc or CD122, and Table 1 of Example 1 summarizes the affinities of the bispecific antibodies to bind to the respective proteins, as determined by surface plasmon resonance analysis.

[0786] Example 5 and Figure 21B demonstrate that the bispecific anti-yc, anti-CD122 antibodies bind efficiently to CD56+ NK cells, CD19+ B cells, and CD14+ / CD16+ monocytes, but do not bind to CD4+ or CD8+ T cells with high affinity.

[0787] Example 6.1 and Figure 22 demonstrate that treatment of NK92 cells with the bispecific anti-yc, anti-CD122 antibody in vitro induces signaling through the STAT5 and Akt pathways, which are known intracellular signaling pathways triggered by IL-2. The molecule does not significantly upregulate phosphorylation of ERK, indicating that it does not activate the ERK pathway (another intracellular signaling pathway triggered by IL-2). Example 6.1 and Figure 23 demonstrate that the bispecific anti-yc, anti-CD122 antibody triggers signaling in IL-2 receptor-expressing cells (as determined by STAT5 phosphorylation analysis) that activates signaling in NK and CD8+ T cells to a greater extent than the activation of signaling in Tregs, particularly compared to the activation of signaling in Tregs.

[0788] Example 6.2 and Figure 24 demonstrate that the bispecific anti-yc, anti-CD122 antibody promotes in vitro proliferation of NK92 cells, and that bispecific molecules comprising one arm and an unrelated second arm do not stimulate such cell proliferation.

[0789] Example 6.2 and Figures 25B and 25C demonstrate that bispecific molecules comprising a yc binding arm and a CD122 binding arm (provided in scFv-Fc(KiH)-scFv or tandem scFv-scFv format and with various different lengths of linker (i.e., between the scFv portion and the constant region in the KiH format molecules, and between the two scFv portions in the tandem scFv-scFv format molecules)) induce proliferation of NK92 cells in vitro with similar efficiency.

[0790] Example 6.3 and Figure 26 show that the bispecific anti-yc, anti-CD122 antibody stimulates intracellular IL-2 receptor-mediated signaling (as determined by STAT5 phosphorylation analysis) in cynomolgus monkey splenocytes.

[0791] Example 7.1 and Figures 27A-27D demonstrate that the bispecific anti-yc, anti-CD122 antibody stimulates proliferation of primary human T cells in a dose-dependent manner (i.e., anti-CD3 / CD28 bead-stimulated) with a CD8:CD4 cell ratio in the expanded cell population comparable to that achieved following stimulation with recombinant human IL-2. Example 7.2 and Figure 28 show that stimulation with IL-2 under these conditions promotes expansion of Tregs, but the bispecific antibody does not trigger expansion of this Treg population. In addition, Example 7.3 and Figure 29 demonstrate that the bispecific antibody promotes expansion of effector memory CD8+ cells to a greater extent, and expansion of central memory T cells and naive T cells to a lesser extent, than stimulation with IL-2 under these conditions.

[0792] Example 8.1 and Figures 30A-30D demonstrate that the bispecific anti-yc, anti-CD122 antibody stimulates proliferation of primary human T cells within LCL-stimulated PBMC populations obtained from EBV seropositive donors. This molecule elicits an increase in the number of CD8+ T cells to a greater extent than recombinant IL-2, thus resulting in a greater CD8:CD4 T cell ratio. Example 8.2 and Figures 31A-31C show that under these conditions, the bispecific antibody favors expansion of CD8+ T cells over CD8+ memory cells, compared to IL-2, and also results in increased expansion of the CD8+ PD-1+ subpopulation, and decreased expansion of Tregs, compared to stimulation with IL-2. Example 8.3 and Figure 32 demonstrate that cytotoxicity against LCL is elicited by expanded T cells stimulated with the bispecific antibody.

[0793] Example 9 and FIG. 33A, FIG. 33B, FIG. 34A, and FIG. 34B disclose the generation of framework region variants of a yc-binding clone and a CD122-binding clone that have higher thermal stability relative to their respective parental clones. FIG. 35A and FIG. 35B demonstrate that the preferred, thermally stable variant clones retain dose-dependent binding to their respective target antigens.

