Tumor-targeted il2 receptor agonists and multispecific t-cell engagers

Abstract

The present disclosure relates to combinations of tumor-targeted IL2 receptor agonists and multispecific T-cell engagers, e.g., for use in stimulating T-cells against cancer cells or treatment of cancer. The tumor-targeted IL2 receptor agonists comprise a tumor-associated antigen targeting moiety and an IL2 moiety. The multispecific T-cell engagers comprise a tumor-associated antigen targeting moiety and a T-cell receptor complex targeting moiety.

Description

IMMUNE CELL SYNAPSE TARGETED THERAPEUTICS1. CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority benefit of U.S. provisional application no. 63 / 701 ,707, filed on October 1 , 2024, the contents of which are incorporated herein in their entirety by reference thereto.2. SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically and is hereby incorporated by reference in its entirety. Said copy, created on September 24, 2025, is named RGN-047WO_SL.xml and is 78,415 bytes in size.3. BACKGROUND

[0003] Interleukin 2 (IL-2 or IL2) is a pluripotent cytokine produced primarily by CD4+ helper T-cells. It stimulates the proliferation and differentiation of T cells, induces the generation of cytotoxic T lymphocytes (CTLs) and the differentiation of peripheral blood lymphocytes to cytotoxic cells and lymphokine-activated killer (LAK) cells, promotes cytokine and cytolytic molecule expression by T cells, facilitates the proliferation and differentiation of B-cells and the synthesis of immunoglobulin by B-cells, and stimulates the generation, proliferation and activation of natural killer (NK) cells (see Waldmann, 2009, Nat Rev Immunol 6:595-601 and Malek, 2008, Annu Rev Immunol 26:453-79).

[0004] Due to its pleotropic effects, IL2 is not optimal for inhibiting tumor growth. The use of IL2 as an antineoplastic agent has been limited by the serious toxicities that accompany the doses necessary for a tumor response. Proleukin® (marketed by Prometheus Laboratories, San Diego, Calif.) is a recombinant form of IL2 that is approved for the treatment of metastatic melanoma and metastatic renal cancer, but its side effects are so severe that its use is only recommended in a hospital setting with access to intensive care. Patients receiving high-dose IL2 treatment frequently experience severe cardiovascular, pulmonary, renal, hepatic, gastrointestinal, neurological, cutaneous, hematological and systemic adverse events, which require intensive monitoring and in-patient management. The major side effect of IL2 therapy is vascular leak syndrome (VLS), which leads to the accumulation of interstitial fluid in the lungs and liver resulting in pulmonary edema and liver damage. There is no treatment for VLS other than withdrawal of IL2. Low-dose IL2 regimens have been tested in patients to avoid VLS, however, at the expense of suboptimal therapeutic results. It has been shown that IL2-induced pulmonary edema resulted from direct binding of IL2 to lung endothelial cells, which express low to intermediate levels of functional high affinity IL2 receptors (Krieg et al., 2010, Proc Nat Acad Sci USA 107:11906-11).

[0005] A variety of IL2 variants and prodrugs have been generated with the aim of reducing the toxicity of IL2 cancer therapy. However, it has been surprisingly discovered that such molecules have poor therapeutic indices for cancer therapy. For example, the PEGylated IL2 prodrug bempegaldesleukin failed to improve on the therapeutic efficacy of a PD1 checkpoint inhibitor in melanoma patients in phase 3 clinical studies (Mullard, 2022, Nature Reviews Drug Discovery 21 .321 (doi: https: / / doi.org / 10.1038 / d41573-022-00069-3).

[0006] Thus, there is a need in the art for novel IL2-based therapies with improved therapeutic efficacy and safety profiles.4. SUMMARY

[0007] The present disclosure provides combinations comprising tumor-targeted IL2 receptor agonists and multispecific T-cell engagers, e.g., for use in cancer therapy.

[0008] In certain aspects, the combinations address the drawbacks of IL2 therapy and are characterized by improved therapeutic profiles by virtue of improved efficacy and / or improved safety profiles.

[0009] The combinations of the disclosure can be formulated in a single formulation or separate formulations comprising a tumor-targeted IL2 receptor agonist and a multispecific T-cell engager. An overview of combinations comprising tumor-targeted IL2 receptor agonists and multispecific T-cell engagers is disclosed in Section 6.2 and exemplary combinations comprising tumor-targeted IL2 receptor agonist and multispecific T-cell engager are set forth in numbered embodiments 1 to 7.

[0010] Tumor-targeted IL2 receptor agonists comprise a tumor associated antigen (“TAA”) targeting moiety and an IL2 moiety. Exemplary tumor-targeted IL2 receptor agonists are disclosed in Section 6.3 and numbered embodiments 15 to 79, 90 to 93, and 81 to 101 .

[0011] Multispecific T-cell engagers comprise a TAA targeting moiety and a T-cell targeting moiety. Exemplary multispecific T-cell engagers are disclosed in Section 6.4 and numbered embodiments 80 to 90, 94 to 100, and 102 to 104.

[0012] The disclosure further provides nucleic acids encoding the combinations and their components. The nucleic acids can be in the form of a single nucleic acid (e.g., a vector encoding all components of a tumor-targeted IL2 receptor agonist and a multispecific T-cell engager) or a plurality of nucleic acids (e.g., two or more vectors encoding the different components and / or their individual polypeptide chains). The disclosure further provides host cells and cell lines engineered to express the nucleic acids and tumor-targeted IL2 receptor agonist and / or multispecific T-cell engager of the disclosure. The disclosure further provides methods of producing a tumor-targeted IL2 receptor agonist and a multispecific T-cellengager of the disclosure. Exemplary nucleic acids, host cells, cell lines, and methods of production are described in Section 6.8.

[0013] The disclosure further provides pharmaceutical compositions comprising the tumor- targeted IL2 receptor agonists and / or multispecific T-cell engagers of the disclosure. Exemplary pharmaceutical compositions are described in Section 6.9.

[0014] Further provided herein are methods of using the combinations of the disclosure, e.g., for eliciting anti-tumor cytotoxicity and treating cancerous conditions. Exemplary methods are described in Section 6.10 and numbered embodiments 8 to 104, infra.5. BRIEF DESCRIPTION OF THE FIGURES

[0015] FIGS. 1A-1C are cartoons that show the constituent components of tumor-targeted interleukin-2 (IL2) receptor agonists and multispecific T-cell engager constructs and formation of a cell-cell linkage between a tumor cell and a T-cell by a tumor-targeted IL2 receptor agonist and a multispecific T-cell engager construct. FIG. 1 A depicts the components of an exemplary tumor-targeted IL2 receptor agonist construct comprising a tumor associated antigen (TAA) targeting moiety (TTM), a connector moiety (CM), and an IL2 moiety (IM). FIG. 1B depicts the components of an exemplary multispecific T-cell engager comprising a TAA targeting moiety (TTM), a connector moiety (CM), and a T-cell receptor complex targeting moiety (TCRTM). FIG. 1C depicts a tumor cell and a T-cell linked by an exemplary combination of a tumor-targeted IL2 receptor agonist and a multispecific T- cell engager. The tumor-targeted IL2 receptor agonist binds to an antigen on the surface of the tumor cell via its TAA targeting moiety and to an IL2 receptor on the surface of the T-cell via its IL2 moiety, whereas the multispecific T-cell engager binds to an antigen on the surface of the tumor cell via its TAA targeting moiety and to a T-cell receptor complex on the surface of the T-cell. As a result, the tumor cell-T-cell cytotoxic synapse and activity is enhanced.

[0016] FIGS. 2A-2C are illustrations that depict an exemplary tumor-targeted IL2 receptor agonist, its constituent components and mechanism of action. FIG. 2A shows representations of the individual components used in the construct depicted in FIG. 2B, wherein IM stands for IL2 moiety, MM stands for masking moiety, CM stands for connector moiety, and TTM stands for TAA targeting moiety. FIG. 2B is an exemplary structure of a tumor-targeted IL2 receptor agonist, comprising TAA targeting moieties in Fab format located N-terminally to a connector moiety in an Fc format and masking moieties (e.g., IL2Ra masking moieties) and IL2 moieties located C-terminally to the connector moiety. Although the TAA targeting moieties are illustrated as Fabs, they can be in other formats, e.g., scFvs or other formats described in Sections 6.6.2 and 6.6.3, respectively. Similarly, theconnector moiety can be in other formats, such as those described in Section 6.7. FIG. 2C illustrates the mechanism of action of masking the IL2 moiety, in which there is an equilibrium between masked and unmasked species.

[0017] FIGS. 3A-3C show exemplary multispecific T-cell engager constructs. FIG. 3A shows a bispecific T-cell engager construct which has a TAA targeting moiety and a T-cell receptor (e.g., CD3) targeting moiety, both in Fab formats, located N-terminally to a connector moiety in Fab format. Although the TAA targeting moieties and T-cell receptor complex targeting moieties are illustrated as Fabs, they can be in other formats, e.g., scFvs or other formats described in Sections 6.6.2 and 6.6.3, respectively. Similarly, the connector moieties can be in other formats, such as those described in Section 6.7. FIG. 3B shows a bispecific T-cell engager construct in a CrossMabCH CLformat, which comprises a domain crossover between the CH1 and CL domains of one of the Fabs. Although the domain crossover is illustrated between the CH1 and CL domains of the T-cell receptor complex targeting moiety, it can be present between the CH1 and CL domains of the TAA targeting moiety instead of or in addition to the domain crossover illustrated here. FIG. 3C shows a bispecific T-cell engager in BiTE format, which comprises a T-cell receptor complex targeting moiety and a TAA targeting moiety, both in scFv format, connected via a linker, e.g., a linker described in Section 6.7.3.

[0018] FIGS. 4A - 4B are cartoon illustrations depicting the improved trans-activity of tumor- targeted IL2 receptor agonist with a masked IL2 moiety in the presence of multispecific T-cell engager. FIG. 4A illustrates a tumor cell and T-cell that are unlinked in the absence of a multispecific T-cell engager. In this exemplary depiction, a tumor-targeted IL2 receptor agonist is bound to the antigens on the surface of the tumor cell via its TAA targeting moieties, whereas its IL2 and masking moieties remain unbound. FIG. 4B illustrates a tumor cell and T-cell that are linked by a multispecific (e.g., a bispecific) T-cell engager, allowing the tumor-targeted IL2 receptor agonist to more efficiently engage and activate IL2 receptors on T cell. In the top portion of FIG. 4B, the multispecific (e.g., a bispecific) T-cell engager binds to an antigen on the surface of the tumor cell via its TAA targeting moiety and to a T- cell receptor complex on the surface of the T-cell via its T-cell receptor complex targeting moiety, bringing the tumor cell and T-cell into proximity and stabilizing the tumor cell-T-cell synapse, thereby allowing the IL2 moiety in the IL2 receptor agonist to become unmasked and activate the IL2 receptor as illustrated in the bottom portion of FIG. 4B.

[0019] FIGS. 5A-5B are graphs that show the T-cell mediated MUC16+tumor cell killing upon treatment with a tumor-targeted IL2 receptor agonist or control molecules in the presence of a bispecific T-cell engager, MUC16 x CD3. FIG. 5A shows the T-cell mediatedtumor cell killing by MUC16-targeted IL2 or control constructs, obtained after the unbound constructs were washed out. FIG. 5B shows the T-cell mediated tumor cell killing by MUC16-targeted IL2 or control constructs, obtained without washing out the unbound constructs.

[0020] FIGS. 6A-6H are graphs that show the effect of MUC16-targeted IL2 and MUC16 x CD3 combination or control treatments on tumor growth and percent change in weight in an ID8-VEGF tumor model. FIG. 6A shows the changes in mean tumor volume values over time in mice treated with a combination of 2.5 mpk MUC16 x CD3 and 2.5 mpk MUC16- targeted IL2 or with control treatments. FIG. 6B shows the change in tumor volume over time in individual mice treated with 2.5 mpk NT x CD3. FIG. 6C shows the change in tumor volume over time in individual mice treated with 2.5 mpk MUC16 x CD3. FIG. 6D shows the change in tumor volume over time in individual mice treated with 2.5 mpk MUC16 x CD3 + 0.5 mpk NT-IL2. FIG. 6E shows the change in tumor volume over time in individual mice treated with 2.5 mpk MUC16 x CD3 + 2.5 mpk NT-IL2. FIG. 6F shows the change in tumor volume over time in individual mice treated with 2.5 mpk MUC16 x CD3 + 0.5 mpk MUC16(NC)-IL2. FIG. 6G shows the change in tumor volume overtime in individual mice treated with 2.5 mpk MUC16 x CD3 + 2.5 mpk MUC16(NC)-IL2. FIG. 6H shows percent weight changes over time in the same group of mice in FIGS. 6A-6G. The arrows in FIGS. 6A-6H represent delivery of dosing. NT= non-targeted; NC = non-competing.

[0021] FIGS. 7A-7B are graphs that show the effect of MSLN-targeted IL2 and MUC16 x CD3 combination or control treatments on tumor volume and percent change in weight in an ID8-VEGF tumor model. FIG. 7A shows the changes in mean tumor volume values over time in mice treated with a combination of 2.5 mpk MUC16 x CD3 and 2.5 mpk MSLN- targeted IL2 or with control treatments. FIG. 7B shows percent weight changes over time in mice treated with a combination of 2.5 mpk MUC16 x CD3 and 2.5 mpk MSLN-targeted IL2 or with control treatments. The arrows in FIGS. 7A-7B represent delivery of dosing. NT= non-targeted; NC = non-competing.

[0022] FIGS. 8A-8B are graphs that show the effect of MUC16-targeted IL2 + MUC16 x CD3 combination, and the trispecific construct MUC16-IL2 x CD3-IL2 on tumor growth and percent change in weight in an ID8-VEGF tumor model. FIG. 8A shows the changes in mean tumor volume values overtime in mice treated with a combination of 1 mpk MUC16 x CD3 and 1.5 mpk MUC16-targeted IL2 or with control treatments. FIG. 8B shows percent weight changes over time in the same groups of mice. The arrows in FIGS. 8A-8B represent delivery of dosing.

[0023] FIGS. 9A-9D show the results of tumor rechallenge in mice treated with MUC16- targeted IL2 and MUC 16 x CD3 combination or controls. FIG. 9A is a schematic depiction of the tumor rechallenge protocol. FIG. 9B is a chart that shows the numbers of tumor-free mice post-implantation and post-reimplantation. FIG. 9C is a graph that shows the change in tumor volume over time in individual control mice. FIG. 9D is a graph that shows the growth of rechallenged ID8 / parental tumors in individual naive control mice, and mice previously cleared ID8 / MUC16 tumors after treatment with a combination of 1 mpk MUC16-CD3 and 1 .5 mpk MUC16-targeted IL2. NC = non-competing.

[0024] FIGS. 10A-10I show T-cell expansion and systemic cytokine levels in blood collected from mice treated with a combination of MUC 16 x CD3 and MUC16-targeted IL2 or with control treatments. FIG. 10A is a schematic depiction of the experimental protocol. FIG. 10B is a graph that shows the change in tumor volume overtime in mice treated with a combination of 1 mpk MUC16-CD3 and 2.5 mpk MUC16-targeted IL2 or dose matched control treatments. FIG. 10C is a graph that shows the percent change in body weight over time in mice treated with a combination of 1 mpk MUC16-CD3 and 2.5 mpk MUC16-targeted IL2 or control treatments. FIG. 10D is a graph that shows the total white blood cell count. FIG. 10E is a graph that shows the number of T-cells. FIG. 10F is a graph that shows the number of Treg cells. FIG. 10G is a graph that shows systemic IFNy levels. FIG. 10H is a graph that shows systemic TNFa levels. FIG. 101 is a graph that shows systemic IL10 levels.6. DETAILED DESCRIPTION6.1. DEFINITIONS

[0025] About, Approximately: The terms “about”, “approximately” and the like are used throughout the specification in front of a number to show that the number is not necessarily exact (e.g., to account for fractions, variations in measurement accuracy and / or precision, timing, etc.). It should be understood that a disclosure of “about X” or “approximately X” where X is a number is also a disclosure of “X.” Thus, for example, a disclosure of an embodiment in which one sequence has “about X% sequence identity” to another sequence is also a disclosure of an embodiment in which the sequence has “X% sequence identity” to the other sequence.

[0026] And, or: Unless indicated otherwise, an “or” conjunction is intended to be used in its correct sense as a Boolean logical operator, encompassing both the selection of features in the alternative (A or B, where the selection of A is mutually exclusive from B) and the selection of features in conjunction (A or B, where both A and B are selected). In some places in the text, the term “and / or” is used for the same purpose, which shall not be construed to imply that “or” is used with reference to mutually exclusive alternatives.

[0027] Associated: As used herein, the term “associated” refers to a functional relationship between two or more polypeptide chains. In particular, the term “associated” means that two or more polypeptides are associated with one another, e.g., non-covalently through molecular interactions or covalently through one or more disulfide bridges or chemical crosslinkages, so as to produce a functional tumor-targeted IL2 receptor agonist or a functional multispecific T-cell engager. Examples of associations that might be present in a tumor- targeted IL2 receptor agonist or a multispecific T-cell engager of the disclosure include (but are not limited to) associations between Fc domains to form an Fc region (homodimeric or heterodimeric as described in Section 6.7.1), associations between VH and VL regions in a Fab or Fv, and associations between CH1 and CL in a Fab.

[0028] Bispecific: As used herein, the term “bispecific” refers to molecules (e.g., T-cell engager molecules) comprising two different targeting moieties. For instance, each targeting moiety of a bispecific molecule can bind to a different target antigen (e.g., one targeting moiety binds to a tumor associated antigen and the other targeting moiety binds to a T-cell antigen) or targeting moieties of a bispecific molecule can bind to two different portions of the same target antigen (or, in some cases, different variants of the same target antigen).

[0029] Bispecific T-Cell Engager: The term “bispecific T-cell engager” as used herein refers to a molecule (e.g., a molecule comprising multiple polypeptide chains) which specifically bind to a T-cell (e.g., to a TCR complex of a T-cell) and one other target. In some embodiments, the bispecific T-cell engager binds to a target on a T-cell (e.g., one or more different epitopes of a TCR complex of a T-cell) and a target on a tumor cell (e.g., a tumor- associated antigen present on a tumor cell).

[0030] Bivalent: The term “bivalent” as used herein in reference to a tumor-targeted IL2 receptor agonist means that the tumor-targeted IL2 receptor agonist has two IL2 moieties and / or targeting moieties. Typically, tumor-targeted IL2 receptor agonist molecules that are bivalent for an IL2 moiety and / or a targeting moiety are dimeric (either homodimeric or heterodimeric).

[0031] Cancer: The term “cancer” refers to a disease characterized by the uncontrolled (and often rapid) growth of aberrant cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers are described herein and include but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, adrenal gland cancer, autonomic ganglial cancer, biliary tract cancer, bone cancer, endometrial cancer, eye cancer, fallopian tube cancer, genital tract cancers, large intestinal cancer, cancer of the meninges, esophageal cancer, peritonealcancer, pituitary cancer, penile cancer, placental cancer, pleura cancer, salivary gland cancer, small intestinal cancer, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, upper aerodigestive cancers, urinary tract cancer, vaginal cancer, vulva cancer, lymphoma, leukemia, lung cancer and the like.

[0032] Connector Moiety: As used herein, the term “connector moiety” refers to an amino acid sequence that connects two components of a protein, e.g., a TAA targeting moiety and an IL2 moiety in a tumor-targeted IL2 receptor agonist or a TAA targeting moiety and a T-cell receptor complex targeting moiety in a multispecific T-cell engager. In some embodiments, the connector moiety is an Fc domain or an Fc region formed by the association of two Fc domains. The term connector moiety need not connect the two components directly. For example, in a multispecific T-cell engager, an Fc region formed by association of two Fc domains, one operably linked to a TAA targeting moiety (or portion thereof, e.g., a VH domain or VH-CH1 domain) and a T-cell receptor complex targeting moiety (or portion thereof, e.g., a VH domain or VH-CH1 domain), is considered a connector moiety connecting the TAA targeting moiety and the T-cell receptor complex targeting moiety.

[0033] Epitope: An epitope, or antigenic determinant, is a portion of an antigen recognized by an antibody or other antigen-binding moiety as described herein. An epitope can be linear or conformational.

[0034] Fab: The term “Fab” refers to a pair of polypeptide chains, the first comprising a variable heavy (VH) domain of an antibody operably linked (typically N-terminal to) to a first constant domain (referred to herein as C1), and the second comprising variable light (VL) domain of an antibody N-terminal operably linked (typically N-terminal) to a second constant domain (referred to herein as C2) capable of pairing with the first constant domain. In a native antibody, the VH is N-terminal to the first constant domain (CH1) of the heavy chain and the VL is N-terminal to the constant domain of the light chain (CL). The Fabs of the disclosure can be arranged according to the native orientation or include domain substitutions or swaps that facilitate correct VH and VL pairings. For example, it is possible to replace the CH1 and CL domain pair in a Fab with a CH3-domain pair to facilitate correct modified Fab-chain pairing in heterodimeric molecules. It is also possible to reverse CH1 and CL, so that the CH1 is attached to VL and CL is attached to the VH, a configuration generally known as Crossmab. The term “Fab” encompasses single chain Fabs.

[0035] Fc Domain and Fc Region: The term “Fc domain” refers to a portion of the heavy chain that pairs with the corresponding portion of another heavy chain. The term “Fc region” refers to the region formed by association of two heavy chain Fc domains. The two Fc domains within the Fc region may be the same or different from one another. In a nativeantibody the Fc domains are typically identical, but one or both Fc domains might be modified to allow for heterodimerization, e.g., via a knob-in-hole interaction.

[0036] Host Cell or Recombinant Host Cell: The terms “host cell” or “recombinant host cell” refer to a cell that has been genetically engineered, e.g., through introduction of a heterologous nucleic acid. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein. A host cell may carry the heterologous nucleic acid transiently, e.g., on an extrachromosomal heterologous expression vector, or stably, e.g., through integration of the heterologous nucleic acid into the host cell genome. For purposes of expressing a IL2 proprotein of the disclosure, a host cell is preferably a cell line of mammalian origin or mammalian-like characteristics, such as monkey kidney cells (COS, e.g., COS-1 , COS-7), HEK293 ), baby hamster kidney (BHK, e.g., BHK21), Chinese hamster ovary (CHO), NSO, PerC6, BSC-1 , human hepatocellular carcinoma cells (e.g., Hep G2), SP2 / 0, HeLa, Madin-Darby bovine kidney (MDBK), myeloma and lymphoma cells, or derivatives and / or engineered variants thereof. The engineered variants include, e.g., derivatives that grow at higher density than the original cell lines and / or glycan profile modified derivatives and / or site- specific integration site derivatives.

[0037] IL2 Moiety: As used herein, the terms “IL2 moiety” and “interleukin-2 moiety” refer to a polypeptide that has IL2 activity and comprises an amino acid sequence having at least 70% sequence identity, e.g., at least 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, to a mammalian (e.g., human or murine) wild type IL2 amino acid sequence as described in Section 6.3.2.

[0038] IL2Ra Moiety: As used herein, the term “IL2Ra moiety” and related terms “interleukin-2 receptor subunit alpha moiety”, “IL2 receptor alpha moiety” and the like refer to a polypeptide comprising an amino acid sequence having at least 70% sequence identity, e.g., at least 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, to an IL2 binding portion of a mammalian (e.g., human or murine) IL2 receptor subunit alpha (IL2Ra) as described in Section 6.3.3. In some embodiments, the IL2 binding portion of IL2Ra comprises or consists of the extracellular domain of the receptor subunit or a portion thereof.

[0039] Multispecific T-Cell Engager: The term “multispecific T-cell engager” as used herein refers to a molecule (e.g., a molecule comprising multiple polypeptide chains) which specifically bind to a T-cell (e.g., to a TCR complex of a T-cell) and at least one other target. In some embodiments, a multispecific T-cell engager binds to at least two different epitopes (and in some instances three, four, or more different epitopes). A multispecific T-cell engager of the disclosure may be bivalent, trivalent, tetravalent, or otherwise multivalent, and may be monospecific, bispecific, or otherwise multispecific. A multispecific T-cell engager of the disclosure may specifically bind to one or more epitopes on a T-cell (e.g., one or more different epitopes of a TCR complex of a T-cell) and / or one, two, three, four, or more different antigens in addition to an epitope on a T-cell (e.g., one or more different epitopes of a TCR complex of a T-cell). In some embodiments, the multispecific T-cell engager binds to one or more epitopes on a T-cell (e.g., one or more different epitopes of a TCR complex of a T-cell) and on one or more epitopes on a tumor cell (e.g., a tumor-associated antigen present on a tumor cell).

[0040] Multispecific: As used herein, the term “multispecific” refers to molecules (e.g., T- cell engager molecules) comprising two or more different targeting moieties. For instance, targeting moieties in a multispecific molecule can bind to two or more different portions of the same target antigen (or, in some cases, different variants of the same target antigen) or each targeting moiety can bind to a different target antigen (e.g., a tumor associated antigen, a T-cell antigen, etc.).

