Tumor-targeted split il12 receptor agonists

Abstract

The present disclosure relates to tumor-targeted split IL12 receptor agonists with improved therapeutic profiles.

Description

TUMOR-TARGETED SPLIT IL12 RECEPTOR AGONISTS1. CROSS-REFERNECE TO RELATED APPLICATIONS

[0001] This application claims the priority benefit of U.S. provisional application no. 63 / 651 ,855, filed May 24, 2024 and U.S. provisional application no. 63 / 730,129, filed December 10, 2024, the contents of each of which are incorporated herein in their entireties 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 May 20, 2025, is named RGN-052WO_SL.xml and is 208,693 bytes in size.3. BACKGROUND

[0003] Interleukin 12 (IL- 12 or I L12) is a pro-inflammatory cytokine having an important role in both innate and adaptive immunity. Hamza et al., 2010, Int. J. Mol. Sci., 11(3):789-806. I L12 functions primarily as a 70 kDa heterodimer consisting of disulfide-linked p35 and p40 subunits. Id. A variety of different immune cells, including B cells, dendritic cells, macrophages, monocytes, and neutrophils express IL12 when stimulated (Tugues et al., 2015, Cell Death Differ., 22:237-246), with the active heterodimer forming following protein synthesis. Binding of IL12 to the IL12 receptor complex on T and natural killer (NK) cells leads to signaling via signal transducer and activator of transcription 4 (STAT4) and signal transducer and activator of transcription 3 (STAT3), and subsequent interferon gamma (IFN-y) production and secretion. Ullrich et al., 2020, EXCLI J., 19:1563-1589. Signaling downstream of IFN-y includes activation of T-box transcription factor TBX21 (Tbet) and induces pro-inflammatory functions of T helper 1 (TH1 ) cells. Id.

[0004] Due to its ability to activate NK cells and cytotoxic T-cells, IL12 has been studied as an anti-cancer therapeutic since the early 1990’s. Lasek et al., 2014, Cancer Immunol. Immunother. 63(5):419-435. However, in most patients, repeated administration of IL12 led to adaptive response and a progressive decline of IL12-induced IFN-y blood levels. Id. Further, severe toxicity resulted from the concomitant induction of IFN-y along with other cytokines (e.g., TNF-a) and / or chemokines (IP-10 or MIG). Id. Different dosing and timing protocols were developed in an attempt to minimize IFN-y toxicity and improve IL12 efficacy. Id. These approaches had minimal effect and have not significantly improved patient survival. Id.

[0005] Despite the general acceptance in the field of I L12 therapies being developed for immunotherapy, including anticancer therapy, IL12 molecules have generally displayed poor therapeutic indices, with high, toxic doses required to confer modest anti-cancer effects.

[0006] Thus, there is a need in the art for novel I L12 therapies with improved therapeutic efficacy and safety profiles.4. SUMMARY

[0007] The present disclosure provides tumor-targeted split IL12 receptor agonists.

[0008] In certain aspects, the tumor-targeted split IL12 receptor agonists address the drawbacks of IL12 therapy and are characterized by improved therapeutic profiles by virtue of efficacy and / or improved safety profiles. The tumor-targeted split IL12 receptor agonists of the disclosure typically comprise two components, or a “combination”, formulated in a single formulation or separate formulations, comprising a tumor-targeted I L12Rp 1 agonist and a tumor-targeted I L12R|32 agonist. Exemplary tumor-targeted split IL12 receptor agonists are disclosed in Section 6.2 and numbered embodiments 1 to 9 and 19 to 136. Exemplary tumor- targeted IL12Rp1 agonists are disclosed in Section 6.3 and numbered embodiments 19 to 44, 60 to 97, 106 to 107, and 110 to 118. Exemplary tumor-targeted IL12R 2 agonists are disclosed in Section 6.4 and numbered embodiments 19 to 29, 45 to 79, 98 to 105, 108 to 112, and 125 to 136.

[0009] The disclosure further provides nucleic acids encoding the tumor-targeted split IL12 receptor agonists of the disclosure and their components. The nucleic acids can be in the form of a single nucleic acid (e.g., a vector encoding all components of the tumor-targeted split IL12 receptor agonists) 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 the tumor-targeted split IL12 receptor agonists of the disclosure. The disclosure further provides methods of producing a tumor-targeted split IL12 receptor agonist of the disclosure. Exemplary nucleic acids, host cells, cell lines, and methods of producing tumor-targeted split I L12 receptor agonists are described in Section 6.10.

[0010] The disclosure further provides pharmaceutical compositions comprising the tumor- targeted split IL12 receptor agonists of the disclosure. Exemplary pharmaceutical compositions are described in Section 6.11.

[0011] Further provided herein are methods of using the tumor-targeted split IL12 receptor agonists, e g., for eliciting anti-tumor cytotoxicity and treating cancerous conditions. Exemplary methods are described in Section 6.12 and numbered embodiments 10 to 142, infra.

[0012] Also described are combination methods using the tumor-targeted split IL12 receptor agonists in combination with one or more additional therapeutic agents, such as a multispecific T-cell engager as described in Section 6.6. Exemplary combination therapy methods are described in Section 6.13 and numbered embodiments 137 to 142, infra.

[0013] Also provided are tumor-targeted IL12R 1 agonists for use in a method (e.g., a method described in Section 6.12 or 6.13) comprising administrating to a subject the tumor-targeted I L12Rp 1 agonist (e.g., as described in Section 6.3) and a tumor-targeted I L12R 2 agonist (e.g., as described in Section 6.4). Exemplary tumor-targeted I L12Rp1 agonists for use in such methods are described in numbered embodiments 153 to 160.

[0014] Further provided are tumor-targeted IL12RP2 agonists for use in a method (e.g., a method described in Section 6.12 or 6.13) comprising administrating to a subject the tumor- targeted IL12Rp2 agonist (e.g., as described in Section 6.4) and a tumor-targeted IL12Rp1 agonist (e.g., as described in Section 6.3). Exemplary tumor-targeted IL12RP2 agonists for use in such methods are described in numbered embodiments 161 to 168.5. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] FIGS. 1A-1L show exemplary tumor-targeted IL12Rp1 agonist structures. FIG. 1A shows a tumor-targeted I L12RP1 agonist comprising: (a) a tumor targeting moiety in Fab format (e.g., a Fab derived from an antibody against a tumor-associated antigen) connected to the N- terminus of a first Fc domain via a first linker (7), and (b) an IL12RP1 binding moiety comprising an IL12 moiety connected to the N-terminus of a second Fc domain via a second linker (2). The p35 and p40 moieties of the IL12 moiety are connected to one another via a third linker (3).Mutation(s) in the p35 moiety are represented by a star. FIG. 1 B shows a tumor-targetedI L12Rp 1 agonist comprising (a) a tumor targeting moiety in Fab format (e.g., a Fab derived from an antibody against a tumor-associated antigen) connected to the N-terminus of a first Fc domain via a first linker (1), and (b) an I L12RP1 binding moiety in Fab format (e.g., a Fab derived from an antibody against I L12RP1 ) connected to the N-terminus of a second Fc domain via a second linker (2). FIG. 1C shows a tumor-targeted I L12RP1 agonist comprising (a) a tumor targeting moiety in Fab format (e.g., a Fab derived from an antibody against a tumor-associated antigen) connected to the N-terminus of a first Fc domain via a first linker (1), and (b) an IL12Rp1 binding moiety in single domain antibody (sdAb) format (e.g., an sdAb againstI L12R|31 ) connected to the N-terminus of a second Fc domain via a second linker (2). FIG. 1D shows a tumor-targeted I L12R|31 agonist comprising (a) a tumor targeting moiety in sdAb format (e.g., an sdAb against a tumor-associated antigen) connected to the N-terminus of a first Fc domain via a first linker (1), and (b) an IL12R 1 binding moiety comprising an I L12 moiety connected to the N-terminus of a second Fc domain via a second linker (2). FIG. 1E shows a tumor-targeted IL12R 1 agonist comprising (a) a tumor targeting moiety in a sdAb format (e.g., an sdAb against a tumor-associated antigen) connected to the N-terminus of a first Fc domain via a first linker (1), and (b) an I L12R|31 binding moiety in Fab format (e.g., a Fab derived from an antibody against I L12R|31 ) connected to the N-terminus of a second Fc domain via a second linker (2). FIG. 1F shows a tumor-targeted I L12R|31 agonist comprising (a) a tumor targeting moiety in sdAb format (e.g., an sdAb against a tumor-associated antigen) connected to the N- terminus of a first Fc domain via a first linker (1) and (b) an I L12R|31 binding moiety in sdAb format (e.g., an sdAb against IL12R 1) connected to the N-terminus of a second Fc domain via a second linker (2). FIG. 1G shows a tumor-targeted I L12R 1 agonist comprising (a) a tumor targeting moiety in scFv format (e.g., an scFv against a tumor-associated antigen) connected to the N-terminus of a first Fc domain via a first linker (1) and an IL12R 1 binding moiety in sdAb format (e.g., an sdAb against I L12R|31 ) connected to the N-terminus of a second Fc domain via a second linker (2). FIG. 1 H shows a tumor-targeted IL12R|31 agonist comprising (a) a tumor targeting moiety in scFv format (e.g., an scFv derived from an antibody against a tumor- associated antigen) connected to the N-terminus of a first Fc domain via a first linker (1) and an I L12R|31 binding moiety in scFv format (e.g., an scFv derived from an antibody against I L12Rp 1 ) connected to the N-terminus of a second Fc domain via a second linker (2). FIG. 11 shows a tumor-targeted I L12Rp 1 agonist comprising (a) a tumor targeting moiety in scFv format (e.g., an scFv derived from an antibody against a tumor-associated antigen) connected to the N- terminus of a first Fc domain via a first linker (1) and (b) an I L12Rp 1 binding moiety comprising an IL12 moiety connected to the N-terminus of a second Fc domain via a second linker (2). The p35 and p40 moieties of the IL12 moiety are connected to one another via a third linker (3). Mutation(s) in the p35 moiety are represented by a star. FIG. 1 J shows a tumor-targetedI L12Rp 1 agonist comprising (a) a tumor targeting moiety in sdAb format (e.g., an sdAb against a tumor-associated antigen) connected to the N-terminus of a first Fc domain via a first linker (1) and an I L12R|31 binding moiety in scFv format (e.g., an scFv derived from an antibody against I L12Rp 1 ) connected to the N-terminus of a second Fc domain via a second linker (2). FIG. 1K shows a tumor-targeted I L12R|31 agonist comprising (a) a tumor targeting moiety in Fab format (e.g., a Fab derived from an antibody against a tumor-associated antigen) connected to the N-terminus of a first Fc domain via a first linker (1) and an I L12R 1 binding moiety in scFv format (e g., an scFv derived from an antibody against I L12R|31 ) connected to the N-terminus of a second Fc domain via a second linker (2). FIG. 1L shows a tumor-targeted I L12R[31 agonist comprising (a) a tumor targeting moiety in scFv format (e.g., an scFv derived from an antibody against a tumor-associated antigen) connected to the N-terminus of a first Fc domain via a first linker (1) and an IL12R 1 binding moiety in Fab format (e.g., a Fab derived from an antibody against I L12R|31 ) connected to the N-terminus of a second Fc domain via a second linker (2).

[0016] FIGS. 2A-2L show exemplary tumor-targeted IL12RP2 agonist structures. FIG. 2A shows a tumor-targeted I L12R|32 agonist comprising: (a) a tumor targeting moiety in Fab format (e.g., a Fab derived from an antibody against a tumor-associated antigen) connected to the N- terminus of a first Fc domain via a first linker (7), and (b) an IL12R|32 binding moiety comprising an IL12 moiety, connected to the N-terminus of a second Fc domain via a second linker (2). The p35 and p40 moieties of the IL12 moiety are connected to one another via a third linker (3). Mutation(s) in the p40 moiety are represented by a star. FIG. 2B shows a tumor-targetedI L12Rp2 agonist comprising (a) a tumor targeting moiety in Fab format (e.g., a Fab derived from an antibody against a tumor-associated antigen) connected to the N-terminus of a first Fc domain via a first linker (1), and (b) an IL12R 2 binding moiety in Fab format (e.g., a Fab derived from an antibody against IL12RP2) connected to the N-terminus of a second Fc domain via a second linker (2). FIG. 2C shows a tumor-targeted I L12R|32 agonist comprising (a) a tumor targeting moiety in Fab format (e.g., a Fab derived from an antibody against a tumor-associated antigen) connected to the N-terminus of a first Fc domain via a first linker (1), and (b) an IL12R|32 binding moiety in single domain antibody (sdAb) format (e.g., an sdAb againstI L12R|32) connected to the N-terminus of a second Fc domain via a second linker (2). FIG. 2D shows a tumor-targeted IL12RP2 agonist comprising (a) a tumor targeting moiety in sdAb format (e.g., an sdAb against a tumor-associated antigen) connected to the N-terminus of a first Fc domain via a first linker (1), and (b) an IL12RP2 binding moiety comprising an IL12 moiety connected to the N-terminus of a second Fc domain via a second linker (2). FIG. 2E shows a tumor-targeted I L12R|32 agonist comprising (a) a tumor targeting moiety in a sdAb format (e.g., an sdAb against a tumor-associated antigen) connected to the N-terminus of a first Fc domain via a first linker (1), and (b) an IL12R|32 binding moiety in Fab format (e.g., a Fab derived from an antibody against I L12R|32) connected to the N-terminus of a second Fc domain via a second linker (2). FIG. 2F shows a tumor-targeted I L12R|32 agonist comprising (a) a tumor targeting moiety in sdAb format (e.g., an sdAb against a tumor-associated antigen) connected to the N- terminus of a first Fc domain via a first linker (1) and (b) an I L12R|32 binding moiety in sdAbformat (e.g., an sdAb against IL12R 2) connected to the N-terminus of a second Fc domain via a second linker (2). FIG. 2G shows a tumor-targeted I L12R|32 agonist comprising (a) a tumor targeting moiety in scFv format (e.g., an scFv against a tumor-associated antigen) connected to the N-terminus of a first Fc domain via a first linker (1) and an IL12R 2 binding moiety in sdAb format (e.g., an sdAb against I L12R|32) connected to the N-terminus of a second Fc domain via a second linker (2). FIG. 2H shows a tumor-targeted I L12R|32 agonist comprising (a) a tumor targeting moiety in scFv format (e.g., an scFv derived from an antibody against a tumor- associated antigen) connected to the N-terminus of a first Fc domain via a first linker (1) and an I L12R|32 binding moiety in scFv format (e.g., an scFv derived from an antibody against I L12Rp2) connected to the N-terminus of a second Fc domain via a second linker (2). FIG. 2I shows a tumor-targeted IL12RP2 agonist comprising (a) a tumor targeting moiety in scFv format (e.g., an scFv derived from an antibody against a tumor-associated antigen) connected to the N- terminus of a first Fc domain via a first linker (1) and (b) an IL12R 2 binding moiety comprising an IL12 moiety connected to the N-terminus of a second Fc domain via a second linker (2). The p35 and p40 moieties of the IL12 moiety are connected to one another via a third linker (3). Mutation(s) in the p40 moiety are represented by a star. FIG. 2J shows a tumor-targetedI L12Rp2 agonist comprising (a) a tumor targeting moiety in sdAb format (e.g., an sdAb against a tumor-associated antigen) connected to the N-terminus of a first Fc domain via a first linker (1) and an I L12R|32 binding moiety in scFv format (e.g., an scFv derived from an antibody against I L12Rp2) connected to the N-terminus of a second Fc domain via a second linker (2). FIG. 2K shows a tumor-targeted I L12R|32 agonist comprising (a) a tumor targeting moiety in Fab format (e.g., a Fab derived from an antibody against a tumor-associated antigen) connected to the N- terminus of a first Fc domain via a first linker (1) and an I L12Rp2 binding moiety in scFv format (e.g., an scFv derived from an antibody against IL12RP2) connected to the N-terminus of a second Fc domain via a second linker (2). FIG. 2L shows a tumor-targeted I L12R|32 agonist comprising (a) a tumor targeting moiety in scFv format (e.g., an scFv derived from an antibody against a tumor-associated antigen) connected to the N-terminus of a first Fc domain via a first linker (1) and an IL12R 2 binding moiety in Fab format (e.g., a Fab derived from an antibody against I L12R|32) connected to the N-terminus of a second Fc domain via a second linker (2).

[0017] FIGS. 3A-3B are cartoon illustrations depicting the cell-cell linkage between a tumor cell and a T-cell facilitated by combinations of tumor-targeted IL12R|31 and IL12RP2 agonists. FIG. 3A illustrates a combination of a tumor-targeted IL12Rp1 and IL12RP2 agonists, each binding to a tumor-associated antigen via a Fab domain. The tumor-targeted IL12Rp1 and I L12Rp2 agonists bind to IL12RP1 or to IL12Rp2 via their IL12 moieties, respectively. 1: Tumor-associated antigen (TAA); 2: IL12R|31 ; 3: IL12R 2. FIG. 3B illustrates a combination of a tumor- targeted I L12Rp1 and I L12R|32 agonists, each binding to a tumor-associated antigen via a Fab domain. The tumor-targeted IL12R|31 and IL12R 2 agonists bind to IL12R|31 or to IL12R|32 via anti-IL12R|31 Fab or anti-IL12Rp2 Fab domains, respectively. 1 : Tumor-associated antigen (TAA)); 2: 11_12R|31 ; 3: IL12R|32. FIG. 3C illustrates a combination of a tumor-targeted I L12R|31 and IL12RP2 agonists, each binding to a different tumor-associated antigen via a Fab domain. The tumor-targeted IL12R 1 and I L12R|32 agonists bind to IL12R 1 or to IL12R|32 via their IL12 moieties, respectively. 1a: First tumor-associated antigen (TAA); 1b: Second tumor-associated antigen (TAA); 2: IL12R|31 ; 3: IL12R|32. FIG. 3D illustrates a combination of a tumor-targeted I L12Rp 1 and I L12R|32 agonists, each binding to a different tumor-associated antigen via a Fab domain. The tumor-targeted IL12Rp1 and IL12R|32 agonists bind to IL12Rp1 or to IL12R|32 via anti-IL12R|31 Fab or anti-IL12R|32 Fab domains, respectively. 1a: First tumor-associated antigen (TAA); 1 b: Second tumor-associated antigen (TAA); 2: I L12Rp1 ; 3: IL12R|32.

[0018] FIGS. 4A-4D are graphs that show STAT3-Luc activity associated with IL12 moiety- comprising tumor-targeted IL12Rp1 and I L12Rp2 agonists in combinations. FIG. 4A is a graph that shows the ST AT3-Luc activity of tumor-targeted IL12Rp1 and IL12Rp2 agonists in NK92 / STAT3-Luc cells that were not co-cultured with other cells. FIG. 4B is a close-up of the graph displayed in FIG. 4A, showing an enhanced view of the non-hlL12 data points. FIG. 4C is a graph that shows the STAT3-Luc activity of tumor-targeted IL12Rp1 and IL12RP2 agonists in NK92 / STAT3-Luc cells co-cultured with Raji / hPSMA cells. FIG. 4D is a close-up of the graph displayed in FIG. 4C, showing a closer view of the non-hlL12 data points.