[0794] Example 10 and FIG. 36A, FIG. 36B, FIG. 37A, and FIG. 37B demonstrate that scFv-Fc(KiH)-scFv format molecules comprising different short linkers between the scFv portions and the hinge region bind to recombinant yc or CD122 with similar affinity in ELISA, independent of the sequence of the short linker, and similar or improved affinity compared to the parental molecules.

[0795] Example 11 and FIG. 38A and FIG. 38B demonstrate that PBMC of EBV-positive, seropositive donors stimulated in vitro with LCL in the presence of a bispecific molecule comprising a yc-binding arm and a CD122-binding arm expand antigen-specific CD8+ T cells to a greater extent than stimulation in the presence of recombinant human IL-2 instead. Stimulation with the bispecific antibody also achieves expanded populations with a higher CD8:CD4 T cell ratio than using IL-2. FIG. 39A and FIG. 39B demonstrate that under antigen-specific and non-specific stimulation conditions, use of the bispecific anti-yc, anti-CD122 antibody is associated with significantly less Treg expansion than when IL-2 is used.

[0796] Example 12 discloses that intravenous administration of the bispecific anti-yc, anti-CD122 antibody to cynomolgus monkeys is associated with a significant proliferation of CD4+ and CD8+ T cells and NK cell populations (FIG. 41A-FIG. 41C, FIG. 42A and FIG. 42B) and is not associated with a significant increase in inflammatory cytokine levels (FIG. 40A-FIG. 40E). Expansion of CD4+ and CD8+ T cells and NK cells is observed after a single dose of the bispecific antibody, where continuous infusion / repeated doses of recombinant IL-2 are required to achieve such expansion, indicating that the bispecific antibody has a longer half-life than IL-2.

[0797] 1.2 WO 2019 / 092181 Al

[0798] Example 1 of WO 2019 / 092181 A1 discloses the generation of bispecific anti-yc, anti-CD122 antibodies in various different formats, in particular scFv-FcKiH-scFv format, CrossMab format, and Duobody format.

[0799] Example 2.1 and Figure 1 A and Figure 1Example 2.2 and Example 2.3 and

[0800] Example 2.2 and Example 2.3 and Figure 2 Example 2.2 and Example 2.3 and Figure 2 Example 2.2 and Example 2.3 and Figure 4 Example 2.2 and Example 2.3 and Figure 4 Example 2.2 and Example 2.3 and Example 2.2 and Example 2.3 and

[0801] Example 2.2 and Example 2.3 and Figure 3 Example 2.2 and Example 2.3 and Figure 3 Example 2.2 and Example 2.3 and Figure 5 Example 2.2 and Example 2.3 and Example 2.2 and Example 2.3 and

[0802] Example 3.1 and Example 3.2 and Figure 6 Example 3.1 and Example 3.2 and Figure 6 Example 3.1 and Example 3.2 and

[0803] Example 3.2 and Figures 7A to 7L demonstrate that the bispecific anti-gc, anti-CD122 antibody stimulates proliferation of primary human CD8+ T cells that were non-specifically activated (i.e., anti-CD3 / CD28 stimulation) while causing only minimal expansion of Tregs. The absolute number of Tregs was reduced 10-fold after treatment with the bispecific anti-gc, anti-CD122 antibody compared to treatment with IL-2. CD8+ T effector memory cells reacted most strongly to stimulation with the bispecific anti-gc, anti-CD122 antibody, and proliferation of CD4+ T effector memory cells was also observed. Figures 8A to 8G show that the bispecific anti-gc, anti-CD122 antibody proliferated non-specifically activated T cells in a dose-dependent manner, with expansion of CD8+ T cells being more pronounced than that of CD4+ T cells. The bispecific anti-gc, anti-CD122 antibody did not induce significant proliferation of Tregs, and it resulted in expanded populations with a higher ratio of CD8+ T cells to Tregs compared to populations expanded with IL-2 or IL-15. Figure 8H shows that pre-expanded human Tregs did not expand by treatment with the bispecific anti-gc, anti-CD122 antibody, while they responded to treatment with IL-2 or IL-15 to expand in a dose-dependent manner. Example 3.3 and Figures 9A to 9I similarly disclose preferential expansion of CD8+ T cells relative to Tregs and CD4+ T proliferation upon treatment of non-specifically activated primary human PBMCs treated with the bispecific anti-gc, anti-CD122 antibody.