[0041] Multivalent: The term “multivalent” as used herein in reference to a tumor-targeted IL2 receptor agonist means that the tumor-targeted IL2 receptor agonist has two or more IL2 moieties and / or targeting moieties.

[0042] Operably linked: The term “operably linked” refers to a functional relationship between two or more peptide or polypeptide domains or nucleic acid (e.g., DNA) segments. In the context of a fusion protein or other polypeptide, the term “operably linked” means that two or more amino acid segments are linked so as to produce a functional polypeptide. For example, in the context of a tumor-targeted IL2 receptor agonist of the disclosure, separate components (e.g., an Fc domain and an IL2Ra moiety) can be operably linked directly or through peptide linker sequences. In the context of a nucleic acid encoding a polypeptide chain, such as a polypeptide chain of a tumor-targeted IL2 receptor agonist of the disclosure, “operably linked” means that the two nucleic acids are joined such that the amino acid sequences encoded by the two nucleic acids remain in-frame. In the context of transcriptional regulation, the term refers to the functional relationship of a transcriptional regulatory sequence to a transcribed sequence. For example, a promoter or enhancersequence is operably linked to a coding sequence if it stimulates or modulates the transcription of the coding sequence in an appropriate host cell or other expression system.

[0043] Polypeptide, Peptide and Protein: The terms “polypeptide”, “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues.

[0044] Single Chain Fv or scFv: The term “single-chain Fv” or “scFv” as used herein refers to targeting moieties comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Preferably, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen-binding. For a review of scFv see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. (1994), Springer-Verlag, New York, pp. 269-315. The VH and VL and be arranged in the N- to C- terminal order VH-VL orVL-VH, typically separated by a linker, for example a linker described in Section 6.7.3.

[0045] Single Domain Antibody or sdAb: The term “single domain antibody” or “sdAb” as used herein refers to an antibody or antigen binding fragment thereof comprising a single binding domain (e.g., heavy chain variable region) capable of binding a target molecule without pairing with a corresponding CDR-containing polypeptide (e.g., a light chain). An sdAb or sdAb fragment can be derived from a VHH or from a non-antibody scaffold protein, for example a designed ankyrin repeat protein (darpin), an avimer, an anticalin / lipocalin, a centyrin or a fynomer. A sdAb typically lacks a CH1 domain and thus cannot associate with a light chain. In some embodiments, an sdAb is a sdVH. In some embodiments, an sdAb is a VHH.

[0046] Single Domain VH Antibody or sdVH: The term “single domain VH” or “sdVH” as used herein refers to a variable region of an sdAb that is not of camelid or cartilaginous fish origin. An sdVH can be, for example, of human or non-human mammalian origin. A basic sdVH has the following structure from the N-terminus to the C-terminus: FR1-CDR1-FR2- CDR2-FR3-CDR3-FR4, in which FR1 to FR4 refer to framework regions 1 to 4, respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3.

[0047] Specifically (or Selectively) Binds: The term “specifically (or selectively) binds” to an antigen or an epitope refers to a binding reaction that is determinative of the presence of a cognate antigen or an epitope in a heterogeneous population of proteins and other molecules. The binding reaction can be but need not be mediated by an antibody or antibody fragment. The term “specifically binds” does not exclude cross-species reactivity. For example, an antigen-binding domain (e.g., an antigen-binding fragment of an antibody) that “specifically binds” to an antigen from one species may also “specifically bind” to that antigenin one or more other species. Thus, such cross-species reactivity does not itself alter the classification of an antigen-binding domain as a “specific” binder. In certain embodiments, an antigen-binding domain of the disclosure that specifically binds to a human antigen has cross-species reactivity with one or more non-human mammalian species, e.g., a primate species (including but not limited to one or more of Macaca fascicularis, Macaca mulatta, and Macaca nemestrina) or a rodent species, e.g., Mus musculus.

[0048] Subject: The term “subject” includes human and non-human animals. Non-human animals include all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dog, cow, chickens, amphibians, and reptiles. In preferred embodiments, the subject is human.

[0049] Targeting Moiety: The term “targeting moiety” as used herein refers to any molecule or binding portion (e.g., an immunoglobulin or an antigen binding fragment) thereof that can bind to a cell surface or extracellular matrix molecule at a site to which a tumor- targeted IL2 receptor agonist or a multispecific T-cell engager of the disclosure is to be localized, for example on tumor cells or on lymphocytes in the tumor microenvironment. In some embodiments, the targeting moiety binds to a tumor associated antigen. In other embodiments, the targeting moiety binds to a T-cell receptor complex. The targeting moiety can also have a functional activity in addition to localizing a tumor-targeted IL2 receptor agonist or a multispecific T-cell engager to a particular site. For example, a targeting moiety that is a CD3 targeting moiety (e.g., an anti-CD3 antibody or an antigen binding portion thereof) may facilitate clustering and activation of CD3 on a surface of a T-cell.

[0050] T-cell Receptor: As used herein, the “T-cell receptor” or “TCR” refer to the component of a T-cell that is responsible for interacting with and sensing the targets of T-cell adaptive immunity. In general terms, the T-cell receptor is comprised of a heterodimeric protein complex presented on the cell surface. T-cells may be broadly classified as ap or y6 according to the somatically rearranged T-cell receptor form they express at their surface. There exist two T-cell receptor chain pair forms; TCRa and TCRp pairs TCRy and TCR6. Mature ap and y6 T-cell receptor chain pairs are presented at the cell surface in a complex with a number of accessory CD3 subunits, denoted E, y, 6 and These subunits associate with ap or y6 T-cell receptors as three dimers (Ey, s6, ( ). This T-cell receptor complex forms the unit for initiation of cellular signaling responses upon engagement of a TCRap or TCRyd with cognate antigen. The terms “T-cell receptor complex” and “TCR complex” refer to complexes of TCR ap or TCRyG and CD3.

[0051] T-cell Receptor Complex Targeting Moiety, TCR Targeting Moiety: The terms “T- cell receptor complex targeting moiety” and “TCR targeting moiety” as used herein refers toany molecule or binding portion (e.g., an immunoglobulin or an antigen binding fragment) thereof that can bind to a T-cell receptor complex at a site to which a T-cell engager of the disclosure is to be localized, for example on lymphocytes in the tumor microenvironment. The TCR targeting moiety can also have a functional activity in addition to localizing a multispecific T-cell engager to a particular site. For example, a targeting moiety that is a CD3 targeting moiety (e.g., an anti-CD3 antibody or an antigen binding portion thereof) may facilitate clustering and activation of CD3 on a surface of a T-cell.

[0052] Tumor: The term “tumor” is used interchangeably with the term “cancer” herein, e.g., both terms encompass solid and liquid, e.g., diffuse or circulating, tumors. As used herein, the term “cancer” or “tumor” includes premalignant, as well as malignant cancers and tumors.

[0053] Tumor Associated Antigen, TAA: As used herein, the term “tumor associated antigen” refers to a molecule (typically a protein, carbohydrate, lipid or some combination thereof) that is expressed on the surface of a cancer cell, either entirely or as a fragment (e.g., MHC / peptide), and which is useful for the preferential targeting of a pharmacological agent to the cancer cell. In some embodiments, a tumor associated antigen is a marker expressed by both normal cells and cancer cells, e.g., a lineage marker. In some embodiments, a tumor associated antigen is a cell surface molecule that is overexpressed in a cancer cell in comparison to a normal cell, for instance, 1-fold overexpression, 2-fold overexpression, 3-fold overexpression or more in comparison to a normal cell. In some embodiments, a tumor associated antigen is a cell surface molecule that is inappropriately synthesized in the cancer cell, for instance, a molecule that contains deletions, additions or mutations in comparison to the molecule expressed on a normal cell. In some embodiments, a tumor associated antigen will be expressed exclusively on the cell surface of a cancer cell, entirely or as a fragment (e.g., MHC / peptide), and not synthesized or expressed on the surface of a normal cell. Accordingly, the term “tumor associated antigen” encompasses antigens that are specific to cancer cells, sometimes known in the art as tumor-specific antigens (“TSAs”).

[0054] Tumor-Targeted IL2 Receptor Agonist : The term “tumor-targeted IL2 receptor agonist” as used herein refers to a molecule comprising a tumor-associated antigen (“TAA”) targeting moiety, an IL2 moiety and, optionally, a connector moiety.

[0055] Tumor Associated Antigen Targeting Moiety, TAA Targeting Moiety: The terms “tumor associated antigen targeting moiety” and “TAA targeting moiety” as used herein refer to any molecule or binding portion (e.g., an immunoglobulin or an antigen binding fragment) thereof that can bind to a tumor cell surface molecule at a site to which a tumor-targeted IL2receptor agonist or a multispecific T-cell engager of the disclosure is to be localized, for example on tumor cells in the tumor microenvironment.

[0056] Treat Treatment, Treating: As used herein, the terms “treat”, “treatment” and “treating” refer to the reduction or amelioration of the progression, severity and / or duration of a proliferative disorder, or the amelioration of one or more symptoms (preferably, one or more discernible symptoms) of a proliferative disorder resulting from the administration of one or more tumor-targeted IL2 receptor agonist molecules of the disclosure in combination with one or more multispecific T-cell engagers of the disclosure. In specific embodiments, the terms “treat”, “treatment” and “treating” refer to the amelioration of at least one measurable physical parameter of a proliferative disorder, such as growth of a tumor, not necessarily discernible by the patient. In other embodiments the terms “treat”, “treatment” and “treating” refer to the inhibition of the progression of a proliferative disorder, either physically by, e.g., stabilization of a discernible symptom, physiologically by, e.g., stabilization of a physical parameter, or both. In other embodiments the terms “treat”, “treatment” and “treating” refer to the reduction or stabilization of tumor size or cancerous cell count.

[0057] Universal Light Chain, ULC: The term “universal light chain” or “ULC” as used herein refers to a light chain variable region (VL) that can pair with more than one heavy chain variable region (VH). In the context of a targeting moiety, the term “universal light chain” or “ULC” refers to a light chain polypeptide capable of pairing with the heavy chain region of the targeting moiety and also capable of pairing with other heavy chain regions. ULCs can also include constant domains, e.g., a CL domain of an antibody. Universal light chains are also known as “common light chains.

[0058] VHH: The term “VHH” refers to a variable region of an antibody consisting of only a heavy chain, e.g., an antibody of camelid or cartilaginous fish origin. A VHH variable region can bind to a target molecule in the absence of a light chain. A basic VHH has the following structure from the N-terminus to the C-terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, in which FR1 to FR4 refer to framework regions 1 to 4, respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3.

[0059] VH: The term “VH” refers to the variable region of an immunoglobulin heavy chain of an antibody, including the heavy chain of an Fv, scFv, dsFv or Fab.

[0060] VL: The term “VL” refers to the variable region of an immunoglobulin light chain, including the light chain of an Fv, scFv, dsFv or Fab.6.2. Tumor-Targeted lnterleukin-2 (IL2) Receptor Agonist and Multispecific T-Cell Engager Combinations

[0061] The present disclosure relates to combinations comprising a tumor-targeted interleukin-2 (IL2) receptor agonist and a multispecific T-cell engager for use as a combination therapy.

[0062] Generally, and as depicted in FIGS. 1A-1C, the combinations of the present disclosure typically comprise:(a) a tumor-targeted IL2 receptor agonist comprising:(i) a TAA targeting moiety;(ii) an IL2 moiety; and(iii) optionally, a first connector moiety separating the first TAA targeting moiety and the IL2 moiety; and(b) a multispecific T-cell engager comprising:(i) a TAA targeting moiety;(ii) a T-cell receptor complex targeting moiety; and(iii) optionally, a second connector moiety and / or a third connector moiety separating the second TAA targeting moiety and the T-cell receptor complex targeting moiety.

[0063] Exemplary tumor-targeted IL2 receptor agonists are disclosed in Section 6.3 and illustrated in FIGS. 2A-2B. As depicted in FIG. 2B, the IL2 moiety in the tumor-targeted IL2 receptor agonist can be masked, e.g., with an IL2 receptor alpha based mask.

[0064] Exemplary multispecific T-cell engagers are disclosed in Section 6.4 and illustrated in FIGS. 3A-3C.

[0065] Without being bound by theory, it is the inventors’ belief that engaging a T-cell and a tumor cell via both a tumor-targeted IL2 receptor agonist and a multispecific T-cell engager strengthens the immune synapse, leading to enhanced anti-tumor immunity. This is illustrated in FIG. 4B.

[0066] In some embodiments, the TAA targeting moiety of the tumor-targeted IL2 component recognizes a TAA recognized by the TAA targeting moiety of the multispecific T- cell engager in the same combination, whether on the same epitope or on different epitopes. If the TAA targeting moieties recognize different epitopes, they preferably can bind to the cancer cell simultaneously and / or in a non-competing manner.

[0067] In some embodiments, the TAA targeting moiety of the tumor-targeted IL2 component recognizes a different TAA from that recognized by the TAA targeting moiety ofthe multispecific T-cell engager in the same combination. Preferably, the two TAAs are expressed on the same cancer cell. Exemplary TAA targeting moieties are disclosed in Section 6.5.

[0068] Targeting moiety formats suitable for use in the TAA targeting moieties of the tumor- targeted IL2 receptor agonists and multispecific T-cell engagers as well as the TOR targeting moieties of the multispecific T-cell engagers are disclosed in Section 6.6.

[0069] Connector moieties suitable for use in the tumor-targeted IL2 receptor agonist and multispecific T-cell engager of a combination of the disclosure are disclosed in Section 6.7.

[0070] The tumor-targeted IL2 receptor agonist and multispecific T-cell engager can be formulated together in a single composition. Alternatively, they can be formulated in different compositions for simultaneous, sequential or separate use. Exemplary pharmaceutical compositions comprising one or both components of a combination of the disclosure are described in Section 6.9 and method of use of the combinations of the disclosure are set forth in Section 6.10.

[0071] Nucleic acids encoding the tumor-targeted IL2 receptor agonist and multispecific T- cell engager and their components, together with recombinant host cells and methods of production, are disclosed in Section 6.8.6.3. Tumor-Targeted IL2 Receptor Agonists

[0072] The tumor-targeted IL2 receptor agonist component of a combination of the disclosure comprises a TAA targeting moiety, an optional connector moiety, and an IL2 moiety.

[0073] Exemplary TAA targeting moieties are disclosed in Sections 6.3.1 and 6.5, and suitable formats for the TAA targeting moieties are disclosed in Section 6.6.

[0074] Exemplary IL2 moieties are disclosed in Section 6.3.2.

[0075] In some embodiments, the IL2 moiety of a tumor-targeted IL2 receptor agonist is masked by a masking moiety, e.g., as described in Section 6.3.3. An exemplary masking moiety is an IL2 receptor alpha (IL2Ra) moiety.

[0076] The TAA targeting moiety, the IL2 moiety and the optional masking moiety can be connected by one or more connector moieties. Exemplary connector moieties are described in Section 6.7.

[0077] In certain aspects, the TAA targeting moiety and the IL2 moiety are connected, directly or indirectly, via one or more Fc domains. Exemplary Fc domains are described in Section 6.7.1.

[0078] A tumor-targeted IL2 receptor agonist can comprise additional components. For example, a tumor-targeted IL2 receptor agonist can comprise a hinge sequence (e.g., as part of the Fc domain). Exemplary hinge sequences are disclosed in Section 6.7.2. A tumor- targeted IL2 receptor agonist can also comprise a peptide linker, e.g., to connect an Fc domain and an IL2 moiety, an Fc domain and a masking moiety, or an IL2 moiety and a masking moiety. Exemplary linkers are disclosed in Section 6.7.3.

[0079] In some embodiments, a tumor-targeted IL2 receptor agonist comprises a polypeptide chain having, in N- to C-terminal orientation, a TAA targeting moiety (or a component thereof, e.g., a VH, associated with another component, e.g., a VL, on a separate polypeptide chain), an Fc domain and an IL2 moiety. Due to the Fc domain, the polypeptide chain can dimerize with another polypeptide chain comprising a suitable Fc domain. In some embodiments, the polypeptide chain dimerized with another polypeptide chain comprising, in N- to C-terminal orientation a TAA targeting moiety (or a component thereof, e.g., a VH, associated with another component, e.g., a VL, on a separate polypeptide chain), an Fc domain and an IL2 moiety. The Fc dimer can be an Fc homodimer or Fc heterodimer.

[0080] Where the IL2 moiety is masked, the tumor-targeted IL2 receptor agonist can further comprise a masking moiety, e.g., an IL2Ra moiety, between the Fc domain and the IL2 moiety. Thus, an exemplary tumor-targeted IL2 receptor agonist comprises two polypeptide chains, each having, in N- to C-terminal orientation, a TAA targeting moiety (or a component thereof, e.g., a VH, associated with another component, e.g., a VL, on a separate polypeptide chain), an Fc domain, an IL2Ra moiety and an IL2 moiety, with an optional linker separating the Fc domain and the IL2Ra moiety and another optional linker separating the IL2Ra moiety and the IL2 moiety. An illustrative tumor-targeted IL2 receptor agonist having this configuration is illustrated in FIG. 2B.

[0081] Although the illustrative tumor-targeted IL2 receptor agonist is bivalent for both the TAA targeting moiety and the IL2 moiety, in alternative embodiments a tumor-targeted IL2 receptor can be (a) monovalent for the IL2 moiety and / or (b) monovalent for the TAA targeting moiety. A tumor-targeted IL2 receptor agonist that is monovalent for one or both of the IL2 moiety and the TAA targeting moiety can include an Fc heterodimer, with one Fc domain operably linked to both a TAA targeting moiety and an IL2 moiety and another Fc domain linked to only one (or neither) of a TAA targeting moiety or IL2 moiety or two Fc domains, one operably linked to a TAA targeting moiety and the other operably linked to an IL2 moiety.6.3.1. Tumor Associated Antigen Targeting Moiety

[0082] The tumor-targeted IL2 receptor agonist component of a combination of the disclosure comprises a tumor associated antigen (“TAA”) targeting moiety.

[0083] Typically, the TAA recognized by the TAA targeting moiety of the tumor-targeted IL2 agonist is expressed on the same cancer cell as the TAA recognized by the multispecific T- cell engager in the combination.

[0084] Suitable TAA targeting moieties are described in Section 6.5 and suitable formats for the TAA targeting moieties are described in Section 6.6. The TAA targeting moiety is preferably an antigen binding moiety, e.g., a Fab, as described in Section 6.6.1 , an scFv, as described in Section 6.6.2, or a single domain antibody, as described in Section 6.6.3. In some embodiment, the TAA targeting moiety of the tumor-targeted IL2 receptor agonist comprises a universal light chain.6.3.2. IL2 Moiety

[0085] The tumor-targeted IL2 receptor agonist component of a combination of the disclosure comprises an IL2 moiety.

[0086] In eukaryotic cells human IL2 is synthesized as a precursor polypeptide of 153 amino acids, from which 20 amino acids are removed to generate mature secreted IL2 (Taniguchi et al., 1983, Nature 302(5906):305-10). Mature human IL2 has the following amino acid sequence:APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELK HLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYA DETATIVEFLNRWITFCQS I ISTLT ( SEQ ID NO : 1 )

[0087] In various embodiments, the IL2 moiety comprises a wild type or variant IL2 domain that is capable of binding the IL2 receptor, e.g., the intermediate affinity dimeric IL2 receptor (IL2R) consisting of IL2RP (CD122) and IL2Ry (CD132) or the high affinity trimeric high- affinity IL2R consisting of IL2Ra (CD25), IL2Rp (CD122) and IL2Ry (CD132).

[0088] In certain embodiments, the IL2 moiety does not include any amino acid substitutions that alter its affinity to one or more of the IL2R subunits.

[0089] In other embodiments, the IL2 moiety comprises one or more amino acid substitutions in the IL2 moiety that reduce binding affinity to IL2Rp, resulting in IL2Ra directed IL2 moieties. Optionally, the IL2 moiety’s binding affinity to IL2Ra is preserved. An exemplary amino acid substitution is N88D. Other amino acid substitutions that reduce the affinity of IL2 to IL2RP are D20T, N88R, and Q126D (see e.g., US Patent Publication No. US 2007 / 0036752).

[0090] In one embodiment, the IL2 moiety comprises one or more amino acid substitutions that reduce affinity to IL2Ra and preserve, or reduces affinity to a lesser degree, to IL2R|3, resulting in IL2Rp directed IL2 moieties. Exemplary IL2RP directed IL2 moieties are those comprising both H16A and F42A substitutions. Accordingly, in some embodiments, the IL2 moiety comprises the amino acid sequence of human IL2 with H16A and F42A substitutions, as shown below:APTSSSTKKTQLQLEALLLDLQMILNGINNYKNPKLTRMLTAKF YMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLI SN INVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSI ISTL T ( SEQ ID NO : 29 )

[0091] In certain embodiments, the IL2 moiety comprises an amino acid substitution which eliminates the O-glycosylation site of IL2 at a position corresponding to residue 3 of human IL2. Exemplary amino acid substitutions at T3 are T3A, T3G, T3Q, T3E, T3N, T3D, T3R, T3K, and T3P. In a specific embodiment, the substitution is T3A.

[0092] The IL2 moiety preferably comprises the amino acid sequence of a full-length IL2 molecule, e.g., a human IL2 molecule.

[0093] C125 in IL2 can be substituted with S, V, or A to reduce protein aggregation, as described in U.S. Patent No. 4,518,584.

[0094] In some embodiments, the IL2 moiety can comprise a deletion of the N-terminal alanine residue of IL2, resulting in des-A1 IL2.

[0095] Further, the IL2 moiety may include a substitution of methionine 104 with a neutral amino acid such as alanine, as described in U.S. Patent No. 5,206,344.

[0096] Accordingly, the IL2 moieties of the disclosure can have amino acid deletions and I or substitutions selected from des-A1 M104A IL2, des-A1 M104A C125S IL2, M104A IL2, M104A C125A IL2, des-A1 M104A C125A IL2, or M104A C125S IL2, in addition to other variations alter the binding of IL2 to its receptor. These and other mutants may be found in U.S. Patent No. 5,116,943 and in Weiger et al., 1989, Eur J Biochem 180:295-300.

[0097] In various aspects, any of the foregoing IL2 moieties comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, about at least 93%, at least about 94%, at eat least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity to the amino acid sequence of mature human IL2 (SEQ ID NO:1).6.3.3. Masking Moiety

[0098] The tumor-targeted IL2 receptor agonists of the disclosure may further include a masking moiety configured to mask the IL2 moiety. Without intending to be bound by theory, the inventors believe that masking an IL2 moiety can serve to reduce its off-target effects.

[0099] In some embodiments, the masking moiety comprises an IL2 binding domain ofIL2Ra (referred to as an “IL2Ra moiety” for convenience), e.g., the extracellular domain of an IL2Ra or an I L2-binding portion thereof.

[0100] The sequence of the mature human IL2Ra extracellular domain (corresponding to amino acids 22-272 of human IL2Ra) is:ELCDDDPPEI PHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSS WDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPP PWENEATERIYHFWGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLI CTGEMETSQFPGEEKPQASPEGRPESETSCLVTTTDFQIQTEMAATMETS IFTT EYQVAVAGCVFLLISVLLLSGLTWQRRQRKSRRTI ( SEQ ID NO : 3 )

[0101] The sequence of an IL2 binding portion of the human IL2Ra extracellular domain comprising the two “sushi” domains and corresponding to amino acids 22-186 of humanIL2Ra, is:ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQ CTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYH FWGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLI CTG ( SEQ ID NO : 19 )

[0102] The sequence of an alternative IL2 binding portion of the human IL2Ra extracellular domain which corresponds to amino acids 22-240 of human IL2Ra is:ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQ CTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYH FWGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLI CTGEMETSQFPGEEKPQA SPEGRPESETSCLVTTTDFQIQTEMAATMETSIFTTEYQ ( SEQ ID NO : 20 )

[0103] In various embodiments, the IL2Ra moiety has an amino acid sequence with at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity to any of the sequences above, i.e., any one of amino acids 22-186 of IL2Ra, amino acids 22-240 of IL-Ra, amino acids 22-272 of IL2Ra, or any IL2 binding portion thereof.

[0104] In some embodiments, the IL2Ra moiety has an amino acid sequence with at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity to SEQ ID NO: 19. In some embodiments, the IL2Ra moiety has an amino acid sequence with at least about 90%, at least about 95%, atleast about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity to SEQ ID NO:20.