[0019] FIGS. 5A-5B are graphs that show STAT3-Luc activity associated with tumor-targeted IL12Rp1 and I L12Rp2 agonists in combinations in NK92 / STAT3-Luc cells co-cultured with Raji cells that do not express PSMA or PSMA-expressing Raji cells. FIG. 5A shows STAT3-Luc activity associated with tumor-targeted I L12Rpi and I L12Rp2 agonists alone or in combinations in NK92 / STAT3-Luc cells co-cultured with Raji cells that do not express PSMA. FIG. 5B shows STAT3-Luc activity associated with tumor-targeted I L12Rpi and I L12Rp2 agonists alone or in combinations in NK92 / STAT3-Luc cells co-cultured with PSMA-expressing Raji cells.

[0020] FIG. 6A-6D are graphs that show the release of IFNy from primary human T-cells upon treatment with tumor-targeted IL12Rp1 and IL12RP2 agonists in combination or control treatments, in the presence of a tumor-targeting CD3 bispecific Ab. FIGS. 6A and 6C show the release of IFNy from primary human T-cells in the presence of Raji cells that do not expressPSMA. FIGS. 6B and 6D show the release of I FNy from primary human T-cells in the presence of PSMA-expressing Raji cells.

[0021] FIGS. 7A-7B are graphs that show the activation of phospho-STAT4 (pSTAT4) signaling in primary human T-cells upon treatment with tumor-targeted IL12R|31 and I L12R[32 agonists. FIG. 7A shows percent pSTAT4 activation in primary human T-cells in the absence of tumor cells. FIG. 7B shows percent pSTAT4 activation in primary human T-cells in the presence of PSMA-expressing C4-2 tumor cells.

[0022] FIGS. 8A-8D show target cell killing and I FNy release from primary human T-cells upon treatment with tumor-targeted IL12Rp1 and IL12R|32 agonists in combination or control treatments, in the presence of a hMUC16-targeting CD3 bispecific Ab. FIG. 8A is a graph that shows the extent of killing of PSMA-expressing HEK293 / hMUC16 cells. FIG. 8B is a graph that shows the extent of killing of HEK293 / hMUC16 cells that do not express PSMA. FIG. 8C is a graph that shows I FNy release from primary human T-cells in the presence of PSMA-expressing HEK293 / hMUC16 cells. FIG. 8D is a graph that shows IFNy release from primary human T-cells in the presence of HEK293 / hMUC16 cells that do not express PSMA.

[0023] FIGS. 9A-9D are graphs that show STAT3-Luc activity associated with tumor-targeted IL12Rp1 and IL12RP2 agonists in combination. FIG. 9A shows STAT3-Luc activity associated with a first set of PSMA-targeted I L12R|31 and IL12R 2 agonists alone or in combination in NK92 / STAT3-Luc cells co-cultured with Jurkat cells that do not express PSMA. FIG. 9B shows STAT3-Luc activity associated with the first set of PSMA-targeted I L12R|31 and I L12R|32 agonists alone or in combination in NK92 / STAT3-Luc cells co-cultured with PSMA-expressing Jurkat cells. FIG. 9C shows STAT3-Luc activity associated with a second set of PSMA-targeted I L12Rp 1 and IL12RP2 agonists alone or in combination in NK92 / STAT3-Luc cells co-cultured with Jurkat cells that do not express PSMA. FIG. 9D shows STAT3-Luc activity associated with the second set of PSMA-targeted IL12Rp1 and IL12RP2 agonists alone or in combination in NK92 / STAT3-Luc cells co-cultured with PSMA-expressing Jurkat cells.

[0024] FIGS. 10A-10D are graphs that show target cell killing and IFNy release from primary human T-cells upon treatment with PSMA-targeted IL12R|31 and IL12R|32 agonists, alone or in combination, in the presence of a hSTEAPI-targeting CD3 bispecific antibody. FIG. 10A shows the extent of killing of PSMA-expressing LNCaP cells associated with a first set of PSMA- targeted IL12R|31 and IL12R 2 agonists alone or in combination. FIG. 10B shows IFNy release from PSMA-expressing LNCaP cells associated with the first set of PSMA-targeted I L12R|31 and I L12R|32 agonists alone or in combination. FIG. 10C shows the extent of killing of PSMA-expressing LNCaP cells associated with a second set of PSMA-targeted IL12R 1 and I L12R 2 agonists alone or in combination. FIG. 10D shows IFNy release from PSMA-expressing LNCaP cells associated with the second set of PSMA-targeted I L12R|31 and I L12R|32 agonists alone or in combination.

[0025] FIGS. 11A-11 B are graphs that show the activation of phospho-STAT4 (pSTAT4) signaling in primary human T-cells upon treatment with PSMA-targeted IL12Rp1 and IL12R 2 agonists. FIG. 11A shows percent pSTAT4 activation in primary human T-cells in the presence of PSMA-expressing C4-2 tumor cells associated with a first set of PSMA-targeted IL12R 1 and IL12Rp2 agonists alone or in combination. FIG. 11 B shows percent pSTAT4 activation in primary human T-cells in the presence of PSMA-expressing C4-2 tumor cells associated with a second set of PSMA-targeted I L12R|31 and I L12Rp2 agonists alone or in combination.

[0026] FIG. 12A shows activation of phospho-STAT4 (pSTAT4) signaling in primary human T- cells in the presence OVCAR3 tumor cells upon treatment with MUC16-targeted IL12Rp1 and IL12R 2 agonists alone or in combination. FIG. 12B shows target cell killing upon treatment with MUC16-targeted I L12R|31 and IL12RJ32 agonists, alone or in combination, in the presence of a hMSLN-targeting CD3 bispecific antibody.

[0027] FIG. 13A shows activation of phospho-STAT4 (pSTAT4) signaling in primary human T- cells in the presence of OVCAR3 tumor cells upon treatment with MSLN-targeted IL12R 1 and IL12Rp2 agonists alone or in combination. FIG. 13B shows target cell killing upon treatment with MSLN-targeted I L12Rpi and I L12Rp2 agonists, alone or in combination, in the presence of a hMUC16-targeting CD3 bispecific antibody.

[0028] FIG. 14A-14B are graphs that show STAT3-Luc activity associated with tumor-targeted I L12Rp 1 and IL12R 2 agonists in combination. FIG. 14A shows STAT3-Luc activity associated with sets of PSMA-targeted I L12RP1 and I L12Rp2 agonists in combination in NK92 / STAT3-Luc cells co-cultured with Raji cells that do not express PSMA. FIG. 14B shows STAT3-Luc activity associated with sets of PSMA-targeted I L12Rp 1 and I L12RP2 agonists in combination in NK92 / STAT3-Luc cells co-cultured with PSMA-expressing Raji cells.

[0029] FIG. 15A-15B show activation of pSTAT4 signaling in primary human T-cells upon treatment with tumor-targeted IL12Rp1 and IL12RP2 agonists in combination. FIG. 15A shows activation of pSTAT4 signaling in primary human T-cells in the absence of tumor cells upon treatment with PSMA-targeted IL12RP1 and IL12RJ32 agonists in combination. FIG. 15B showsactivation of pSTAT4 signaling in primary human T-cells in the presence of C4-2 tumor cells upon treatment with PSMA-targeted IL12Rp1 and IL12Rp2 agonists in combination.6. DETAILED DESCRIPTION6.1. Definitions

[0030] 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.

[0031] 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.

[0032] Antigen Binding Domain or ABD: The term “antigen binding domain” or “ABD” as used herein refers to the portion of a targeting moiety that is capable of specific, non-covalent, and reversible binding to a target molecule.

[0033] Associated: The term “associated” in the context of a protein or protein component (e.g., a tumor-targeted IL12R 1 agonist; a tumor-targeted IL12R 2 agonist; a targeting moiety such as a Fab) refers to a functional relationship between two amino acid sequences on one or more polypeptide chains. In particular, the term “associated” means that two or more sequences or polypeptide chains are associated with one another, e.g., non-covalently through molecular interactions or covalently through one or more disulfide bridges or chemical cross-linkages, so as to produce a functional protein or protein component. Examples of associations that might be present in a tumor-targeted split IL12 receptor agonist of the disclosure include (but are not limited to) associations between p40 and p35 moieties, associations between homodimeric or heterodimeric Fc domains in an Fc region, associations between VH and VL regions in a Fab or scFv, associations between CH1 and CL in a Fab, and associations between CH3 and CH3 in a domain substituted Fab.

[0034] 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, oesophageal cancer, peritoneal cancer, 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.

[0035] Complementarity Determining Region or CDR: The terms “complementarity determining region” or “CDR,” as used herein, refer to the sequences of amino acids within antibody variable regions which confer antigen specificity and binding affinity. In general, there are three CDRs in each heavy chain variable region (CDR-H1, CDR-H2, CDR-H3) and three CDRs in each light chain variable region (CDR1-L1 , CDR-L2, CDR-L3). Though most naturally occurring antibodies are composed of heavy chains and light chains, camelids (e.g., camels, dromedaries, llamas, and alpacas) and some sharks produce antibodies that consist only of heavy chains. These antibodies bind antigenic epitopes using a single variable domain known as VHH and contain only heavy chain CDRs (CDR-H1 , CDR-H2, CDR-H3). Exemplary conventions that can be used to identify the boundaries of CDRs include, e.g., the Kabat definition, the Chothia definition, the ABM definition and the IMGT definition. See, e.g., Kabat, 1991 , “Sequences of Proteins of Immunological Interest,” National Institutes of Health, Bethesda, Md. (Kabat numbering scheme); Al-Lazikani et al., 1997, J. Mol. Biol. 273:927-948 (Chothia numbering scheme); Martin et al., 1989, Proc. Natl. Acad. Sci. USA 86:9268-9272 (ABM numbering scheme); and Lefranc et al., 2003, Dev. Comp. Immunol. 27:55-77 (IMGT numbering scheme). Public databases are also available for identifying CDR sequences within an antibody.

[0036] EC50: The term “EC50” refers to the half maximal effective concentration of a molecule or combination of molecules (such as a tumor-targeted split IL12 receptor agonist) which induces a response halfway between the baseline and maximum after a specified exposure time. The EC50 essentially represents the concentration of an antibody or Tumor-targeted splitIL12 receptor agonist where 50% of its maximal effect is observed. In certain embodiments, the EC50 value equals the concentration of a tumor-targeted split I L12 receptor agonist that gives half-maximal STAT3 activation in an assay as described in Section 8.1.2.

[0037] Epitope: An epitope, or antigenic determinant, is a portion of an antigen (e.g., target molecule) recognized by an antibody or other antigen-binding moiety as described herein. An epitope can be linear or conformational.

[0038] Fab: The term “Fab” in the context of a targeting moiety of the disclosure refers to a pair of polypeptide chains, the first comprising a variable heavy (VH) domain of an antibody N- terminal to a first constant domain (referred to herein as C1), and the second comprising variable light (VL) domain of an antibody 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.

[0039] 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 of antibody-based binding molecules 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 native antibody the Fc domains are typically identical, but one or both Fc domains might advantageously be modified to allow for heterodimerization, e.g., via a knob-in-hole interaction.

[0040] Host cell: The term “host cell” as used herein refers to cells into which a nucleic acid of the disclosure has been introduced. The terms “host cell” and “recombinant host cell” are used interchangeably herein. It is understood that such terms refer to the particular subject cell and to the progeny or potential 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 as used herein. Typical host cells are eukaryotic host cells, such as mammalian host cells. Exemplary eukaryotic host cells include yeast and mammalian cells, for example vertebratecells such as a mouse, rat, monkey or human cell line, for example HKB11 cells, PER.C6 cells, HEK cells or CHO cells.

[0041] IL12 Moiety: The term “I L12 moiety” refers to an amino acid sequence comprising a p35 moiety and a p40 moiety, which may be on a single polypeptide chain. The p35 moiety may be N- or C-terminal to the p40 moiety. In some embodiments, the p35 moiety and the p40 moiety are configured to associate with one another. In some embodiments, the p35 moiety and the p40 moiety are connected via a linker. The related term “I L12 moiety linker” refers to a linker connecting a p35 moiety and a p40 moiety.

[0042] IL12 p35 moiety or p35 moiety: An IL12 p35 moiety or a p35 moiety is 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 I L12R|32 binding portion of a mammalian, e.g., human or murine, p35 (sometimes referred to as the alpha subunit of IL12 or I L12a), optionally with one or amino acid substitutions as defined in Section 6.3.2.2 or Section 6.4.2.2 below.

[0043] In eukaryotic cells, the human IL12 p35 subunit is synthesized as a precursor polypeptide of 219 amino acids, from which 22 amino acids are removed to generate mature IL12 p35. In some embodiments, the mammalian p35 is full-length human p35. In other embodiments, the mammalian p40 is mature human p35. The sequence of human p35 has the Uniprot identifier P29459 (uniprot.org / uniprot / P29459). In some embodiments, the mammalian p35 is full-length murine p35. In some embodiments, the mammalian p35 is mature murine p40. The sequence of murine p40 has the Uniprot identifier P43431 (uniprot.org / uniprot / P43431).

[0044] Full-length human I L12 p35 has the following amino acid sequence (signal sequence = underlined):MCPARSLLLVATLVLLDHLSLARN LP VATPD PGM FPCLHHSQN LLRAVS N M LQ KARQT LEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFM MALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVP QKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS (SEQ ID NO: 1).

[0045] p35 comprises a signal sequence (at amino acids 1-22 of human p35). Thus, amino acid 23 of full-length human p35 is amino acid 1 of mature human p35.

[0046] In native I L12, p35 has four conserved cysteine residues that form two inter-strand disulfide bonds, which bridge C64 and C96 as well as C85 and C123 of human p35. p35 alsoincludes a cysteine (C74 of human p35) that forms an inter-chain bond with p40 (at amino acid C177 of human p40)).

[0047] The p35 moiety preferably comprises an amino acid sequence comprising 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 mature a mammalian p35, e.g., human or murine p35 (corresponding to amino acids 23-219 of human p35), optionally with one or amino acid substitutions as defined in Section 6.3.2 below.

[0048] IL12 p40 moiety or p40 moiety: An IL12 p40 moiety or a p40 moiety is an amino acid sequence comprising 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 I L12F?|31 binding portion of a mammalian, e.g., human or murine, p40 (sometimes referred to as the beta subunit of IL12 or I L12|3), optionally with one or amino acid substitutions as defined in Section 6.3.2.2 or Section 6.4.2.2 below.

[0049] In eukaryotic cells, the human IL12 p40 subunit is synthesized as a precursor polypeptide of 328 amino acids, from which 22 amino acids are removed to generate mature IL12 p40. The sequence of human p40 has the Uniprot identifier P29460 (uniprot.org / uniprot / P29460). In some embodiments, the mammalian p40 is full-length murine p40. In some embodiments, the mammalian p40 is mature murine p40. The sequence of murine p40 has the Uniprot identifier P43432 (uniprot.org / uniprot / P43432).

[0050] In some embodiments, the p40 moiety comprises p40 D2 and D3 domains, to the exclusion of the p40 D1 domain. In other embodiments, the p40 moiety comprises p40 D1 , D2, and D3 domains.

[0051] Full-length human I L12 p40 has the following amino acid sequence (signal sequence = underlined; D1 domain = italicized; D2 domain = bold; D3 domain = bold and underlined):MCHQQLVISWFSLVFLASPLVA / I4 / E / .K / <D VYV VELD WYPDAPGEMVVL TCD TPEEDGIT WTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILK DQKEPK / WTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATL SAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIK PDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVF TDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS (SEQ ID NO: 5).

[0052] p40 comprises a signal sequence at amino acids 1-22 of human p40. Thus, amino acid 23 of full-length human p40 is amino acid 1 of mature human p40.

[0053] The sequence of human p40 has the Uniprot identifier P29460 (uniprot.org / uniprot / P29460). The sequence of murine p40 has the Uniprot identifier P43432 (uniprot.org / uniprot / P43432). p40 comprises an Ig-like C2-type domain referred to as D1 (at amino acids 23 to 106 of human p40), a first fibronectin type-l 11 domain referred to as D2 (at amino acids 107 to 236 of human p40) and a second fibronectin type-ill domain referred to as D3 (at amino acids 237 to 328 of human p40). In native IL12, the D2 domain of p40 has four conserved cysteine residues which form two inter-strand disulfide bonds, which bridge C109 and C120 and C148 and C171 in human p40 and the D3 domain also contains an inter-strain disulfide bond, which bridges C278 and C305 in human p40. D2 also includes a cysteine (C177 in human p40) that forms an inter-chain bond with p35 (at amino acid C74 of human p35). D3 also contains the highly conserved WSXWS motif (SEQ ID NO: 44) (WSEWAS (SEQ ID NO: 45) in human p40).

[0054] The p40 moiety preferably includes a D2 domain and a D3 domain (or an amino acid sequence comprising 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 the D2 and D3 domains) of a mammalian, e.g., human or murine, p40, optionally with one or amino acid substitutions as defined in Section 6.3.2.2 or Section 6.4.2.2 below.

[0055] The p40 moiety can also include a D1 domain or an amino acid sequence comprising 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 the D1 domain of a mammalian, e.g., human or murine, p40, optionally with one or amino acid substitutions as defined in Section 6.3.2.2 or Section 6.4.2.2 below.

[0056] In various embodiments, the p40 moiety of an IL12 moiety of the disclosure retains any combination of (a) none, any one, any two or all three inter-strand disulfide bonds and / or (b) the cysteine that forms an inter-chain bond with p35 and / or (c) the conserved WSXWS motif (SEQ ID NO: 44).

[0057] IL12RB1 : I L12R[31 is the I L12 receptor subunit beta-1 (I L12Rp1), which binds to IL12 p40. The sequence of human I L12R[31 has the Uniprot identifier P42701 (uniprot.org / uniprot / P42701), with amino acids 24 to 545 making up the extracellular domain.The sequence of murine IL12R 1 has the Uniprot identifier Q60837 (uniprot.org / uniprot / Q60837), with amino acids 20 to 565 making up the extracellular domain. I L12Rp 1 comprises a signal sequence (at amino acids 1-23 of human I L12R|31 ) , an extracellular p40-binding domain (at amino acids 24 to 545 of human I L12Rp1 ), a helical transmembrane domain (at amino acids 546 to 570 of human I L12Rp1 ) and a cytoplasmic domain (at amino acids 571 to 662 of human IL12R 1).

[0058] IL12RB2: I L12R|32 is the I L12 receptor subunit beta-2 (I L12R02), which binds to IL12 p30. The sequence of human I L12R|3 has the Uniprot identifier Q99665 (uniprot.org / uniprot / Q99665), with amino acids 24 to 622 making up the extracellular domain. The sequence of murine IL12Rp2 has the Uniprot identifier P97378 (uniprot.org / uniprot / Q60837), with amino acids 24 to 637 making up the extracellular domain. I L12R|32 comprises a signal sequence (at amino acids 1-23 of human I L12R|32) , an extracellular p40-binding domain (at amino acids 24 to 622 of human I L12Rp2), a helical transmembrane domain (at amino acids 623 to 643 of human IL12R 2) and a cytoplasmic domain (at amino acids 644 to 862 of human IL12RP2).

[0059] IL12 Variant or Variant IL12: An “IL12 variant” or “variant IL12” is an IL12 moiety composed or one or more polypeptide chains comprising an I L12 p35 (referred to as “p35”) moiety and an IL12 p40 (“p40”) moiety in association with one another and which varies from native IL12 by the primary amino acid sequence of its p35 moiety (a “variant p35 moiety” or “variant p35”) and / or p40 moiety (a “variant p40 moiety” or “variant p40”) a relative to wild type p35 and / or p40, respectively.