[0804] Example 3.4 and Figures 10A to 10G demonstrate that the bispecific anti-gc, anti-CD122 antibody stimulates in vitro proliferation of antigen-specific (in particular EBV-specific) CD4+ and CD8+ T cells in a dose-dependent manner and also induces proliferation of NK cells within the viral-specific T cell population.

[0805] Example 3.5 and Figures 11A to 11K show that the bispecific anti-gc, anti-CD122 antibody stimulates dose-dependent proliferation of cynomolgus monkey CD4+ effector memory T cells, CD8+ naive T cells, CD8+ effector memory T cells, and NK cells that were non-specifically activated (i.e., anti-CD3 / CD28 stimulation). The bispecific anti-gc, anti-CD122 antibody did not induce proliferation of cynomolgus monkey Tregs, while IL-2 did.

[0806] Example 3.6 and Figures 12A to 12N similarly show that the bispecific anti-gc, anti-CD122 antibody induced in vitro proliferation of all cynomolgus monkey CD4+ and CD8+ T cell subsets under non-specific activation conditions, with preferential expansion of CD8+ T cells relative to CD4+ T cells.

[0807] Example 3.7 and Figures 27A-27D demonstrate that the bispecific anti-gc, anti-CD122 antibody also induces proliferation of pre-activated NK cells in a dose-dependent manner.

[0808] Example 3.8 and Figures 28A-28D further demonstrate that the bispecific anti-gc, anti-CD122 antibody induces proliferation of CAR-expressing T cells, with CD8+ CAR-T cells appearing to be more responsive to this stimulation than CD4+ CAR-T cells.

[0809] Example 4.1 and Figure 13 demonstrate that the bispecific anti-gc, anti-CD122 antibody induces STAT5 phosphorylation in NK92 cells, while Example 4.2 and Figures 14A-14H similarly demonstrate that the bispecific anti-gc, anti-CD122 antibody induces STAT5 phosphorylation in primary human T cells and NK cells in a dose-dependent manner. Example 4.3 and Figures 15A-15C demonstrate that the bispecific anti-gc, anti-CD122 antibody induces STAT5 phosphorylation in primary human CD4+, CD8+ T cell subsets as well as NK cells in a dose-dependent manner, in the presence of non-specific activation (i.e., anti-CD3 / CD28 stimulation). Example 4.6 and Figures 18A-18C demonstrate the induction of STAT5 phosphorylation by the bispecific molecule in EBV-specific T cells. Example 4.4 and 4.5 and Figure 16 demonstrate the time-dependent induction of STAT5 phosphorylation by the bispecific anti-gc, anti...

Claims

1. An antigen binding molecule, optionally isolated, comprising: (i) a yc binding moiety, and (ii) an IL-21Ra binding moiety.

2. The antigen binding molecule of any one claim 1, wherein the antigen binding molecule is an agonist of a yc-containing cytokine receptor, or an antagonist of a yc-containing cytokine receptor.

3. The antigen binding molecule of claim 1 or claim 2, wherein the antigen binding molecule increases signaling mediated by the antigen binding molecule bound yc-containing cytokine receptor, or decreases signaling mediated by the antigen binding molecule bound yc-containing cytokine receptor.

4. The antigen binding molecule of any one of claims 1 to 3, wherein the antigen binding molecule increases signaling mediated by a yc:IL-21Ra receptor, or decreases signaling mediated by a yc:IL-21Ra receptor.