[0105] In certain aspects, the IL2Ra moiety comprises or consists of an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity to an IL2 binding portion of human IL2Ra, optionally wherein the binding portion has an amino acid sequence of (a) at least 160 amino acids, at least 161 amino acids, at least 162 amino acids, at least 164 amino acids or at least 165 amino acids and / or (b) up to 251 , up to 240, up to 230, up to 220, up to 210, up to 200, up to 190, up to 180 or up to 170 amino acids of the extracellular domain of human IL2-Ra. In particular embodiments, the portion of human IL2Ra is bounded by any one of (a) and (b) in the preceding sentence, e.g., at least 160 and up to 180 amino acids from human IL2Ra, at least 162 and up to 200 amino acids from human IL2Ra, at least 160 and up to 220 amino acids from human IL2Ra, at least 164 and up to 190 amino acids from human IL2Ra, and so on and so forth.

[0106] In some embodiments, the IL2Ra moiety comprises or consists of an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity to amino acids 22- 186, with or without an additional up to 5 amino acids, up to 10 amino acids, up to 15 amino acids, up to 20 amino acids, up to 30 amino acids, or up to 40 amino acids C-terminal to amino acid residue 186, of IL2Ra.

[0107] In certain embodiments, the IL2Ra moiety has at least one fewer O-glycosylation and / or N-glycosylation compared to the extracellular domain of native IL2Ra, for example by a substitution at one or more of amino acid N49, amino acid N68, amino acid T74, amino acid T85, amino acid T197, amino acid T203, amino acid T208, and amino acid T216. In some embodiments, the one or more substitutions are from asparagine to an amino acid selected from the group consisting of alanine, threonine, serine, arginine, aspartic acid, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, tryptophan, tyrosine, and valine. In some embodiments, the one or more substitutions are from threonine to an amino acid selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, tryptophan, tyrosine, and valine. In some embodiments, the one or more substitutions are at amino acid S50 (e.g., S50P), amino acid S51 (e.g., S51 R, S51 N, S51 D, S51C, S51Q, S51 E, S51G, S51 H, S511, S51L, S51 K, S51M, S51 F, S51 P, S51W, S51Y, or S51V), amino acid T69 (e.g., T69P), amino acid T70 (e.g., T70R, T70N, T70D, T70C, T70Q, T70E, T70G, T70H, T70I,T70L, T70K, T70M, T70F, T70P, T70W, T70Y, or T70V, amino acid C192 (e.g., C192R, C192N, C192D, C192Q, C192E, C192G, C192H, C192I, C192L, C192K, C192M, C192F, C192P, C192W, C192Y, or C192V), or any combination thereof.

[0108] In some embodiments, the IL2Ra moiety is N-terminal to the IL2 moiety, e.g., as depicted in FIG. 2B. In other embodiments, the IL2Ra moiety is C-terminal to the IL2 moiety.

[0109] Optionally, the IL2Ra moiety and the IL2 moiety are separated by a linker, e.g., a linker as described in Section 6.7.3.6.4. Multispecific T-Cell Engagers

[0110] The combinations of the disclosure comprise, in addition to a tumor-targeted IL2 receptor agonist, a multispecific T-cell engager.

[0111] A multispecific T-cell engager comprises at least one TAA targeting moiety and at least at least one T-cell receptor (TCR) complex targeting moiety, typically connected via a connector moiety.

[0112] Exemplary TAA targeting moieties are disclosed in Sections 6.3.1 and 6.5, and suitable formats for the TAA targeting moieties are disclosed in Section 6.6.

[0113] Exemplary TCR complex targeting moieties are disclosed in Section 6.4.2.

[0114] The TAA targeting moiety and the T-cell receptor complex targeting moiety can be connected by one or more connector moieties. Exemplary connector moieties are described in Section 6.7.

[0115] In certain aspects, the TAA targeting moiety and T-cell receptor complex targeting moiety are connected, directly or indirectly, via one or more Fc domains. Exemplary Fc domains are described in Section 6.7.1 .

[0116] A multispecific T-cell engager can comprise additional components. For example, a multispecific T-cell engager can comprise a hinge sequence (e.g., as part of the Fc domain). Exemplary hinge sequences are disclosed in Section 6.7.2.

[0117] In some embodiments, a multispecific T-cell engager comprises a first polypeptide chain having, in N- to C-terminal orientation, a TAA targeting moiety (or a component thereof, e.g., a VH, associated with another component, e.g., a VL, on a separate polypeptide chain) and an Fc domain and a second polypeptide chain having, in N- to C- terminal orientation, a T-cell receptor complex targeting moiety and an Fc domain. The Fc domains can associate with one another to form an Fc dimer, e.g., an Fc heterodimer. In certain aspects, a multispecific T-cell engager is a bispecific T-cell engager. Illustrativebispecific T-cell engager formats are depicted in FIGS. 3A-3C, and illustrative bispecific T- cell engagers are disclosed in Section 6.4.3.6.4.1. Tumor Associated Antigen Targeting Moiety

[0118] The T-cell receptor complex targeting moiety component of a combination of the disclosure comprises a tumor associated antigen (“TAA”) targeting moiety.

[0119] Typically, the TAA recognized by the TAA targeting moiety of the tumor-targeted IL2 agonist is expressed on the same cancer cell as the TAA recognized by the tumor-targeted IL2 receptor agonist in the combination.

[0120] Suitable TAA targeting moieties are described in Section 6.5.

[0121] Suitable formats for TAA targeting moieties and T-cell receptor complex targeting moieties are described in Section 6.6. The TAA targeting moiety is preferably an antigen binding moiety, e.g., a Fab, for example as described in Section 6.6.1 , an scFv, for example as described in Section 6.6.2, or a single domain antibody, for example as described in Section 6.6.3. In some embodiment, the TAA targeting moiety of the multispecific T-cell engager comprises a universal light chain.6.4.2. T-Cell Receptor Complex Targeting Moieties

[0122] The multispecific T-cell engager component of a combination of the disclosure comprises, in addition to a TAA targeting moiety, a T-cell receptor (TCR) complex targeting moiety.

[0123] The TCR complex targeting moiety is preferably an antigen binding moiety, e.g., a Fab, as described in Section 6.6.1 , an scFv, as described in Section 6.6.2, or a single domain antibody, as described in Section 6.6.3. In some embodiment, the TCR complex targeting moiety of the multispecific T-cell engager comprises a universal light chain.

[0124] The TCR complex targeting moiety generally binds to any component of the TCR complex. Example targets for a TCR complex targeting moiety of the disclosure include, but are not limited to, CD3 and the T-cell receptor (e.g., TCRap or TCRyS). In some embodiments, the target for the TCR complex targeting moiety is CD3. In some embodiments, the target for the TCR complex targeting moiety is the T-cell receptor (e.g., TCRap or TCRyS). The epitope of the TCR complex targeting moiety can be an individual polypeptide (e.g., CD3 epsilon) or a multimeric component of a protein complex (e.g., the TCRap dimer or the TCRyQ dimer of the T-cell receptor complex).

[0125] In particular embodiments, a TCR complex targeting moiety of the present disclosure is a CD3 targeting moiety and / or a TCR targeting moiety. A CD3 targeting moiety may be orcomprise an antigen-binding domain from an anti-CD3 antibody. A TCR targeting moiety may be or comprise an antigen-binding domain from an anti-TCR antibody.

[0126] Exemplary anti-CD3 and anti-TCR antibodies or antibody sequences are set forth in Table G below, upon which the TCR complex targeting moiety can be based.

[0127] In some aspects, the TCR complex targeting moiety competes with an antibody set forth in Table G for binding to the target (e.g., CD3 or a T-cell receptor). In further aspects, the TCR complex targeting moiety comprises CDRs having CDR sequences of an antibodyset forth in Table G. In some embodiments, the TOR complex targeting moiety comprises all 6 CDR sequences of an antibody set forth in Table G. In other embodiments, the TCR complex targeting moiety comprises at least the heavy chain CDR sequences (CDR-H1 , CDR-H2, CDR-H3) or an antibody set forth in Table G and the light chain CDR sequences of a universal light chain. In further aspects, a TCR complex targeting moiety comprises a VH comprising the amino acid sequence of the VH of an antibody set forth in Table G. In some embodiments, the TCR complex targeting moiety further comprises a VL comprising the amino acid sequence of the VL of an antibody set forth in Table G. In other embodiments, the TCR complex targeting moiety further comprises a universal light chain VL sequence.6.4.3. Bispecific T-Cell Engagers

[0128] In some embodiments, the multispecific T-cell engager component of a combination of the disclosure is a bispecific T-cell engager.

[0129] Certain example bispecific T-cell engagers are provided in Table K. In some embodiments, a bispecific T-cell engager useful in combination with a tumor-targeted IL2 receptor agonist of the disclosure is a bispecific T-cell engager of Table K. In some embodiments, a bispecific T-cell engager comprises one or more CDR, VH, and / or VL sequences from a bispecific T-cell engager of Table K.6.5. Tumor-Associated Antigen Targeting Moieties

[0130] The tumor-targeted IL2 receptor agonist and the multispecific T-cell engager of the disclosure each comprise one or more tumor-associated antigen (“TAA”) targeting moieties. Typically, the TAA recognized by the TAA targeting moiety of the tumor-targeted IL2 receptor agonist and the TAA recognized by the TAA targeting moiety the multispecific T-cell engager is expressed on the same cancer cell and may be the same TAA or different TAAs. If the TAA targeting moiety of the tumor-targeted IL2 receptor agonist and the TAA targeting moiety of the multispecific T-cell engager are the same, in some embodiments binding the TAA targeting moiety of the tumor-targeted IL2 receptor agonist and the TAA targeting moiety of the multispecific T-cell engager bind to the TAA in a non-competing fashion such that both the tumor-targeted IL2 receptor agonist and the multispecific T-cell engager can bind to the same cell concurrently.

[0131] Without being bound by theory, the inventors believe that the incorporation of TAA targeting moieties that bind to the same tumor cell in both the tumor-targeted IL2 receptor agonist and the multispecific T-cell engager permits the delivery of high concentrations of IL2 into the tumor microenvironment while engaging tumor reactive lymphocytes, resulting in enhancement of the cytotoxic response against tumor cells with a concomitant reduction of systemic exposure.

[0132] Suitable TAA targeting moiety formats are described in Section 6.6. The TAA targeting moiety is preferably an antigen binding moiety, for example an antibody or an antigen-binding portion of an antibody, e.g., a Fab, as described in Section 6.6.1 , an scFv, as described in Section 6.6.2, or a single domain antibody, as described in Section 6.6.3.

[0133] Exemplary target molecules recognized by the TAA targeting moieties of the tumor- targeted IL2 receptor agonist and / or the bispecific T-cell engager are Fibroblast ActivationProtein (FAP), the A1 domain of Tenascin-C (TNC A1), the A2 domain of Tenascin-C (TNC A2), the Extra Domain B of Fibronectin (EDB), the Melanoma-associated Chondroitin Sulfate Proteoglycan (MCSP), MART-1 / Melan-A, gp1OO, Dipeptidyl peptidase IV (DPPIV), adenosine deaminase-binding protein (ADAbp), cyclophilin b, colorectal associated antigen (CRC)-C017-1A / GA733, Carcinoembryonic Antigen (CEA) and its immunogenic epitopes CAP-1 and CAP-2, etv6, aml1, prostate-specific membrane antigen (PSMA), T-cell receptor / CD3-zeta chain, GAGE-family of tumor antigens (e.g., GAGE-1 , GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9), BAGE, RAGE, LAGE-1 , NAG, GnT-V, MUM-1 , CDK4, tyrosinase, p53, MUC family, HER2 / neu, p21 ras, RCAS1 , a- fetoprotein, E-cadherin, a-catenin, p-catenin and y-catenin, p120ctn, gp1OO Pmel117, PRAME, NY-ESO-1 , cdc27, adenomatous polyposis coli protein (APC), fodrin, Connexin 37, Ig-idiotype, p15, gp75, GM2 and GD2 gangliosides, viral products such as human papilloma virus proteins, Smad family of tumor antigens, Imp-1 , P1 A, EBV-encoded nuclear antigen (EBNA)-1 , brain glycogen phosphorylase, SSX-1 , SSX-2 (HOM-MEL-40), SSX-1 , SSX-4, SSX-5, SCP-1 and CT-7, c-erbB-2, Her2, Her3, EGFR, IGF-1 R, CD2 (T-cell surface antigen), CD3 (heteromultimer associated with the TCR), CD22 (B-cell receptor), CD23 (low affinity IgE receptor), CD30 (cytokine receptor), CD33 (myeloid cell surface antigen), CD20, MCSP, PDGFpR (p-platelet-derived growth factor receptor), ErbB2 epithelial cell adhesion molecule (EpCAM), EGFR variant III (EGFRvlll), CD19, disialoganglioside GD2, ductal- epithelial mucine, gp36, TAG-72, glioma-associated antigen, p-human chorionic gonadotropin, alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulin, MN-CA IX, human telomerase reverse transcriptase, RU1 , RU2 (AS), intestinal carboxyl esterase, mut hsp70-2, M-CSF, PAP, LAGA-1a, prostein, survivin and telomerase, prostate-carcinoma tumor antigen-1 (PCTA-1), ELF2M, neutrophil elastase, ephrin B2, insulin growth factor (IGF1 )-l, IGF-II, IGFI receptor, 5T4, ROR1 , Nkp30, NKG2D, tumor stromal antigens, CA166-9, the extra domain A (EDA) and extra domain B (EDB) of fibronectin and the A1 domain of tenascin-C (TnC A1).

[0134] In some embodiments, the target molecule recognized by the TAA targeting moiety of the tumor-targeted IL2 receptor agonist and / or the multispecific T-cell engager is BCMA. In some embodiments, the target molecule recognized by the TAA targeting moiety of the tumor-targeted IL2 receptor agonist and / or the multispecific T-cell engager is CD20. In some embodiments, the target molecule recognized by the TAA targeting moiety of the tumor- targeted IL2 receptor agonist and / or the multispecific T-cell engager is EGFR. In some embodiments, the target molecule recognized by the TAA targeting moiety of the tumor- targeted IL2 receptor agonist and / or the multispecific T-cell engager is PSMA. In some embodiments, the target molecule recognized by the TAA targeting moiety of the tumor-targeted IL2 receptor agonist and / or the multispecific T-cell engager is CA9. In some embodiments, the target molecule recognized by the TAA targeting moiety of the tumor- targeted IL2 receptor agonist and / or the multispecific T-cell engager is MSLN. In some embodiments, the target molecule recognized by the TAA targeting moiety of the tumor- targeted IL2 receptor agonist and / or the multispecific T-cell engager is EPCAM. In some embodiments, the target molecule recognized by the TAA targeting moiety of the tumor- targeted IL2 receptor agonist and / or the multispecific T-cell engager is B7H3. In some embodiments, the target molecule recognized by the TAA targeting moiety of the tumor- targeted IL2 receptor agonist and / or the multispecific T-cell engager is HER2 / HER3. In some embodiments, the target molecule recognized by the TAA targeting moiety of the tumor- targeted IL2 receptor agonist and / or the multispecific T-cell engager is STEAP1 . In some embodiments, the target molecule recognized by the TAA targeting moiety of the tumor- targeted IL2 receptor agonist and / or the multispecific T-cell engager is CEACAM5. In some embodiments, the target molecule recognized by the TAA targeting moiety of the tumor- targeted IL2 receptor agonist and / or the multispecific T-cell engager is MUC16.

[0135] In some embodiments, the targeting moieties target the exemplary target molecules set forth in Table T 1 below, which provides references to exemplary antibodies or antibody sequences upon which the targeting moiety can be based.

[0136] In some aspects, the TAA targeting moiety competes with an antibody set forth in Table T 1 for binding to the target molecule. In further aspects, the TAA targeting moiety comprises CDRs having CDR sequences of an antibody set forth in Table T1. In some embodiments, the targeting moiety comprises all 6 CDR sequences of the antibody set forth in Table T1. In other embodiments, the targeting moiety comprises at least the heavy chain CDR sequences (CDR-H1 , CDR-H2, CDR-H3) of such antibody and the light chain CDR sequences of a universal light chain. In further aspects, a targeting moiety comprises a VH comprising the amino acid sequence of the VH of an antibody set forth in Table T1 . In some embodiments, the targeting moiety further comprises a VL comprising the amino acidsequence of the VL of the antibody set forth in Table T1 . In other embodiments, the targeting moiety further comprises a universal light chain VL sequence.

[0137] In some embodiments, the targeting moieties target the exemplary target molecules set forth in Table T2 below, which provides references to exemplary single domain antibodies or antibody sequences upon which the targeting moiety can be based.

[0138] In some aspects, the TAA targeting moiety competes with an antibody set forth above in Table T2, for binding to the target molecule. In further aspects, the TAA targeting moiety comprises CDRs having CDR sequences of an antibody set forth in Table T2. In some embodiments, the targeting moiety comprises all 3 CDR sequences of the antibody set forth in Table T2. In further aspects, a targeting moiety comprises a VH (e.g., a VHH) comprising the amino acid sequence of the VH of an antibody set forth in Table T2.

[0139] Additional target molecules that can be targeted by the tumor-targeted IL2 receptor agonist and / or multispecific T-cell engagers are disclosed in Table I below and in, e.g.,Hafeez et al., 2020, Molecules 25:4764, doi:10.3390 / molecules25204764, particularly inTable 1. Table 1 of Hafeez et al. is incorporated by reference in its entirety herein.6.5.1. MUC16 Targeting Moieties

[0140] In certain aspects, the TAA targeting moiety of the tumor-targeted IL2 receptor agonist and / or the multispecific T-cell engager is a MUC16 targeting moiety. In some embodiments, the MUC16 targeting moiety is or comprises an antigen-binding domain from an anti-MUC16 antibody.

[0141] Exemplary anti-MUC16 antibodies or antibody sequences are set forth in Tables M1 and M2 below, upon which the TAA targeting moiety can be based.

[0142] In some aspects, the MUC16 targeting moiety competes with an antibody set forth in Table M1 for binding to MUC16. In further aspects, the MUC16 targeting moiety comprises CDRs having CDR sequences of an anti-MUC16 antibody set forth in Table M1 . In some embodiments, the MUC16 targeting moiety comprises all 6 CDR sequences of an anti- MUC16 antibody set forth in Table M1. In other embodiments, the MUC16 targeting moiety comprises at least the heavy chain CDR sequences (CDR-H1 , CDR-H2, CDR-H3) of an anti- MUC16 antibody set forth in Table M1 and the light chain CDR sequences of a universal light chain. In further aspects, the MUC16 targeting moiety comprises a VH comprising the amino acid sequence of the VH of an anti-MUC16 antibody set forth in Table M1. In some embodiments, the MUC16 targeting moiety further comprises a VL comprising the amino acid sequence of the VL of an anti-MUC16 antibody set forth in Table M1 . In other embodiments, the MUC16 targeting moiety further comprises a universal light chain VL sequence.

[0143] In some aspects, the MUC16 targeting moiety competes with an antibody set forth in Table M2 for binding to MUC16. In further aspects, the MUC16 targeting moiety comprises CDRs having CDR sequences of an anti-MUC16 antibody set forth in Table M2. In some embodiments, the MUC16 targeting moiety comprises all 6 CDR sequences of an anti- MUC16 antibody set forth in Table M2. In other embodiments, the MUC16 targeting moiety comprises at least the heavy chain CDR sequences (CDR-H1 , CDR-H2, CDR-H3) of an anti- MUC16 antibody set forth in Table M2 and the light chain CDR sequences of a universal light chain. In further aspects, the MUC16 targeting moiety comprises a VH comprising the amino acid sequence of the VH of an anti-MUC16 antibody set forth in Table M2. In some embodiments, the MUC16 targeting moiety further comprises a VL comprising the amino acid sequence of the VL of an anti-MUC16 antibody set forth in Table M2. In other embodiments, the MUC16 targeting moiety further comprises a universal light chain VL sequence.

[0144] In certain aspects, the MUC16 targeting moiety is or comprises a single domain antibody (sdAb). Exemplary anti-MUC16 sdAbs or antibody sequences are set forth in Tables M3 below, upon which the TAA targeting moiety can be based.

[0145] In some aspects, the MUC16 targeting moiety competes with a sdAb set forth in Table M3 for binding to MUC16. In further aspects, the MUC16 targeting moiety comprises CDRs having CDR sequences of an anti-MUC16 sdAb set forth in Table M3. In some embodiments, the MUC16 targeting moiety comprises the CDR3 sequence of an anti- MUC16 sdAb set forth in Table M3. In some embodiments, the MUC16 targeting moiety comprises all 3 CDR sequences of an anti-MUC16 sdAb set forth in Table M3.6.6. Targeting Moiety Formats

[0146] In certain aspects, a targeting moiety (e.g., a TAA targeting moiety, or a TCR complex targeting moiety) can be any type of antibody or fragment thereof that retains specific binding to an antigenic determinant. In one embodiment the antigen binding moiety is a full-length antibody. In one embodiment the antigen binding moiety is an immunoglobulin molecule, particularly an IgG class immunoglobulin molecule, more particularly an IgG 1 or lgG4 immunoglobulin molecule. In another embodiment, the antigen binding moiety is single domain antibody. Antibody fragments include, but are not limited to, VH (or VH) fragments, VL (or VL) fragments, Fab fragments, F(ab')2 fragments, scFv fragments, Fv fragments, VHH domains, minibodies, diabodies, triabodies, and tetrabodies.

[0147] In some embodiments, the TAA targeting moiety of the tumor-targeted IL2 receptor agonist and the TAA targeting moiety of the multispecific T-cell engager share the same format (e.g., Fab, scFv or sdAb). In another embodiment, the TAA targeting moiety of the tumor-targeted IL2 receptor agonist and the TAA targeting moiety of the multispecific T-cell engager do not share the same format.

[0148] In some embodiments the TAA targeting moieties of the tumor-targeted IL2 receptor agonist and / or the multispecific T-cell engager are Fabs. In other embodiments, the TAA targeting moieties of the tumor-targeted IL2 receptor agonist and / or the multispecific T-cell engager are scFvs. In yet other embodiments, the TAA targeting moieties of the tumor- targeted IL2 receptor agonist and / or the multispecific T-cell engager are sdAbs.

[0149] In some embodiments the TCR complex targeting moieties are Fabs. In other embodiments, the TCR complex targeting moieties are scFvs. In yet other embodiments, the TCR complex targeting moieties are sdAbs.

[0150] In some embodiments, the TAA targeting moieties and the TCR complex targeting moieties share the same format (e.g., Fab, scFv or sdAb). In other embodiments, the TAA targeting moieties and the TCR complex targeting moieties do not share the same format(e.g., the TAA targeting moieties are Fabs and the TOR complex targeting moieties are sdAbs or vice versa).6.6.1. Fabs

[0151] Fab domains were traditionally produced by proteolytic cleavage of immunoglobulin molecules using enzymes such as papain. In the tumor-targeted IL2 receptor agonist and the multispecific T-cell engagers of the disclosure, the Fab domains can be recombinantly expressed as part of the tumor-targeted IL2 receptor agonist and / or the multispecific T-cell engager.

[0152] The Fab domains can comprise constant domain and variable region sequences from any suitable species, and thus can be murine, chimeric, human or humanized.

[0153] Fab domains typically comprise a CH1 domain attached to a VH domain which pairs with a CL domain attached to a VL domain. In a wild-type immunoglobulin, the VH domain is paired with the VL domain to constitute the Fv region, and the CH1 domain is paired with the CL domain to further stabilize the binding module. A disulfide bond between the two constant domains can further stabilize the Fab domain.

[0154] For the tumor-target IL2 of the disclosure, particularly when the light chain is not a common or universal light chain, it is advantageous to use Fab heterodimerization strategies to permit the correct association of Fab domains belonging to the same ABD and minimize aberrant pairing of Fab domains belonging to different ABDs. For example, the Fab heterodimerization strategies shown in Table F below can be used:

[0155] Accordingly, in certain embodiments, correct association between the two polypeptides of a Fab is promoted by exchanging the VL and VH domains of the Fab for each other or exchanging the CH1 and CL domains for each other, e.g., as described in WO 2009 / 080251.

[0156] Correct Fab pairing can also be promoted by introducing one or more amino acid modifications in the CH1 domain and one or more amino acid modifications in the CL domain of the Fab and / or one or more amino acid modifications in the VH domain and one or more amino acid modifications in the VL domain. The amino acids that are modified are typically part of the VH:VL and CH1 :CL interface such that the Fab components preferentially pair with each other rather than with components of other Fabs.

[0157] In one embodiment, the one or more amino acid modifications are limited to the conserved framework residues of the variable (VH, VL) and constant (CH1 , CL) domains as indicated by the Kabat numbering of residues. Almagro, 2008, Frontiers In Bioscience 13:1619-1633 provides a definition of the framework residues on the basis of Kabat, Chothia, and IMGT numbering schemes.

[0158] In one embodiment, the modifications introduced in the VH and CH1 and / or VL and CL domains are complementary to each other. Complementarity at the heavy and light chain interface can be achieved on the basis of steric and hydrophobic contacts, electrostatic / charge interactions or a combination of the variety of interactions. The complementarity between protein surfaces is broadly described in the literature in terms oflock and key fit, knob into hole, protrusion and cavity, donor and acceptor etc., all implying the nature of structural and chemical match between the two interacting surfaces.