[0060] In some embodiments, the variant IL12 has increased relative affinity to the I L12R|31 receptor vs. the I L12Rp2 receptor as compared to wild-type IL12, for example through one or more mutations in p40 that increase binding to I L12Rp1 and / or through one or more mutations in p35 that reduce binding to IL12R[32. Such variants are sometimes referred to herein as “IL12R 1 ligands,” “IL12R[31-skewed IL12 variants,” and the like.

[0061] In some embodiments, the variant IL12 has increased relative affinity to the I L12R|32 receptor vs. the I L12R|31 receptor as compared to wild-type IL12, for example through one or more mutations in p35 that increase binding to I L12Rp2 and / or through one or more mutations in p40 that reduce binding to I L12R|31. Such variants are sometimes referred to herein as “IL12RP2 ligands,” “IL12Rp2-skewed IL12 variants,” and the like.

[0062] Binding affinity of p40 to IL12R|31 and of p35 to I L12R|32 can be assayed by surface plasmon resonance (SPR) techniques (analyzed on a Biacore instrument) (Liljeblad et al., 2000, Glyco J 17:323-329).

[0063] The variant IL12 can thus comprise a p35 and / or p40 moiety with one or more amino acid substitutions, deletions and / or insertions compared to wild type p35 and / or p40. Exemplary mutations, e.g., substitutions, are disclosed, inter alia, in Sections 6.3.2.2 and 6.4.2.2.

[0064] Operably linked: The term “operably linked” as used herein refers to a functional relationship between two or more regions of a polypeptide chain in which the two or more regions are linked so as to produce a functional polypeptide, or two or more nucleic acid sequences, e.g., to produce an in-frame fusion of two polypeptide components or to link a regulatory sequence to a coding sequence.

[0065] Single Chain Fv or scFv: The term “single chain Fv” or “scFv” as used herein refers to a polypeptide chain comprising the VH and VL domains of antibody, where these domains are present in a single polypeptide chain.

[0066] 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 VH, 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.

[0067] 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.

[0068] Specifically (or selectively) binds: The term “specifically (or selectively) binds” as used herein means that a targeting moiety, e.g., an antibody, or antigen binding domain (“ABD”) thereof, forms a complex with a target molecule that is relatively stable under physiologic conditions. Specific binding can be characterized by a KDof about 5x10'2M or less (e.g., less than 5x10'2M, less than 10'2M, less than 5x10'3M, less than 10'3M, less than 5x10'4M, less than10’4M, less than 5x10’5M, less than 10-5M, less than 5x10’6M, less than 16M, less than 5x1 O’7M, less than 1O’7M, less than 5x10’8M, less than 10’8M, less than 5x10’9M, less than 10’9M, or less than 10’10M). Methods for determining the binding affinity of an antibody or an antibody fragment, e.g., a tumor-targeted split IL12 receptor agonist or a component targeting moiety, to a target molecule are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance (e.g., Biacore assays), fluorescent-activated cell sorting (FACS) binding assays and the like. A tumor-targeted split IL12 receptor agonist of the disclosure comprising a targeting moiety or an ABD thereof that specifically binds a target molecule from one species can, however, have cross- reactivity to the target molecule from one or more other species.

[0069] 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. Except when noted, the terms “patient” or “subject” are used herein interchangeably.

[0070] Target Molecule: The term “target molecule” as used herein refers to any biological molecule (e.g., protein, carbohydrate, lipid or combination thereof) expressed on a cell surface or in the extracellular matrix that can be specifically bound by a targeting moiety in a tumor- targeted split IL12 receptor agonist of the disclosure. In various embodiments of the tumor- targeted split IL12 receptor agonists of the disclosure, a target molecule can be tumor- associated antigen, I L12R|31 or I L12R|32.

[0071] 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 molecule, e.g., at a site to which a tumor-targeted split IL12 receptor agonist of the disclosure is to be localized. In some embodiments, a targeting moiety binds to a cell surface molecule on tumor cells or on lymphocytes in the tumor microenvironment. The targeting moiety can also have a functional activity in addition to localizing a molecule to a particular site. For example, a targeting moiety in a tumor-targeted split IL12 receptor that is an anti-l L12R|31 or anti- 1 L12R|32 antibody or an antigen binding portion thereof can modulate (e.g., agonize) IL12 signaling in T-lymphocytes.

[0072] 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 moreTumor-targeted split IL12 receptor agonists 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.

[0073] 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.

[0074] Tumor-Associated Antigen: The term “tumor-associated antigen” or “TAA” 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, and which is useful for the preferential targeting of a pharmacological agent to the cancer cell. In some embodiments, a TAA is a marker expressed by both normal cells and cancer cells, e.g., a lineage marker, e.g., CD19 on B cells. In some embodiments, a TAA is a cell surface molecule that is overexpressed in a cancer cell in comparison to a normal cell, for instance, 1-fold over expression, 2-fold overexpression, 3-fold overexpression or more in comparison to a normal cell. In some embodiments, a TAA 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 TAA will be expressed exclusively on the cell surface of a cancer cell, entirely or as a fragment, and not synthesized or expressed on the surface of a normal cell. Accordingly, the term “TAA” encompasses antigens that are specific to cancer cells, sometimes known in the art as tumor-specific antigens (“TSAs”).

[0075] Tumor-targeted IL12RB1 agonist: The term “tumor-targeted I L12R[31 agonist” as used herein refers to a molecule comprising a tumor-associated antigen (“TAA”) targeting moiety and an I L12R|31 binding moiety. The word “agonist” is used for convenience only and is not intended to imply that the molecule need possess I L12 signaling or other activity, whether alone or in combination with another molecule (e.g., a tumor-targeted I L12R|32 agonist). In some embodiments, the combination of a tumor-targeted IL12R 1 agonist and a tumor-targetedI L12R|32 agonist results in signaling via the IL12 receptor and / or clustering of I L12R|31 and I L12Rp2 receptor subunits.

[0076] Tumor-targeted IL12RB2 agonist: The term “tumor-targeted I L12R|32 agonist” as used herein refers to a molecule comprising a tumor-associated antigen (“TAA”) targeting moiety and an IL12RB2 binding moiety. The word “agonist” is used for convenience only and is not intended to imply that the molecule need possess I L12 signaling or other activity, whether alone or in combination with another molecule (e.g., a tumor-targeted I L12R|31 agonist). In some embodiments, the combination of a tumor-targeted I L12R|31 agonist and a tumor-targeted I L12Rp2 agonist results in signaling via the IL12 receptor and / or clustering of I L12Rp1 and I L12R|32 receptor subunits.

[0077] Universal Light Chain: The term “universal light chain” as used herein in the context of a targeting moiety refers to a light chain polypeptide capable of pairing with the heavy chain region of an antibody or antibody fragment, e.g., a targeting moiety, and also capable of pairing with other heavy chain regions. Universal light chains are also known as “common light chains.”

[0078] 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.

[0079] VH: The term “VH” refers to the variable region of an immunoglobulin heavy chain of an antibody, including the heavy chain of an scFv or a Fab.

[0080] VL: The term “VL” refers to the variable region of an immunoglobulin light chain, including the light chain of an scFv or a Fab.6.2. Tumor-Targeted Split IL12 Receptor Agonists

[0081] The present disclosure provides tumor-targeted split IL12 receptor agonists. Tumor- targeted split I L12 receptor agonists comprise two components, a tumor-targeted I L12R|31 agonist and a tumor-targeted I L12R|32 agonist. Thus, a tumor-targeted split IL12 receptor agonist of the disclosure is sometimes referred to herein as a “combination.”

[0082] The tumor-targeted I L12R|31 agonist and a tumor-targeted I L12R|32 agonists each comprises a tumor-associated antigen (“TAA”) targeting moiety, and the TAA targeting moiety ofthe tumor-targeted I L12 R|31 agonist and the TAA targeting moiety of the tumor-targeted I L12Rp2 agonist are typically capable of binding to the same cell, e.g., a tumor cell.

[0083] The tumor-targeted IL12R|31 agonist further comprises an IL12Rp1 binding moiety, and the tumor-targeted I L12Rp2 agonist further comprises an I L12Rp2 binding moiety. The IL12R 1 and IL12R 2 binding moieties can each be a targeting moiety (e.g., an antigen binding fragment of an anti-l L12R 1 or anti-l L12Rp2 antibody, respectively), or it can be a ligand (e.g., an IL12 moiety with preferential binding to the I L12R|31 or IL12RJ32 receptor, respectively).

[0084] When the tumor-targeted IL12R 1 agonist and tumor-targeted IL12R 2 agonist are in proximity of a tumor cell recognized by the TAA targeting moiety and a cell harboring the IL12 receptor such as a cytotoxic T-lymphocyte, the tumor-targeted split IL12 receptor agonist can cross-link the tumor cell and cytotoxic T-lymphocyte, thereby triggering a cytotoxic immune response against the tumor cell. Accordingly, in some embodiments, disclosed are tumor- targeted I L12Rp 1 agonists for use in a method of triggering a cytotoxic immune response against a tumor cell, the method comprising administration of the tumor-targeted IL12R 1 agonist and a tumor-targeted I L12R|32 agonist. Also disclosed, in some embodiments, are tumor-targeted IL12RP2 agonists for use in a method of triggering a cytotoxic immune response against a tumor cell, the method comprising administration of the tumor-targeted IL12Rp2 agonist and a tumor-targeted IL12R 1 agonist. The tumor-targeted IL12R 1 agonist and the tumor-targeted IL12R 2 agonist may be administered simultaneously or sequentially, and may be administered in the same composition or different compositions.

[0085] The use of singular terms, such as “tumor-targeted split IL12 receptor agonist” and “combination” is for convenience only and does not necessitate that the tumor-targeted IL12R 1 agonist and tumor-targeted I L12R|32 agonist be present in the same composition, but merely that the two components be capable of being used with one another, e.g., to trigger a cytotoxic immune response against a tumor cell.

[0086] Examples of tumor-targeted IL12R|31 agonists and their components are described in Section 6.3 and subsections thereof.

[0087] Examples of tumor-targeted IL12R|32 agonists and their components are described in Section 6.4 and subsections thereof.

[0088] Suitable TAA targeting moieties for inclusion in the tumor-targeted I L12Rp1 agonists and tumor-targeted I L12R|32 agonists in a tumor-targeted split IL12 receptor agonist are exemplified in Section 6.5.

[0089] Suitable formats of the targeting moieties in a tumor-targeted split IL12 receptor agonist (e g., a TAA targeting moiety, an I L12R|31 targeting moiety, or an IL12RP2 targeting moiety) are disclosed in Section 6.6.

[0090] The tumor-targeted I L12R|31 agonist and a tumor-targeted IL12R|32 agonist typically contain Fc domains to which the TAA targeting moieties and the IL12R|31 or IL12R 1 binding moieties are operably linked. Suitable Fc domains are disclosed in Section 6.8. Suitable arrangements of Fc domain, TAA targeting moiety and I L12R|31 binding moiety in a tumor- targeted IL12Rp1 agonist are disclosed in FIG. 1 (FIGS. 1A-1 L). Suitable arrangements of Fc domain, TAA targeting moiety and I L12R 2 binding moiety in a tumor-targeted I L12R 2 agonist are disclosed in FIG. 2 (FIGS. 2A-2L).

[0091] One or more domains in a tumor-targeted I L12R|31 agonist and / or a tumor-targeted IL12Rp2 agonist may be connected to one another via one or more linkers. Suitable linkers are disclosed in Section 6.9.

[0092] Nucleic acids encoding and host cells capable of expressing a tumor-targeted I L12RP1 agonist and / or a tumor-targeted IL12Rp2 agonist are disclosed in Section 6.10.

[0093] Pharmaceutical compositions comprising the tumor-targeted IL12Rp1 agonists, tumor- targeted I L12Rp2 agonists and tumor-targeted split I L12 receptor agonists are disclosed in Section 6.11.

[0094] Methods of using the tumor-targeted split IL12 receptor agonists, e.g., to treat cancer or elicit anti-cancer immunity, are disclosed in Section 6.12.

[0095] Combination methods using the targeted split IL12 receptor agonists in combination with an additional therapeutic agent (e.g., a multispecific T-cell engager as described in Section 6.6), e.g., to treat cancer or elicit anti-cancer immunity, are disclosed in Section 6.13.6.3. Tumor-Targeted IL12R 1 Agonist6.3.1. Tumor-Associated Antigen Targeting Moieties

[0096] The tumor-targeted I L12R|31 agonist of the tumor-targeted split I L12 receptor agonists of the disclosure comprise a tumor-associated antigen (“TAA”) targeting moiety. Typically, the TAA recognized by the TAA targeting moiety of the tumor-targeted I L12RP1 agonist is expressed on the same cancer cell as the TAA recognized by the TAA targeting moiety of the tumor-targeted IL12Rp2 agonist.

[0097] In some embodiments, both TAA targeting moieties recognize the same TAA, whether on the same epitope or on different epitopes. If the TAA targeting moieties recognize differentepitopes, they preferably can bind to the cancer cell simultaneously and / or in a non-competing manner. When the TAA(s) recognized by both TAA targeting moieties are expressed on the same cancer cell, the TAA(s) may be the same TAA or different TAAs.

[0098] In some embodiments, both TAA targeting moieties recognize different TAAs expressed on the same cancer cell.

[0099] Suitable TAA targeting moieties are described in Section 6.5.6.3.2. IL12RP1 Binding Moieties6.3.2.1. IL12RP1 Targeting Moieties

[0100] In some embodiments, the tumor-targeted IL12R|31 agonist of the tumor-targeted split IL12 receptor agonists of the disclosure comprises an I L12F?|31 targeting moiety as an I L12Rp 1 binding moiety.

[0101] The I L12RP1 targeting moiety typically is or comprises an antigen binding domain of an antibody. The I L12R|31 targeting moiety can be any format, e.g., as disclosed in Section 6.6 or subsections thereof. In some embodiments, the I L12RJ31 targeting moiety is a Fab. In some embodiments, the I L12R|31 targeting moiety is an scFv. In some embodiments, the I L12R 1 targeting moiety is a sdAb (e.g., a VHH or an sdVH).

[0102] In some embodiments, the I L12R[31 targeting moiety is based on an antibody comprising both heavy and light chain variable regions. Exemplary anti-l L12R 1 antibodies comprising both heavy and light chain variable regions are set forth in Table R1 below.

[0103] In some aspects, the I L12Rp1 targeting moiety competes with an antibody set forth in Table R1 for binding to I L12R|31. In further aspects, the I L12R|31 targeting moiety comprises CDRs having CDR sequences of an antibody set forth in Table R1. In some embodiments, the targeting moiety comprises all 6 CDR sequences of an antibody set forth in Table R1. In other embodiments, the I L12R|31 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, an I L12 Rp 1 targeting moiety comprises a VH comprising the amino acid sequence of the VH of an antibody set forth in Table R1 . In some embodiments, the I L12R 1 targeting moiety further comprises a VL comprising the amino acid sequence of the VL of the antibody set forth above in Table R1 . In other embodiments, the I L12Rp 1 targeting moiety further comprises a universal light chain VL sequence.

[0104] In some embodiments, the I L12R i targeting moiety is based on a single domain antibody. In some embodiments, the I L12R 1 targeting moieties are based on the exemplary anti-l L12R 1 single domain antibodies or antibody sequences set forth in Table R2 below.

[0105] In some aspects, the I L12Rp1 targeting moiety competes with an antibody set forth above in Table R2 for binding to I L12R 1. In further aspects, the I L12R 1 targeting moiety comprises CDRs having CDR sequences of an antibody set forth in Table R2. In some embodiments, the I L12R|31 targeting moiety comprises all 3 CDR sequences of an antibody set forth in Table R2. In further aspects, an I L12R|32 targeting moiety comprises a VH (e.g., a VHH) comprising the amino acid sequence of the VH of an antibody set forth in Table R2.

[0106] In some embodiments, the I L12R|31 targeting moiety binds an epitope at similar proximity to cell membrane as the IL12R|32 targeting of the tumor-targeted split I L12 receptor agonist. In some embodiments, if the IL12Rp2 targeting moiety binds to the D1 domain of IL12R 2, then the IL12R 1 targeting moiety binds to D2 domain of IL12R 1.6.3.2.2. IL12R 1 Ligands

[0107] In some embodiments, the I L12R|31 binding domain of a tumor-targeted split IL12 receptor agonist is an IL12R 1 ligand. An I L12Rpi ligand refers to a variant IL12 moiety which has preferential binding to IL12R|31 vs. IL12R 2 as compared to wild-type IL12.

[0108] In some embodiments, the preferential binding to I L12R|31 is achieved through mutations in the p35 moiety that reduce its binding to I L12R|32, while maintaining or increasing the binding of the p40 moiety to IL12Rp1.

[0109] In some embodiments, the I L12R|31 ligand comprises a p35 moiety whose amino acid sequence has 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 I L12R[32 binding portion of a mammalian, e.g., human or murine, p35 (sometimes referred to as the alpha subunit of IL12 or IL12a). For example, the p35 moiety can comprise an amino acid 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 the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:5. In some embodiments, the p35 moiety is a variant p35 moiety having an amino acid comprising one ormore mutations that reduce I L12R|32 binding as compared to the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:5. For example, in some embodiments, the variant p35 moiety can have up to 1 ,000-fold attenuated binding to human I L12R|32 as compared to wild type human p35. In some embodiments, the variant p35 moiety can have up to 100-fold, up to 50-fold, up to 25-fold, up to 20-fold, up to 15-fold, up to 10-fold, or up to 5-fold attenuated binding to human I L12R|32 as compared to wild type human I L12 p35.

[0110] Other characteristics of useful IL12 p35 variants may include the ability to destabilize dimerization with I L12 p40.

[0111] Exemplary amino acid substitutions include, but are not limited to substitutions at N21 , Q35, E38, E45, D55, N71 , L75, N76, E79, N85, L89, F96, M97, L124, M125, Q130, Q135, N136, E143, Q146, Y167, 1171 , and R189, wherein amino acid positions are relative to the mature human IL12 p35 amino acid sequence, excluding the 22-amino acid signal sequence. Corresponding amino acid positions in the full-length human sequence, full-length murine sequence, and mature murine sequence are provided in Table B1. Table B1 also provides exemplary substitutions at each noted positions.

[0112] An exemplary amino acid substitution at mature human N21 is N21 D.

[0113] An exemplary amino acid substitution at mature human Q35 is Q35D.

[0114] An exemplary amino acid substitution at mature human E38 is E38Q.

[0115] An exemplary amino acid substitution at mature human E45 is E45Q.

[0116] Exemplary amino acid substitutions at mature human D55 include D55Q and D55K.

[0117] An exemplary amino acid substitution at mature human N71 is N71 D.

[0118] An exemplary amino acid substitution at mature human L75 is L75A.

[0119] An exemplary amino acid substitution at mature human N76 is N76D.

[0120] An exemplary amino acid substitution at mature human E79 is E79Q.

[0121] Exemplary amino acid substitutions at mature human N85 include N85D and N85Q.

[0122] An exemplary amino acid substitution at mature human L89 is L89A.

[0123] An exemplary amino acid substitution at mature human F96 is F96A.

[0124] An exemplary amino acid substitution at mature human M97 is M97A.

[0125] An exemplary amino acid substitution at mature human L124 is L124A.

[0126] An exemplary amino acid substitution at mature human M 125 is M125A.

[0127] An exemplary amino acid substitution at mature human Q130 is Q130E.

[0128] An exemplary amino acid substitution at mature human Q135 is Q135E.

[0129] An exemplary amino acid substitution at mature human N 136 is N136D.