5. The antigen binding molecule of any one of claims 1 to 4, wherein the IL-21Ra binding moiety comprises a single domain antibody sequence incorporating: (i) a CDR1 having the amino acid sequence of SEQ ID NO: 378 a CDR2 having the amino acid sequence of SEQ ID NO: 379 a CDR3 having the amino acid sequence of SEQ ID NO: 380; (ii) a CDR1 having the amino acid sequence of SEQ ID NO: 386 a CDR2 having the amino acid sequence of SEQ ID NO: 387 a CDR3 having the amino acid sequence of SEQ ID NO: 388; or (iii) a CDR1 having the amino acid sequence of SEQ ID NO: 394 a CDR2 having the amino acid sequence of SEQ ID NO: 395 a CDR3 having the amino acid sequence of SEQ ID NO:

396.

6. The antigen binding molecule of claim 5, wherein the IL-21Ra binding moiety comprises or consists of an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 385, 393 or 401.

7. The antigen binding molecule of claim 5 or claim 6, wherein the IL-21Ra binding moiety comprises a single domain antibody sequence incorporating: (i) a FR1 having the amino acid sequence of SEQ ID NO: 381 a FR2 having the amino acid sequence of SEQ ID NO: 382 a FR3 having the amino acid sequence of SEQ ID NO: 383 a FR4 having the amino acid sequence of SEQ ID NO: 384; (ii) a FR1 having the amino acid sequence of SEQ ID NO: 389 a FR2 having the amino acid sequence of SEQ ID NO: 390 a FR3 having the amino acid sequence of SEQ ID NO: 391 a FR4 having the amino acid sequence of SEQ ID NO: 392; or (iii) a FR1 having the amino acid sequence of SEQ ID NO: 398 a FR2 having the amino acid sequence of SEQ ID NO: 399 a FR3 having the amino acid sequence of SEQ ID NO: 400 a FR4 having the amino acid sequence of SEQ ID NO:

401. (iii) FR1 having the amino acid sequence of SEQ ID NO:397 FR2 having the amino acid sequence of SEQ ID NO:398 FR3 having the amino acid sequence of SEQ ID NO:399 FR4 having the amino acid sequence of SEQ ID NO:

400.

8. The antigen binding molecule of any one of claims 1 to 7, wherein the yc binding moiety comprises a single domain antibody sequence incorporating the following CDRs: CDR1 having the amino acid sequence of SEQ ID NO:370 CDR2 having the amino acid sequence of SEQ ID NO:371 CDR3 having the amino acid sequence of SEQ ID NO:

372.

9. The antigen binding molecule of claim 8, wherein the yc binding moiety comprises or consists of an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:

377.

10. The antigen binding molecule of claim 8 or claim 9, wherein the yc binding moiety comprises a single domain antibody sequence incorporating the following FRs: FR1 having the amino acid sequence of SEQ ID NO:373 FR2 having the amino acid sequence of SEQ ID NO:374 FR3 having the amino acid sequence of SEQ ID NO:375 FR4 having the amino acid sequence of SEQ ID NO:

376.

11. The antigen binding molecule of any one of claims 1 to 10, wherein 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.

12. The antigen binding molecule of claim 11, wherein the target antigen other than a yc containing cytokine receptor polypeptide is a disease associated antigen or an antigen expressed by an immune cell.

13. A chimeric antigen receptor (CAR) comprising the antigen binding molecule of any one of claims 1 to 12.

14. A nucleic acid or plurality of nucleic acids, optionally isolated, encoding the antigen binding molecule of any one of claims 1 to 12 or the CAR of claim 13.

15. An expression vector or plurality of expression vectors comprising the nucleic acid or plurality of nucleic acids of claim 14.

16. A cell comprising the antigen binding molecule of any one of claims 1 to 12, the CAR of claim 13, the nucleic acid or plurality of nucleic acids of claim 14, or the expression vector or plurality of expression vectors of claim 15.

17. A method comprising culturing a cell of claim 15 under conditions suitable for the cell to express the antigen binding molecule or CAR.