[0159] In one embodiment, the one or more introduced modifications introduce a new hydrogen bond across the interface of the Fab components. In one embodiment, the one or more introduced modifications introduce a new salt bridge across the interface of the Fab components. Exemplary substitutions are described in WO 2014 / 150973 and WO 2014 / 082179, the contents of which are hereby incorporated by reference.

[0160] In some embodiments, the Fab domain comprises a 192E substitution in the CH1 domain and 114A and 137K substitutions in the CL domain, which introduces a salt-bridge between the CH1 and CL domains (see, e.g., Golay et al., 2016, J Immunol 196:3199-211).

[0161] In some embodiments, the Fab domain comprises 143Q and 188V substitutions in the CH1 domain and 113T and 176V substitutions in the CL domain, which serves to swap hydrophobic and polar regions of contact between the CH1 and CL domain (see, e.g., Golay et al., 2016, J Immunol 196:3199-211).

[0162] In some embodiments, the Fab domain can comprise modifications in some or all of the VH, CH1 , VL, CL domains to introduce orthogonal Fab interfaces which promote correct assembly of Fab domains (Lewis et al., 2014 Nature Biotechnology 32:191-198). In an embodiment, 39K, 62E modifications are introduced in the VH domain, H172A, F174G modifications are introduced in the CH1 domain, 1 R, 38D, (36F) modifications are introduced in the VL domain, and L135Y, S176W modifications are introduced in the CL domain. In another embodiment, a 39Y modification is introduced in the VH domain and a 38R modification is introduced in the VL domain.

[0163] Fab domains can also be modified to replace the native CH1 :CL disulfide bond with an engineered disulfide bond, thereby increasing the efficiency of Fab component pairing. For example, an engineered disulfide bond can be introduced by introducing a 126C in the CH1 domain and a 121 C in the CL domain (see, e.g., Mazor et al., 2015, MAbs 7:377-89).

[0164] Fab domains can also be modified by replacing the CH1 domain and CL domain with alternative domains that promote correct assembly. For example, Wu et al., 2015, MAbs 7:364-76, describes substituting the CH1 domain with the constant domain of the T-cell receptor and substituting the CL domain with the b domain of the T-cell receptor, and pairing these domain replacements with an additional charge-charge interaction between the VL and VH domains by introducing a 38D modification in the VL domain and a 39K modification in the VH domain.

[0165] In lieu of, or in addition to, the use of Fab heterodimerization strategies to promote correct VH-VL pairings, the VL of common light chain (also referred to as a universal light chain) can be used for each Fab VL region of a tumor-targeted IL2 receptor agonist and / or a multispecific T-cell engager of the disclosure. In various embodiments, employing a common light chain as described herein reduces the number of inappropriate species of tumor- targeted IL2 receptor agonist and / or multispecific T-cell engagers as compared to employing original cognate VLs. In various embodiments, the VL domains of the tumor-targeted IL2 receptor agonist and / or multispecific T-cell engagers are identified from monospecific antibodies comprising a common light chain. In various embodiments, the VH regions of the tumor-targeted IL2 receptor agonist and / or the multispecific T-cell engagers comprise human heavy chain variable gene segments that are rearranged in vivo within mouse B cells that have been previously engineered to express a limited human light chain repertoire, or a single human light chain, cognate with human heavy chains and, in response to exposure with an antigen of interest, generate an antibody repertoire containing a plurality of human VHs that are cognate with one or one of two possible human VLs, wherein the antibody repertoire specific for the antigen of interest. Common light chains are those derived from a rearranged human VK1 -39JK5 sequence or a rearranged human VK3-20JK1 sequence, and include somatically mutated (e.g., affinity matured) versions. See, for example, U.S. Patent No. 10,412,940.6.6.2. scFvs

[0166] Single chain Fv or “scFv” antibody fragments comprise the VH and VL domains of an antibody in a single polypeptide chain, are capable of being expressed as a single chain polypeptide and retain the specificity of the intact antibodies from which they are derived. Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domain that enables the scFv to form the desired structure for target binding. Examples of linkers suitable for connecting the VH and VL chains of an scFv are the linkers identified in Section 6.7.3.

[0167] Unless specified, as used herein an scFv may have the VL and VH variable regions in either order, e.g., with respect to the N-terminal and C-terminal ends of the polypeptide, the scFv may comprise VL-linker-VH or may comprise VH-linker-VL.

[0168] The scFv can comprise VH and VL sequences from any suitable species, such as murine, human or humanized VH and VL sequences.

[0169] To create an scFv-encoding nucleic acid, the VH and VL-encoding DNA fragments are operably linked to another fragment encoding a linker, e.g., encoding any of the linkers described in Section 6.7.3 (typically a repeat of a sequence containing the amino acidsglycine and serine, such as the amino acid sequence (Gly4~Ser)3(SEQ ID NO:77), such that the VH and VL sequences can be expressed as a contiguous single-chain protein, with the VL and VH regions joined by the flexible linker (see, e.g., Bird et al., 1988, Science 242:423- 426; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., 1990, Nature 348:552-554).6.6.3. Single Domain Antibodies

[0170] In some embodiments, a targeting moiety e.g., a TAA targeting moiety or a TCR complex targeting moiety) is a single-domain antibody. A single-domain antibody (sdAb) describes a single antigen-binding domain capable of binding to a cognate antigen. sdAbs are often derived from heavy-chain only antibodies, however they also include single VH domains capable of binding to their cognate antigen in the absence of an associated light chain.

[0171] Heavy-chain only antibodies lack both light chains and a functional CH1 domain and thus rely exclusively on a heavy chain variable domain for antigen binding. Heavy-chain only antibodies are produced naturally in the Camelidae family (e.g., camels, dromedaries, llamas, vicunas, guanaco, and alpacas) as well as in cartilaginous fish (e.g., sharks). In addition to natural sources, transgenic mammals (e.g., mice) have been engineered to express heavy-chain only antibodies. Such transgenic mammals include, for example, transgenic animals described in U.S. Patent Publications 2015 / 0289489 A1 , 2023 / 0270086 A1 , and 2023 / 0062964 A1 , and 2020 / 0267951 A1 , each of which is incorporated herein by reference.

[0172] In some embodiments, an sdAb is generated by immunizing an animal that produces heavy-chain only antibodies, including a natural producer (e.g., camelids, sharks) or an engineered non-human mammal (e.g., a transgenic mouse), to obtain heavy-chain only antibodies. Such antibodies may be screened to identify those having desirable properties (e.g., target affinity). Once produced and identified, the variable region of the antibody heavy chain is cloned to construct a single domain antibody consisting of only one heavy chain variable region.

[0173] sdAbs can also be obtained by immunizing animals that generate traditional antibodies (e.g., rabbits) followed by screening for VHs having high binding affinity in the absence of their cognate light chain (see e.g., Shinozaki et al., 2017, Scientific Reports, 7(1):5794).

[0174] sdAbs can be humanized by replacing natural (e.g., camelid) framework sequences with human sequences (see, e.g., Vincke, 2009, The Journal of Biological Chemistry,285(5) :3273-3284; Murakami et al., 2022, Antibodies, 11 (1):10; and U.S. Patent Publication No. 2016 / 0237142 A1 , incorporated herein by reference).

[0175] Fully human sdAbs can also be obtained using human VH single domains (see, e.g., Rouet et al., 2015, The Journal of Biological Chemistry, 290(19):11905-11917).

[0176] Additional methods for producing heavy-chain only antibodies and / or sdAbs are recognized in the art and include, for example, those described in Muyldermans, 2021 , The FEBS journal, 288(7):2084-2102.

[0177] In some cases, an sdAb is engineered to enhance certain properties. For example, in some embodiments, a disulfide bond is introduced within a VHH to increase stability (see e.g., Hagihara et al., 2007, The Journal of Biological Chemistry, 282(50) :36489-36495).6.7. Connector Moiety

[0178] One or both of the tumor-targeted IL2 receptor agonist and multispecific T-cell engager of a combination of the disclosure may comprise a connector moiety. The term connector moiety need not connect the two components directly. For example, in a multispecific T-cell engager, an Fc region formed by association of two Fc domains, one operably linked to a TAA targeting moiety (or portion thereof, e.g., a VH domain or VH-CH1 domain) and a T-cell receptor complex targeting moiety (or portion thereof, e.g., a VH domain or VH-CH1 domain), is considered a connector moiety connecting the TAA targeting moiety and the T-cell receptor complex targeting moiety.6.7.1. Fc Domains and Fc Regions

[0179] Several embodiments of the tumor-targeted IL2 receptor agonist and the multispecific T-cell engager components of a combination of the disclosure comprise an Fc region formed by the association of two Fc domains.

[0180] In some embodiments, an Fc domain serves as a connector moiety, e.g., connecting a TAA targeting moiety and an IL2 moiety as depicted in FIG. 2B.

[0181] In some embodiments, an Fc region serves as a connector moiety, e.g., connecting a TAA targeting moiety and a T-cell receptor complex targeting moiety as depicted in FIG. 3A.

[0182] In some embodiments, each of the tumor-targeted IL2 receptor agonist and the multispecific T-cell engager comprises a pair of Fc domains associated to form an Fc region.

[0183] In one embodiment, the Fc domains of a tumor-targeted IL2 receptor agonist and / or a multispecific T-cell engager are derived from a human Fc domain.

[0184] The Fc domains that can be incorporated into a tumor-targeted IL2 receptor agonist and / or a multispecific T-cell engager can be derived from any suitable class of antibody,including IgA (including subclasses lgA1 and lgA2), IgD, IgE, IgG (including subclasses lgG1 , lgG2, lgG3 and lgG4), and IgM.

[0185] In some embodiments, the tumor-targeted IL2 receptor agonist and the multispecific T-cell engager comprises a pair of Fc domains.

[0186] In one embodiment, the Fc domains of both the tumor-targeted IL2 receptor agonist and the multispecific T-cell engager are derived from IgG 1 , lgG2, lgG3 or lgG4.

[0187] In one embodiment, the Fc domains of one or both of the tumor-targeted IL2 receptor agonist and the multispecific T-cell engager are derived from IgG 1 .

[0188] In one embodiment, the Fc domains of one or both of the tumor-targeted IL2 receptor agonist and the multispecific T-cell engager are derived from lgG4.

[0189] In one embodiment, the Fc domains of one of the tumor-targeted IL2 receptor agonist and the multispecific T-cell engager are derived from lgG1 and the Fc domains of the other are derived from lgG4.

[0190] The two Fc domains within the Fc region of the tumor-targeted IL2 receptor agonist and / or multispecific T-cell engager can be the same or different from one another. In a native antibody the Fc domains are typically identical, but for the purpose of producing molecules with different binding domains (e.g., a TAA targeting moiety and a TOR complex targeting moiety), the Fc domains might advantageously be different to allow for heterodimerization, as described in Section 6.7.1.2 below.

[0191] In native antibodies, the heavy chain Fc domain of IgA, IgD and IgG is composed of two heavy chain constant domains (CH2 and CH3) and that of IgE and IgM is composed of three heavy chain constant domains (CH2, CH3 and CH4). These dimerize to create an Fc region.

[0192] In the tumor-targeted IL2 receptor agonist and / or multispecific T-cell engager of a combination of the present disclosure, the Fc region, and / or the Fc domains within it, can comprise heavy chain constant domains from one or more different classes of antibody, for example one, two or three different classes.

[0193] In one embodiment the Fc region of one or both of a tumor-targeted IL2 receptor agonist and / or a multispecific T-cell engager comprises CH2 and CH3 domains derived from igGi .

[0194] In one embodiment the Fc region of one or both of a tumor-targeted IL2 receptor agonist and / or a multispecific T-cell engager comprises CH2 and CH3 domains derived from !gG2.

[0195] In one embodiment the Fc region of one or both of a tumor-targeted IL2 receptor agonist and / or a multispecific T-cell engager comprises CH2 and CH3 domains derived from igG3.

[0196] In one embodiment the Fc region of one or both of a tumor-targeted IL2 receptor agonist and / or a multispecific T-cell engager comprises CH2 and CH3 domains derived from igG4.

[0197] In one embodiment the Fc region of one or both of a tumor-targeted IL2 receptor agonist and / or a multispecific T-cell engager comprises a CH4 domain from IgM. The IgM CH4 domain is typically located at the C-terminus of the CH3 domain.

[0198] In one embodiment the Fc region of one or both of a tumor-targeted IL2 receptor agonist and / or a multispecific T-cell engager comprises CH2 and CH3 domains derived from IgG and a CH4 domain derived from IgM.

[0199] It will be appreciated that the heavy chain constant domains for use in producing an Fc region for a tumor-targeted IL2 receptor agonist and / or a multispecific T-cell engager of a combination of the disclosure may include variants of the naturally occurring constant domains described above. Such variants may comprise one or more amino acid variations compared to wild type constant domains. In one example the Fc region of the present disclosure comprises at least one constant domain that varies in sequence from the wild type constant domain. It will be appreciated that the variant constant domains may be longer or shorter than the wild type constant domain. Preferably the variant constant domains are at least 60% identical or similar to a wild type constant domain. In another example the variant constant domains are at least 70% identical or similar. In another example the variant constant domains are at least 80% identical or similar. In another example the variant constant domains are at least 90% identical or similar. In another example the variant constant domains are at least 95% identical or similar.

[0200] IgM and IgA occur naturally in humans as covalent multimers of the common H2L2 antibody unit. IgM occurs as a pentamer when it has incorporated a J-chain, or as a hexamer when it lacks a J-chain. IgA occurs as monomer and dimer forms. The heavy chains of IgM and IgA possess an 18 amino acid extension to the C-terminal constant domain, known as a tailpiece. The tailpiece includes a cysteine residue that forms a disulfide bond between heavy chains in the polymer and is believed to have an important role in polymerization. The tailpiece also contains a glycosylation site. In certain embodiments, the tumor-targeted IL2 receptor agonist and / or multispecific T-cell engager of a combination of the disclosure do not comprise a tailpiece.

[0201] In some embodiments, a tumor-targeted IL2 receptor agonist and / or multispecific T- cell engager comprises one or a pair of Fc domains that has at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:4. In some embodiments, the tumor-targeted IL2 receptor agonist and / or the multispecific T-cell engager comprises one or a pair of Fc domains that has the amino acid sequence of SEQ ID NO:4, optionally with one more mutations that facilitate heterodimerization or purification, e.g., (a) knob or hole mutations and / or (b) star mutations.

[0202] In some embodiments, a tumor-targeted IL2 receptor agonist and / or multispecific T- cell engager comprises one or a pair of Fc domains that has at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:5. In some embodiments, the tumor-targeted IL2 receptor agonist and / or the multispecific T-cell engager comprises one or a pair of Fc domains that has the amino acid sequence of SEQ ID NO:5, optionally with one more mutations that facilitate heterodimerization or purification, e.g., (a) knob or hole mutations and / or (b) star mutations.

[0203] In some embodiments, a tumor-targeted IL2 receptor agonist and / or multispecific T- cell engager comprises one or a pair of Fc domains that has at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:6. In some embodiments, the tumor-targeted IL2 receptor agonist and / or the multispecific T-cell engager comprises one or a pair of Fc domains that has the amino acid sequence of SEQ ID NO:6, optionally with one more mutations that facilitate heterodimerization or purification, e.g., (a) knob or hole mutations and / or (b) star mutations.

[0204] In some embodiments, a tumor-targeted IL2 receptor agonist and / or multispecific T- cell engager comprises one or a pair of Fc domains that has at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:7. In some embodiments, the tumor-targeted IL2 receptor agonist and / or the multispecific T-cell engager comprises one or a pair of Fc domains that has the amino acid sequence of SEQ ID NO:7, optionally with one more mutations that facilitate heterodimerization or purification, e.g., (a) knob or hole mutations and / or (b) star mutations.

[0205] In some embodiments, a tumor-targeted IL2 receptor agonist and / or multispecific T- cell engager comprises one or a pair of Fc domains that has at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:8. In some embodiments, the tumor-targeted IL2 receptor agonist and / or the multispecific T-cell engager comprises one or a pair of Fc domains that has the amino acid sequence of SEQ ID NO:8, optionally with one more mutations that facilitate heterodimerization or purification, e.g., (a) knob or hole mutations and / or (b) star mutations.

[0206] In some embodiments, a tumor-targeted IL2 receptor agonist and / or multispecific T- cell engager comprises one or a pair of Fc domains that has at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:9. In some embodiments, the tumor-targeted IL2 receptor agonist and / or the multispecific T-cell engager comprises one or a pair of Fc domains that has the amino acid sequence of SEQ ID NO:9, optionally with one more mutations that facilitate heterodimerization or purification, e.g., (a) knob or hole mutations and / or (b) star mutations.

[0207] In some embodiments, a tumor-targeted IL2 receptor agonist and / or multispecific T- cell engager comprises one or a pair of Fc domains that has at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 10. In some embodiments, the tumor-targeted IL2 receptor agonist and / or the multispecific T-cell engager comprises one or a pair of Fc domains that has the amino acid sequence of SEQ ID NO: 10, optionally with one more mutations that facilitate heterodimerization or purification, e.g., (a) knob or hole mutations and / or (b) star mutations.

[0208] In some embodiments, a tumor-targeted IL2 receptor agonist and / or multispecific T- cell engager comprises one or a pair of Fc domains that has at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:11. In some embodiments, the tumor-targeted IL2 receptor agonist and / or the multispecific T-cell engager comprises one or a pair of Fc domains that has the amino acid sequence of SEQ ID NO:11 , optionally with one more mutations that facilitate heterodimerization or purification, e.g., (a) knob or hole mutations and / or (b) star mutations.

[0209] In some embodiments, a tumor-targeted IL2 receptor agonist and / or multispecific T- cell engager comprises one or a pair of Fc domains that has at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the tumor-targeted IL2 receptor agonist and / or the multispecific T-cell engager comprises one or a pair of Fc domains that has the amino acid sequence of SEQ ID NO: 12, optionally with one more mutations that facilitate heterodimerization or purification, e.g., (a) knob or hole mutations and / or (b) star mutations.

[0210] In some embodiments, a tumor-targeted IL2 receptor agonist and / or multispecific T- cell engager comprises one or a pair of Fc domains that has at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 13. In some embodiments, the tumor-targeted IL2 receptor agonist and / or the multispecific T-cell engager comprises one or a pair of Fc domains that has the amino acid sequence of SEQ ID NO: 13, optionally with one more mutations that facilitate heterodimerization or purification, e.g., (a) knob or hole mutations and / or (b) star mutations.

[0211] In some embodiments, a tumor-targeted IL2 receptor agonist and / or multispecific T- cell engager comprises one or a pair of Fc domains that has at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 14. In some embodiments, the tumor-targeted IL2 receptor agonist and / or the multispecific T-cell engager comprises one or a pair of Fc domains that has the amino acid sequence of SEQ ID NO: 14, optionally with one more mutations that facilitate heterodimerization or purification, e.g., (a) knob or hole mutations and / or (b) star mutations.

[0212] In some embodiments, a tumor-targeted IL2 receptor agonist and / or multispecific T- cell engager comprises one or a pair of Fc domains that has at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 15. In some embodiments, the tumor-targeted IL2 receptor agonist and / or the multispecific T-cell engager comprises one or a pair of Fc domains that has the amino acid sequence of SEQ ID NO: 15, optionally with one more mutations that facilitate heterodimerization or purification, e.g., (a) knob or hole mutations and / or (b) star mutations.

[0213] In some embodiments, a tumor-targeted IL2 receptor agonist and / or multispecific T- cell engager comprises one or a pair of Fc domains that has at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 16. In some embodiments, the tumor-targeted IL2 receptor agonist and / or the multispecific T-cell engager comprises one or a pair of Fc domains that has the amino acid sequence of SEQ ID NO: 16, optionally with one more mutations that facilitate heterodimerization or purification, e.g., (a) knob or hole mutations and / or (b) star mutations.

[0214] In some embodiments, a tumor-targeted IL2 receptor agonist and / or multispecific T- cell engager comprises one or a pair of Fc domains that has at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 17. In some embodiments, the tumor-targeted IL2 receptor agonist and / or the multispecific T-cell engager comprises one or a pair of Fc domains that has the amino acid sequence of SEQ ID NO: 17, optionally with one more mutations that facilitate heterodimerization or purification, e.g., (a) knob or hole mutations and / or (b) star mutations.

[0215] In some embodiments, a tumor-targeted IL2 receptor agonist and / or multispecific T- cell engager comprises one or a pair of Fc domains that has at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 18. In some embodiments, the tumor-targeted IL2 receptor agonist and / or the multispecific T-cell engager comprises one or a pair of Fc domains that has the amino acid sequence of SEQ ID NO: 18, optionally with one more mutations that facilitate heterodimerization or purification, e.g., (a) knob or hole mutations and / or (b) star mutations.

[0216] The Fc domains that are incorporated into the tumor-targeted IL2 receptor agonist and / or the multispecific T-cell engager of a combination of the present disclosure may comprise one or more modifications that alter the functional properties of the proteins, for example, binding to Fc-receptors such as FcRn or leukocyte receptors, binding to complement, modified disulfide bond architecture, or altered glycosylation patterns. Exemplary Fc modifications that alter effector function are described in Section 6.7.1 .1 .

[0217] The Fc domains can also be altered to include modifications that improve manufacturability of asymmetric tumor-targeted IL2 receptor agonist and / or multispecific T- cell engagers, for example by allowing heterodimerization, which is the preferential pairing of non-identical Fc domains over identical Fc domains. Heterodimerization permits the production of tumor-targeted IL2 receptor agonist and / or multispecific T-cell engagers in which different polypeptide components are connected to one another by an Fc region containing Fc domains that differ in sequence. Examples of heterodimerization strategies are exemplified in Section 6.7.1.2.

[0218] It will be appreciated that any of the modifications mentioned above can be combined in any suitable manner to achieve the desired functional properties and / or combined with other modifications to alter the properties of the tumor-targeted IL2 receptor agonist and / or multispecific T-cell engagers.6.7.1.1. Fc Domains with Altered Effector Function

[0219] In some embodiments, the Fc domain(s) of a tumor-targeted IL2 receptor agonist and / or multispecific T-cell engager comprises one or more amino acid substitutions that reduces binding to an Fc receptor and / or effector function.

[0220] In a particular embodiment the Fc receptor is an Fey receptor. In one embodiment the Fc receptor is a human Fc receptor. In one embodiment the Fc receptor is an activating Fc receptor. In a specific embodiment the Fc receptor is an activating human Fey receptor, more specifically human FcyRllla, FcyRI or FcyRlla, most specifically human FcyRllla. In one embodiment the effector function is one or more selected from the group of complement dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and cytokine secretion. In a particular embodiment, the effector function is ADCC.

[0221] In one embodiment, the Fc domain (e.g., an Fc domain of a tumor-targeted IL2 receptor agonist and / or multispecific T-cell engager) or the Fc region (e.g., one or both Fc domains of a tumor-targeted IL2 receptor agonist and / or multispecific T-cell engager that can associate to form an Fc region) comprises an amino acid substitution at a position selected from the group of E233, L234, L235, N297, P331 and P329 (numberings according to KabatEU index). In a more specific embodiment, the Fc domain or the Fc region comprises an amino acid substitution at a position selected from the group of L234, L235 and P329 (numberings according to Kabat EU index). In some embodiments, the Fc domain or the Fc region comprises the amino acid substitutions L234A and L235A (numberings according to Kabat EU index). In one such embodiment, the Fc domain or region is an Igd Fc domain or region, particularly a human Igd Fc domain or region. In one embodiment, the Fc domain or the Fc region comprises an amino acid substitution at position P329. In a more specific embodiment, the amino acid substitution is P329A or P329G, particularly P329G (numberings according to Kabat EU index). In one embodiment, the Fc domain or the Fc region comprises an amino acid substitution at position P329 and a further amino acid substitution at a position selected from E233, L234, L235, N297 and P331 (numberings according to Kabat EU index). In a more specific embodiment, the further amino acid substitution is E233P, L234A, L235A, L235E, N297A, N297D or P331S. In particular embodiments, the Fc domain or the Fc region comprises amino acid substitutions at positions P329, L234 and L235 (numberings according to Kabat EU index). In more particular embodiments, the Fc domain comprises the amino acid mutations L234A, L235A and P329G (“P329G LALA”, “PGLALA” or “LALAPG”).

[0222] Typically, the same one or more amino acid substitution is present in each of the two Fc domains of an Fc region. Thus, in a particular embodiment, each Fc domain of the Fc region comprises the amino acid substitutions L234A, L235A and P329G (Kabat EU index numbering), i.e. in each of the first and the second Fc domains in the Fc region the leucine residue at position 234 is replaced with an alanine residue (L234A), the leucine residue at position 235 is replaced with an alanine residue (L235A) and the proline residue at position 329 is replaced by a glycine residue (P329G) (numbering according to Kabat EU index).

[0223] In one embodiment, the Fc domain is an IgG 1 Fc domain, particularly a human IgG 1 Fc domain. In some embodiments, the IgG 1 Fc domain is a variant IgG 1 comprising D265A, N297A mutations (EU numbering) to reduce effector function.