[0130] An exemplary amino acid substitution at mature human E143 is E143Q.

[0131] An exemplary amino acid substitution at mature human Q146 is Q146E.

[0132] Exemplary amino acid substitutions at mature human Y167 include Y167A, Y167V, Y167R, and Y167E. In some embodiments, the substitution is Y167E (corresponding to Y189E in the context of full length p35). An exemplary variant p35 moiety with the substitution at Y167E in the context of mature human p35 or Y189E in the context of full length p35 is provided herein as SEQ ID NO:40. For convenience, this variant is sometimes referred to as a “Y189E” variant, wherein the position of the substitution is referred to in the context of the full length, rather than the mature, human p35 sequence.

[0133] Exemplary amino acid substitutions at mature human 1171 include 1171 A, 1171V, and I171E.

[0134] In certain embodiments, an amino acid substitution at mature human R189 destabilizes the p40 / p35 heterodimer by preventing formation of a disulfide bond between the two subunits. Exemplary amino acid substitutions at mature human R189 include R189A and R189K.

[0135] In some embodiments, the I L12R|31 ligand comprises a p40 moiety whose amino acid sequence has 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 IL12R[31 binding portion of a mammalian, e.g., human or murine, p40 (sometimes referred to as the beta subunit of IL12 or IL12P). For example, the p40 moiety can comprise an amino acid 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 the amino acid sequence of any one of SEQ ID NO:6, SEQ ID NO:7,SEQ ID N0:9 and SEQ ID NO:10. In some embodiments, the p40 moiety comprises an amino acid having the amino acid sequence of any one of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9 or SEQ ID NQ:10 and / or a variant of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9 or SEQ ID NQ:10 that does not have reduced IL12R 1 binding as compared to the amino acid sequence of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9 or SEQ ID NQ:10, respectively.6.4. Tumor-Targeted IL12RP2 Agonist6.4.1. Tumor-Associated Antigen Targeting Moieties

[0136] The tumor-targeted I L12R|32 agonist of the tumor-targeted split I L12 receptor agonists of the disclosure comprise a tumor-associated antigen (“TAA”) targeting moiety. Typically, the TAA recognized by the TAA targeting moiety of the tumor-targeted IL12RP2 agonist is expressed on the same cancer cell as the TAA recognized by the TAA targeting moiety of the tumor-targeted IL12Rp1 agonist.

[0137] In some embodiments, both TAA targeting moieties recognize the same TAA, 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 simultaneous and / or in a non-competing manner. TAA targeting moieties both expressed on the same cancer cell and may be the same TAA or different TAAs.

[0138] In some embodiments, both TAA targeting moieties recognize different TAAs expressed on the same cancer cell.

[0139] Suitable TAA targeting moieties are described in Section 6.5.6.4.2. IL12R 2 Binding Moieties6.4.2.1. IL12R 2 Targeting Moieties

[0140] In some embodiments, the tumor-targeted IL12R|32 agonist of the tumor-targeted split IL12 receptor agonists of the disclosure comprise an IL12R 2 targeting moiety as an I L12R|32 binding moiety.

[0141] The IL12RP2 targeting moiety typically is or comprises an antigen binding domain of an antibody. The I L12Rp2 targeting moiety can be any format, e.g., as disclosed in Section 6.6 or subsections thereof. In some embodiments, the I L12R|32 targeting moiety is a Fab. In some embodiments, the IL12R 2 targeting moiety is an scFv. In some embodiments, the I L12Rp2 targeting moiety is a sdAb.

[0142] In some embodiments, the I L12R|32 targeting moiety is based on an antibody comprising both heavy and light chain variable regions. Exemplary anti-l L12Rp2 antibodies are set forth in Table R3 below.

[0143] In some aspects, the I L12Rp2 targeting moiety competes with an antibody set forth in Table R3 for binding to I L12Rp2. In further aspects, the I L12Rp2 targeting moiety comprises CDRs having CDR sequences of an antibody set forth in Table R3. In some embodiments, the targeting moiety comprises all 6 CDR sequences of an antibody set forth in Table R3. 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, an I L12R|32 targeting moiety comprises a VH comprising the amino acid sequence of the VH of an antibody set forth in Table R3. In some embodiments, theI L12Rp2 targeting moiety further comprises a VL comprising the amino acid sequence of the VL of an antibody set forth above in Table R3. In other embodiments, the IL12R02 targeting moiety further comprises a universal light chain VL sequence.

[0144] In some embodiments, the IL12R02 targeting moieties are based on the exemplary anti- I L12Rp2 single domain antibodies or antibody sequences set forth in Table R4 below.

[0145] In some aspects, the I L12R|32 targeting moiety competes with an antibody set forth above in Table R4, for binding to the IL12R|32. In further aspects, the IL12R[32 targeting moiety comprises CDRs having CDR sequences of an antibody set forth in Table R4. In some embodiments, the IL12RP2 targeting moiety comprises all 3 CDR sequences of the antibody set forth in Table R4. In further aspects, an IL12RP2 targeting moiety comprises a VH (e.g., a VHH) comprising the amino acid sequence of the VH of an antibody set forth in Table R4.

[0146] In some embodiments, the I L12R|32 targeting moiety binds an epitope at similar proximity to cell membrane as the IL12Rp1 targeting of the tumor-targeted split I L12 receptor agonist. In some embodiments, if the IL12R|31 targeting moiety binds to the D2 domain of IL12Rp1 , then the I L12R|32 targeting moiety binds to D1 domain of I L12R|32.6.4.2.2. IL12R 2 Ligands

[0147] In some embodiments, the I L12R|32 binding domain of a tumor-targeted split IL12 receptor agonist is an I L12R|32 ligand. An IL12R 2 ligand refers to a variant IL12 moiety which has preferential binding to IL12RJ32 vs. IL12Rp1 as compared to wild-type IL12.

[0148] In some embodiments, the preferential binding to I L12R[32 is achieved through mutations in the p40 moiety that reduce its binding to I L12R|31 , while maintaining or increasing the binding of the p35 moiety to I L12Rp2.

[0149] In some embodiments, the IL12R 2 ligand comprises a p35 moiety whose amino acid sequence has 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 I L12R|32 binding portion of a mammalian, e.g., human or murine, p35 (sometimes referred to as the alpha subunit of IL12 or IL12a). For example, the p35 moiety can comprise an amino acid 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 the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:5. In some embodiments, the p35 moiety comprises an amino acid having the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:5 and / or a variant of SEQ ID NO:3 or SEQ ID NO:5 that does not have reduced IL12R|32 binding as compared to the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:5.

[0150] In some embodiments, the IL12R 2 ligand comprises a p40 moiety whose amino acid sequence has 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 IL12R|31 binding portion of a mammalian, e.g., human or murine, p40 (sometimes referred to as the beta subunit of IL12 or I L12|3). For example, the p40 moiety can comprise an amino acid 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 the amino acid sequence of any one of SEQ ID NO:6, SEQ ID NO:7,SEQ ID N0:9 and SEQ ID NO:10. In some embodiments, the p40 moiety is a variant p40 moiety having an amino acid comprising one or more mutations that reduce I L12R|31 binding as compared to the amino acid sequence of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9 or SEQ ID NQ:10. For example, in some embodiments, the variant p40 moiety can have up to 1 ,000-fold attenuated binding to human I L12R|31 as compared to wild type human p40. In some embodiments, the variant p40 moiety can have up to 100-fold, up to 50-fold, up to 25-fold, up to 20-fold, up to 15-fold, up to 10-fold, or up to 5-fold attenuated binding to human I L12R|31 as compared to wild type human IL12 p40.

[0151] Other characteristics of useful IL12 p40 variants may include the ability to destabilize dimerization with I L12 p35.

[0152] Exemplary amino acid substitutions include, but are not limited to substitutions at positions K6, W15, D18, E32, E33, D34, Q42, S43, E45, Q56, E59, F60, D62, E73, K84, D87, D93, K96, K99, E100, N103, K104, N113, Q144, R159, D161 , K163, E187, N200, N218, Q229, E235, Y246, C252, Q256, K258, K260, E262, K264, N281 , Y292, and E299, wherein amino acid positions, unless otherwise noted, are relative to the mature human IL12 p40 amino acid sequence, excluding the 22-amino acid signal sequence. Corresponding amino acid positions in the full-length human sequence, full-length murine sequence, and mature murine sequence are provided in Table B2. Table B2 also provides exemplary substitutions at each noted position.

[0153] An exemplary amino acid substitution at mature human K6 is K6A.

[0154] An exemplary amino acid substitution at mature human W15 is W15A.

[0155] Exemplary amino acid substitutions at mature human D18 include D18N, D18K, and D18A.

[0156] Exemplary amino acid substitutions at mature human E32 include E32Q and E32A.

[0157] Exemplary amino acid substitutions at mature human E33 include E33Q and E33A.

[0158] Exemplary amino acid substitutions at mature human D34 include D34N, D34K, andD34A.

[0159] An exemplary amino acid substitution at mature human Q42 is Q42E.

[0160] Exemplary amino acid substitutions at mature human S43 include S43E and S34K.

[0161] An exemplary amino acid substitution at mature human E45 is E45Q.

[0162] An exemplary amino acid substitution at mature human Q56 is Q56E.

[0163] Exemplary amino acid substitutions at mature human E59 include E59K, E59Q, and E59A.

[0164] An exemplary amino acid substitution at mature human F60 is F60A.

[0165] An exemplary amino acid substitution at mature human D62 is D62N.

[0166] An exemplary amino acid substitution at mature human E73 is E73Q.

[0167] An exemplary amino acid substitution at mature human K84 is K84A.

[0168] An exemplary amino acid substitution at mature human D87 is D87N.

[0169] An exemplary amino acid substitution at mature human D93 is D93A.

[0170] An exemplary amino acid substitution at mature human K96 is E93A.

[0171] Exemplary amino acid substitutions at mature human K99 include K99E, K99Y, and K99A.

[0172] An exemplary amino acid substitution at mature human E100 is E100 Q.

[0173] Exemplary amino acid substitutions at mature human N103 include N103D and N103Q.

[0174] An exemplary amino acid substitution at mature human K104 is K104A.

[0175] Exemplary amino acid substitutions at mature human N113 include N113D and N113Q.

[0176] An exemplary amino acid substitution at mature human Q144 is Q144E.

[0177] An exemplary amino acid substitution at mature human R159 is R159 E.

[0178] An exemplary amino acid substitution at mature human D161 is D161 N.

[0179] An exemplary amino acid substitution at mature human K163 is K163E.

[0180] An exemplary amino acid substitution at mature human E187 is E187Q.

[0181] Exemplary amino acid substitutions at mature human N200 include N200D and N200Q.

[0182] An exemplary amino acid substitution at mature human N218 is N218Q.

[0183] An exemplary amino acid substitution at mature human Q229 is Q229E.

[0184] An exemplary amino acid substitution at mature human E235 is E235Q.

[0185] Exemplary amino acid substitutions at mature human Y246 include Y246V and Y246F.

[0186] An exemplary amino acid substitution at mature human C252 is C252S.

[0187] An exemplary amino acid substitution at mature human Q256 is Q256N.

[0188] An exemplary amino acid substitution at mature human K258 is K258E.

[0189] An exemplary amino acid substitution at mature human K260 is K260E.

[0190] An exemplary amino acid substitution at mature human E262 is E262Q.

[0191] An exemplary amino acid substitution at mature human K264 is K264E.

[0192] Exemplary amino acid substitutions at mature human N281 include N281 D and N281Q.

[0193] An exemplary amino acid substitution at mature human Y292 is Y292F.

[0194] An exemplary amino acid substitution at mature human E299 is E299Q.

[0195] In certain embodiments, amino acid substitutions at mature human Y246 and / or Y292 destabilize the p40 / p35 heterodimer by preventing formation of a disulfide bond between the two subunits. Exemplary amino acid substitutions at Y246 include Y246V and Y246F. An exemplary amino acid substitution at Y292 is Y292F.6.5. Tumor-Associated Antigen Targeting Moieties

[0196] The tumor-targeted I L12R|31 agonist and the tumor-targeted IL12R|32 of the tumor- targeted split IL12 receptor agonists of the disclosure both comprise a tumor-associated antigen (“TAA”) targeting moiety. Typically, the TAA recognized by the TAA targeting moiety of the tumor-targeted I L12R 1 agonist and the TAA recognized by the TAA targeting moiety of the tumor-targeted I L12R|32 agonist are both expressed on the same cancer cell and may be the same TAA or different TAAs. If the TAA targeting moiety of the tumor-targeted IL12Rp1 agonist and the TAA targeting moiety of the tumor-targeted 11_12R|32 agonist are the same, in some embodiments binding the TAA targeting moiety of the tumor-targeted I L12R 1 agonist and the TAA targeting moiety of the tumor-targeted IL12RP2 agonist bind to the TAA in a non-competing fashion such that both the tumor-targeted IL12R|31 agonist and the tumor-targeted IL12R 2 agonist can bind to the same cell concurrently.

[0197] 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 IL12Rp1 agonist and the tumor-targeted I L12Rp2 agonist permits the delivery of high concentrations of IL12 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.

[0198] Suitable TAA targeting moiety formats are described in Section 6.7. 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 6.7.1 , an scFv, as described in Section 6.7.2, or a single domain antibody, as described in Section 6.7.3.

[0199] Exemplary target molecules recognized by the TAA targeting moieties of the tumor- targeted I L12Rp 1 agonist and / or the tumor-targeted I L12Rp2 agonist are Fibroblast Activation Protein (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, 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 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, [3-catenin and y-catenin, p120ctn, gp100 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, melanotransferrin (MELTF; CD228), MCSP, PDGF[3R (P-platelet-derived growth factor receptor), ErbB2 epithelial cell adhesion molecule (EpCAM), EGFR variant III (EGFRvll I), CD19, disialoganglioside GD2, ductal-epithelial mucine, gp36, TAG-72, glioma-associated antigen, [3- 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 (I GF1)- I, 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).

[0200] In some embodiments, the target molecule recognized by the TAA targeting moiety of the tumor-targeted I L12 Rp 1 agonist and / or the tumor-targeted IL12R 2 agonist is BCMA. In some embodiments, the target molecule recognized by the TAA targeting moiety of the tumor- targeted IL12R|31 agonist and / or the tumor-targeted IL12Rp2 agonist is CD20. In some embodiments, the target molecule recognized by the TAA targeting moiety of the tumor-targeted IL12R|31 agonist and / or the tumor-targeted IL12R|32 agonist is EGFR. In some embodiments,the target molecule recognized by the TAA targeting moiety of the tumor-targeted I L12R|31 agonist and / or the tumor-targeted IL12Rp2 agonist is PSMA. In some embodiments, the target molecule recognized by the TAA targeting moiety of the tumor-targeted I L12R|31 agonist and / or the tumor-targeted IL12R[32 agonist is CA9. In some embodiments, the target molecule recognized by the TAA targeting moiety of the tumor-targeted I L12R|31 agonist and / or the tumor-targeted IL12RP2 agonist is MSLN. In some embodiments, the target molecule recognized by the TAA targeting moiety of the tumor-targeted IL12R 1 agonist and / or the tumor-targeted I L12R|32 agonist is EPCAM. In some embodiments, the target molecule recognized by the TAA targeting moiety of the tumor-targeted IL12R 1 agonist and / or the tumor-targeted IL12RP2 agonist is B7H3. In some embodiments, the target molecule recognized by the TAA targeting moiety of the tumor-targeted IL12Rp1 agonist and / or the tumor-targeted IL12RP2 agonist is HER2 / HER3. In some embodiments, the target molecule recognized by the TAA targeting moiety of the tumor-targeted I L12Rp1 agonist and / or the tumor-targeted I L12R 2 agonist is STEAP1. In some embodiments, the target molecule recognized by the TAA targeting moiety of the tumor-targeted IL12R|31 agonist and / or the tumor-targeted I L12R|32 agonist is CEACAM5.

[0201] In some embodiments, the targeting moieties target the exemplary target molecules set forth in Table T1 below, which provides references to exemplary antibodies or antibody sequences upon which the targeting moiety can be based.

[0202] 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 acid sequence 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.

[0203] 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.

[0204] 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 inTable 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.

[0205] Additional target molecules that can be targeted by the IL12 receptor agonists are disclosed in Table I below and in, e.g., Hafeez et al., 2020, Molecules 25:4764, doi:10.3390 / molecules25204764, particularly in Table 1. Table 1 of Hafeez et al. is incorporated by reference in its entirety herein.6.6. Multispecific T-Cell Engagers

[0206] Aspects of the present disclosure are directed to combinations comprising (a) a tumor- targeted split IL12 receptor agonist (comprising a tumor-targeted I L12F?|31 agonist and a tumor- targeted I L12Rp2 agonist) and (b) a multispecific T-cell engager, as well as to methods comprising administration of a tumor-targeted split IL12 receptor agonist and a multispecific T- cell engager simultaneously, sequentially or separately. The tumor-targeted I L12R 1 agonist and the tumor-targeted IL12Rp2 agonist of the tumor-targeted split I L12 receptor agonist can be in the same or separate compositions, and the multispecific T-cell engager can be in the same or separate composition as the tumor-targeted I L12R|31 agonist and the tumor-targeted IL12Rp2 agonist.

[0207] As used herein, a “multispecific T-cell engager” describes a molecule comprising: (a) at least one TAA targeting moiety (e.g., as described in Section 6.5); and (b) at least one T-cell receptor (TCR) complex targeting moiety (e.g., as described below). In general, “multispecific T- cell engager,” as used herein, describes a molecule that does not comprise either an I L12Rp 1 targeting moiety or an I L12Rp2 targeting moiety. Also disclosed are pharmaceutical compositions comprising such multispecific T-cell engagers, in some cases together also comprising a tumor-targeted IL12Rp1 agonist and / or a tumor-targeted IL12Rp2 agonist. Further disclosed are methods for use of such multispecific T-cell engagers in treatment of cancer in combination with a tumor-targeted split IL12 receptor agonist of the disclosure.

[0208] Suitable targeting moiety formats (useful for both the TAA targeting moiety and the TCR complex targeting moiety) are described in Section 6.7. The targeting moiety is preferably an antigen binding moiety, e.g., a Fab, as described in Section 6.7.1 , an scFv, as described in Section 6.7.2, or a single domain antibody, as described in Section 6.7.3.

[0209] Example TAA targeting moieties which may be included in a multispecific T-cell engager disclosed herein are described in Section 6.5. In some embodiments, the TAA targeting moiety of a multispecific T-cell engager targets the same TAA as one or both TAA targeting moieties of the tumor-targeted split IL12 receptor agonist. In some embodiments, the TAA targeting moietyof a multispecific T-cell engager targets a different TAA from both TAA targeting moieties of the tumor-targeted split I L12 receptor agonist. In some embodiments, the TAA targeting moiety of a multispecific T-cell engager targets the same TAA as the TAA targeting moiety of the tumor- targeted IL12Rp1 receptor agonist. In some embodiments, the TAA targeting moiety of a multispecific T-cell engager targets the same TAA as the TAA targeting moiety of the tumor- targeted IL12RP2 receptor agonist. In some embodiments, the TAA targeting moiety of a multispecific T-cell engager targets a different TAA as the TAA targeting moiety of the tumor- targeted IL12Rp1 receptor agonist. In some embodiments, the TAA targeting moiety of a multispecific T-cell engager targets a different TAA as the TAA targeting moiety of the tumor- targeted IL12Rp2 receptor agonist.