18. A composition comprising the antigen binding molecule of any one of claims 1 to 12, the CAR of claim 13, the nucleic acid or plurality of nucleic acids of claim 14, the expression vector or plurality of expression vectors of claim 15, or the cell of claim 16, and a pharmaceutically acceptable carrier, diluent, excipient, or adjuvant.

19. The antigen binding molecule of any one of claims 1 to 12, the CAR of claim 13, the nucleic acid or plurality of nucleic acids of claim 15, the expression vector or plurality of expression vectors of claim 15, the cell of claim 16, or the composition of claim 18, for use in a method of treatment or prevention.

20. Use of the antigen binding molecule of any one of claims 1 to 12, the CAR of claim 13, the nucleic acid or plurality of nucleic acids of claim 15, the expression vector or plurality of expression vectors of claim 15, the cell of claim 16, or the composition of claim 18, in the manufacture of a medicament for use in a method of treatment or prevention.

21. A method of treatment or prevention comprising administering to a subject in need thereof a therapeutically or prophylactically effective amount of the antigen binding molecule of any one of claims 1 to 12, the CAR of claim 13, the nucleic acid or plurality of nucleic acids of claim 15, the expression vector or plurality of expression vectors of claim 15, the cell of claim 16, or the composition of claim 18.

22. The antigen binding molecule, nucleic acid or plurality of nucleic acids, expression vector or plurality of expression vectors, cell or composition for use according to claim 19, the use according to claim 20, or the method according to claim 21, wherein the method of treatment or prevention is a method of treatment or prevention of a disease / condition characterized by T cell dysfunction, cancer, infectious disease, or autoimmune disease.

23. The antigen binding molecule, nucleic acid or plurality of nucleic acids, expression vector or plurality of expression vectors, cell or composition for use, use, or method according to claim 22, wherein the cancer is selected from the group consisting of colon cancer, colon cancer, colorectal cancer, nasopharyngeal cancer, cervical cancer, oropharyngeal cancer, gastric cancer, hepatocellular cancer, 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.

24. An in vitro complex, optionally isolated, comprising the antigen binding molecule of any one of claims 1 to 12, the CAR of claim 13, bound to yc and IL-21Ra.

25. A method for generating or expanding a population of cells expressing a yc-containing cytokine receptor, comprising contacting a cell expressing a yc-containing cytokine receptor in vitro, in vivo, or ex vivo with the antigen binding molecule of any one of claims 1 to 12.

26. A method for increasing proliferation, survival, and / or effector activity of a cell expressing a yc-containing cytokine receptor, comprising contacting a cell expressing a yc-containing cytokine receptor in vitro, in vivo, or ex vivo with the antigen binding molecule of any one of claims 1 to 12.

27. A method for reducing the number / proportion of cells expressing a yc-containing cytokine receptor, comprising contacting a cell expressing a yc-containing cytokine receptor in vitro, in vivo, or ex vivo with the antigen binding molecule of any one of claims 1 to 12 or a cell comprising the CAR of claim 13.

28. A method for decreasing proliferation, survival, and / or effector activity of a cell expressing a yc-containing cytokine receptor, comprising contacting a cell expressing a yc-containing cytokine receptor in vitro, in vivo, or ex vivo with the antigen binding molecule of any one of claims 1 to 12.

29. The method of any one of claims 25 to 28, wherein the cell is an effector immune cell.

30. The method of any one of claims 25 to 29, wherein the cell is a T cell or an NK cell.

31. The method of any one of claims 25 to 30, wherein the yc-containing cytokine receptor is a yc:IL-21Ra receptor.

32. A method of promoting heteromultimerization of yc and IL-21Ra, comprising contacting yc and IL-21Ra in vitro, in vivo, or ex vivo with the antigen binding molecule of any one of claims 1 to 12, or the CAR of claim 13.

33. A method of inhibiting heteromultimerization of yc and IL-21Ra, comprising contacting yc and IL-21Ra in vitro, in vivo, or ex vivo with the antigen binding molecule of any one of claims 1 to 12, or the CAR of claim 13.

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