[0224] In another embodiment, the Fc domain is an lgG4 Fc domain with reduced binding to Fc receptors. Exemplary lgG4 Fc domains with reduced binding to Fc receptors may comprise an amino acid sequence selected from Table C below: In some embodiments, the Fc domain includes only the bolded portion of the sequences shown below:

[0225] In a particular embodiment, the lgG4 with reduced effector function comprises the bolded portion of the amino acid sequence of SEQ ID NO:12 (SEQ ID NO:31 of W02014 / 121087), sometimes referred to herein as lgG4s or hlgG4s.

[0226] For heterodimeric Fc regions, it is possible to incorporate a combination of the variant lgG4 Fc sequences set forth above, for example an Fc region comprising an Fc domain comprising the amino acid sequence of SEQ ID NO: 11 (SEQ ID NO:30 of W02014 / 121087) (or the bolded portion thereof) and an Fc domain comprising the amino acid sequence of SEQ ID NO:13 (SEQ ID NO:37 of WQ2014 / 121087) (or the bolded portion thereof) or an Fc region comprising an Fc domain comprising the amino acid sequence of SEQ ID NO:12 (SEQ ID NO:31 of WQ2014 / 121087) (or the bolded portion thereof) and an Fc domain comprising the amino acid sequence of SEQ ID NO:14 (SEQ ID NO:38 of WQ2014 / 121087) (or the bolded portion thereof).6.7.1.2. Fc Heterodimerization Variants

[0227] In some embodiments, a component of a combination of the disclosure, e.g., a multispecific T-cell engager, comprises different heavy chain polypeptide chains that entail dimerization between two Fc domains that, unlike a native immunoglobulin, are operably linked to non-identical N-terminal regions, e.g., one Fc domain connected to a TAA targeting moiety and the other Fc domain connected to a TCR complex targeting moiety. Inadequate heterodimerization of two Fc domains to form an Fc region has can be an obstacle for increasing the yield of desired heterodimeric molecules and represents challenges for purification. A variety of approaches available in the art can be used in for enhancing dimerization of Fc domains that might be present in the multispecific T-cell engagers of the disclosure, for example as disclosed in EP 1870459A1 ; U.S. Patent No. 5,582,996; U.S.Patent No. 5,731 ,168; U.S. Patent No. 5,910,573; U.S. Patent No. 5,932,448; U.S. Patent No. 6,833,441 ; U.S. Patent No. 7,183,076; U.S. Patent Application Publication No. 2006204493A1 ; and PCT Publication No. WO 2009 / 089004A1 .

[0228] The present disclosure provides combinations comprising tumor-targeted IL2 receptor agonist and / or multispecific T-cell engagers comprising Fc heterodimers, i.e., Fc regions comprising heterologous, non-identical Fc domains. Typically, each Fc domain in the Fc heterodimer comprises a CH3 domain of an antibody. The CH3 domains are derived from the constant region of an antibody of any isotype, class or subclass, and preferably of IgG (lgG1 , lgG2, lgG3 and lgG4) class, as described in the preceding section.

[0229] Heterodimerization of the two different heavy chains at CH3 domains give rise to the desired tumor-targeted IL2 receptor agonist and / or multispecific T-cell engager, while homodimerization of identical heavy chains will reduce yield of the desired tumor-targeted IL2 receptor agonist and / or multispecific T-cell engager. Thus, in a preferred embodiment, the polypeptides that associate to form a tumor-targeted IL2 receptor agonist and / or amultispecific T-cell engager of the disclosure will contain CH3 domains with modifications that favor heterodimeric association relative to unmodified Fc domains.

[0230] In a specific embodiment said modification promoting the formation of Fc heterodimers is a so-called “knob-into-hole” or “knob-in-hole” modification, comprising a “knob” modification in one of the Fc domains and a “hole” modification in the other Fc domain. The knob-into-hole technology is described e.g., in U.S. Patent No. 5,731 ,168; US 7,695,936; Ridgway et al., 1996, Prot Eng 9:617-621 , and Carter, 2001 , Immunol Meth 248:7-15. Generally, the method involves introducing a protuberance (“knob”) at the interface of a first polypeptide and a corresponding cavity (“hole”) in the interface of a second polypeptide, such that the protuberance can be positioned in the cavity so as to promote heterodimer formation and hinder homodimer formation. Protuberances are constructed by replacing small amino acid side chains from the interface of the first polypeptide with larger side chains (e.g., tyrosine or tryptophan). Compensatory cavities of identical or similar size to the protuberances are created in the interface of the second polypeptide by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine).

[0231] Accordingly, in some embodiments, an amino acid residue in the CH3 domain of the first subunit of the Fc domain is replaced with an amino acid residue having a larger side chain volume, thereby generating a protuberance within the CH3 domain of the first subunit which is positionable in a cavity within the CH3 domain of the second subunit, and an amino acid residue in the CH3 domain of the second subunit of the Fc domain is replaced with an amino acid residue having a smaller side chain volume, thereby generating a cavity within the CH3 domain of the second subunit within which the protuberance within the CH3 domain of the first subunit is positionable. Preferably said amino acid residue having a larger side chain volume is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W). Preferably said amino acid residue having a smaller side chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T), and valine (V). The protuberance and cavity can be made by altering the nucleic acid encoding the polypeptides, e.g., by site-specific mutagenesis, or by peptide synthesis. An exemplary substitution is Y470T.

[0232] In a specific such embodiment, in the first Fc domain the threonine residue at position 366 is replaced with a tryptophan residue (T366W), and in the Fc domain the tyrosine residue at position 407 is replaced with a valine residue (Y407V) and optionally the threonine residue at position 366 is replaced with a serine residue (T366S) and the leucine residue at position 368 is replaced with an alanine residue (L368A) (numbering according toKabat EU index). In a further embodiment, in the first Fc domain additionally the serine residue at position 354 is replaced with a cysteine residue (S354C) or the glutamic acid residue at position 356 is replaced with a cysteine residue (E356C) (particularly the serine residue at position 354 is replaced with a cysteine residue), and in the second Fc domain additionally the tyrosine residue at position 349 is replaced by a cysteine residue (Y349C)(numbering according to Kabat EU index). In a particular embodiment, the first Fc domain comprises the amino acid substitutions S354C and T366W, and the second Fc domain comprises the amino acid substitutions Y349C, T366S, L368A and Y407V (numbering according to Kabat EU index).

[0233] In some embodiments, electrostatic steering (e.g., as described in Gunasekaran et al., 2010, J Biol Chem 285(25): 19637-46) can be used to promote the association of the first and the second Fc domains of the Fc region.

[0234] As an alternative, or in addition, to the use of Fc domains that are modified to promote heterodimerization, an Fc domain can be modified to allow a purification strategy that enables selections of Fc heterodimers. In one such embodiment, one polypeptide comprises a modified Fc domain that abrogates its binding to Protein A, thus enabling a purification method that yields a heterodimeric protein. See, for example, U.S. Patent No. 8,586,713. As such, the tumor-targeted IL2 receptor agonist or the multispecific T-cell engagers comprise a first CH3 domain and a second Ig CH3 domain, wherein the first and second Ig CH3 domains differ from one another by at least one amino acid, and wherein at least one amino acid difference reduces binding of the tumor-targeted IL2 receptor agonist and / or multispecific T-cell engager to Protein A as compared to a corresponding tumor- targeted IL2 receptor agonist and / or multispecific T-cell engager lacking the amino acid difference. In one embodiment, the first CH3 domain binds Protein A and the second CH3 domain contains a mutation / modification that reduces or abolishes Protein A binding such as an H95R modification (by IMGT exon numbering; H435R by EU numbering). The second CH3 may further comprise a Y96F modification (by IMGT; Y436F by EU). This class of modifications is referred to herein as “star” mutations.

[0235] In some embodiments, the Fc can contain one or more mutations (e.g., knob and hole mutations) to facilitate heterodimerization as well as star mutations to facilitate purification.6.7.2. Hinge Sequences

[0236] In other embodiments, the tumor-targeted IL2 receptor agonist and / or the multispecific T-cell engagers of the disclosure comprise a linker that is a hinge region. In particular, where a tumor-targeted IL2 receptor agonist and / or a multispecific T-cell engagercontain an immunoglobulin-based targeting moiety, the hinge can be used to connect the targeting moiety, e.g., a Fab domain, to a connector domain, e.g., an Fc domain. The hinge region can be a native or a modified hinge region. Hinge regions are typically found at the N- termini of Fc regions. The term “hinge region”, unless the context dictates otherwise, refers to a naturally or non-naturally occurring hinge sequence that in the context of a single or monomeric polypeptide chain is a monomeric hinge domain and in the context of a dimeric polypeptide (e.g., a heterodimeric multispecific T-cell engager formed by the association of two Fc domains) can comprise two associated hinge sequences on separate polypeptide chains.

[0237] A native hinge region is the hinge region that would normally be found between Fab and Fc domains in a naturally occurring antibody. A modified hinge region is any hinge that differs in length and / or composition from the native hinge region. Such hinges can include hinge regions from other species, such as human, mouse, rat, rabbit, shark, pig, hamster, camel, llama or goat hinge regions. Other modified hinge regions may comprise a complete hinge region derived from an antibody of a different class or subclass from that of the heavy chain Fc domain or Fc region. Alternatively, the modified hinge region may comprise part of a natural hinge or a repeating unit in which each unit in the repeat is derived from a natural hinge region. In a further alternative, the natural hinge region may be altered by converting one or more cysteine or other residues into neutral residues, such as serine or alanine, or by converting suitably placed residues into cysteine residues. By such means the number of cysteine residues in the hinge region may be increased or decreased. Other modified hinge regions may be entirely synthetic and may be designed to possess desired properties such as length, cysteine composition and flexibility.

[0238] A number of modified hinge regions have already been described for example, in U.S. Patent No. 5,677,425, WO 99 / 15549, WO 2005 / 003170, WO 2005 / 003169, WO 2005 / 003170, WO 98 / 25971 and WO 2005 / 003171 and these are incorporated herein by reference.

[0239] In one embodiment, a tumor-targeted IL2 receptor agonist of the disclosure comprises an Fc region in which one or both Fc domains possesses an intact hinge region at its N-terminus.

[0240] In one embodiment, a multispecific T-cell engager of the disclosure comprises an Fc region in which one or both Fc domains possesses an intact hinge region at its N-terminus.

[0241] In various embodiments, positions 233-236 within a hinge region may be G, G, G and unoccupied; G, G, unoccupied, and unoccupied; G, unoccupied, unoccupied, and unoccupied; or all unoccupied, with positions numbered by EU numbering.

[0242] In some embodiments, the tumor-targeted IL2 receptor agonist and / or the multispecific T-cell engager of the disclosure comprise a modified hinge region that reduces binding affinity for an Fey receptor relative to a wild-type hinge region of the same isotype (e.g., human IgG 1 or human lgG4).

[0243] In one embodiment, the tumor-targeted IL2 receptor agonist and the multispecific T- cell engager of the disclosure comprise an Fc region in which each Fc domain possesses an intact hinge region at its N-terminus, where each Fc domain and hinge region is derived from lgG4, and each hinge region comprise the modified sequence CPPC (SEQ ID NO:21). The core hinge region of human lgG4 contains the sequence CPSC (SEQ ID NO:22) compared to IgG 1 that contains the sequence CPPC (SEQ ID NO:21). The serine residue present in the lgG4 sequence leads to increased flexibility in this region, and therefore a proportion of molecules form disulfide bonds within the same protein chain (an intrachain disulfide) rather than bridging to the other heavy chain in the IgG molecule to form the interchain disulfide. (Angel et al., 1993, Mol Immunol 30(1):105-108). Changing the serine residue to a proline to give the same core sequence as IgG 1 allows complete formation of inter-chain disulfides in the lgG4 hinge region, thus reducing heterogeneity in the purified product. This altered isotype is termed lgG4P.6.7.2.1. Chimeric Hinge Sequences

[0244] The hinge region can be a chimeric hinge region.

[0245] For example, a chimeric hinge may comprise an “upper hinge” sequence, derived from a human IgG 1 , a human lgG2 or a human lgG4 hinge region, combined with a “lower hinge” sequence, derived from a human lgG1 , a human lgG2 or a human lgG4 hinge region.

[0246] In particular embodiments, a chimeric hinge region comprises the amino acid sequence EPKSCDKTHTCPPCPAPPVA (SEQ ID NO:23) (previously disclosed as SEQ ID NO:8 of WQ2014 / 121087, which is incorporated by reference in its entirety herein) or ESKYGPPCPPCPAPPVA (SEQ ID NO:24(previously disclosed as SEQ ID NO:9 of W02014 / 121087). Such chimeric hinge sequences can be suitably linked to an lgG4 CH2 region (for example by incorporation into an lgG4 Fc domain, for example a human or murine Fc domain, which can be further modified in the CH2 and / or CH3 domain to reduce effector function, for example as described in Section 6.7.1.1).6.7.2.2. Hinge Sequences with Reduced Effector Function

[0247] In further embodiments, the hinge region can be modified to reduce effector function, for example as described in W02016161010A2, which is incorporated by reference in its entirety herein. In various embodiments, the positions 233-236 of the modified hinge region are G, G, G and unoccupied; G, G, unoccupied, and unoccupied; G, unoccupied,unoccupied, and unoccupied; or all unoccupied, with positions numbered by EU numbering (as shown in FIG. 1 of W02016161010A2). These segments can be represented as GGG-, GG--, G— or -— with representing an unoccupied position.

[0248] Position 236 is unoccupied in canonical human lgG2 but is occupied by in other canonical human IgG isotypes. Positions 233-235 are occupied by residues other than G in all four human isotypes (as shown in FIG. 1 of W02016161010A2).

[0249] The hinge modification within positions 233-236 can be combined with position 228 being occupied by P. Position 228 is naturally occupied by P in human IgG 1 and lgG2 but is occupied by S in human lgG4 and R in human lgG3. An S228P mutation in an lgG4 antibody is advantageous in stabilizing an lgG4 antibody and reducing exchange of heavy chain light chain pairs between exogenous and endogenous antibodies. Preferably positions 226-229 are occupied by C, P, P and C respectively.

[0250] Exemplary hinge regions have residues 226-236, sometimes referred to as middle (or core) and lower hinge, occupied by the modified hinge sequences designated GGG-(233- 236), GG-(233-236), G— (233-236) and no G(233-236). Optionally, the hinge domain amino acid sequence comprises CPPCPAPGGG-GPSVF (SEQ ID NO:25) (previously disclosed as SEQ ID NO:1 of W02016161010A2), CPPCPAPGG-GPSVF (SEQ ID NO: 26) (previously disclosed as SEQ ID NO:2 of W02016161010A2), CPPCPAPG— GPSVF (SEQ ID NO:27) (previously disclosed as SEQ ID NO:3 of W02016161010A2), or CPPCPAP— -GPSVF (SEQ ID NO:28) (previously disclosed as SEQ ID NO:4 of W02016161010A2).

[0251] The modified hinge regions described above can be incorporated into a heavy chain constant region, which typically include CH2 and CH3 domains, and which may have an additional hinge segment (e.g., an upper hinge) flanking the designated region. Such additional constant region segments present are typically of the same isotype, preferably a human isotype, although can be hybrids of different isotypes. The isotype of such additional human constant regions segments is preferably human lgG4 but can also be human lgG1 , lgG2, or lgG3 or hybrids thereof in which domains are of different isotypes. Exemplary sequences of human lgG1 , lgG2 and lgG4 are shown in FIGS. 2-4 of WQ2016161010A2.

[0252] In specific embodiments, the modified hinge sequences can be linked to an lgG4 CH2 region (for example by incorporation into an lgG4 Fc domain, for example a human or murine Fc domain, which can be further modified in the CH2 and / or CH3 domain to reduce effector function, for example as described in Section 6.7.1.1).

[0253] The linkers useful in the tumor-targeted IL2 receptor agonist and / or the multispecific T-cell engagers of the disclosure are typically non-cleavable linkers. A non-cleavable linker is one whose amino acid sequences lacks a (canonical) substrate sequence a for aprotease, for example a substrate as set forth in Table B on pages 45-49 of international patent publication no. W02024040249A1 and / or a protease as set forth in Table A on pages 43-44 of international application publication no. W02024040249A1 . The contents of Tables A and B of W02024040249A1 are incorporated by reference herein.6.7.3. Linkers

[0254] In certain aspects, the present disclosure provides cell synapse targeted therapeutics comprising tumor-targeted IL2 receptor agonist and multispecific T-cell engager constructs in which two or more components are connected to one another by a peptide linker. By way of example and not limitation, linkers can be used to connect (a) an IL2 moiety and a connector moiety; (b) an IL2 moiety and a targeting moiety; (c) a targeting moiety and a connector moiety (e.g., a Fab domain and an Fc domain); (d) an IL2 moiety and a masking moiety (e.g., an IL2 moiety and an IL2Ra domain); or (e) different domains within a targeting moiety (e.g., the VH and VL domains in a scFv).

[0255] A peptide linker can range from 2 amino acids to 60 or more amino acids, and in certain aspects a peptide linker ranges from 3 amino acids to 50 amino acids, from 4 to 30 amino acids, from 5 to 25 amino acids, from 10 to 25 amino acids, 10 amino acids to 60 amino acids, from 12 amino acids to 20 amino acids, from 20 amino acids to 50 amino acids, or from 25 amino acids to 35 amino acids in length.

[0256] In particular aspects, a peptide linker is at least 5 amino acids, at least 6 amino acids or at least 7 amino acids in length and optionally is up to 30 amino acids, up to 40 amino acids, up to 50 amino acids or up to 60 amino acids in length.

[0257] In some embodiments of the foregoing, the linker ranges from 5 amino acids to 50 amino acids in length, e.g., ranges from 5 to 50, from 5 to 45, from 5 to 40, from 5 to 35, from 5 to 30, from 5 to 25, or from 5 to 20 amino acids in length. In other embodiments of the foregoing, the linker ranges from 6 amino acids to 50 amino acids in length, e.g., ranges from 6 to 50, from 6 to 45, from 6 to 40, from 6 to 35, from 6 to 30, from 6 to 25, or from 6 to 20 amino acids in length. In yet other embodiments of the foregoing, the linker ranges from 7 amino acids to 50 amino acids in length, e.g., ranges from 7 to 50, from 7 to 45, from 7 to 40, from 7 to 35, from 7 to 30, from 7 to 25, or from 7 to 20 amino acids in length.

[0258] Charged (e.g., charged hydrophilic linkers) and / or flexible linkers are particularly preferred.

[0259] Examples of flexible linkers that can be used in the IL2 agonists of the disclosure include those disclosed by Chen et al., 2013, Adv Drug Deliv Rev. 65(10): 1357-1369 and Klein et al., 2014, Protein Engineering, Design & Selection 27(10): 325-330. Particularly useful flexible linkers are or comprise repeats of glycines and serines, e.g., a monomer ormultimer of GnS (SEQ ID NO:68) or SGn(SEQ ID NO:69]), where n is an integer from 1 to 10, e.g., 1 2, 3, 4, 5, 6, 7, 8, 9 or 10. In one embodiment, the linker is or comprises a monomer or multimer of repeat of G4S e.g., (GGGGS)n(SEQ ID NO:70).

[0260] Polyglycine linkers can suitably be used in the IL2 agonists of the disclosure. In some embodiments, a peptide linker comprises two consecutive glycines (2Gly) , three consecutive glycines (3Gly), four consecutive glycines (4Gly) (SEQ ID NO:71), five consecutive glycines (5Gly) (SEQ ID NO:72), six consecutive glycines (6Gly) (SEQ ID NO:73), seven consecutive glycines (7Gly) (SEQ ID NO:74), eight consecutive glycines (8Gly) (SEQ ID NO:75) or nine consecutive glycines (9Gly) (SEQ ID NO:76).

[0261] The linkers useful in the components of a combination of the disclosure (e.g., a linker in a tumor-targeted IL2 receptor agonist and / or a multispecific T-cell engager) are typically non-cleavable linkers. A non-cleavable linker is one whose amino acid sequences lacks a (canonical) substrate sequence for a protease, for example a substrate as set forth in Table B on pages 45-49 of international patent publication no. WQ2024040249A1 and / or a protease as set forth in Table A on pages 43-44 of international application publication no. WQ2024040249A1 . The contents of Tables A and B of WQ2024040249A1 are incorporated by reference herein.6.8. Nucleic Acids and Host Cells

[0262] The disclosure provides nucleic acids encoding one or more components of a combination of the disclosure, e.g., a tumor-targeted IL2 receptor agonist and / or a multispecific T-cell engager and / or their individual polypeptide chains.

[0263] In some embodiments, a tumor-targeted IL2 receptor agonist and / or a multispecific T-cell engager is encoded by a single nucleic acid. In other embodiments, for example in the case of a heterodimeric molecule or a molecule comprising a targeting moiety composed of more than one polypeptide chain, the tumor-targeted IL2 receptor agonist and / or the multispecific T-cell engager can be encoded by a plurality (e.g., two, three, four or more) nucleic acids.

[0264] A single nucleic acid can encode a tumor-targeted IL2 receptor agonist and / or a multispecific T-cell engager. If the tumor-targeted IL2 receptor agonist and / or multispecific T-cell engager comprises more than one polypeptide chain, two or more open reading frames encoding two or more polypeptide chains can be under the control of separate transcriptional regulatory elements (e.g., promoters and / or enhancers). The open reading frames encoding two or more polypeptide chains can also be controlled by the same transcriptional regulatory elements and separated by internal ribosome entry site (IRES) sequences allowing for translation into separate polypeptide chains.

[0265] In some embodiments, a tumor-targeted IL2 receptor agonist and / or a multispecific T-cell engager construct comprising two or more polypeptide chains is encoded by two or more nucleic acids. The number of nucleic acids encoding a tumor-targeted IL2 receptor agonist and / or a multispecific T-cell engager construct can be equal to or less than the number of polypeptide chains in the tumor-targeted IL2 receptor agonist and / or the multispecific T-cell engager (for example, when more than one polypeptide chains are encoded by a single nucleic acid).

[0266] The nucleic acids of the disclosure can be DNA or RNA (e.g., mRNA).

[0267] In another aspect, the disclosure provides host cells and vectors containing one or more nucleic acids of the disclosure. The nucleic acids may be present in a single vector or separate vectors present in the same host cell or separate host cell, as described in more detail herein below.6.8.1. Vectors

[0268] The disclosure provides vectors comprising nucleotide sequences encoding a tumor- targeted IL2 receptor agonist and / or a multispecific T-cell engager described herein or a component of a tumor-targeted IL2 receptor agonist and / or a multispecific T-cell engager, for example one or two or more polypeptide chains present in a tumor-targeted IL2 receptor agonist and / or multispecific T-cell engager. The vectors include, but are not limited to, a virus, plasmid, cosmid, lambda phage or a yeast artificial chromosome (YAC).

[0269] Numerous vector systems can be employed. For example, one class of vectors utilizes DNA elements which are derived from animal viruses such as, for example, bovine papilloma virus, polyoma virus, adenovirus, vaccinia virus, baculovirus, retroviruses (Rous Sarcoma Virus, MMTV or MOMLV) or SV40 virus. Another class of vectors utilizes RNA elements derived from RNA viruses such as Semliki Forest virus, Eastern Equine Encephalitis virus and Flaviviruses.

[0270] Additionally, cells which have stably integrated the DNA into their chromosomes can be selected by introducing one or more markers which allow for the selection of transfected host cells. The marker may provide, for example, prototropy to an auxotrophic host, biocide resistance (e.g., antibiotics), or resistance to heavy metals such as copper, or the like. The selectable marker gene can be either directly linked to the DNA sequences to be expressed, or introduced into the same cell by co-transformation. Additional elements may also be needed for optimal synthesis of mRNA. These elements may include splice signals, as well as transcriptional promoters, enhancers, and termination signals.

[0271] Once the expression vector or DNA sequence containing the constructs has been prepared for expression, the expression vectors can be transfected or introduced into anappropriate host cell. Various techniques may be employed to achieve this, such as, for example, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, lipid-based transfection or other conventional techniques. Methods and conditions for culturing the resulting transfected cells and for recovering the expressed polypeptides are known to those skilled in the art and may be varied or optimized depending upon the specific expression vector and mammalian host cell employed, based upon the present description.6.8.2. Cells

[0272] The disclosure also provides host cells comprising a nucleic acid of the disclosure.

[0273] In one embodiment, the host cells are genetically engineered to comprise one or more nucleic acids described herein, e.g., to express a tumor-targeted IL2 receptor agonist and / or a multispecific T-cell engager.

[0274] In one embodiment, the host cells are genetically engineered by using an expression cassette. The phrase “expression cassette,’’ refers to nucleotide sequences, which are capable of affecting expression of a gene in hosts compatible with such sequences. Such cassettes may include a promoter, an open reading frame with or without introns, and a termination signal. Additional factors necessary or helpful in effecting expression may also be used, such as, for example, an inducible promoter.