[0210] Certain example tumor-associated antigens and associated antibodies (or antibody sequences) are provided in Table T1. In some embodiments, a multispecific T-cell engager comprises a TAA targeting moiety that specifically binds to a TAA of Table T1. In some embodiments, the TAA is BCMA. In some embodiments, the TAA is CD19. In some embodiments, the TAA is CD20. In some embodiments, the TAA is CD22. In some embodiments, the TAA is EGFR. In some embodiments, the TAA is PSMA. In some embodiments, the TAA is MUC16. In some embodiments, the TAA is CA9. In some embodiments, the TAA is mesothelin (MSLN). In some embodiments, the TAA is EPCAM. In some embodiments, the TAA is B7H3. In some embodiments, the TAA is HER2 / HER3. In some embodiments, the TAA is STEAP1. In some embodiments, the TAA is CEACAM5.

[0211] A multispecific T-cell engager comprises, in addition to a TAA targeting moiety, a T-cell receptor (TCR) complex targeting moiety. 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 TCRyb). 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 TCRyO). 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 TCRyd dimer of the T-cell receptor complex).

[0212] 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 or comprise 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.

[0213] Exemplary anti-CD3 and anti-TCR antibodies or antibody sequences are set forth in Table G below, upon which the TOR complex targeting moiety can be based.

[0214] 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 antibody set forth in Table G. In some embodiments, the TCR 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.

[0215] In some embodiments, the multispecific T-cell engager is a bispecific T-cell engager. 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 split IL12 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.6.7. Targeting Moiety Formats

[0216] In certain aspects, a targeting moiety (e.g., a TAA targeting moiety, an IL12R 1 targeting moiety, an I L12Rp2 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 lgG1 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.

[0217] In some embodiments, the TAA targeting moiety of the tumor-targeted IL12R 1 agonist and the TAA targeting moiety of the tumor-targeted I L12R|32 agonist share the same format (e.g., Fab, scFv or sdAb). In another embodiment, the TAA targeting moiety of the tumor- targeted IL12Rp1 agonist and the TAA targeting moiety of the tumor-targeted I L12R|32 agonist do not share the same format.

[0218] In some embodiments the TAA targeting moieties of the tumor-targeted I L12Rp1 agonist and the tumor-targeted IL12RP2 agonist are Fabs. In other embodiments, the TAA targeting moieties of the tumor-targeted I L12Rp1 agonist and the tumor-targeted I L12R|32 agonist arescFvs. In yet other embodiments, and the TAA targeting moieties of the tumor-targeted I L12 R|31 agonist and the tumor-targeted I L12R|32 agonist are sdAbs.

[0219] In some embodiments, where the IL12Rp1 and IL12R|32 binding moieties of a tumor- targeted split IL12 receptor agonist are I L12Rp 1 and IL12RP2 targeting moieties, the I L12Rp1 and I L12R|32 targeting moieties share the same format (e.g., Fab, scFv or sdAb). In other embodiments, where the I L12Rp1 and IL12RP2 binding moieties of a tumor-targeted split IL12 receptor agonist are IL12R|31 and IL12RP2 targeting moieties, the IL12Rp1 and IL12R|32 targeting moieties do not share the same format.

[0220] In some embodiments the IL12Rp1 and I L12R|32 targeting moieties are Fabs. In other embodiments, the I L12R|31 and IL12R 2 targeting moieties are scFvs. In yet other embodiments, and the I L12R|31 and IL12RP2 targeting moieties are sdAbs.

[0221] In some embodiments, the TAA targeting moieties and the IL12R 1 and IL12RP2 targeting moieties share the same format (e.g., Fab, scFv or sdAb). In other embodiments, the the TAA targeting moieties and the I L12Rp 1 and I L12R|32 targeting moieties do not share the same format (e.g., the TAA targeting moieties are Fabs and the I L12R|31 and I L12R|32 targeting moieties are sdAbs or vice versa).6.7.1. Fabs

[0222] Fab domains were traditionally produced by proteolytic cleavage of immunoglobulin molecules using enzymes such as papain. In the tumor-targeted split IL12 receptor agonists of the disclosure, the Fab domains can be recombinantly expressed as part of the tumor-targeted IL12Rp1 agonist and / or the tumor-targeted IL12R|32 agonist.

[0223] The Fab domains can comprise constant domain and variable region sequences from any suitable species, and thus can be murine, chimeric, human or humanized.

[0224] 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.

[0225] For the tumor-target split IL12 receptor agonists 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 thesame 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:

[0226] 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.

[0227] 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.

[0228] 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.

[0229] 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 of lock 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.

[0230] 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.

[0231] 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).

[0232] In some embodiments, the Fab domain comprises a 143Q and 188V substitutions in the CH1 domain and 113T and 176V substitutions in the CL domain, which serves to swaphydrophobic and polar regions of contact between the CH1 and CL domain (see, e.g., Golay et al., 2016, J Immunol 196:3199-211).

[0233] 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.

[0234] 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).

[0235] 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.

[0236] 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 an I L12 receptor agonist of the disclosure. In various embodiments, employing a common light chain as described herein reduces the number of inappropriate species of IL12 receptor agonists as compared to employing original cognate VLs. In various embodiments, the VL domains of the I L12 receptor agonists are identified from monospecific antibodies comprising a common light chain. In various embodiments, the VH regions of the IL12 receptor agonists 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 chainsare 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. 10,412,940.6.7.2. scFvs

[0237] 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 6.9.

[0238] 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.

[0239] The scFv can comprise VH and VL sequences from any suitable species, such as murine, human or humanized VH and VL sequences.

[0240] 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 6.5.3 (typically a repeat of a sequence containing the amino acids glycine and serine, such as the amino acid sequence (Gly4~Ser)3(SEQ ID NO:51), 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).6.7.3. Single domain antibodies

[0241] In some embodiments, a targeting moiety e.g., a TAA targeting moiety, an I L12R 1 targeting moiety, or an I L12F?|32 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. In some embodiments, sdAbs also include single VL domains capable of binding to their cognate antigen in the absence of an associated light chain. Single VH or VL domains mayhave amino acid changes relative to native VH or VL sequences that stabilize the domains and / or reduce or eliminate aggregation.

[0242] 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 heavychain 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.

[0243] 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.

[0244] 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).

[0245] 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).

[0246] 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).

[0247] 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.

[0248] 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).6.8. Fc Domains

[0249] The tumor-targeted I L12R|31 agonist and the tumor-targeted I L12Rp2 agonist include an Fc domain of the tumor-targeted split IL12 receptor agonists of the disclosure each comprises a pair of Fc domains to which the TAA targeting moiety and the I L12 R|31 binding moiety (in the case of the tumor-targeted IL12R|31 agonist) or the TAA targeting moiety and the I L12R|32 binding moiety (in the case of the tumor-targeted I L12 R|32 agonist) are operably linked.

[0250] In some embodiments, the tumor-targeted IL12R|31 agonist comprises an Fc region formed by the association of an Fc pair, one comprising a TAA targeting moiety at its N- terminus and the other comprising an I L12R|31 binding moiety (e.g., an I L12R|31 targeting moiety or an IL12R|31 ligand) at its N-terminus.

[0251] In some embodiments, the tumor-targeted IL12Rp2 agonist comprises an Fc region formed by the association of an Fc pair, one comprising a TAA targeting moiety at its N- terminus and the other comprising an I L12Rp2 binding moiety (e.g., an I L12R|32 targeting moiety or an I L12R|32 ligand) at its N-terminus.

[0252] In one embodiment the Fc domains of the tumor-targeted I L12Rp1 agonist and the tumor-targeted I L12R|32 agonist are derived from a human Fc domain.

[0253] The Fc domains that can be incorporated into a tumor-targeted I L12R|31 agonist and / or a tumor-targeted IL12R|32 agonist 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. In one embodiment, the Fc domains of both the tumor-targeted IL12RJ31 agonist and the tumor-targeted IL12RP2 agonist are derived from lgG1, lgG2, lgG3 or lgG4. In one embodiment, one or both pairs of Fc domains are derived from lgG1. In one embodiment, one or both pairs of Fc domains are derived from lgG4.

[0254] The two Fc domains within the Fc region of the tumor-targeted I L12R 1 agonist and / or a tumor-targeted I L12R|32 agonist 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 an I L12Rp 1 binding moiety or an I L12Rp2 binding moiety, the Fc domains might advantageously be different to allow for heterodimerization, as described in Section 6.8.2 below.

[0255] 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.

[0256] In the tumor-targeted IL12R|31 agonists and / or tumor-targeted IL12R|32 agonists of the present disclosure, the Fc region, and I 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.

[0257] In one embodiment the Fc region comprises CH2 and CH3 domains derived from lgG1.

[0258] In one embodiment the Fc region comprises CH2 and CH3 domains derived from lgG2.

[0259] In one embodiment the Fc region comprises CH2 and CH3 domains derived from lgG3.

[0260] In one embodiment the Fc region comprises CH2 and CH3 domains derived from lgG4.

[0261] In one embodiment the Fc region comprises a CH4 domain from IgM. The IgM CH4 domain is typically located at the C-terminus of the CH3 domain.

[0262] In one embodiment the Fc region comprises CH2 and CH3 domains derived from IgG and a CH4 domain derived from IgM.

[0263] It will be appreciated that the heavy chain constant domains for use in producing an Fc region for the tumor-targeted IL12R|31 agonists and / or tumor-targeted IL12R|32 agonists of the present 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.

[0264] 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 tailpiecealso contains a glycosylation site. In certain embodiments, the tumor-targeted IL12R 1 agonists and / or tumor-targeted I L12R|32 agonists of the present disclosure do not comprise a tailpiece.

[0265] In some embodiments, a tumor-targeted IL12R|31 agonist and / or tumor-targetedI L12Rp2 agonist comprises an Fc domain 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 a tumor-targeted I L12R 1 agonist and / or tumor-targeted IL12RP2 agonist comprises an Fc domain 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.

[0266] In some embodiments, a tumor-targeted IL12R|31 agonist and / or tumor-targeted I L12Rp2 agonist comprises an Fc domain 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 a tumor-targeted I L12R 1 agonist and / or tumor-targeted I L12R|32 agonist comprises an Fc domain 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.

[0267] In some embodiments, a tumor-targeted IL12R|31 agonist and / or tumor-targeted IL12R 2 agonist comprises an Fc domain 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 a tumor-targeted IL12R 1 agonist and / or tumor-targeted IL12R 2 agonist comprises an Fc domain 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.

[0268] In some embodiments, a tumor-targeted IL12R|31 agonist and / or tumor-targeted I L12Rp2 agonist comprises an Fc domain 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 a tumor-targeted I L12R|31 agonist and / or tumor-targeted IL12RP2 agonist comprises an Fc domain 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.

[0269] In some embodiments, a tumor-targeted IL12R|31 agonist and / or tumor-targeted I L12Rp2 agonist comprises an Fc domain that has at least 95%, at least 96%, at least 97%, atleast 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 15. In some embodiments, the a tumor-targeted I L12R|31 agonist and / or tumor-targeted IL12RP2 agonist comprises an Fc domain 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.

[0270] In some embodiments, a tumor-targeted IL12R|31 agonist and / or tumor-targeted I L12Rp2 agonist comprises an Fc domain 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:41. In some embodiments, the a tumor-targeted IL12R 1 agonist and / or tumor-targeted IL12R 2 agonist comprises an Fc domain that has the amino acid sequence of SEQ ID NO:41, optionally with one more mutations that facilitate heterodimerization or purification, e.g., (a) knob or hole mutations and / or (b) star mutations.

[0271] In some embodiments, a tumor-targeted IL12R|31 agonist and / or tumor-targeted IL12R 2 agonist comprises an Fc domain 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:42. In some embodiments, the a tumor-targeted IL12R 1 agonist and / or tumor-targeted IL12RP2 agonist comprises an Fc domain that has the amino acid sequence of SEQ ID NO:42, optionally with one more mutations that facilitate heterodimerization or purification, e.g., (a) knob or hole mutations and / or (b) star mutations.

[0272] In some embodiments, a tumor-targeted IL12R|31 agonist and / or tumor-targeted I L12R|32 agonist comprises an Fc domain 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:43. In some embodiments, the a tumor-targeted I L12R 1 agonist and / or tumor-targeted IL12RP2 agonist comprises an Fc domain that has the amino acid sequence of SEQ ID NO:43, optionally with one more mutations that facilitate heterodimerization or purification, e.g., (a) knob or hole mutations and / or (b) star mutations.

[0273] The Fc domains that are incorporated into the tumor-targeted IL12R 1 agonists and / or tumor-targeted I L12R|32 agonists 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 6.8.1.

[0274] The Fc domains can also be altered to include modifications that improve manufacturability of asymmetric tumor-targeted IL12F?|31 agonists and / or tumor-targeted IL12Rp2 agonists, 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 IL12R|31 agonists and / or tumor-targeted IL12Rp2 agonists 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 6.8.2.

[0275] 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 IL12R|31 agonists and / or tumor- targeted IL12Rp2 agonists.6.8.1. Fc Domains with Altered Effector Function

[0276] In some embodiments, the Fc domain comprises one or more amino acid substitutions that reduces binding to an Fc receptor and / or effector function.

[0277] 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), antibodydependent cellular phagocytosis (ADCP), and cytokine secretion. In a particular embodiment, the effector function is ADCC.

[0278] In one embodiment, the Fc domain (e.g., an Fc domain of an IL12 monomer) or the Fc region (e.g., one or both Fc domains of a tumor-targeted I L12Rp1 agonists and / or tumor- targeted IL12Rp2 agonist 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 Kabat EU 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 Fcdomain 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”).

[0279] 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).

[0280] In one embodiment, the Fc domain is an lgG1 Fc domain, particularly a human lgG1 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.

[0281] 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:

[0282] In a particular embodiment, the lgG4 with reduced effector function comprises the bolded portion of the amino acid sequence of SEQ ID NO: 19 (SEQ ID NO:31 of W02014 / 121087), sometimes referred to herein as lgG4s or hlgG4s.

[0283] 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 domaincomprising the amino acid sequence of SEQ ID NO:18 (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:20 (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: 19 (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:21 (SEQ ID NO:38 of WQ2014 / 121087) (or the bolded portion thereof).6.8.2. Fc Heterodimerization Variants

[0284] Certain tumor-targeted IL12R[31 agonists and / or tumor-targeted 11_12R|32 agonists 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 an I L12R|31 or I L12R|32 binding 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 tumor-targeted I L12R 1 agonists and / or tumor-targeted I L12R|32 agonists 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.

[0285] The present disclosure provides tumor-targeted IL12R|31 agonists and / or tumor-targeted I L12Rp2 agonists comprising Fc heterodimers, i.e., Fc regions comprising heterologous, nonidentical 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 (IgG 1 , lgG2, lgG3 and lgG4) class, as described in the preceding section.

[0286] Heterodimerization of the two different heavy chains at CH3 domains give rise to the desired tumor-targeted IL12R|31 agonist and / or tumor-targeted IL12Rp2 agonist, while homodimerization of identical heavy chains will reduce yield of the desired tumor-targeted IL12RP1 agonist and / or tumor-targeted IL12RP2 agonist. Thus, in a preferred embodiment, the polypeptides that associate to form a tumor-targeted I L12R 1 agonist and / or tumor-targeted IL12R 2 agonist of the disclosure will contain CH3 domains with modifications that favor heterodimeric association relative to unmodified Fc domains.

[0287] 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).

[0288] 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.

[0289] 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 to Kabat 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 acysteine 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).

[0290] 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.

[0291] 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 IL12 receptor agonists 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 I L12Rp 1 agonist and / or tumor-targeted I L12R|32 agonist to Protein A as compared to a corresponding tumor-targeted IL12Rp1 agonist and / or tumor-targeted IL12Rp2 agonist 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.

[0292] 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.6.9. Linkers

[0293] In certain aspects, the present disclosure provides tumor-targeted split I L12 receptor agonists in which two or more components of a tumor-targeted I L12R|31 agonist and / or a tumor- targeted I L12Rp2 agonist are connected to one another by a peptide linker. By way of example and not limitation, linkers can be used to connect (a) an I L12R|31 binding moiety or a IL12R 2 binding moiety (e.g., an IL12 moiety or an anti-l L12R|31 or anti-l L12Rp2 Fab, scFv, or sdAb) and an Fc domain; (b) a tumor targeting moiety and an Fc domain; (c) different domains withinan I L12R(31 binding moiety or a I L12R|32 binding moiety (e.g., different domains within an IL12 moiety, such as a p35 moiety and a p40 moiety); or (d) different domains within a tumor targeting moiety (e.g., the VH and VL domains in a scFv).

[0294] 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.

[0295] 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.

[0296] 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.

[0297] Charged (e.g., charged hydrophilic linkers) and / or flexible linkers are particularly preferred.

[0298] Examples of flexible linkers that can be used in the tumor-targeted split IL12 receptor 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 or multimer of GnS (SEQ ID NO: 23) or SGn, where n is an integer from 1 to 10, e.g., 1 2, 3, 4, 5, 6, 7, 8, 9 or 10 (SEQ ID NO: 24). In one embodiment, the linker is or comprises a monomer or multimer of repeat of G4S (SEQ ID NO: 25) e.g., (GGGGS)n (SEQ ID NO: 25).

[0299] Polyglycine linkers can suitably be used in the tumor-targeted split IL12 receptor 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: 27), five consecutive glycines (5Gly) (SEQ ID NO: 28), six consecutive glycines (6Gly)(SEQ ID NO: 29), seven consecutive glycines (7Gly) (SEQ ID NO: 30), eight consecutive glycines (8Gly) (SEQ ID NO: 31) or nine consecutive glycines (9Gly) (SEQ ID NO: 22).6.9.1. Hinge Sequences

[0300] In other embodiments, the tumor-targeted split IL12 receptor agonists of the disclosure comprise a linker that is a hinge region. In particular, where a tumor-targeted IL12R 1 agonist and / or a tumor-targeted I L12F?|32 of a tumor-targeted split IL12 receptor agonist contains an immunoglobulin-based targeting moiety, the hinge can be used to connect the targeting moiety, e.g., a Fab domain, to a multimerization 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 tumor-targeted IL12R 1 agonist or a tumor-targeted I L12R|32 agonist formed by the association of two Fc domains) can comprise two associated hinge sequences on separate polypeptide chains.

[0301] 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.

[0302] 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.

[0303] In one embodiment, a tumor-targeted I L12 R 1 agonist of the disclosure comprises an Fc region in which one or both Fc domains possesses an intact hinge region at its N-terminus.

[0304] In one embodiment, a tumor-targeted I L12 R|32 agonist of the disclosure comprises an Fc region in which one or both Fc domains possesses an intact hinge region at its N-terminus.

[0305] 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.

[0306] In some embodiments, the tumor-targeted I L12Rp 1 agonists and / or the tumor-targeted IL12R|32 agonists 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 lgG1 or human lgG4).

[0307] In one embodiment, the tumor-targeted IL12R|31 agonists and / or the tumor-targeted IL12Rp2 agonists 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: 32). The core hinge region of human lgG4 contains the sequence CPSC (SEQ ID NO: 33) compared to lgG1 that contains the sequence CPPC (SEQ ID NO: 32). 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.6.9.1.1. Chimeric Hinge Sequences

[0308] The hinge region can be a chimeric hinge region.

[0309] For example, a chimeric hinge may comprise an “upper hinge” sequence, derived from a human lgG1 , 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.

[0310] In particular embodiments, a chimeric hinge region comprises the amino acid sequence EPKSCDKTHTCPPCPAPPVA (SEQ ID NO: 34) (previously disclosed as SEQ ID NO:8 of WQ2014 / 121087, which is incorporated by reference in its entirety herein) or ESKYGPPCPPCPAPPVA (SEQ ID NO: 35) (previously disclosed as SEQ ID NO:9 ofWQ2014 / 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 6.6.1.1).6.9.1.2. Hinge Sequences with Reduced Effector Function

[0311] 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.

[0312] 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).

[0313] 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.

[0314] 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: 36) (previously disclosed as SEQ ID NO:1 of W02016161010A2), CPPCPAPGG-GPSVF (SEQ ID NO: 37) (previously disclosed as SEQ ID NO:2 of W02016161010A2), CPPCPAPG— GPSVF (SEQ ID NO: 38) (previously disclosed as SEQ ID NO:3 of W02016161010A2), or CPPCPAP — GPSVF (SEQ ID NO: 39) (previously disclosed as SEQ ID NO:4 of W02016161010A2).

[0315] 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 W02016161010A2.

[0316] 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 6.6.1.1).

[0317] The linkers useful in the tumor-targeted split IL12 receptor agonists 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 a protease, 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.6.10. Nucleic Acids and Host Cells

[0318] In another aspect, the disclosure provides nucleic acids encoding the tumor-targeted split I L12 receptor agonists of the disclosure and / or their individual components (the tumor- targeted IL12Rp1 agonists and the tumor-targeted I L12R|32 agonists). In some embodiments, the tumor-targeted split IL12 receptor agonists, the tumor-targeted I L12Rp1 agonists and / or the tumor-targeted IL12R|32 agonists are encoded by a single nucleic acid. In other embodiments, the tumor-targeted I L12Rp1 agonists and the tumor-targeted IL12R|32 agonists are encoded by separate nucleic acids. In other embodiments, for example in the case of a heterodimeric tumor- targeted I L12Rpi agonists and / or tumor-targeted IL12RP2 agonist, one or both of the tumor- targeted IL12Rp1 agonists and / or the tumor-targeted IL12R|32 agonists, e.g., when comprising an Fc heterodimer or a targeting moiety composed of more than one polypeptide chain, the tumor-targeted I L12R|31 agonists and / or the tumor-targeted IL12R[32 agonist are encoded by a plurality of (e.g., two, three, four or more) nucleic acids.

[0319] A single nucleic acid can encode a tumor-targeted I L12R[31 agonist and / or a tumor- targeted IL12Rp2 agonist that comprises a single polypeptide chain, a tumor-targeted IL12Rp1 agonists and / or a tumor-targeted I L12 R|32 agonist that comprises two or more polypeptide chains, or a portion of a tumor-targeted I L12R|31 agonist and / or a tumor-targeted I L12 R|32 agonist that comprises more than two polypeptide chains (for example, a single nucleic acid canencode two polypeptide chains of a tumor-targeted I L12R|31 agonists and / or a tumor-targeted I L12Rp2 agonist comprising three, four or more polypeptide chains, or three polypeptide chains of a tumor-targeted I L12R|31 agonist and / or a tumor-targeted I L12R|32 agonist comprising four or more polypeptide chains). For separate control of expression, the 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 polypeptides can also be controlled by the same transcriptional regulatory elements, and separated by internal ribosome entry site (IRES) sequences allowing for translation into separate polypeptides.

[0320] In some embodiments, a tumor-targeted IL12R|31 agonist and / or a tumor-targeted I L12R|32 agonist comprising two or more polypeptide chains is encoded by two or more nucleic acids. The number of nucleic acids encoding an IL12 receptor agonist can be equal to or less than the number of polypeptide chains in the IL12 receptor agonist (for example, when more than one polypeptide chains are encoded by a single nucleic acid).

[0321] The nucleic acids of the disclosure can be DNA or RNA (e.g., mRNA).

[0322] In another aspect, the disclosure provides host cells and vectors containing the 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.6.10.1. Vectors

[0323] The disclosure provides vectors comprising nucleotide sequences encoding a tumor- targeted I L12Rp1 agonist and / or a tumor-targeted I L12Rp2 agonist or component thereof described herein, for example one or two of the polypeptide chains of a tumor-targeted IL12R|31 agonists and / or a tumor-targeted I L12R|32 agonist. The vectors include, but are not limited to, a virus, plasmid, cosmid, lambda phage or a yeast artificial chromosome (YAC).

[0324] 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.

[0325] 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, biocideresistance (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.

[0326] Once the expression vector or DNA sequence containing the constructs has been prepared for expression, the expression vectors can be transfected or introduced into an appropriate 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.6.10.2. Cells

[0327] The disclosure also provides host cells comprising a nucleic acid of the disclosure.

[0328] In one embodiment, the host cells are genetically engineered to comprise one or more nucleic acids described herein.

[0329] 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.

[0330] The disclosure also provides host cells comprising the vectors described herein.

[0331] 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.6.11. Pharmaceutical Compositions

[0332] The tumor-targeted split IL12 receptor agonists of the disclosure may be in the form of compositions comprising the tumor-targeted IL12R|31 agonists and / or the tumor-targetedI L12R|32 agonists and 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 tumor-targeted split IL12 receptor agonist and, for therapeutic uses, the mode of administration.

[0333] 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.

[0334] Pharmaceutical compositions can be conveniently presented in unit dosage forms containing a predetermined amount of a tumor-targeted I L12R 1 receptor agonist and / or tumor- targeted IL12Rp2 receptor agonist per dose. The quantity of the tumor-targeted I L12R|31 receptor agonist and / or tumor-targeted I L12R|32 receptor agonist 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 the tumor- targeted IL12Rp1 receptor agonist and / or tumor-targeted IL12Rp2 receptor agonist 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 the tumor-targeted I L12R|31 receptor agonist and / or tumor- targeted I L12Rp2 receptor agonist suitable for a single administration.

[0335] The pharmaceutical compositions may also be supplied in bulk from containing quantities of the tumor-targeted IL12R|31 agonist and / or the tumor-targeted IL12R|32 agonist suitable for multiple administrations.

[0336] When formulated into a single formulation, the tumor-targeted I L12R|31 receptor agonist and tumor-targeted IL12RP2 receptor agonist can be used in approximately equimolar quantities.

[0337] Pharmaceutical compositions may be prepared for storage as lyophilized formulations or aqueous solutions by mixing a tumor-targeted I L12R[31 agonist and / or a tumor-targeted I L12R|32 agonist 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 nontoxic to the recipients at the dosages and concentrations employed.

[0338] 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.

[0339] 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 disclosureand include polyhydric sugar alcohols, for example trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol and mannitol. Stabilizers refer to 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 IL12 receptor agonist.

[0340] 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.

[0341] Additional miscellaneous excipients include bulking agents (e.g., starch), chelating agents (e.g., EDTA), antioxidants (e.g., ascorbic acid, methionine, vitamin E), and cosolvents.6.12. Therapeutic Indications and Methods of Treatment

[0342] The present disclosure provides methods for using and applications for the tumor- targeted split IL12 receptor agonists of the disclosure.

[0343] Tumor-targeted split IL12 receptor agonists 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.

[0344] Disease states for which the tumor-targeted split I L12 receptor agonists 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 tumor-targeted split I L12 receptor agonists of the present disclosure can be employed include cancer, including breast cancer, prostate cancer, and colorectal cancer. The tumor-targeted split IL12 receptor agonists of the disclosure may be administered per se or in any suitable pharmaceutical composition.

[0345] In various embodiments, the tumor-targeted split I L12 receptor agonists 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.

[0346] In one aspect, tumor-targeted split I L12 receptor agonists of the disclosure for use as a medicament are provided. In further aspects, tumor-targeted split IL12 receptor agonists of the disclosure for use in treating a disease are provided. In certain embodiments, tumor-targeted split IL12 receptor agonists of the disclosure for use in a method of treatment are provided. In one embodiment, the disclosure provides a tumor-targeted split I L12 receptor agonist as described herein for use in the treatment of a disease in a subject in need thereof. In certain embodiments, the disclosure provides a tumor-targeted split IL12 receptor agonist for use in a method of treating a subject having a disease comprising administering to the individual a therapeutically effective amount of the tumor-targeted split IL12 receptor agonist. In certain embodiments the disease to be treated is a proliferative disorder. In a preferred embodiment the disease is cancer. In certain embodiments the method further comprises administering to the individual a therapeutically effective amount of at least one additional therapeutic agent, e.g., an anti-cancer agent if the disease to be treated is cancer. In further embodiments, the disclosure provides a tumor-targeted split I L12 receptor agonist for use in stimulating the immune system. In certain embodiments, the disclosure provides a tumor-targeted split I L12 receptor agonist for use in a method of stimulating the immune system in a subject comprising administering to the individual an effective amount of the tumor-targeted split IL12 receptor agonist to stimulate the immune system. An “individual” according to any of the above embodiments is a mammal, preferably a human. “Stimulation of the immune system” according to any of the above embodiments may include any one or more of a general increase in immune function, an increase in T-cell function, an increase in B cell function, a restoration of lymphocyte function,an increase in the expression of IL12 receptors, an increase in T-cell responsiveness, an increase in natural killer cell activity or lymphokine-activated killer (LAK) cell activity, and the like.

[0347] In a further aspect, the disclosure provides for the use of a tumor-targeted split IL12 receptor agonist of the disclosure in the manufacture or preparation of a medicament 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 medicament. In certain embodiments the disease to be treated is a proliferative disorder. In a preferred embodiment the disease is cancer. In one such embodiment, the method further comprises administering to the individual a therapeutically effective amount of at least one additional therapeutic agent, e.g., an anti-cancer agent if the disease to be treated is cancer. In a further embodiment, the medicament is for stimulating the immune system. In a further embodiment, the medicament is for use in a method of stimulating the immune system in a subject comprising administering to the individual an amount effective of the medicament to stimulate the immune system. An “individual” according to any of the above embodiments may be a mammal, preferably a human. “Stimulation of the immune system” according to any of the above embodiments may include any one or more of a general increase in immune function, an increase in T-cell function, an increase in B cell function, a restoration of lymphocyte function, an increase in the expression of IL12 receptors, an increase in T-cell responsiveness, an increase in natural killer cell activity or lymphokine-activated killer (LAK) cell activity, and the like.

[0348] 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 tumor- targeted split IL12 receptor agonist of the disclosure (with the tumor-targeted I L12R|31 agonist and the tumor-targeted I L12R|32 agonist in separate pharmaceutical preparations or the same pharmaceutical preparation. In one embodiment one or two compositions comprising a tumor- targeted I L12Rp1 agonist and a tumor-targeted IL12RP2 agonist in a pharmaceutically acceptable form, e.g., in equimolar amounts, are administered to said individual. In certain embodiments the disease to be treated is a proliferative disorder. In a preferred embodiment the disease is cancer. In certain embodiments the method further comprises administering to the individual a therapeutically effective amount of at least one additional therapeutic agent, e.g., an anti-cancer agent if the disease to be treated is cancer. In a further aspect, the disclosure provides a method for stimulating the immune system in a subject, comprising administering tothe individual an effective amount of a tumor-targeted split I L12 receptor agonist to stimulate the immune system. An “individual” according to any of the above embodiments may be a mammal, preferably a human. “Stimulation of the immune system” according to any of the above embodiments may include any one or more of a general increase in immune function, an increase in T-cell function, an increase in B cell function, a restoration of lymphocyte function, an increase in the expression of I L12 receptors, an increase in T-cell responsiveness, an increase in natural killer cell activity or lymphokine-activated killer (LAK) cell activity, and the like.

[0349] In certain aspects, the disclosure provides a method of treating cancer, comprising administering to a subject in need thereof a tumor-targeted split IL12 receptor agonist or (a) pharmaceutical composition(s) comprising the tumor-targeted IL12R 1 agonist and the tumor- targeted I L12Rp2 agonist as described herein.

[0350] In some embodiments, the disclosure provides a method of treating cancer with a tumor- targeted split I L12 receptor agonist that is targeted to cancer tissue, comprising administering to a subject in need thereof a tumor-targeted split IL12 receptor agonist or (a) pharmaceutical composition(s) comprising the tumor-targeted I L12R|31 agonist and the tumor-targeted IL12R 2 agonist as described herein, with the tumor-targeted I L12Rp1 agonist and the tumor-targeted I L12Rp2 agonist each comprising a targeting moiety that recognizes a target molecule that is expressed on the cancer cells.

[0351] The present disclosure further provides a method of localized delivery of a tumor- targeted split IL12 agonist, comprising administering to a subject a tumor-targeted split IL12 receptor agonist or (a) pharmaceutical composition(s) comprising the tumor-targeted I L12R|31 agonist and the tumor-targeted IL12RP2 agonist as described herein, where the tumor-targeted I L12Rp 1 agonist and the tumor-targeted I L12Rp2 agonist comprise a targeting moiety that recognizes a target molecule that is expressed by a tissue to which the tumor-targeted split IL12 receptor agonist is to be locally delivered. As used herein, the term “locally delivered” does not require local administration but rather indicates that the tumor-targeted IL12 receptor agonist be selectively localized to a tissue of interest following administration.

[0352] The present disclosure further provides a method of administering to the subject IL12 therapy with reduced systemic exposure and / or reduced systemic toxicity and / or an improved therapeutic index, comprising administering to a subject the IL12 therapy in the form of a tumor- targeted split IL12 receptor agonist or (a) pharmaceutical composition(s) comprising the tumor- targeted I L12Rp1 agonist and the tumor-targeted IL12R 2 agonist as described herein.Accordingly, the foregoing methods permit IL12 therapy with reduced off-target side effects by virtue of preferential targeting of an IL12 receptor agonist to a particular target tissue and / or improved anti-tumor cytotoxicity at the site of intended activity.

[0353] The present disclosure further provides method of locally inducing an immune response in a target tissue, comprising administering to a subject a tumor-targeted split IL12 receptor agonist or (a) pharmaceutical composition(s) comprising the tumor-targeted I L12R 1 agonist and the tumor-targeted I L12R[32 agonist as described herein, where the tumor-targeted I L12Rp 1 agonist and the tumor-targeted I L12R|32 agonist each comprise a targeting moiety capable of binding a target molecule expressed in the target tissue. The tumor-targeted split IL12 receptor agonist 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.

[0354] 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.

[0355] 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 split I L12 receptor agonist 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 IL12 receptor agonists 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.

[0356] Table I below shows exemplary indications for which tumor-targeted split IL12 receptor agonists targeting particular target molecules can be used.

[0357] Additional target molecules and corresponding indications are disclosed in, e.g., Hafeez et al., 2020, Molecules 25:4764, doi:10.3390 / molecules25204764, particularly in Table 1. Table 1 is incorporated by reference in its entirety here.

[0358] A skilled artisan readily recognizes that in many cases the tumor-targeted split IL12 receptor agonists 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 tumor-targeted split IL12 receptor agonist that provides a physiological change is considered an “effective amount” or a “therapeutically effective amount”.

[0359] The subject, patient, or individual in need of treatment is typically a mammal, more specifically a human.

[0360] The appropriate dosage of a tumor-targeted split IL12 receptor agonist of the disclosure (when used alone or in combination with one or more other additional therapeutic agents, e.g., a multispecific T-cell engager) will depend on the type of disease to be treated, the route of administration, the body weight of the patient, the particular tumor-targeted I L12F?|31 agonist and tumor-targeted I L12Rf52 agonist in the tumor-targeted split I L12 receptor agonist, the severity and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, previous or concurrent therapeutic interventions, the patient's clinical history and response to the tumor-targeted split IL12 receptor agonist, and the discretion of the attending physician. In some embodiments, the tumor-targeted IL12R|31 agonist and tumor-targeted I L12R|32 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.

[0361] A single administration of unconjugated IL12 can range from about 50,000 lU / kg to about 1 ,000,000 lU / kg or more, more typically about 600,000 lU / kg of IL12. This may be repeated several times a day (e.g., 2-3 times.), for several days (e.g., about 3-5 consecutive days) and then may be repeated one or more times following a period of rest (e.g., about 7-14 days). Thus, a therapeutically effective amount may comprise only a single administration or many administrations over a period of time (e.g., about 20-30 individual administrations of about 600,000 lU / kg of I L12 each given over about a 10-20 day period).

[0362] Similarly, the tumor-targeted split IL12 receptor agonist is suitably administered to the patient at one time or over a series of treatments, each comprising administration of both a tumor-targeted I L12R|31 agonist and a tumor-targeted I L12R|32 agonist. Depending on the type and severity of the disease, about 1 g / kg to 15 mg / kg (e.g., 0.1 mg / kg-10 mg / kg) of each of thetumor-targeted I L12R 1 agonist and tumor-targeted I L12R 2 agonist can be an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. One typical daily dosage might range from about 1 pg / kg to 100 mg / kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment would generally be sustained until a desired suppression of disease symptoms occurs. One exemplary dosage of the IL12 receptor agonist would be in the range from about 0.005 mg / kg to about 10 mg / kg. In other non-limiting examples, a dose may also comprise from about 1 pg / kg / body weight, about 5 pg / kg / body weight, about 10 pg / kg / body weight, about 50 pg / kg / body weight, about 100 pg / kg / body weight, about 200 pg / kg / body weight, about 350 pg / kg / body weight, about 500 pg / kg / body weight, about 1 mg / kg / body weight, about 5 mg / kg / body weight, about 10 mg / kg / body weight, about 50 mg / kg / body weight, about 100 mg / kg / body weight, about 200 mg / kg / body weight, about 350 mg / kg / body weight, about 500 mg / kg / body weight, to about 1000 mg / kg / body weight or more per administration, and any range derivable therein. In non-limiting examples of a derivable range from the numbers listed herein, a range of about 5 mg / kg / body weight to about 100 mg / kg / body weight, about 5 pg / kg / body weight to about 500 mg / kg / body weight, etc., can be administered, based on the numbers described above. Thus, one or more doses of about 0.5 mg / kg, 2.0 mg / kg, 5.0 mg / kg or 10 mg / kg (or any combination thereof) may be administered to the patient. Such doses may be administered intermittently, e.g., every week or every three weeks (e.g., such that the patient receives from about two to about twenty, or e.g., about six doses of the tumor-targeted split I L12 receptor agonist). An initial higher loading dose, followed by one or more lower doses may be administered. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.

[0363] 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 ECso as determined in cell culture. Such information can be used to more accurately determine useful doses in humans.

[0364] 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.

[0365] Dosage amount and interval may be adjusted individually to provide plasma levels of the tumor-targeted I L12Rf51 agonist and tumor-targeted IL12R|32 agonist which are sufficient tomaintain therapeutic effect. Usual patient dosages for administration by injection range from about 0.1 to 50 mg / kg / day, typically from about 0.5 to 1 mg / kg / day. Therapeutically effective plasma levels may be achieved by administering multiple doses each day. Levels in plasma may be measured, for example, by ELISA HPLC.

[0366] In cases of local administration or selective uptake, the effective local concentration of the tumor-targeted I L12R[31 agonist and tumor-targeted IL12R 2 agonist may not be related to plasma concentration. One having skill in the art will be able to optimize therapeutically effective local dosages without undue experimentation.

[0367] Due to lower toxicity, the tumor-targeted split I L12 receptor agonists of the disclosure can have higher maximum therapeutic doses than wild type IL12, although, the tumor-targeted split I L12 receptor agonists are typically administered at lower doses than wild type IL12 due to the prolonged half-lives.6.13. Combination Therapy

[0368] The tumor-targeted split IL12 receptor agonists disclosed herein may be administered in combination with one or more other agents in therapy. For instance, a tumor-targeted split IL12 receptor agonist of the disclosure may be co-administered 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. In particular embodiments, the additional therapeutic is a multispecific T- cell engager as described in Section 6.6, including but not limited to the multispecific T-cell engagers set forth in Table K.