[0275] The disclosure also provides host cells comprising the vectors described herein.

[0276] The cell can be, but is not limited to, a eukaryotic cell, a bacterial cell, an insect cell, or a human cell. Suitable eukaryotic cells include, but are not limited to, Vero cells, HeLa cells, COS cells, CHO cells, HEK293 cells, BHK cells and MDCKII cells. Suitable insect cells include, but are not limited to, Sf9 cells.6.9. Pharmaceutical Compositions

[0277] The combinations of tumor-targeted IL2 receptor agonist and multispecific T-cell engager constructs of the disclosure may be in the form of individual compositions comprising the tumor-targeted IL2 receptor agonist or multispecific T-cell engager constructs together one or more carriers, excipients and / or diluents. The compositions may be formulated for specific uses, such as for veterinary uses or pharmaceutical uses in humans. The form of the composition (e.g., dry powder, liquid formulation, etc.) and the excipients, diluents and / or carriers used will depend upon the intended uses of the combination comprising the tumor-targeted IL2 receptor agonist and multispecific T-cell engager and, for therapeutic uses, the mode of administration.

[0278] The combinations of tumor-targeted IL2 receptor agonist and multispecific T-cell engager constructs of the disclosure may be in the form of combined compositions comprising both the tumor-targeted IL2 receptor agonist and multispecific T-cell engager constructs together one or more carriers, excipients and / or diluents. The compositions may be formulated for specific uses, such as for veterinary uses or pharmaceutical uses in humans. The form of the composition (e.g., dry powder, liquid formulation, etc.) and the excipients, diluents and / or carriers used will depend upon the intended uses of the combination comprising tumor-targeted IL2 receptor agonist and multispecific T-cell engager and, for therapeutic uses, the mode of administration.

[0279] For therapeutic uses, the compositions may be supplied as part of a sterile, pharmaceutical composition that includes a pharmaceutically acceptable carrier. This composition can be in any suitable form (depending upon the desired method of administering it to a patient). The pharmaceutical composition can be administered to a patient by a variety of routes such as orally, transdermally, subcutaneously, intranasally, intravenously, intramuscularly, intratumorally, intrathecally, topically or locally. The most suitable route for administration in any given case will depend on the particular antibody, the subject, and the nature and severity of the disease and the physical condition of the subject. Typically, the pharmaceutical composition will be administered intravenously or subcutaneously.

[0280] Pharmaceutical compositions can be conveniently presented in unit dosage forms containing a predetermined amount of a tumor-targeted IL2 receptor agonist and / or multispecific T-cell engager of the disclosure per dose. The quantity of a tumor-targeted IL2 receptor agonist and / or a multispecific T-cell engager included in a unit dose will depend on the disease being treated, as well as other factors as are well known in the art. Such unit dosages may be in the form of a lyophilized dry powder containing an amount of a tumor- targeted IL2 receptor agonist and / or a multispecific T-cell engager suitable for a single administration, or in the form of a liquid. Dry powder unit dosage forms may be packaged in a kit with a syringe, a suitable quantity of diluent and / or other components useful for administration. Unit dosages in liquid form may be conveniently supplied in the form of a syringe pre-filled with a quantity of tumor-targeted IL2 receptor agonist and / or multispecific T-cell engager suitable for a single administration.

[0281] The pharmaceutical compositions may also be supplied in bulk from containing quantities of a tumor-targeted IL2 receptor agonist and / or multispecific T-cell engager suitable for multiple administrations.

[0282] Pharmaceutical compositions may be prepared for storage as lyophilized formulations or aqueous solutions by mixing a tumor-targeted IL2 receptor agonist and / or a multispecific T-cell engager having the desired degree of purity with optional pharmaceutically-acceptable carriers, excipients or stabilizers typically employed in the art (all of which are referred to herein as “carriers”), i.e., buffering agents, stabilizing agents, preservatives, isotonifiers, non-ionic detergents, antioxidants, and other miscellaneous additives. See, Remington’s Pharmaceutical Sciences, 16th edition (Osol, ed. 1980). Such additives should be non-toxic to the recipients at the dosages and concentrations employed.

[0283] Buffering agents help to maintain the pH in the range which approximates physiological conditions. They may be present at a wide variety of concentrations but will typically be present in concentrations ranging from about 2 mM to about 50 mM. Suitable buffering agents for use with the present disclosure include both organic and inorganic acids and salts thereof such as citrate buffers (e.g., monosodium citrate-disodium citrate mixture, citric acid-trisodium citrate mixture, citric acid-monosodium citrate mixture, etc.), succinate buffers (e.g., succinic acid-monosodium succinate mixture, succinic acid-sodium hydroxide mixture, succinic acid-disodium succinate mixture, etc.), tartrate buffers (e.g., tartaric acid- sodium tartrate mixture, tartaric acid-potassium tartrate mixture, tartaric acid-sodium hydroxide mixture, etc.), fumarate buffers (e.g., fumaric acid-monosodium fumarate mixture, fumaric acid-disodium fumarate mixture, monosodium fumarate-disodium fumarate mixture, etc.), gluconate buffers (e.g., gluconic acid-sodium glyconate mixture, gluconic acid-sodium hydroxide mixture, gluconic acid-potassium glyconate mixture, etc.), oxalate buffer (e.g., oxalic acid-sodium oxalate mixture, oxalic acid-sodium hydroxide mixture, oxalic acid- potassium oxalate mixture, etc.), lactate buffers (e.g., lactic acid-sodium lactate mixture, lactic acid-sodium hydroxide mixture, lactic acid-potassium lactate mixture, etc.) and acetate buffers (e.g., acetic acid-sodium acetate mixture, acetic acid-sodium hydroxide mixture, etc ). Additionally, phosphate buffers, histidine buffers and trimethylamine salts such as Tris can be used.

[0284] Preservatives may be added to retard microbial growth and can be added in amounts ranging from about 0.2%-1 % (w / v). Suitable preservatives for use with the present disclosure include phenol, benzyl alcohol, meta-cresol, methyl paraben, propyl paraben, octadecyldimethylbenzyl ammonium chloride, benzalconium halides (e.g., chloride, bromide, and iodide), hexamethonium chloride, and alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, and 3-pentanol. Isotonicifiers sometimes known as “stabilizers” can be added to ensure isotonicity of liquid compositions of the present disclosure and include polyhydric sugar alcohols, for example trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol and mannitol. Stabilizers referto a broad category of excipients which can range in function from a bulking agent to an additive which solubilizes the therapeutic agent or helps to prevent denaturation or adherence to the container wall. Typical stabilizers can be polyhydric sugar alcohols (enumerated above); amino acids such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, threonine, etc., organic sugars or sugar alcohols, such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myoinisitol, galactitol, glycerol and the like, including cyclitols such as inositol; polyethylene glycol; amino acid polymers; sulfur containing reducing agents, such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, a-monothioglycerol and sodium thio sulfate; low molecular weight polypeptides (e.g., peptides of 10 residues or fewer); proteins such as human serum albumin, bovine serum albumin, gelatin or immunoglobulins; hydrophylic polymers, such as polyvinylpyrrolidone monosaccharides, such as xylose, mannose, fructose, glucose; disaccharides such as lactose, maltose, sucrose and trehalose; and trisaccacharides such as raffinose; and polysaccharides such as dextran. Stabilizers may be present in amounts ranging from 0.5 to 10 wt % per wt of tumor-targeted IL2 receptor agonist and / or multispecific T-cell engager.

[0285] Non-ionic surfactants or detergents (also known as “wetting agents”) may be added to help solubilize the glycoprotein as well as to protect the glycoprotein against agitation- induced aggregation, which also permits the formulation to be exposed to shear surface stressed without causing denaturation of the protein. Suitable non-ionic surfactants include polysorbates (20, 80, etc.), polyoxamers (184, 188 etc.), and pluronic polyols. Non-ionic surfactants may be present in a range of about 0.05 mg / mL to about 1 .0 mg / mL, for example about 0.07 mg / mL to about 0.2 mg / mL.

[0286] Additional miscellaneous excipients include bulking agents (e.g., starch), chelating agents (e.g., EDTA), antioxidants (e.g., ascorbic acid, methionine, vitamin E), and cosolvents.6.10. Therapeutic Indications and Methods of Use

[0287] The present disclosure provides methods for using and applications for combinations of the disclosure comprising tumor-targeted IL2 receptor agonist and multispecific T-cell engagers.

[0288] The combinations of the disclosure are useful in treating disease states where stimulation of the immune system of the host is beneficial, in particular conditions where an enhanced cellular immune response is desirable. These may include disease states where the host immune response is insufficient or deficient.

[0289] Disease states for which the combinations of the disclosure can be administered comprise, for example, a tumor or infection where a cellular immune response would be a critical mechanism for specific immunity. Specific disease states for which the combinations of the present disclosure can be employed include cancer, including breast cancer, prostate cancer, and colorectal cancer. The combinations of the disclosure may be administered per se or in any suitable pharmaceutical composition.

[0290] In various embodiments, the combinations of the disclosure are useful for the treatment of cancer, for the prevention or treatment of metastasis, for stimulating the formation, stability and / or activity of a cytotoxic immune synapse, for inducing tumor cytolysis, for inducing anti-tumor cytotoxicity, for stimulating an immune response against a tumor, or any combination of two or more of the foregoing uses.

[0291] In one aspect, a combination of the disclosure for use as a medicament is provided. In further aspects, a combination of the disclosure for use in treating a disease is provided. In certain embodiments, a combination of the disclosure for use in a method of treatment is provided. In one embodiment, the disclosure provides a combination as described herein for use in the treatment of a disease in a subject in need thereof. In certain embodiments, the disclosure provides a combination as described herein for use in a method of treating a subject having a disease comprising administering to the individual a therapeutically effective amount of a combination of a tumor-targeted IL2 receptor agonist and a multispecific T-cell engager. In certain embodiments the disease to be treated is a proliferative disorder. In a preferred embodiment the disease is cancer. In certain embodiments the method further comprises administering to the individual a therapeutically effective amount of at least one additional therapeutic agent, e.g., an anti-cancer agent if the disease to be treated is cancer. In further embodiments, the disclosure provides a combination as described herein for use in stimulating the immune system. In certain embodiments, the disclosure provides a combination as disclosed herein for use in a method of stimulating the immune system in a subject comprising administering to the individual an effective amount of the combination to stimulate the immune system. An “individual” according to any of the above embodiments is a mammal, preferably a human. “Stimulation of the immune system” according to any of the above embodiments may include any one or more of a general increase in immune function, an increase in T-cell function, an increase in B-cell function, a restoration of lymphocyte function, an increase in the expression of IL2 receptors, an increase in T-cell responsiveness, an increase in natural killer (NK) cell activity or lymphokine-activated killer (LAK) cell activity, and the like.

[0292] In a further aspect, the disclosure provides for the use of a combination of the disclosure in the manufacture or preparation of one or more medicaments for the treatment of a disease in a subject in need thereof. In one embodiment, the medicament is for use in a method of treating a disease comprising administering to a subject having the disease a therapeutically effective amount of the one or more medicaments. In certain embodiments the disease to be treated is a proliferative disorder. In a preferred embodiment the disease is cancer. In one such embodiment, the method further comprises administering to the individual a therapeutically effective amount of at least one additional therapeutic agent, e.g., an anti-cancer agent if the disease to be treated is cancer. In a further embodiment, the one or more medicaments are for stimulating the immune system. In a further embodiment, one or more medicaments are for use in a method of stimulating the immune system in a subject comprising administering to the individual an amount effective of the one or more medicaments to stimulate the immune system. An “individual” according to any of the above embodiments may be a mammal, preferably a human. “Stimulation of the immune system” according to any of the above embodiments may include any one or more of a general increase in immune function, an increase in T-cell function, an increase in B-cell function, a restoration of lymphocyte function, an increase in the expression of IL2 receptors, an increase in T-cell responsiveness, an increase in natural killer (NK) cell activity or lymphokine-activated killer (LAK) cell activity, and the like.

[0293] In a further aspect, the disclosure provides a method for treating a disease in a subject, comprising administering to said individual a therapeutically effective amount of a combination of the disclosure.

[0294] In some embodiments, the disclosure provides a method of treating cancer with a combination as described herein in which the TAA targeting moiety of the tumor-targeted IL2 receptor agonist and the TAA targeting moiety of the multispecific T-cell engager recognize the cancer tissue to be treated

[0295] The present disclosure further provides a method of localized delivery of a combination as described herein, comprising administering the combination to the subject, where the TAA targeting moiety of the tumor-targeted IL2 receptor agonist and the TAA targeting moiety of the multispecific T cell engager recognize a tissue to which the combination is to be locally delivered. As used herein, the term “locally delivered” does not require local administration but rather indicates that the components of the combination be selectively localized to a tissue of interest following administration.

[0296] The present disclosure further provides a method of administering to the subject IL2 therapy with reduced systemic exposure and / or reduced systemic toxicity and / or animproved therapeutic index, comprising administering to a subject the IL2 therapy in the form of a cell combination of the disclosure. Accordingly, the foregoing methods permit IL2 therapy with reduced off-target side effects by virtue of preferential targeting of an IL2 receptor agonist to a particular target tissue and / or improved anti-tumor cytotoxicity at the site of intended activity.

[0297] The present disclosure further provides method of locally inducing an immune response in a target tissue, comprising administering to a subject a combination as described herein, where the TAA targeting moiety of the tumor-targeted IL2 receptor agonist and the TAA targeting moiety of the multispecific T-cell engager are capable of binding a target molecule expressed in the target tissue. The tumor-targeted IL2 receptor agonist and multispecific T-cell engager can then induce the immune response against at least one cell type in the target tissue. In some embodiments, the target tissue is cancer tissue.

[0298] In some embodiments, the administration is not local to the tissue. For example, when the target tissue is cancer tissue, the administration can be systemic or subcutaneous.

[0299] In each of the foregoing applications and uses of the combinations of the disclosure, the tumor-targeted IL2 receptor agonist and the multispecific T-cell engager can be administered in separate pharmaceutical preparations or the same pharmaceutical preparation. When administered in separate pharmaceutical preparations, the preparations can be administered simultaneously, sequentially or separately. It is appreciated that the administration of the tumor-targeted IL2 receptor agonist and the multispecific T-cell engager is timed such both agents are capable of acting on the target tissue at the same time.

[0300] In certain embodiments the disease to be treated is a proliferative disorder, preferably cancer. Non-limiting examples of cancers include bladder cancer, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, colon cancer, colorectal cancer, rectal cancer, gastric cancer, prostate cancer, blood cancer, skin cancer, squamous cell carcinoma, bone cancer, and kidney cancer. Other cell proliferation disorders that can be treated using a tumor-targeted IL2 receptor agonist and multispecific T-cell engager of the present disclosure include, but are not limited to neoplasms located in the: abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid), eye, head and neck, nervous system (central and peripheral), lymphatic system, pelvic, skin, soft tissue, spleen, thoracic region, and urogenital system. Also included are pre-cancerous conditions or lesions and cancer metastases. In certain embodiments the cancer is chosen from the group consisting of renal cell cancer, skin cancer, lung cancer, colorectal cancer, breast cancer,brain cancer, head and neck cancer. Similarly, other cell proliferation disorders can also be treated by the tumor-targeted IL2 receptor agonist and multispecific T-cell engagers of the present disclosure. Examples of such cell proliferation disorders include, but are not limited to: hypergammaglobulinemia, lymphoproliferative disorders, paraproteinemias, purpura, sarcoidosis, Sezary Syndrome, Waldenstron's Macroglobulinemia, Gaucher's Disease, histiocytosis, and any other cell proliferation disease, besides neoplasia, located in an organ system listed above.

[0301] Table I below shows exemplary indications for which combinations of the disclosure targeting particular target molecules can be used.

[0302] Additional target molecules and corresponding indications are disclosed in, e.g., Hafeez et a!., 2020, Molecules 25:4764, doi:10.3390 / molecules25204764, particularly in Table 1. Table 1 is incorporated by reference in its entirety here.

[0303] A skilled artisan readily recognizes that in many cases a combination of the disclosure may not provide a cure but may only provide partial benefit. In some embodiments, a physiological change having some benefit is also considered therapeutically beneficial. Thus, in some embodiments, an amount of combination (or individual components thereof) that provides a physiological change is considered an “effective amount” or a “therapeutically effective amount”.

[0304] The subject, patient, or individual in need of treatment is typically a mammal, more specifically a human.

[0305] The appropriate dosage of a combination of the disclosure (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the route of administration, the body weight of the patient, the particular tumor-targeted IL2 receptor agonist and multispecific T-cell engager, the severity and course of the disease, whether the combination is administered for preventive or therapeutic purposes, previous or concurrent therapeutic interventions, the patient's clinical history and response to the tumor-targeted IL2 receptor agonist and multispecific T-cell engager, and the discretion of the attending physician. In some embodiments, the components of a combination of the disclosure are administered concurrently and / or in equimolar amounts. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject. Various dosing schedules including but not limited to single or multiple administrations over various time-points, bolus administration, and pulse infusion are contemplated herein.

[0306] A therapeutically effective amount of a combination of the disclosure may comprise only a single administration of each component or many administrations of each component over a period of time. Thus, a combination of the disclosure is suitably administered to the patient at one time or over a series of treatments, each comprising administration of both a tumor-targeted IL2 receptor agonist and a multispecific T-cell engager.

[0307] For systemic administration, a therapeutically effective dose can be estimated initially from in vitro assays, such as cell culture assays. A dose can then be formulated in animal models to achieve a circulating concentration range that includes the EC5o as determined in cell culture. Such information can be used to more accurately determine useful doses in humans.

[0308] Initial dosages can also be estimated from in vivo data, e.g., animal models, using techniques that are well known in the art. One having ordinary skill in the art could readily optimize administration to humans based on animal data.6.11. Combination Therapy

[0309] The cell synapse targeted therapeutic combinations disclosed herein may be administered in combination with one or more other agents in therapy. For instance, a tumor- targeted IL2 receptor agonist and multispecific T-cell engager of the disclosure may be coadministered with at least one additional therapeutic agent. The term “therapeutic agent” encompasses any agent administered to treat a symptom or disease in a subject in need of such treatment. Such additional therapeutic agent may comprise any active ingredients suitable for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. In certain embodiments, an additional therapeutic agent is an immunomodulatory agent, a cytostatic agent, an inhibitor of cell adhesion, a cytotoxic agent, an activator of cell apoptosis, or an agent that increases the sensitivity of cells to apoptotic inducers. In a particular embodiment, the additional therapeutic agent is an anti-cancer agent, for example a microtubule disruptor, an antimetabolite, a topoisomerase inhibitor, a DNA intercalator, an alkylating agent, a hormonal therapy, a kinase inhibitor, a receptor antagonist, an activator of tumor cell apoptosis, or an antiangiogenic agent

[0310] Such other agents are suitably present in combination in amounts that are effective for the purpose intended. The effective amount of such other agents depends on the amount of tumor-targeted IL2 receptor agonist and multispecific T-cell engagers used, the type of disorder or treatment, and other factors discussed above. The tumor-targeted IL2 receptor agonist and multispecific T-cell engagers are generally used in the same dosages and with administration routes as described herein, or about from 1 to 99% of the dosages described herein, or in any dosage and by any route that is empirically / clinically determined to be appropriate.

[0311] Such combination therapies noted above encompass combined administration and separate administration, in which case, administration of the tumor-targeted IL2 receptor agonist and multispecific T-cell engagers can occur prior to, simultaneously, and / or following, administration of the additional therapeutic agent and / or adjuvant.

[0312] Tumor-targeted IL2 and multispecific T-cell engagers of the disclosure can also be used in combination with radiation therapy.7. SPECIFIC EMBODIMENTS