[0369] 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 split IL12 receptor agonists used, the type of disorder or treatment, and other factors discussed above. The tumor-targeted split IL12 receptor agonists are generally used inthe 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 empi rically / cl inically determined to be appropriate.

[0370] Such combination therapies noted above encompass combined administration (where two or more therapeutic agents are included in the same or separate compositions), and separate administration, in which case, administration of the tumor-targeted split IL12 receptor agonists can occur prior to, simultaneously, and / or following, administration of the additional therapeutic agent and / or adjuvant. Tumor-targeted split IL12 receptor agonists of the disclosure can also be used in combination with radiation therapy.7. SPECIFIC EMBODIMENTS

[0371] While various specific embodiments have been illustrated and described, it will be appreciated that various changes can be made without departing from the spirit and scope of the disclosure(s). The present disclosure is exemplified by the numbered embodiments set forth below. Unless otherwise specified, features of any of the concepts, aspects and / or embodiments described in the detailed description above are applicable mutatis mutandis to any of the following numbered embodiments.1. A combination comprising:(a) a tumor-targeted IL12R 1 agonist comprising:(i) a first tumor-targeting moiety (e.g., an antibody or antibody fragment comprising means for binding a tumor-associated antigen); and(ii) an I L12Rp1 binding moiety (e.g., an antibody or antibody fragment comprising means for binding I L12R|31); and(b) a tumor-targeted IL12RP2 agonist comprising:(i) a second tumor-targeting moiety (e.g., an antibody or antibody fragment comprising means for binding a tumor-associated antigen); and(ii) a I L12R 2 binding moiety (e.g., an antibody or antibody fragment comprising means for binding I L12Rp2); for use as a combination therapy, optionally for 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 clustering of I L12R 1 and I L12Rp2 receptor subunits in a lymphocyte, for eliciting signaling through the IL12 receptor in alymphocyte, 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 combination therapy for clustering of I L12R 1 and I L12R(32 receptor subunits in a lymphocyte.6. The combination of any one of embodiments 1 to 5, for use as combination therapy for eliciting signaling through the I L12 receptor in a lymphocyte.7. The combination of any one of embodiments 1 to 6, for use as a combination therapy for inducing tumor cytolysis.8. The combination of any one of embodiments 1 to 7, for use as a combination therapy for inducing anti-tumor cytotoxicity.9. The combination of any one of embodiments 1 to 8, for use as a combination therapy for stimulating an immune response against a tumor.10. A method comprising administering to a subject in need thereof a combination comprising:(a) a tumor-targeted IL12R|31 agonist (“R1 agonist”) comprising:(i) a first tumor-targeting moiety (e.g., an antibody or antibody fragment comprising means for binding a tumor-associated antigen); and(ii) an I L12R 1 binding moiety (e.g., an antibody or antibody fragment comprising means for binding I L12Rf31); and(b) a tumor-targeted IL12F?|32 agonist (“R2 agonist”) comprising:(i) a second tumor-targeting moiety (e.g., an antibody or antibody fragment comprising means for binding a tumor-associated antigen); and(ii) a IL12RP2 binding moiety (e.g., an antibody or antibody fragment comprising means for binding I L12Rp2), 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 for clustering of I L12R|31 and I L12R|32 receptor subunits in a lymphocyte, a method for eliciting signaling through the IL12 receptor in a lymphocyte, 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.11. The method of embodiment 10, which is a method for the treatment of cancer.12. The method of embodiment 10 or embodiment 11 , which is a method for the prevention or treatment of metastasis.13. The method of any one of embodiments 10 to 12, which is a method for stimulating the formation, stability and / or activity of a cytotoxic immune synapse.14. The method of any one of embodiments 10 to 13, which is a method for clustering of I L12Rp 1 and I L12R|32 receptor subunits in a lymphocyte.15. The method of any one of embodiments 10 to 14, which is a method for eliciting signaling through the IL12 receptor in a lymphocyte.16. The method of any one of embodiments 10 to 15, which is a method for inducing tumor cytolysis.17. The method of any one of embodiments 10 to 16, which is a method for inducing antitumor cytotoxicity.18. The method of any one of embodiments 10 to 17, which is a method for stimulating an immune response against a tumor.19. The combination of any one of embodiments 1 to 9 or the method of any one of embodiments 10 to 18, wherein the IL12Rp1 binding moiety and the IL12R 2 binding moiety each comprises or consists of an antigen binding domain of an antibody.20. The combination or method of embodiment 19, wherein the IL12R 1 binding moiety and the IL12RP2 binding moiety are Fabs.21. The combination or method of embodiment 19, wherein the I L12R|31 binding moiety and the IL12R 2 binding moiety are scFvs.22. The combination or method of embodiment 19, wherein the I L12Rp1 binding moiety and the I L12R|32 binding moiety are sdAbs.23. The combination or method of embodiment 22, wherein the IL12R 1 binding moiety and the I L12R|32 binding moiety are sdVHs.24. The combination or method of any one of embodiments 19 to 22, wherein the antigen binding domain of an antibody of the I L12R|31 binding moiety comprises means for binding human IL12R|31 .25. The combination or method of any one of embodiments 19 to 22, wherein the antigen binding domain of an antibody of the I L12R|32 binding moiety comprises means for binding human IL12R|32.26. The combination or method of any one of embodiments 19 to 22, wherein the I L12Rp 1 binding moiety binds to the D2 domain of I L12R|31 and the IL12R|32 binding moiety binds to the D1 domain of IL12RJ32.27. The combination or method of any one of embodiments 19 to 22, wherein the I L12R|31 binding moiety comprises means for binding to the D2 domain of I L12R(31 and the I L12R|32 binding moiety comprises means for binding to the D1 domain of I L12R(32.28. The combination of any one of embodiments 1 to 9 or the method of any one of embodiments 10 to 18, wherein:(a) the I L12R|31 binding moiety is a first IL12 moiety comprising a first p40 moiety optionally associated with a first p35 moiety; and(b) the IL12RP2 binding moiety is a second I L12 moiety comprising a second p35 moiety optionally associated with a second p40 moiety.29. The combination or method of embodiment 28, wherein:(a) the first IL12 moiety has greater selectivity to I L12Rpi than I L12R|32 as compared to wild-type IL12 (e.g., wild-type human IL12); and / or(b) the second I L12 moiety has greater selectivity to IL12RP2 than I L12R|31 as compared to wild-type IL12 (e.g., wild-type human IL12).30. The combination or method of embodiment 28 or embodiment 29, wherein the first IL12 moiety comprises a first p35 moiety and a first p40 moiety.31. The combination or method of any one of embodiments 28 to 30, wherein the first p35 moiety comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:2.32. The combination or method of any one of embodiments 28 to 30, wherein the first p35 moiety comprises an amino acid sequence having at least 96% sequence identity to the amino acid sequence of SEQ ID NO:2.33. The combination or method of any one of embodiments 28 to 30, wherein the first p35 moiety comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:2.34. The combination or method of any one of embodiments 28 to 30, wherein the first p35 moiety comprises an amino acid sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID NO:2.35. The combination or method of any one of embodiments 28 to 30, wherein the first p35 moiety comprises an amino acid sequence having at least 99% sequence identity to the amino acid sequence of SEQ ID NO:2.36. The combination or method of any one of embodiments 28 to 35, wherein the first p35 moiety is a variant p35 moiety having reduced binding to 11_12R[32 as compared to a wild-type p35 moiety (e.g., a p35 moiety having the amino acid sequence of SEQ ID NO:2).37. The combination or method of embodiment 36, wherein the variant p35 moiety comprises one or more of the mutations set forth in Table B1 , optionally wherein the one or more mutations comprise or consist of Y189E as compared to wild-type p35 (e.g., wherein the variant p35 moiety comprises the amino acid sequence of SEQ ID NQ:40).38. The combination or method of any one of embodiments 28 to 30, wherein the first p35 moiety comprises the amino acid sequence of SEQ ID NO:2.39. The combination or method of any one of embodiments 28 to 38, wherein the first p40 moiety comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:6 or SEQ ID NO:7.40. The combination or method of any one of embodiments 28 to 38, wherein the first p40 moiety comprises an amino acid sequence having at least 96% sequence identity to the amino acid sequence of SEQ ID NO:6 or SEQ ID NO:7.41. The combination or method of any one of embodiments 28 to 38, wherein the first p40 moiety comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:6 or SEQ ID NO:7.42. The combination or method of any one of embodiments 28 to 38, wherein the first p40 moiety comprises an amino acid sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID NO:6 or SEQ ID NO:7.43. The combination or method of any one of embodiments 28 to 38, wherein the first p40 moiety comprises an amino acid sequence having at least 99% sequence identity to the amino acid sequence of SEQ ID NO:6 or SEQ ID NO:7.44. The combination or method of any one of embodiments 28 to 38, wherein the first p40 moiety comprises the amino acid sequence of SEQ ID NO:6 or SEQ ID NO:7.45. The combination or method of any one of embodiments 28 to 44, wherein the second IL12 moiety comprises a second p35 moiety and a second p40 moiety.46. The combination or method of embodiment 45, wherein the second p35 moiety comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:2.47. The combination or method of embodiment 45, wherein the second p35 moiety comprises an amino acid sequence having at least 96% sequence identity to the amino acid sequence of SEQ ID NO:2.48. The combination or method of embodiment 45, wherein the second p35 moiety comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:2.49. The combination or method of embodiment 45, wherein the second p35 moiety comprises an amino acid sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID NO:2.50. The combination or method of embodiment 45, wherein the second p35 moiety comprises an amino acid sequence having at least 99% sequence identity to the amino acid sequence of SEQ ID NO:2.51. The combination or method of embodiment 45, wherein the second p35 moiety comprises the amino acid sequence of SEQ ID NO:2.52. The combination or method of any one of embodiments 45 to 51 , wherein the second p40 moiety comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:6 or SEQ ID NO:7.53. The combination or method of any one of embodiments 45 to 51 , wherein the second p40 moiety comprises an amino acid sequence having at least 96% sequence identity to the amino acid sequence of SEQ ID NO:6 or SEQ ID NO:7.54. The combination or method of any one of embodiments 45 to 51 , wherein the second p40 moiety comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:6 or SEQ ID NO:7.55. The combination or method of any one of embodiments 45 to 51 , wherein the second p40 moiety comprises an amino acid sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID NO:6 or SEQ ID NO:7.56. The combination or method of any one of embodiments 45 to 51 , wherein the second p40 moiety comprises an amino acid sequence having at least 99% sequence identity to the amino acid sequence of SEQ ID NO:6 or SEQ ID NO:7.57. The combination or method of embodiment 56, wherein the second p40 moiety is a variant p40 moiety having reduced binding to I L12R 1 as compared to a wild-type p40 moiety.58. The combination or method of embodiment 57, wherein the variant p40 moiety comprises a D1 domain mutation or a D1 domain deletion.59. The combination or method of any one of embodiments 45 to 58, wherein the second p40 moiety comprises the amino acid sequence of SEQ ID NO:6.60. The combination or method of any one of embodiments 28 to 59, wherein the first p35 moiety and the first p40 moiety are separated by a linker (a first “IL12 moiety linker”) and / or thesecond p35 moiety and the second p40 moiety are separated by a linker (a second “IL12 moiety linker”).61. The combination or method of embodiment 60, wherein the first I L12 moiety linker and / or the second IL12 moiety linker are each at least 5 amino acids in length.62. The combination or method of embodiment 60, wherein the first IL12 moiety linker and / or the second IL12 moiety linker are each least 10 amino acids in length.63. The combination or method of embodiment 60, wherein the first IL12 moiety linker and / or the second IL12 moiety linker are each least 15 amino acids in length.64. The combination or method of any one of embodiments 60 to 63, wherein the first IL12 moiety linker and / or the second IL12 moiety linker are a glycine-serine linker.65. The combination or method of any one of embodiments 60 to 64, wherein the first IL12 moiety linker and / or the second IL12 moiety linker comprises the amino acid sequence G4S.66. The combination or method of embodiment 64, wherein the first IL12 moiety linker and / or the second IL12 moiety linker comprises a multimer of the amino acid sequence G4S.67. The combination or method of embodiment 66, wherein the multimer comprises 2, 3, 4, 5, 6 or more repeats of the amino acid sequence G4S.68. The combination or method of any one of embodiments 60 to 67, wherein the first IL12 moiety linker and / or the second IL12 moiety linker is a non-cleavable linker.69. The combination of any one of embodiments 1 to 9 and 19 to 68 or the method of any one of embodiments 10 to 68, wherein the first tumor-targeting moiety binds to a first tumor- associated antigen and the second tumor-targeting moiety binds to a second tumor-associated antigen.70. The combination or method of embodiment 69, wherein the first tumor-associated antigen and the second tumor-associated antigen are expressed on the same tumor cell.71. The combination or method of embodiment 69 or embodiment 70, wherein the first tumor-associated antigen and the second tumor-associated antigen are different.72. The combination or method of embodiment 69 or embodiment 70, wherein the first tumor-associated antigen and the second tumor-associated antigen are the same.73. The combination or method of embodiment 72, wherein the first tumor-targeting moiety and the second tumor-targeting moiety are the same.74. The combination or method of embodiment 72, wherein the first tumor-targeting moiety and the second tumor-targeting moiety are different, e.g., bind to different epitopes.75. The combination or method of embodiment 72 or embodiment 74, wherein the first tumor-targeting moiety and the second tumor-targeting moiety do not compete for binding to the tumor-associated antigen.76. The combination or method of any one of embodiments 69 to 75, wherein the first tumortargeting moiety and / or the second tumor-targeting moiety are Fabs.77. The combination or method of any one of embodiments 69 to 75, wherein the first tumortargeting moiety and / or the second tumor-targeting moiety are scFvs.78. The combination or method of any one of embodiments 69 to 75, wherein the first tumortargeting moiety and / or the second tumor-targeting moiety are sdAbs.79. The combination or method of embodiment 78, wherein the first tumor-targeting moiety and / or the second tumor-targeting moiety are sdVHs.80. The combination or method of any one of embodiments 69 to 79, wherein the first tumortargeting moiety and / or second tumor-targeting moiety bind(s) to Fibroblast Activation Protein (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, 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 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, [3-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, PDGFfBR ([3-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, -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)-I, IGF-II, IGFI receptor, 5T4, ROR1 , Nkp30, NKG2D, tumor stromal antigens, CA166-9, the extra domain A (EDA) of fibronectin or the A1 domain of tenascin-C(TnC A1).81. The combination or method of any one of embodiments 69 to 79, wherein the first tumortargeting moiety and / or second tumor-targeting moiety bind(s) to human PSMA.82. The combination or method of any one of embodiments 69 to 79, wherein the first tumortargeting moiety and / or second tumor-targeting moiety bind(s) to human MSLN.83. The combination or method of any one of embodiments 69 to 79, wherein the first tumortargeting moiety and / or second tumor-targeting moiety bind(s) to human MUC16.84. The combination or method of any one of embodiments 69 to 79, first tumor-targeting moiety and / or second tumor-targeting moiety comprise means for binding to a tumor antigen.85. The combination or method of any one of embodiments 69 to 79, first tumor-targeting moiety and / or second tumor-targeting moiety comprise means for binding human PSMA.86. The combination or method of any one of embodiments 69 to 79, first tumor-targeting moiety and / or second tumor-targeting moiety comprise means for binding human MSLN.87. The combination or method of any one of embodiments 69 to 79, first tumor-targeting moiety and / or second tumor-targeting moiety comprise means for binding human MUC16.88. The combination of any one of embodiments 1 to 9 and 19 to 87 or the method of any one of embodiments 10 to 87, wherein the tumor-targeted I L12 R|31 agonist comprises(a) a first polypeptide chain comprising, in N- to C-terminal orientation:(i) the first tumor-targeting moiety or component thereof (or a component thereof, e.g., a VH-CH1 or VL-CL), optionally associated with another component thereof on a separate polypeptide chain (or a component thereof, e.g., a VL-CL or VH-CH1);(ii) optionally, a linker (a “TAA-Fc linker”); and(iii) a first Fc domain; and(b) a second polypeptide chain comprising, in N- to C-terminal orientation:(i) the IL12RP1 binding moiety or component thereof (or a component thereof, e.g., a VH-CH1 or VL-CL), optionally associated with another component thereof on a separate polypeptide chain (or a component thereof, e.g., a VL-CL or VH-CH1);(ii) optionally, a linker (e.g., where the IL12R 1 binding moiety is an IL12 moiety as defined in any one of embodiments 28 to 68) (“an IL12-Fc linker”), optionally wherein the IL12-Fc linker is as defined in any one of embodiments 61 to 67); and(iii) a second Fc domain associated with the first Fc domain.89. The combination or method of embodiment 88, wherein the first and second Fc domains are lgG1 , lgG2, lgG3 or lgG4 Fc domains.90. The combination or method of embodiment 88 or embodiment 89, wherein the first Fc domain and second Fc domain each comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:11.91. The combination or method of embodiment 88 or embodiment 89, wherein the first Fc domain and second Fc domain each comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 12.92. The combination or method of embodiment 88 or embodiment 89, wherein the first Fc domain and second Fc domain each comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 13.93. The combination or method of any one of embodiments 88 to 92, wherein the first Fc domain and second Fc domain each comprises a chimeric hinge domain.94. The combination or method of embodiment 93, wherein the first Fc domain and second Fc domain each comprises an amino acid sequence having at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 14.95. The combination or method of embodiment 93, wherein the first Fc domain and second Fc domain each comprises an amino acid sequence having at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 15.96. The combination or method of any one of embodiments 88 to 95, wherein the first Fc domain and second Fc domain each has reduced effector function.97. The combination or method of any one of embodiments 88 to 96, wherein the first Fc domain and second Fc domain form an Fc heterodimer.98. The combination of any one of embodiments 1 to 9 and 19 to 97 or the method of any one of embodiments 10 to 97, wherein the tumor-targeted IL12R 2 agonist comprises(a) a third polypeptide chain comprising, in N- to C-terminal orientation:(i) the second tumor-targeting moiety or component thereof (or a component thereof, e.g., a VH-CH1 or VL-CL), optionally associated with anothercomponent thereof on a separate polypeptide chain (or a component thereof, e.g., a VL-CL or VH-CH1);(ii) optionally, a linker (a “TAA-Fc linker”); and(iii) a third Fc domain; and(b) a third polypeptide chain comprising, in N- to C-terminal orientation:(i) the IL12RP2 binding moiety or component thereof (or a component thereof, e.g., a VH-CH1 or VL-CL), optionally associated with another component thereof on a separate polypeptide chain (or a component thereof, e.g., a VL-CL or VH-CH1);(ii) optionally, a linker (e.g., where the IL12R 2 binding moiety is an IL12 moiety as defined in any one of embodiments 28 to 68) (“an IL12-Fc linker”), optionally wherein the IL12-Fc linker is as defined in any one of embodiments 61 to 67); and(iii) a fourth Fc domain associated with the third Fc domain.99. The combination or method of embodiment 98, wherein the third and fourth Fc domains are lgG1 , lgG2, lgG3 or lgG4 Fc domains.100. The combination or method of embodiment 98 or embodiment 99, wherein the third Fc domain and fourth Fc domain each comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:11.101. The combination or method of embodiment 98 or embodiment 100, wherein the third Fc domain and fourth Fc domain each comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 12.102. The combination or method of embodiment 98 or embodiment 100, wherein the third Fc domain and fourth Fc domain each comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 13.103. The combination or method of any one of embodiments 98 102, wherein the third Fc domain and fourth Fc domain each comprises a chimeric hinge domain.104. The combination or method of any one of embodiments 98 103, wherein the third Fc domain and fourth Fc domain each has reduced effector function.105. The combination or method of any one of embodiments 98 104, wherein the third Fc domain and fourth Fc domain form an Fc heterodimer.106. The combination of any one of embodiments 1 to 9 and 19 to 105 or the method of any one of embodiments 10 to 105, wherein the tumor-targeted I L12R 1 agonist is monovalent for the first tumor-targeting moiety.107. The combination of any one of embodiments 1 to 9 and 19 to 106 or the method of any one of embodiments 10 to 106, wherein the tumor-targeted I L12R|31 agonist is monovalent for the IL12RP1 binding moiety.108. The combination of any one of embodiments 1 to 9 and 19 to 107 or the method of any one of embodiments 10 to 107, wherein the tumor-targeted I L12Rp2 agonist is monovalent for the second tumor-targeting moiety.109. The combination of any one of embodiments 1 to 9 and 19 to 108 or the method of any one of embodiments 10 to 108, wherein the tumor-targeted I L12R|32 agonist is monovalent for the IL12R 2 binding moiety.110. The combination of any one of embodiments 1 to 9 and 19 to 109 or the method of any one of embodiments 10 to 109, wherein the tumor-targeted I L12Rf31 agonist and the tumor- targeted I L12R|32 agonist are both in the form of a pharmaceutical composition comprising the agonist and an excipient.111. The combination or method of embodiment 110, wherein the tumor-targeted I L12R|31 agonist and the tumor-targeted IL12RP2 agonist are in the same pharmaceutical composition.112. The combination or method of embodiment 110, wherein the tumor-targeted I L12 R[31 agonist and the tumor-targeted IL12R 2 agonist are in different pharmaceutical compositions.113. The combination of any one of embodiments 1 to 9 and 19 to 112 or the method of any one of embodiments 10 to 112, wherein tumor-targeted I L12Rp 1 agonist is configured as illustrated in FIG. 1A.114. The combination of any one of embodiments 1 to 9 and 19 to 112 or the method of any one of embodiments 10 to 112, wherein tumor-targeted I L12Rp 1 agonist is configured as illustrated in FIG. 1 B.115. The combination of any one of embodiments 1 to 9 and 19 to 112 or the method of any one of embodiments 10 to 112, wherein tumor-targeted I L12R|31 agonist is configured as illustrated in FIG. 1C.116. The combination of any one of embodiments 1 to 9 and 19 to 112 or the method of any one of embodiments 10 to 112, wherein tumor-targeted I L12Rp 1 agonist is configured as illustrated in FIG. 1 D.117. The combination of any one of embodiments 1 to 9 and 19 to 112 or the method of any one of embodiments 10 to 112, wherein tumor-targeted I L12R|31 agonist is configured as illustrated in FIG. 1 E.118. The combination of any one of embodiments 1 to 9 and 19 to 112 or the method of any one of embodiments 10 to 112, wherein tumor-targeted I L12R|31 agonist is configured as illustrated in FIG. 1 F.119. The combination of any one of embodiments 1 to 9 and 19 to 112 or the method of any one of embodiments 10 to 112, wherein tumor-targeted IL12R 1 agonist is configured as illustrated in FIG. 1G.120. The combination of any one of embodiments 1 to 9 and 19 to 112 or the method of any one of embodiments 10 to 112, wherein tumor-targeted I L12Rp 1 agonist is configured as illustrated in FIG. 1 H.121. The combination of any one of embodiments 1 to 9 and 19 to 112 or the method of any one of embodiments 10 to 112, wherein tumor-targeted I L12Rp 1 agonist is configured as illustrated in FIG. 11.122. The combination of any one of embodiments 1 to 9 and 19 to 112 or the method of any one of embodiments 10 to 112, wherein tumor-targeted I L12Rp 1 agonist is configured as illustrated in FIG. 1J.123. The combination of any one of embodiments 1 to 9 and 19 to 112 or the method of any one of embodiments 10 to 112, wherein tumor-targeted I L12R|31 agonist is configured as illustrated in FIG. 1 K.124. The combination of any one of embodiments 1 to 9 and 19 to 112 or the method of any one of embodiments 10 to 112, wherein tumor-targeted I L12Rp 1 agonist is configured as illustrated in FIG. 1 L.125. The combination of any one of embodiments 1 to 9 and 19 to 124 or the method of any one of embodiments 10 to 124, wherein tumor-targeted I L12R|32 agonist is configured as illustrated in FIG. 2A.126. The combination of any one of embodiments 1 to 9 and 19 to 124 or the method of any one of embodiments 10 to 124, wherein tumor-targeted I L12R|32 agonist is configured as illustrated in FIG. 2B.127. The combination of any one of embodiments 1 to 9 and 19 to 124 or the method of any one of embodiments 10 to 124, wherein tumor-targeted IL12R 2 agonist is configured as illustrated in FIG. 2C.128. The combination of any one of embodiments 1 to 9 and 19 to 124 or the method of any one of embodiments 10 to 124, wherein tumor-targeted I L12Rp2 agonist is configured as illustrated in FIG. 2D.129. The combination of any one of embodiments 1 to 9 and 19 to 124 or the method of any one of embodiments 10 to 124, wherein tumor-targeted I L12Rp2 agonist is configured as illustrated in FIG. 2E.130. The combination of any one of embodiments 1 to 9 and 19 to 124 or the method of any one of embodiments 10 to 124, wherein tumor-targeted I L12Rp2 agonist is configured as illustrated in FIG. 2F.131. The combination of any one of embodiments 1 to 9 and 19 to 124 or the method of any one of embodiments 10 to 124, wherein tumor-targeted I L12R|32 agonist is configured as illustrated in FIG. 2G.132. The combination of any one of embodiments 1 to 9 and 19 to 124 or the method of any one of embodiments 10 to 124, wherein tumor-targeted IL12R 2 agonist is configured as illustrated in FIG. 2H.133. The combination of any one of embodiments 1 to 9 and 19 to 124 or the method of any one of embodiments 10 to 124, wherein tumor-targeted I L12R|32 agonist is configured as illustrated in FIG. 2I.134. The combination of any one of embodiments 1 to 9 and 19 to 124 or the method of any one of embodiments 10 to 124, wherein tumor-targeted I L12R|32 agonist is configured as illustrated in FIG. 2J.135. The combination of any one of embodiments 1 to 9 and 19 to 124 or the method of any one of embodiments 10 to 124, wherein tumor-targeted IL12R 2 agonist is configured as illustrated in FIG. 2K.136. The combination of any one of embodiments 1 to 9 and 19 to 124 or the method of any one of embodiments 10 to 124, wherein tumor-targeted I L12Rp2 agonist is configured as illustrated in FIG. 2L.137. The combination of any one of embodiments 1 to 9 and 19 to 135 or the method of any one of embodiments 10 to 135, wherein the combination further comprises or the method furthercomprises administering a multispecific T-cell engager (e.g., simultaneously, sequentially or separately).138. The combination or method of embodiment 137, wherein the multispecific T-cell engager is a bispecific T-cell engager.139. The combination or method of embodiment 137 or 138, wherein the multispecific T-cell engager comprises means for binding a TAA and means for binding CD3.140. The combination or method of embodiment 137 or 138, wherein the multispecific T-cell engager comprises a TAA targeting moiety (e.g., an antibody or antigen-binding fragment comprising means for binding a TAA) and a CD3 targeting moiety (e.g., an antibody or antigenbinding fragment comprising means for binding CD3).141. The combination or method of embodiment 140, wherein the TAA targeting moiety of the multispecific T-cell engager targets the same TAA as the TAA targeted by the tumor-targeted IL12R|31 agonist and / or the tumor-targeted IL12R|32 agonist.142. The combination or method of embodiment 140, wherein the TAA targeting moiety of the multispecific T-cell engager targets a TAA that is different from the TAA targeted by the tumor- targeted IL12R|31 agonist and / or the tumor-targeted IL12Rp2 agonist.143. The combination of any one of embodiments 1 to 9 and 19 to 142 for use as a medicament.144. The combination for use of embodiment 143 for use in a method for the treatment of cancer.145. The combination for use of embodiment 143 or 144 for use in a method for the prevention or treatment of metastasis.146. The combination for use of any one of embodiments 143 to 145, for use in a method of stimulating the formation, stability and / or activity of a cytotoxic immune synapse.147. The combination for use of any one of embodiments 143 to 146, for use in a method of clustering of I L12Rf31 and I L12Rp2 receptor subunits in a lymphocyte.148. The combination for use of any one of embodiments 143 to 147, for use in a method of eliciting signaling through the I L12 receptor in a lymphocyte.149. The combination for use of any one of embodiments 143 to 148, for use in a method of inducing tumor cytolysis.150. The combination for use of any one of embodiments 143 to 149, for use in a method of inducing anti-tumor cytotoxicity.151. The combination for use of any one of embodiments 143 to 150, for use in a method of stimulating an immune response against a tumor.152. The combination for use of any one of embodiments 143 to 151 , for use in a method for the treatment of cancer.153. A tumor-targeted I L12R|31 agonist for use in a method for the treatment of cancer, wherein(a) the I L12R|31 agonist is defined as in any one of embodiments 1 to 136, and(b) the method comprises administering to a subject in need thereof the tumor- targeted I L12R|31 agonist and a tumor-targeted I L12R(32 agonist as defined in any one of embodiments 1 to 136.154. The tumor-targeted I L12R|31 agonist for use of embodiment 153, wherein the method is a method of combination therapy for the prevention or treatment of metastasis.155. The tumor-targeted I L12R|31 agonist for use of embodiment 153 or 154, wherein the method is a method of combination therapy for stimulating the formation, stability and / or activity of a cytotoxic immune synapse.156. The tumor-targeted IL12R|31 agonist for use of any one of embodiments 153 to155, wherein the method is a method of clustering of I L12R|31 and I L12Rp2 receptor subunits in a lymphocyte.157. The tumor-targeted IL12R|31 agonist for use of any one of embodiments 153 to156, wherein the method is a method of eliciting signaling through the IL12 receptor in a lymphocyte.158. The tumor-targeted IL12R|31 agonist for use of any one of embodiments 153 to157, wherein the method is a method of inducing tumor cytolysis.159. The tumor-targeted IL12R|31 agonist for use of any one of embodiments 153 to158, wherein the method is a method of inducing anti-tumor cytotoxicity.160. The tumor-targeted IL12R|31 agonist for use of any one of embodiments 153 to159, wherein the method is a method of stimulating an immune response against a tumor.161. A tumor-targeted IL12R|32 agonist for use in a method for the treatment of cancer, wherein(a) the I L12R|32 agonist is defined as in any one of embodiments 1 to 136, and(b) the method comprises administering to a subject in need thereof the tumor- targeted I L12R|32 agonist and a tumor-targeted I L12R(31 agonist as defined in any one of embodiments 1 to 136.162. The tumor-targeted I L12R|32 agonist for use of embodiment 161 , wherein the method is a method of combination therapy for the prevention or treatment of metastasis.163. The tumor-targeted I L12R|32 agonist for use of embodiment 161 or 162, wherein the method is a method of combination therapy for stimulating the formation, stability and / or activity of a cytotoxic immune synapse.164. The tumor-targeted I L12R|32 agonist for use of any one of embodiments 161 to 163, wherein the method is a method of clustering of I L12R 1 and I L12Rp2 receptor subunits in a lymphocyte.165. The tumor-targeted I L12R|32 agonist for use of any one of embodiments 161 to164, wherein the method is a method of eliciting signaling through the I L12 receptor in a lymphocyte.166. The tumor-targeted I L12R|32 agonist for use of any one of embodiments 161 to165, wherein the method is a method of inducing tumor cytolysis.167. The tumor-targeted IL12RP2 agonist for use of any one of embodiments 161 to166, wherein the method is a method of inducing anti-tumor cytotoxicity.168. The tumor-targeted I L12R|32 agonist for use of any one of embodiments 161 to167, wherein the method is a method of stimulating an immune response against a tumor.8. EXAMPLES8.1. Materials and Methods8.1.1. Design and Production of Tumor-targeted IL12R 1 and IL12R[32 Agonist Constructs