[0313] The present disclosure is exemplified by the specific embodiments below.1. A combination comprising:(a) a tumor-targeted interleukin-2 (“ IL2”) receptor agonist comprising:(i) a first TAA targeting moiety;(ii) an IL2 moiety; and(iii) optionally, a first connector moiety separating the first TAA targeting moiety and the IL2 moiety; and(b) a multispecific T-cell engager comprising:(i) a second TAA targeting moiety;(ii) a T-cell receptor complex targeting moiety; and(iii) optionally, a second connector moiety and / or a third connector moiety separating the second TAA targeting moiety and the T-cell receptor complex targeting moiety, for use as a combination therapy, optionally or use as a combination therapy for the treatment of cancer, for use as a combination therapy for the prevention or treatment of metastasis, for use as combination therapy for stimulating the formation, stability and / or activity of a cytotoxic immune synapse, for use as combination therapy for inducing tumor cytolysis, for use combination therapy for inducing anti-tumor cytotoxicity, for use combination therapy for stimulating an immune response against a tumor, or any combination of two or more of the foregoing uses.2. The combination of embodiment 1 , for use as a combination therapy for the treatment of cancer.3. The combination of embodiment 1 or embodiment 2, for use as a combination therapy for the prevention or treatment of metastasis.4. The combination of any one of embodiments 1 to 3, for use as combination therapy for stimulating the formation, stability and / or activity of a cytotoxic immune synapse.5. The combination of any one of embodiments 1 to 4, for use as a combination therapy for inducing tumor cytolysis.6. The combination of any one of embodiments 1 to 5, for use as a combination therapy for inducing anti-tumor cytotoxicity.7. The combination of any one of embodiments 1 to 6, for use as a combination therapy for stimulating an immune response against a tumor.8. A method comprising administering to a subject in need thereof a combination comprising:(a) a tumor-targeted interleukin-2 (“IL2”) receptor agonist comprising:(i) a first TAA targeting moiety;(ii) an IL2 moiety; and(iii) optionally, a first connector moiety separating the first TAA targeting moiety and the IL2 moiety; and(b) a multispecific T-cell engager comprising:(i) a second TAA targeting moiety;(ii) a T-cell receptor complex targeting moiety; and(iii) optionally, a second connector moiety separating the second TAA targeting moiety and the T-cell receptor complex targeting moiety, optionally wherein the method is a method of combination therapy for the treatment of cancer, a method of combination therapy for the prevention or treatment of metastasis, a method of combination therapy for stimulating the formation, stability and / or activity of a cytotoxic immune synapse, a method of combination therapy for inducing tumor cytolysis, a method of combination therapy for inducing anti-tumor cytotoxicity, a method of combination therapy for stimulating an immune response against a tumor, or a combination of any two or more of the foregoing methods.9. The method of embodiment 8, which is a method for the treatment of cancer.10. The method of embodiment 8 or embodiment 9, which is a method for the prevention or treatment of metastasis.11 . The method of any one of embodiments 8 to 10, which is a method for stimulating the formation, stability and / or activity of a cytotoxic immune synapse.12. The method of any one of embodiments 8 to 11 , which is a method for inducing tumor cytolysis.13. The method of any one of embodiments 8 to 12, which is a method for inducing antitumor cytotoxicity.14. The method of any one of embodiments 8 to 13, which is a method for stimulating an immune response against a tumor.15. The combination of any one of embodiments 1 to 7 or the method of any one of embodiments 8 to 14, wherein the tumor-targeted IL2 receptor agonist comprises, in N- to C- terminal orientation, the first TAA targeting moiety (or a component thereof, e.g., a VH-CH1 or VL-CL), the first connector moiety and the IL2 moiety.16. The combination or method of embodiment 15, wherein the IL2 moiety comprises an amino acid sequence having:(a) at least 95% sequence identity to the amino acid sequence of SEQ ID NO:1 ,(b) an N-terminal alanine deletion as compared to the amino acid sequence of SEQ ID NO:1 ;(c) an amino acid substitution at position N88 as compared to the amino acid sequence of SEQ ID NO:2, optionally wherein the amino acid substitution is N88D;(d) the amino acid substitution C125S, C125A or C125V as compared to the amino acid sequence of SEQ ID NO:2; or(e) any combination of (a), (b), (c) and / or (d).17. The combination or method of embodiment 15, wherein the IL2 moiety comprises an amino acid sequence having at least 96% sequence identity to the amino acid sequence of SEQ ID NO:1.18. The combination or method of embodiment 15, wherein the IL2 moiety comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:1.19. The combination or method of embodiment 15, wherein the IL2 moiety comprises an amino acid sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID NO:1.20. The combination or method of embodiment 15, wherein the IL2 moiety comprises an amino acid sequence having at least 99% sequence identity to the amino acid sequence of SEQ ID NO:1.21 . The combination or method of embodiment 15, wherein the IL2 moiety comprises an amino acid sequence having at least 99.5% sequence identity to the amino acid sequence of SEQ ID NO:1.22. The combination or method of embodiment 15, wherein the IL2 moiety comprises an amino acid sequence having 100% sequence identity to the amino acid sequence of SEQ ID NO:1.23. The combination or method of any one of embodiments 15 to 22, wherein the IL2 moiety is masked by a masking moiety.24. The combination or method of embodiment 23, wherein the masking moiety is an IL2 receptor (IL2R) moiety.25. The combination or method of embodiment 24, wherein the IL2R moiety is an IL2R alpha (IL2Ra) moiety.26. The combination or method of embodiment 25, wherein the IL2Ra moiety comprises or consists of an amino acid sequence having at least 95% sequence identity to an I L2- binding portion (or all) of SEQ ID NO:3.27. The combination or method of embodiment 25, wherein the IL2Ra moiety comprises or consists of an amino acid sequence having at least 96% sequence identity to an I L2- binding portion (or all) of SEQ ID NO:3.28. The combination or method of embodiment 25, wherein the IL2Ra moiety comprises or consists of an amino acid sequence having at least 97% sequence identity to an I L2- binding portion (or all) of SEQ ID NO:3.29. The combination or method of embodiment 25, wherein the IL2Ra moiety comprises or consists of an amino acid sequence having at least 98% sequence identity to an I L2- binding portion (or all) of SEQ ID NO:3.30. The combination or method of embodiment 25, wherein the IL2Ra moiety comprises or consists of an amino acid sequence having at least 99% sequence identity to an I L2- binding portion (or all) of SEQ ID NO:3.31 . The combination or method of embodiment 25, wherein the IL2Ra moiety comprises or consists of an amino acid sequence having at least 99.5% sequence identity to an I L2- binding portion (or all) of SEQ ID NO:3.32. The combination or method of embodiment 25, wherein the IL2Ra moiety comprises or consists of an amino acid sequence having 100% sequence identity to an I L2-binding portion (or all) of SEQ ID NO:3.33. The combination or method of embodiment 25, wherein the IL2Ra moiety comprises or consists of an amino acid sequence having at least 95% sequence identity to SEQ ID NO:19.34. The combination or method of embodiment 25, wherein the IL2Ra moiety comprises or consists of an amino acid sequence having at least 96% sequence identity to SEQ ID NO:19.35. The combination or method of embodiment 25, wherein the IL2Ra moiety comprises or consists of an amino acid sequence having at least 97% sequence identity to SEQ ID NO:19.36. The combination or method of embodiment 25, wherein the IL2Ra moiety comprises or consists of an amino acid sequence having at least 98% sequence identity to SEQ ID NO:19.37. The combination or method of embodiment 25, wherein the IL2Ra moiety comprises or consists of an amino acid sequence having at least 99% sequence identity to SEQ ID NO:19.38. The combination or method of embodiment 25, wherein the IL2Ra moiety comprises or consists of an amino acid sequence having at least 99.5% sequence identity to SEQ ID NO:19.39. The combination or method of embodiment 25, wherein the IL2Ra moiety comprises or consists of an amino acid sequence having 100% sequence identity to SEQ ID NO:19.40. The combination or method of embodiment 25, wherein the IL2Ra moiety comprises or consists of an amino acid sequence having at least 95% sequence identity to SEQ ID NO:20.41 . The combination or method of embodiment 25, wherein the IL2Ra moiety comprises or consists of an amino acid sequence having at least 96% sequence identity to SEQ ID NO:20.42. The combination or method of embodiment 25, wherein the IL2Ra moiety comprises or consists of an amino acid sequence having at least 97% sequence identity to SEQ ID NO:20.43. The combination or method of embodiment 25, wherein the IL2Ra moiety comprises or consists of an amino acid sequence having at least 98% sequence identity to SEQ ID NQ:20.44. The combination or method of embodiment 25, wherein the IL2Ra moiety comprises or consists of an amino acid sequence having at least 99% sequence identity to SEQ ID NQ:20.45. The combination or method of embodiment 25, wherein the IL2Ra moiety comprises or consists of an amino acid sequence having at least 99.5% sequence identity to SEQ ID NQ:20.46. The combination or method of embodiment 25, wherein the IL2Ra moiety comprises or consists of an amino acid sequence having 100% sequence identity to SEQ ID NQ:20.47. The combination or method of any one of embodiments 25 to 46, wherein the IL2Ra moiety is N-terminal to the IL2 moiety.48. The combination or method of embodiment 47, wherein the IL2 moiety and the IL2Ra moiety are separated by a linker (“IL2 moiety linker”).49. The combination or method of embodiment 48, wherein the IL2 moiety linker is at least 5 amino acids in length.50. The combination or method of embodiment 48, wherein the IL2 moiety linker is at least 10 amino acids in length.51 . The combination or method of embodiment 48, wherein the IL2 moiety linker is at least 15 amino acids in length.52. The combination or method of any one of embodiments 48 to 51 , wherein the IL2 moiety linker is or comprises a glycine-serine linker.53. The combination or method of embodiment 52, wherein the IL2 moiety linker comprises the amino acid sequence G4S (SEQ ID NO:70).54. The combination or method of embodiment 52, wherein the IL2 moiety linker is or comprises a multimer of the amino acid sequence G4S (SEQ ID NO:70).55. The combination or method of embodiment 54, wherein the multimer comprises 2, 3, 4, 5, 6 or more repeats of the amino acid sequence G4S (SEQ ID NQ:70).56. The combination or method of any one of embodiments 48 to 55, wherein the IL2 moiety linker is a non-cleavable linker.57. The combination any one of embodiments 1 to 7 and 15 to 56 or the method of any one of embodiments 8 to 56, wherein the IL2 moiety and the first connector moiety are separated by a linker (“connector moiety linker”).58. The combination or method of embodiment 57, wherein the connector moiety linker is at least 5 amino acids in length.59. The combination or method of embodiment 57, wherein the connector moiety linker is at least 10 amino acids in length.60. The combination or method of embodiment 57, wherein the connector moiety linker is at least 15 amino acids in length.61 . The combination or method of any one of embodiments 57 to 60, wherein the connector moiety linker is or comprises a glycine-serine linker.62. The combination or method of embodiment 61 , wherein the connector moiety linker comprises the amino acid sequence G4S (SEQ ID NQ:70).63. The combination or method of embodiment 61 , wherein the connector moiety linker is or comprises a multimer of the amino acid sequence G4S (SEQ ID NQ:70).64. The combination or method of embodiment 63, wherein the multimer comprises 2, 3, 4, 5, 6 or more repeats of the amino acid sequence G4S (SEQ ID NQ:70).65. The combination or method of any one of embodiments 57 to 65, wherein the connector moiety linker is a non-cleavable linker.66. The combination of any one of embodiments 1 to 7 and 15 to 65 or the method of any one of embodiments 8 to 65, wherein first connector moiety is an Fc domain.67. The combination or method of embodiment 66, wherein the Fc domain is an IgG 1 , lgG2, lgG3 or lgG4 Fc domain.68. The combination or method of embodiment 66 or embodiment 67, wherein the Fc domain comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:4.69. The combination or method of embodiment 66 or embodiment 67, wherein the Fc domain comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:5.70. The combination or method of embodiment 66 or embodiment 67, wherein the Fc domain comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:6.71 . The combination or method of any one of embodiments 66 to 70, wherein the Fc domain comprises a chimeric hinge domain.72. The combination or method of any one of embodiments 66 to 71 , wherein the Fc domain has reduced effector function.73. The combination or method of any one of embodiments 66 to 72, wherein the tumor- targeted IL2 receptor agonist comprises an Fc dimer.74. The combination or method of embodiment 73, wherein the Fc dimer is an Fc homodimer.75. The combination or method of embodiment 74, wherein the tumor-targeted IL2 receptor agonist is bivalent for the TAA targeting moiety, the first connector moiety, the IL2 moiety and, if present, the IL2Ra moiety.76. The combination or method of embodiment 73, wherein the Fc dimer is an Fc heterodimer.77. The combination of any one of embodiments 1 to 7 and 15 to 76 or the method of any one of embodiments 8 to 76, wherein the T-cell receptor complex targeting moiety is a CD3-targeting moiety.78. The combination of any one of embodiments 1 to 7 and 15 to 77 or the method of any one of embodiments 8 to 77, wherein the T-cell receptor complex targeting moiety is a Fab.79. The combination of any one of embodiments 1 to 7 and 15 to 77 or the method of any one of embodiments 8 to 77, wherein the T-cell receptor complex targeting moiety is an scFv.80. The combination of any one of embodiments 1 to 7 and 15 to 79 or the method of any one of embodiments 8 to 79, wherein the multispecific T-cell engager is a bispecific T- cell engager.81 . The combination or method of embodiment 80, wherein the bispecific T-cell engager comprises:(a) a first polypeptide chain comprising, in N- to C-terminal orientation, the second TAA targeting moiety (or a component thereof, e.g., a VH-CH1 or VL- CL) and the second connector moiety; and(b) a second polypeptide chain comprising, in N- to C-terminal orientation, the T- cell receptor complex targeting moiety (or a component thereof, e.g., a VH- CH1 or L-CL) and the third connector moiety.82. The combination of any one of embodiments 1 to 7 and 15 to 81 or the method of any one of embodiments 8 to 81 , wherein the second connector moiety and the third connector moiety are Fc domains.83. The combination or method of embodiment 82, wherein the Fc domains are IgG 1 , lgG2, lgG3 or lgG4 Fc domains.84. The combination or method of embodiment 82 or embodiment 83, wherein the Fc domains comprise an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:4.85. The combination or method of embodiment 82 or embodiment 83, wherein the Fc domains comprise an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:5.86. The combination or method of embodiment 82 or embodiment 83, wherein the Fc domains comprise an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:6.87. The combination or method of any one of embodiments 82 to 86, wherein the Fc domains comprise a chimeric hinge domain.88. The combination or method of any one of embodiments 82 to 87, wherein the Fc domains have reduced effector function.89. The combination or method of any one of embodiments 82 to 88, wherein the Fc domains form an Fc heterodimer.90. The combination of any one of embodiments 1 to 7 and 15 to 89 or the method of any one of embodiments 8 to 89, wherein the first TAA targeting moiety and / or second TAA targeting moiety bind(s) to Fibroblast Activation Protein (FAP), the A1 domain of Tenascin-C (TNG A1), the A2 domain of Tenascin-C (TNC A2), the Extra Domain B of Fibronectin (EDB), the Melanoma-associated Chondroitin Sulfate Proteoglycan (MCSP), MART- 1 / Melan-A, gp100, Dipeptidyl peptidase IV (DPPIV), adenosine deaminase-binding protein (ADAbp), cyclophilin b, colorectal associated antigen (CRC)-C017-1A / GA733, Carcinoembryonic Antigen (CEA) and its immunogenic epitopes CAP-1 and CAP-2, etv6, aml1 , Prostate Specific Antigen (PSA) or an immunogenic epitopes thereoPSA-1 , PSA-2, and PSA-3, prostate-specific membrane antigen (PSMA), T-cell receptor / CD3-zeta chain, MAGE-family of tumor antigens (e.g., MAGE-A1 , MAGE-A2, MAGE-A3, MAGE-A4, MAGE- A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11 , MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-C1 ,MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5), GAGE-family of tumor antigens (e.g., GAGE- 1 , GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9), BAGE, RAGE, LAGE-1 , NAG, GnT-V, MUM-1 , CDK4, tyrosinase, p53, MUC family, HER2 / neu, p21ras, RCAS1 , a-fetoprotein, E-cadherin, a-catenin, b-catenin and g-catenin, p120ctn, gp1OO Pmel117, PRAME, NY-ESO-1 , cdc27, adenomatous polyposis coli protein (APC), fodrin, Connexin 37, Ig-idiotype, p15, gp75, GM2 and GD2 gangliosides, viral products such as human papilloma virus proteins, Smad family of tumor antigens, Imp-1 , P1A, EBV- encoded nuclear antigen (EBNA)-1 , brain glycogen phosphorylase, SSX-1 , SSX-2 (HOM- MEL-40), SSX-1 , SSX- 4, SSX-5, SCP-1 and CT-7, c-erbB-2, Her2, EGFR, IGF-1 R, CD2 (T- cell surface antigen), CD3 (heteromultimer associated with the TCR), CD22 (B-cell receptor), CD23 (low binding affinity IgE receptor), CD30 (cytokine receptor), CD33 (myeloid cell surface antigen), CD40 (tumor necrosis factor receptor), IL-6R-(IL6 receptor), CD20, MCSP, RO RbR (b-platelet-derived growth factor receptor), ErbB2 epithelial cell adhesion molecule (EpCAM), EGFR variant III (EGFRvlll), CD19, disialoganglioside GD2, ductal-epithelial mucine, gp36, TAG-72, glioma- associated antigen, b-human chorionic gonadotropin, alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulin, MN-CA IX, human telomerase reverse transcriptase, RU1 , RU2 (AS), intestinal carboxyl esterase, mut hsp70-2, M-CSF, prostase, prostase specific antigen (PSA), PAP, LAGA-1a, p53, prostein, PSMA, surviving and telomerase, prostate-carcinoma tumor antigen-1 (PCTA-1), ELF2M, neutrophil elastase, ephrin B2, insulin growth factor (IGF1)-I, IGF- II, IGFI receptor, 5T4, ROR1 , Nkp30, NKG2D, tumor stromal antigens, the extra domain A (EDA) or extra domain B (EDB) of fibronectin, or the A1 domain of tenascin-C(TnC A1).91 . The combination of any one of embodiments 1 to 7 and 15 to 89 or the method of any one of embodiments 8 to 89, wherein the first TAA targeting moiety and / or second TAA targeting moiety bind(s) to MUC16 (e.g., human MUC16).92. The combination of any one of embodiments 1 to 7 and 15 to 91 or the method of any one of embodiments 8 to 91 , wherein the first TAA targeting moiety is a Fab.93. The combination of any one of embodiments 1 to 7 and 15 to 91 or the method of any one of embodiments 8 to 91 , wherein the first TAA targeting moiety is an scFv.94. The combination of any one of embodiments 1 to 7 and 15 to 93 or the method of any one of embodiments 8 to 93, wherein the second TAA targeting moiety is a Fab.95. The combination of any one of embodiments 1 to 7 and 15 to 93 or the method of any one of embodiments 8 to 93, wherein the second TAA targeting moiety is an scFv.96. The combination of any one of embodiments 1 to 7 and 15 to 95 or the method of any one of embodiments 8 to 95, wherein the first TAA targeting moiety and the second TAA targeting moiety bind to the same tumor associated antigen.97. The combination or method of embodiment 96, wherein the first TAA targeting moiety and the second TAA targeting moiety bind to different epitopes of the tumor associated antigen.98. The combination or method of embodiment 96, wherein the first TAA targeting moiety and the second TAA targeting moiety bind to the same epitope of the tumor associated antigen.99. The combination or method of embodiment 96 or embodiment 98, wherein the first TAA targeting moiety and the second TAA targeting moiety are identical.100. The combination of any one of embodiments 1 to 7 and 15 to 95 or the method of any one of embodiments 8 to 95, wherein the first TAA targeting moiety and the second TAA targeting moiety bind to different tumor associated antigens expressed on the same tumor cell.101. The combination of any one of embodiments 1 to 7 and 15 to 100 or the method of any one of embodiments 8 to 100, wherein tumor-targeted IL2 receptor agonist is configured as illustrated in FIG. 2B.102. The combination of any one of embodiments 1 to 7 and 15 to 101 or the method of any one of embodiments 8 to 101 , wherein multispecific T-cell engager is configured as illustrated in FIG. 3B.103. The combination of any one of embodiments 1 to 7 and 15 to 101 or the method of any one of embodiments 8 to 101 , wherein multispecific T-cell engager is configured as illustrated in FIG. 3C.104. The combination of any one of embodiments 1 to 7 and 15 to 101 or the method of any one of embodiments 8 to 101 , wherein multispecific T-cell engager is configured as illustrated in FIG. 3D.105. A method comprising administering to a subject:(a) a tumor-targeted interleukin-2 (“ IL2”) receptor agonist comprising:(i) a first TAA targeting moiety;(ii) an IL2 moiety; and(iii) optionally, a first connector moiety separating the first TAA targeting moiety and the IL2 moiety; and(b) a multispecific T-cell engager comprising:(i) a second TAA targeting moiety;(i) a T-cell receptor complex targeting moiety; and(ii) optionally, a second connector moiety and / or a third connector moiety separating the second TAA targeting moiety and the T-cell receptor complex targeting moiety.106. The method of claim 105, wherein the tumor-targeted IL2 receptor agonist comprises, in N- to C-terminal orientation, the first TAA targeting moiety (or a component thereof, e.g., a VH-CH1 or VL-CL), the first connector moiety and the IL2 moiety.107. The method of claim 106, wherein the IL2 moiety comprises an amino acid sequence having:(a) at least 95% sequence identity to the amino acid sequence of SEQ ID NO:1 ,(b) an N-terminal alanine deletion as compared to the amino acid sequence of SEQ ID NO:1 ;(c) an amino acid substitution at position N88 as compared to the amino acid sequence of SEQ ID NO:2, optionally wherein the amino acid substitution is N88D;(d) the amino acid substitution C125S, C125A or C125V as compared to the amino acid sequence of SEQ ID NO:2; or(e) any combination of (a), (b), (c) and / or (d).108. The method of claim 106, wherein the IL2 moiety comprises an amino acid sequence having at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to the amino acid sequence of SEQ ID NO:1 .109. The method of claim 106, wherein the IL2 moiety comprises an amino acid sequence having 100% sequence identity to the amino acid sequence of SEQ ID NO:1.110. The method of any one of claims 106 to 109 wherein the IL2 moiety is masked by a masking moiety.111. The method of claim 110, wherein the masking moiety is an IL2 receptor (IL2R) moiety.112. The method of claim 111 , wherein the IL2R moiety is an IL2R alpha (IL2Ra) moiety.113. The method of claim 112, wherein the IL2Ra moiety comprises or consists of an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 99%, or at least 99.5% sequence identity to an I L2-binding portion (or all) of SEQ ID NO:3.114. The method of claim 112, wherein the IL2Ra moiety comprises or consists of an amino acid sequence having 100% sequence identity to an IL2-binding portion (or all) of SEQ ID NO:3.115. The method of claim 112 or 113, wherein the IL2Ra moiety is N-terminal to the IL2 moiety.116. The method of claim 115, wherein the IL2 moiety and the IL2Ra moiety are separated by a linker (“IL2 moiety linker”).117. The method of claim 116, wherein the IL2 moiety linker is at least 5, at least 10, or at least 15 amino acids in length.118. The method of claim 116 or 117, wherein the IL2 moiety linker is or comprises a glycine-serine linker.119. The method of any one of claims 116 to 118, wherein the IL2 moiety linker is a non- cleavable linker.120. The method of any one of claims 105 to 119, wherein the IL2 moiety and the first connector moiety are separated by a linker (“connector moiety linker”).121. The method of claim 120, wherein the connector moiety linker is at least 5, at least 10, or at least 15 amino acids in length.122. The method of claim 120 or 121 , wherein the connector moiety linker is or comprises a glycine-serine linker.123. The method of any one of claims 120 to 122, wherein the connector moiety linker is a non-cleavable linker.124. The method of any one of claims 105 to 123, wherein first connector moiety is an Fc domain.125. The method of claim 124, wherein the Fc domain is an IgG 1 , lgG2, lgG3 or lgG4 Fc domain.126. The method of claim 124 or claim 125, wherein the Fc domain comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6.127. The method of any one of claims 124 to 126, wherein the Fc domain comprises a chimeric hinge domain.128. The method of any one of claims 124 to 127, wherein the Fc domain has reduced effector function.129. The method of any one of claims 124 to 128, wherein the tumor-targeted IL2 receptor agonist comprises an Fc dimer.130. The method of claim 129, wherein the Fc dimer is an Fc homodimer.131. The method of claim 130, wherein the tumor-targeted IL2 receptor agonist is bivalent for the TAA targeting moiety, the first connector moiety, the IL2 moiety and, if present, the IL2Ra moiety.132. The method of claim 129, wherein the Fc dimer is an Fc heterodimer.133. The method of any one of claims 105 to 132, wherein the T-cell receptor complex targeting moiety is a CD3-targeting moiety.134. The method of any one of claims 105 to 133, wherein the T-cell receptor complex targeting moiety is a Fab.135. The method of any one of claims 105 to 133, wherein the T-cell receptor complex targeting moiety is an scFv.136. The method of any one of claims 105 to 135, wherein the multispecific T-cell engager is a bispecific T-cell engager.137. The method of any one of claims 105 to 136, wherein the second connector moiety and the third connector moiety are Fc domains.138. The method of claim 137, wherein the Fc domains are IgG 1 , lgG2, lgG3 or lgG4 Fc domains.139. The method of claim 137 or claim 138, wherein the Fc domains comprise an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6.140. The method of any one of claims 137 to 139, wherein the Fc domains comprise a chimeric hinge domain.141. The method of any one of claims 137 to 140, wherein the Fc domains have reduced effector function.142. The method of any one of claims 137 to 141 , wherein the Fc domains form an Fc heterodimer.143. The method of any one of claims 105 to 142, wherein the first TAA targeting moiety and / or second TAA targeting moiety bind(s) to Fibroblast Activation Protein (FAP), the A1 domain of Tenascin-C (TNG A1), the A2 domain of Tenascin-C (TNC A2), the Extra Domain B of Fibronectin (EDB), the Melanoma-associated Chondroitin Sulfate Proteoglycan (MCSP), MART-1 / Melan-A, gp100, Dipeptidyl peptidase IV (DPPIV), adenosine deaminase-binding protein (ADAbp), cyclophilin b, colorectal associated antigen (CRC)-C017-1A / GA733,Carcinoembryonic Antigen (CEA) and its immunogenic epitopes CAP-1 and CAP-2, etv6, aml1 , Prostate Specific Antigen (PSA) or an immunogenic epitopes thereoPSA-1 , PSA-2, and PSA-3, prostate-specific membrane antigen (PSMA), T-cell receptor / CD3-zeta chain, MAGE-family of tumor antigens (e.g., MAGE-A1 , MAGE-A2, MAGE-A3, MAGE-A4, MAGE- A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11 , MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-C1 , MAGE-02, MAGE-C3, MAGE-C4, MAGE-C5), GAGE-family of tumor antigens (e.g., GAGE- 1 , GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9), BAGE, RAGE, LAGE-1 , NAG, GnT-V, MUM-1 , CDK4, tyrosinase, p53, MUC family, HER2 / neu, p21ras, RCAS1 , a-fetoprotein, E-cadherin, a-catenin, b-catenin and g-catenin, p120ctn, gp1OO Pmel117, PRAME, NY-ESO-1 , cdc27, adenomatous polyposis coli protein (APC), fodrin, Connexin 37, Ig-idiotype, p15, gp75, GM2 and GD2 gangliosides, viral products such as human papilloma virus proteins, Smad family of tumor antigens, Imp-1 , P1A, EBV- encoded nuclear antigen (EBNA)-1 , brain glycogen phosphorylase, SSX-1 , SSX-2 (HOM- MEL-40), SSX-1 , SSX- 4, SSX-5, SCP-1 and CT-7, c-erbB-2, Her2, EGFR, IGF-1 R, CD2 (T- cell surface antigen), CD3 (heteromultimer associated with the TCR), CD22 (B-cell receptor), CD23 (low binding affinity IgE receptor), CD30 (cytokine receptor), CD33 (myeloid cell surface antigen), CD40 (tumor necrosis factor receptor), IL-6R-(IL6 receptor), CD20, MCSP, ROuRbR (b-platelet-derived growth factor receptor), ErbB2 epithelial cell adhesion molecule (EpCAM), EGFR variant III (EGFRvlll), CD19, disialoganglioside GD2, ductal-epithelial mucine, gp36, TAG-72, glioma- associated antigen, b-human chorionic gonadotropin, alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulin, MN-CA IX, human telomerase reverse transcriptase, RU1 , RU2 (AS), intestinal carboxyl esterase, mut hsp70-2, M-CSF, prostase, prostase specific antigen (PSA), PAP, LAGA-1a, p53, prostein, PSMA, surviving and telomerase, prostate-carcinoma tumor antigen-1 (PCTA-1), ELF2M, neutrophil elastase, ephrin B2, insulin growth factor (IGF1)-I, IGF- II, IGFI receptor, 5T4, ROR1 , Nkp30, NKG2D, tumor stromal antigens, the extra domain A (EDA) or extra domain B (EDB) of fibronectin, or the A1 domain of tenascin-C(TnC A1).144. The method of any one of claims 105 to 142, wherein the first TAA targeting moiety and / or second TAA targeting moiety bind(s) to MUC16 (e.g., human MUC16).145. The method of any one of claims 105 to 144, wherein the first TAA targeting moiety is a Fab.146. The method of any one of claims 105 to 144, wherein the first TAA targeting moiety is an scFv.147. The method of any one of claims 105 to 146, wherein the second TAA targeting moiety is a Fab.148. The method of any one of claims 105 to 146, wherein the second TAA targeting moiety is an scFv.149. The method of any one of claims 105 to 148, wherein the first TAA targeting moiety and the second TAA targeting moiety bind to the same tumor associated antigen.150. The method of claim 149, wherein the first TAA targeting moiety and the second TAA targeting moiety bind to different epitopes of the tumor associated antigen.151. The method of claim 149, wherein the first TAA targeting moiety and the second TAA targeting moiety bind to the same epitope of the tumor associated antigen.152. The method of claim 149 or claim 151 , wherein the first TAA targeting moiety and the second TAA targeting moiety are identical.153. The method of any one of claims 105 to 149, wherein the first TAA targeting moiety and the second TAA targeting moiety bind to different tumor associated antigens expressed on the same tumor cell.8. EXAMPLES8.1. Materials and Methods8.1.1. Design and Production of Tumor-Targeted IL2 Constructs

[0314] An exemplary tumor-targeted IL2 receptor agonist construct as depicted in FIG. 2B was designed to comprise two polypeptides, each polypeptide comprising from N- to C- terminal, a TAA targeting moiety in Fab format, a first linker, a connector moiety in Fc format which enables dimerization, a second linker, and IL2Ra masking moiety, a third linker, and an IL2 moiety.

[0315] The constructs were expressed in suitable cells by transient transfection. Proteins in supernatant were purified using the ProteinMaker system (Protein BioSolutions, Gaithersburg, MD) with either HiTrap™ Protein or MabSelect SuRe columns (GE Healthcare). After single step elution, the constructs were neutralized, dialyzed into a finalbuffer of phosphate buffered saline (PBS) with 5% glycerol, aliquoted and stored at -80 °C until use.8.1.2. Design and Production of Multispecific T-cell Engager Constructs

[0316] An exemplary multispecific T-cell engager construct as depicted in FIG. 3A was designed to comprise two polypeptides, wherein the first polypeptide was designed to comprise from N- to C-terminal, a TAA targeting moiety in Fab format, a linker, a connector moiety in Fc format, and the second polypeptide was designed to comprise from N- to C- terminal, a T-cell receptor complex targeting moiety in Fab format, a linker, and a connector moiety in Fc format which is able to form a dimer with the connector moiety of the first polypeptide.

[0317] The constructs were expressed in suitable cells by transient or stable transfection. Proteins in supernatant were purified using the ProteinMaker system (Protein BioSolutions, Gaithersburg, MD) with either HiTrap™ Protein or MabSelect SuRe columns (GE Healthcare). After single step elution, the constructs were neutralized, dialyzed into a final buffer of phosphate buffered saline (PBS) with 5% glycerol, aliquoted and stored at -80 °C until use.8.1.3. In vitro Target Cell Killing Assay

[0318] CD8+ T-cells were isolated from human donor PBMC. Activated CD8+ T-cells were generated by stimulating CD8+ T-cells with CD3 / CD28 DynabeadsTM (Thermo / 11132D) for 3 days. Altered function CD8+ T-cells were generated by incubating CD8+ T-cells with CD3 beads for 9 days. Target cells for this assay were engineered to express MUC16 and a luminescent tag containing a caspase cleavable domain, such that when caspases are active luminescence is lost. Thus, as target cells die the RLU signal is reduced. Target cells were incubated with a serial dilution of tumor-targeted IL2 receptor agonist or a control construct. In some assessments, the unbound constructs were washed off. Next, target cells and activated T cells were incubated together with a constant dose of MUC16xCD3. Assay was readout after 3 days. For the luminescent readout emitted light was measured in RLU on a multilabel plate reader Envision (PerkinElmer). EC50 values were determined from a 4- parameter logistic equation over dose response curve using GraphPad Prism software.8.1.4. In Vivo Tumor Growth Inhibition and Rechallenge Assay

[0319] ID8-VEGF / hMUC16 tumor cells were implanted subcutaneously (s.c.) into the right flank of female mice genetically engineered to express human CD3, MUC16 and MSLN (huCD3 / huMUC16 / huMSLN mice). Once tumors were established, mice were randomized into treatment groups and injected intraperitoneally (i.p.) or intravenously (i.v.) twice perweek for a total of three injections with tumor-targeted IL2 receptor agonist and / or multispecific T-cell engager constructs or controls at doses indicated in each example.Tumor volume was expressed in mm3using the formula: V = 0.5 x a x b2where a and b were the long and short diameters of the tumor, respectively. Tumor sizes and body weight were monitored twice weekly. All procedures were conducted according to the guidelines of the Regeneron Institutional Animal Care and Use Committee. All data were analyzed using GraphPad Prism and tumor sizes were graphed as mean + SEM.

[0320] For the tumor rechallenge experiment, huCD3 / huMUC16 mice which previously cleared ID8-VEGF / huMUC16 tumors after indicated treatments, along with naive control mice, were subcutaneously implanted on the opposite flank with parental ID8-VEGF tumor cells 113 days after initial tumor implantation. Tumor growth was monitored over time.8.2. Example 1 : Target Cell Killing by the Combination of MUC16-Targeted IL2 and MUC16 x CD3 Bispecific T-Cell Engager

[0321] MUC16-targeted IL2 was designed and produced as described in Section 8.1.1.MUC16 x CD3 bispecific T-cell engager was designed and produced as described in Section8.1.2. The ability of a combination of tumor-targeted IL2 receptor agonist and a bispecific T- cell engager was assessed with an in vitro target cell killing assay as described in Section 8.1.3 using MUC16-targeted IL2 as the tumor-targeted IL2 receptor agonist and the anti- MUC16 and anti-CD3 (MUC16 x CD3) bispecific antibody as the bispecific T-cell engager.

[0322] In the assessments where the unbound MUC16-targeted IL and control constructs were washed out from the target cells before co-culturing with activated T-cells and the addition of the constant concentration of the bispecific T-cell engager MUC16 x CD3, MUC16-targeted IL2 (MUC16-IL2) was associated with a dose-dependent killing of the target cells, whereas the non-targeted IL2 (NT-IL2) displayed nonspecific target cell killing only at the highest doses evaluated (FIG. 5A).

[0323] In the assessments where the unbound MUC16-IL2 and control constructs were not washed out from the target cells, MUC16-targeted IL2 lead to biphasic curve, potentially indicating interference of MUC-IL2 with MUC16 x CD3 at higher concentrations (FIG. 5B).8.3. Example 2: Anti-Tumor Activity of MUC16-Targeted IL2 in Combination with MUC16 x CD3 Bispecific T-Cell Engager

[0324] The activity of the combination of MUC16-targeted IL2 and MUC16 x CD3 was evaluated in an ID8-VEGF tumor model. Mice were randomized into groups after the tumors were established and dosed intraperitoneally with control or test constructs as described in Section 8.1.4 at the doses set forth in Table E1 below.NT= Non-targeted

[0325] The administration of 2.5 mpk MUC16-IL2 together with the bispecific T-cell engager MUC16 x CD3 was associated with the highest level of anti-tumor activity relative to the other treatments (FIG. 6A). None of the mice in control groups 1-4 and the mice that were treated with MUC16 x CD3 and 0.5 mpk of MUC16-IL2 (table E1 , group 6) were tumor-free (FIGS. 6B-6D and 6F). One-third (2 out of 6) of mice treated with a combination of MUC16 x CD3 and 2.5 mpk of NT-IL2 (table E1 , group 5) were tumor-free (FIG. 6E), whereas two- thirds (4 out of 6) of mice treated with a combination of MUC16 x CD3 and 2.5 mpk of MUC16-IL2 (table E1 , group 7) were tumor-free at the time of final assessments (FIG. 6G). No concerning changes to body weight were observed with any of the treatments (FIG. 6H). These results suggest that combinations of tumor-targeted IL2 receptor agonist and bispecific T-cell engagers are more effective against tumors than bispecific T-cell engagers alone.8.4. Example 3: Anti-Tumor Activity of MSLN-Targeted IL2 in Combination with MUC16 x CD3 Bispecific T-Cell Engager

[0326] In Example 2, both the tumor-targeted IL2 receptor agonist and the bispecific T-cell engager were targeting the same tumor associated antigen, MUC16. In this Example, the anti-tumor activity of the combination of a tumor-targeting IL2 and a bispecific T-cell engager with different TAA targeting moieties were assessed. More specifically, the combination of MSLN-targeted IL2 and MUC16 x CD3 was evaluated in an ID8-VEGF tumor model. Micewere randomized into groups after the tumors were established and dosed intraperitoneally with control or test constructs as described in Section 8.1.4 at the doses set forth in T able E2 below.NT= Non-targeted

[0327] The administration of 2.5 mpk MSLN-IL2 together with the bispecific T-cell engager MUC16 x CD3 was associated with the highest level of anti-tumor activity relative to the other treatments (FIG. 7 A). No concerning changes to body weight were observed with any of the treatments (FIG. 7B). These results suggest that combinations of tumor-targeted IL2 receptor agonist and bispecific T-cell engagers that target different tumor associated antigens are effective against tumors.8.5. Example 4: Comparison of the Anti-Tumor Activity of MUC16-Targeted IL2 in Combination with MUC16 x CD3 Bispecific T-Cell Engagerand a Trispecific Construct

[0328] The anti-tumor activity of a combination of a tumor-targeted IL2 receptor agonist and a bispecific T-cell engager was compared to the anti-tumor activity of trispecific constructs that comprise all targeting moieties of the tumor-targeted IL2 receptor agonist and the bispecific T-cell engager in an ID8-VEGF tumor model. Mice were randomized into groups after the tumors were established and dosed intraperitoneally with control or test constructs as described in Section 8.1.4 at the doses set forth in Table E3 below.NT= Non-targeted

[0329] The groups that were administered the trispecific construct MUC16-IL2 x CD3-IL2 displayed better anti-tumor efficacy than the control groups. Nevertheless, the groups that were administered the combination of the MUC 16-IL2 and MUC16 x CD3 were associated with an even greater anti-tumor efficacy, in which the average tumor volume was reduced to zero at around Day 10 after dosing (FIG. 8A). No concerning changes to body weight were observed with any of the treatments (FIG. 8B). These results suggest that the combinations of tumor-targeted IL2 receptor agonist and bispecific T-cell engagers are potentially more effective against tumors than trispecific constructs comprising the same targeting moieties.8.6. Example 5: Induction of anti-tumor immune memory by MUC16-Targeted IL2 in Combination with MUC16 x CD3 Bispecific T-Cell Engagerand a Trispecific Construct

[0330] The protective immunity against tumor rechallenge by a tumor-targeted IL2 receptor agonist and a bispecific T-cell engager or by a trispecific construct that comprise all targeting moieties of the tumor-targeted IL2 receptor agonist and the bispecific T-cell engager was assessed with an ID8-VEGF tumor rechallenge model as described in Section 8.1.4 and illustrated in FIG. 9A. Mice were randomized into groups after the tumors were established following the initial tumor implantation and dosed intraperitoneally with control or test constructs as described in Section 8.1.4 at the doses set forth in Table E3 in Section 8.4.

[0331] Four out of seven mice treated with 1 .5 mpk MUC16-IL2 x CD3-IL2 and four out of seven mice treated with 2.5 mpk MUC16-IL2 x CD3-IL2 were tumor-free after the first round of implantation and one mouse out of four in each of these two groups were tumor-free after the tumor rechallenge (FIG. 9B). In contrast, all seven mice treated with the combination of MUC16 x CD3 and MUC16-IL2 were tumor-free after the first round of implantation and three of the seven mice again became tumor-free after the tumor rechallenge (FIG. 9B and 9D). These results revealed the ability of MUC16 x CD3 and MUC16-IL2 combination therapy to induce protective immunity against future tumors.8.7. Example 6: T-Cell Expansion and Cytokine Release by MUC16-Targeted IL2 in Combination with MUC16 x CD3 Bispecific T-Cell Engager

[0332] The effect of the combination of MUC16 x CD3 and MUC16-IL2 on expansion of T- cell populations and the systemic levels of cytokines were assessed in an ID8 tumor model. Briefly, Mice were randomized into groups after the tumors were established and dosed intraperitoneally for a total of three times with control or test constructs as described in Section 8.1.4 and illustrated in FIG. 10A at the doses set forth in Table E4 below. On day 8 after the start of dosing, blood was collected from each mouse and mice were further monitored for changes in tumor volume and body weight for 7 weeks post-dosing.NT = Non-targeted

[0333] The administration of 1 mpk MUC16-IL2 together with 2.5 mpk MUC16 x CD3 was associated with the highest level of anti-tumor activity relative to the other treatments (FIG. 10B) and there were no concerning changes to body weight in any of the treatment groups (FIG. 10C).

[0334] Only the number of T reg cells per volume was higher in groups of mice treated with MUC16-IL2 alone or MUC16-IL2 and MUC16 x CD3 (FIG. 10F). There were no significant differences in total white blood cell counts, numbers of T-cells per volume, percent CD8 T- cells that were PD1 -positive, and the number of PD1 -positive CD8 T-cells (FIGS. 10D-10F) Furthermore, minimal elevation in systemic levels of IFNy, TNFa, and IL10 were detected (FIGS. 10G-10I). These results suggest that the administration of MUC16-IL2 together withMUC16 x CD3 leads to low systemic immune activation while achieving robust anti-tumor efficacy.9. SEQUENCES

[0335] Certain sequences of the present disclosure are provided in Table S, below. Should any discrepancy or inconsistency exist between the sequences listed in Table S and those presented elsewhere in the specification, the sequences and associated sequence identifiers listed in Table S shall govern.10. CITATION OF REFERENCES

[0336] All publications, patents, patent applications and other documents cited in this application are hereby incorporated by reference in their entireties for all purposes to the same extent as if each individual publication, patent, patent application or other document were individually indicated to be incorporated by reference for all purposes. In the event that there is an inconsistency between the teachings of one or more of the references incorporated herein and the present disclosure, the teachings of the present specification are intended.

Claims

WHAT IS CLAIMED IS:

1. A combination comprising:(a) a tumor-targeted interleukin-2 (“IL2”) receptor agonist comprising:(i) a first TAA targeting moiety;(ii) an IL2 moiety; and(iii) optionally, a first connector moiety separating the first TAA targeting moiety and the IL2 moiety; and(b) a multispecific T-cell engager comprising:(i) a second TAA targeting moiety;(ii) a T-cell receptor complex targeting moiety; and(iii) optionally, a second connector moiety and / or a third connector moiety separating the second TAA targeting moiety and the T-cell receptor complex targeting moiety.

2. The combination of claim 1 , wherein the tumor-targeted IL2 receptor agonist comprises, in N- to C-terminal orientation, the first TAA targeting moiety (or a component thereof, e.g., a VH-CH1 or VL-CL), the first connector moiety and the IL2 moiety.

3. The combination of claim 2, wherein the IL2 moiety comprises an amino acid sequence having:(a) at least 95% sequence identity to the amino acid sequence of SEQ ID NO:1 ,(b) an N-terminal alanine deletion as compared to the amino acid sequence ofSEQ ID NO:1 ;(c) an amino acid substitution at position N88 as compared to the amino acid sequence of SEQ ID NO:2, optionally wherein the amino acid substitution is N88D;(d) the amino acid substitution C125S, C125A or C125V as compared to the amino acid sequence of SEQ ID NO:2; or(e) any combination of (a), (b), (c) and / or (d).

4. The combination of claim 2, wherein the IL2 moiety comprises an amino acid sequence having at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to the amino acid sequence of SEQ ID NO: 1.

5. The combination of claim 2, wherein the IL2 moiety comprises an amino acid sequence having 100% sequence identity to the amino acid sequence of SEQ ID NO:1 .

6. The combination of any one of claims 2 to 5 wherein the IL2 moiety is masked by a masking moiety.

7. The combination of claim 6, wherein the masking moiety is an IL2 receptor (IL2R) moiety.

8. The combination of claim 7, wherein the IL2R moiety is an IL2R alpha (IL2Ra) moiety.

9. The combination of claim 8, wherein the IL2Ra moiety comprises or consists of an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 99%, or at least 99.5% sequence identity to an I L2-binding portion (or all) of SEQ ID NO:3.

10. The combination of claim 8, wherein the IL2Ra moiety comprises or consists of an amino acid sequence having 100% sequence identity to an IL2-binding portion (or all) of SEQ ID NO:3.11 . The combination of claim 8 or 9, wherein the IL2Ra moiety is N-terminal to the IL2 moiety.

12. The combination of claim 11 , wherein the IL2 moiety and the IL2Ra moiety are separated by a linker (“IL2 moiety linker”).

13. The combination of claim 12, wherein the IL2 moiety linker is at least 5, at least 10, or at least 15 amino acids in length.

14. The combination of claim 12 or 13, wherein the IL2 moiety linker is or comprises a glycine-serine linker.

15. The combination of any one of claims 12 to 14, wherein the IL2 moiety linker is a non-cleavable linker.

16. The combination of any one of claims 1 to 15, wherein the IL2 moiety and the first connector moiety are separated by a linker (“connector moiety linker”).

17. The combination of claim 16, wherein the connector moiety linker is at least 5, at least 10, or at least 15 amino acids in length.

18. The combination of claim 16 or 17, wherein the connector moiety linker is or comprises a glycine-serine linker.

19. The combination of any one of claims 16 to 18, wherein the connector moiety linker is a non-cleavable linker.

20. The combination of any one of claims 1 to 19, wherein first connector moiety is an Fc domain.21 . The combination of claim 20, wherein the Fc domain is an IgG 1 , lgG2, lgG3 or lgG4 Fc domain.

22. The combination of claim 20 or claim 21 , wherein the Fc domain comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6.

23. The combination of any one of claims 20 to 22, wherein the Fc domain comprises a chimeric hinge domain.

24. The combination of any one of claims 20 to 23, wherein the Fc domain has reduced effector function.

25. The combination of any one of claims 20 to 24, wherein the tumor-targeted IL2 receptor agonist comprises an Fc dimer.

26. The combination of claim 25, wherein the Fc dimer is an Fc homodimer.

27. The combination of claim 26, wherein the tumor-targeted IL2 receptor agonist is bivalent for the TAA targeting moiety, the first connector moiety, the IL2 moiety and, if present, the IL2Ra moiety.

28. The combination of claim 25, wherein the Fc dimer is an Fc heterodimer.

29. The combination of any one of claims 1 to 28, wherein the T-cell receptor complex targeting moiety is a CD3-targeting moiety.

30. The combination of any one of claims 1 to 29, wherein the T-cell receptor complex targeting moiety is a Fab.31 . The combination of any one of claims 1 to 29, wherein the T-cell receptor complex targeting moiety is an scFv.

32. The combination of any one of claims 1 to 31 , wherein the multispecific T-cell engager is a bispecific T-cell engager.

33. The combination of any one of claims 1 to 32, wherein the second connector moiety and the third connector moiety are Fc domains.

34. The combination of claim 33, wherein the Fc domains are IgG 1 , lgG2, lgG3 or lgG4 Fc domains.

35. The combination of claim 33 or claim 34, wherein the Fc domains comprise an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6.

36. The combination of any one of claims 33 to 35, wherein the Fc domains comprise a chimeric hinge domain.

37. The combination of any one of claims 33 to 36, wherein the Fc domains have reduced effector function.

38. The combination of any one of claims 33 to 37, wherein the Fc domains form an Fc heterodimer.

39. The combination of any one of claims 1 to 38, wherein the first TAA targeting moiety and / or second TAA targeting moiety bind(s) to Fibroblast Activation Protein (FAP), the A1 domain of Tenascin-C (TNG A1), the A2 domain of Tenascin-C (TNC A2), the Extra Domain B of Fibronectin (EDB), the Melanoma-associated Chondroitin Sulfate Proteoglycan (MCSP), MART-1 / Melan-A, gp1OO, Dipeptidyl peptidase IV (DPPIV), adenosine deaminase-binding protein (ADAbp), cyclophilin b, colorectal associated antigen (CRC)-C017-1A / GA733, Carcinoembryonic Antigen (CEA) and its immunogenic epitopes CAP-1 and CAP-2, etv6, aml1 , Prostate Specific Antigen (PSA) or an immunogenic epitopes thereoPSA-1 , PSA-2, and PSA-3, prostate-specific membrane antigen (PSMA), T-cell receptor / CD3-zeta chain,MAGE-family of tumor antigens (e.g., MAGE-A1 , MAGE-A2, MAGE-A3, MAGE-A4, MAGE- A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11 , MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-C1 , MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5), GAGE-family of tumor antigens (e.g., GAGE- 1 , GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9), BAGE, RAGE, LAGE-1 , NAG, GnT-V, MUM-1 , CDK4, tyrosinase, p53, MUC family, HER2 / neu, p21ras, RCAS1 , a-fetoprotein, E-cadherin, a-catenin, b-catenin and g-catenin, p120ctn, gp1OO Pmel117, PRAME, NY-ESO-1 , cdc27, adenomatous polyposis coli protein (APC), fodrin, Connexin 37, Ig-idiotype, p15, gp75, GM2 and GD2 gangliosides, viral products such as human papilloma virus proteins, Smad family of tumor antigens, Imp-1 , P1A, EBV- encoded nuclear antigen (EBNA)-1 , brain glycogen phosphorylase, SSX-1 , SSX-2 (HOM- MEL-40), SSX-1 , SSX- 4, SSX-5, SCP-1 and CT-7, c-erbB-2, Her2, EGFR, IGF-1 R, CD2 (T- cell surface antigen), CD3 (heteromultimer associated with the TCR), CD22 (B-cell receptor), CD23 (low binding affinity IgE receptor), CD30 (cytokine receptor), CD33 (myeloid cell surface antigen), CD40 (tumor necrosis factor receptor), IL-6R-(IL6 receptor), CD20, MCSP, ROuRbR (b-platelet-derived growth factor receptor), ErbB2 epithelial cell adhesion molecule (EpCAM), EGFR variant III (EGFRvlll), CD19, disialoganglioside GD2, ductal-epithelial mucine, gp36, TAG-72, glioma- associated antigen, b-human chorionic gonadotropin, alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulin, MN-CA IX, human telomerase reverse transcriptase, RU1 , RU2 (AS), intestinal carboxyl esterase, mut hsp70-2, M-CSF, prostase, prostase specific antigen (PSA), PAP, LAGA-1a, p53, prostein, PSMA, surviving and telomerase, prostate-carcinoma tumor antigen-1 (PCTA-1), ELF2M, neutrophil elastase, ephrin B2, insulin growth factor (IGF1)-I, IGF- II, IGFI receptor, 5T4, ROR1 , Nkp30, NKG2D, tumor stromal antigens, the extra domain A (EDA) or extra domain B (EDB) of fibronectin, or the A1 domain of tenascin-C(TnC A1).

40. The combination of any one of claims 1 to 38, wherein the first TAA targeting moiety and / or second TAA targeting moiety bind(s) to MUC16 (e.g., human MUC16).41 . The combination of any one of claims 1 to 40, wherein the first TAA targeting moiety is a Fab.

42. The combination of any one of claims 1 to 40, wherein the first TAA targeting moiety is an scFv.

43. The combination of any one of claims 1 to 42, wherein the second TAA targeting moiety is a Fab.

44. The combination of any one of claims 1 to 42, wherein the second TAA targeting moiety is an scFv.

45. The combination of any one of claims 1 to 44, wherein the first TAA targeting moiety and the second TAA targeting moiety bind to the same tumor associated antigen.

46. The combination of claim 45, wherein the first TAA targeting moiety and the second TAA targeting moiety bind to different epitopes of the tumor associated antigen.

47. The combination of claim 45, wherein the first TAA targeting moiety and the second TAA targeting moiety bind to the same epitope of the tumor associated antigen.

48. The combination of claim 45 or claim 47, wherein the first TAA targeting moiety and the second TAA targeting moiety are identical.

49. The combination of any one of claims 1 to 45, wherein the first TAA targeting moiety and the second TAA targeting moiety bind to different tumor associated antigens expressed on the same tumor cell.

50. The combination of any one of claims 1 to 49, for use as a medicament.51 . The combination for use of claim 50, for use in a method of treatment of cancer.

52. A tumor-target IL2 receptor agonist for use in a method of for the treatment of cancer, wherein(a) the IL2 receptor agonist is defined as in any one of claims 1 to 49, and(b) the method comprises administering to a subject in need thereof the tumor- targeted IL2 receptor agonist and a multispecific T cell engager as defined in any one of claims 1 to 49.

53. A multispecific T cell engager for use in a method of for the treatment of cancer, wherein(a) the multispecific T cell engager is defined as in any one of claims 1 to 49, and(b) the method comprises administering to a subject in need thereof the multispecific T cell engager and a tumor-targeted IL2 receptor agonist as defined in any one of claims 1 to 49.

54. A method comprising administering to a subject a combination as defined in any one of claims 1 to 49.

55. The method of claim 54, wherein the method is a method of combination therapy for the treatment of cancer.

56. The combination for use of claim 50 or 51 , the tumor-targeted IL2 receptor agonist for use of claim 52, the multispecific T cell engager for use of claim 53, or the method of claims 54 or 55, wherein the method is a method of combination therapy(a) for the prevention or treatment of metastasis,(b) by stimulating the formation, stability and / or activity of a cytotoxic immune synapse,(c) by inducing tumor cytolysis,(d) by inducing anti-tumor cytotoxicity,(e) by stimulating an immune response against a tumor, or(f) a combination of any two or more of the foregoing uses.

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