[0372] Exemplary tumor-targeted IL12R 1 agonists as depicted in FIG. 1A were designed to comprise two polypeptides herein referred to as the first and second polypeptides, the first polypeptide comprising from N- to C-terminal, a first tumor targeting moiety in Fab format, a linker (“TAA linker”), and a first Fc domain which enables dimerization; and the second polypeptide comprising from N- to C-terminal, an IL12R 1 binding moiety comprising an I L12 moiety in the format of a p40-p35 fusion peptide comprising a wild-type p40 moiety linked to a mutated p35 moiety with an I L12 moiety linker, a linker (“IL12-Fc linker”), and a second Fc domain that forms a dimer with the first Fc domain. Exemplary tumor-targeted IL12R 1 agonists as depicted in FIG. 1 B were designed similarly, this time the I L12RJ31 binding moiety comprising an anti-l L12R 1 Fab moiety.

[0373] Similarly, exemplary tumor-targeted I L12R|32 agonists as depicted in FIG. 2A were designed to comprise two polypeptides herein referred to as the third and fourth polypeptides, the third polypeptide comprising from N- to C-terminal, a second tumor targeting moiety in Fab format, a linker (“TAA linker”), and a third Fc domain which enables dimerization; and the fourth polypeptide comprising from N- to C-terminal, an IL12 moiety in the format of a p40-p35 fusionpeptide comprising a mutated p40 moiety linked to a wild-type p35 moiety with an IL12 moiety linker, a linker (“IL12-Fc linker”), and a fourth Fc domain that forms a dimer with the third Fc domain. Exemplary tumor-targeted ll_12R|32 agonists as depicted in FIG.2B were designed similarly, this time the I L12R|32 binding moiety comprising an anti-l L12R|32 Fab moiety.

[0374] The details of exemplary tumor-targeted I L12R|31 and I L12R|32 agonist constructs produced are provided in Table E1 below.

[0375] The constructs were expressed in Expi293F™ cells by transient transfection following the manufacturer’s protocol (Thermo Fisher Scientific). Proteins in Expi293FTMsupernatant were purified using the ProteinMaker system (Protein BioSolutions, Gaithersburg, MD) with either HiTrap™ Protein or MabSelect SuRe columns (Cytiva). 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.8.1.2. STAT3 Reporter Assay

[0376] A Signal Transducer and Activator of Transcription 3 (STAT3)-driven luciferase-based reporter assay was used to evaluate the ability of IL12 polypeptides to activate STAT3-mediated transcription in the human natural killer cell line, NK92. Briefly, NK92 cells were transduced with a STAT3 response element driven luciferase reporter construct and maintained in alpha minimum essential medium without nucleosides + 2 mM L-glutamine / Pen / Strep + 1.5 g / L sodium bicarbonate + 12.5% horse serum + 12.5% FBS + 0.2 mM inositol + 0.1 mM 2- mercaptoethanol + 0.02 mM folic acid + 200U / mL recombinant hlL-2 + 1mg / ml_ puromycin.

[0377] RPMI1640 supplemented with 10% FBS and P / S / G was used as assay medium to prepare cell suspensions and fusion protein dilutions. A day prior to the assay, cells were spun down and resuspended at 5 x 105cells / mL in alpha minimum essential medium without nucleosides + 2 mM L-glutamine / Pen / Strep + 1.5 g / L sodium bicarbonate + 12.5% horse serum + 12.5% FBS + 0.2 mM inositol + 0.1 mM 2-mercaptoethanol + 0.02 mM folic acid. On the day of the assay, NK92 / STAT3-Luc cells were spun down, resuspended in assay medium and added to plates at 2.5 x 104cells / well. Target cells (Raji Raji / hPSMA, Jurkat, or Jurkat / PSMA) were spun down, resuspended in assay medium and added to plates at 2.5 x 104cells / well. Tumor-targeted IL12R|31 and I L12R|32 agonists were serially diluted (range: 50nM to 29.8fM) alone or in equal molar combination and added to cells for 4 hours at 37 °C and 5% CO2 prior to addition of One-Gio Luciferase Substrate to lyse cells and detect luciferase activity. The emitted light was captured in relative light units (RLU) on a multilabel plate reader Envision (PerkinElmer). All serial dilutions were tested in duplicates. EC50 values of the antibodies were determined using GraphPad Prism™ software from a four-parameter logistic equation over a 10-point dose-response curve.8.1.3. IFNy Release Assay

[0378] X-Vivo 15 supplemented with 10% FBS, HEPES, NEAA, Sodium Pyruvate and 10 uM BME was used as assay medium to prepare cell suspensions and protein dilutions. Human peripheral blood mononuclear cells (PBMCs) were isolated from healthy donor leukocyte packs using the EasySep™ Direct Human PBMC Isolation Kit and following the manufacturers recommended protocol. Subsequently, CD3+ T-cells were isolated using an EasySepTM Human CD3+ T Cell Isolation Kit from StemCell Technologies and following the manufacturer’s recommended instructions. T-cells were spun down, resuspended in assay medium and added to plates at 3 x 104cells / well. Target cells (Raji, Raji / hPSMA, or LNCaP) were spun down, resuspended in assay medium and added to plates at 5 x 103cells / well. A CD3xCD20 antibody (for Raji and Raji / PSMA cells) or CD3xSTEAP1 antibody (for LNCaP cells) was diluted in assay medium and added to plates at a constant amount of 30nM or 750pM, respectively. Tumor- targeted I L12Rp1 and IL12R 2 agonists were serially diluted (range: 50nM to 29.8fM) alone or in equal molar combination and added to cells, and plates were incubated for 72 hours at 37 °C and 5% CO2. On Day 3, 5 pL of supernatant was removed from each well and IFNy levels were detected using IFNyalphaLISA (Perkin Elmer). All serial dilutions were tested in duplicates. EC50 values of the antibodies were determined using GraphPad Prism™ software from a four- parameter logistic equation over a 10-point dose-response curve.8.1.4. pSTAT4 Assay

[0379] Human peripheral blood mononuclear cells (PBMCs) were pre-activated by culturing for 72 hours in assay medium (RPMI + 10%FBS + 2 mM L-glutamine / Pen / Strep) in the presence of CD3 / CD28 Dynabeads™ (Thermo / 11132D) at a 1 :2 (beads: PBMC) ratio and in the presence of 30 U / mL human IL-2 (proleukin). Beads were removed and activated PBMCs were rested in assay medium for 1 hour at 37 °C. C4-2 or OVCAR3 tumor cells were spun down, resuspended in assay medium and added to plates at 5 x 104cells / well. Tumor-targeted IL12R|31 and I L12Rp2 agonists were serially diluted (range: 100nM to 47.7fM) alone or in equal molar combination and added to the tumor cells, followed by addition of activated PBMCs at 5 x 104cells / well. Plates were incubated for 1 hour at 37 °C before fixation with Cytofix buffer (BD / 554655) for 12 minutes at 37 °C. Cells were then permeabilization with pre-chilled Perm Buffer III (BD / 558050) for 20 mins on ice and washed 2 times. Cells were then stained with aCD3(BD / 563918), aCD8 (BD / 563795), aCD4(BD / 612936), aCD25 (BD / 562442), and pSTAT4 (BD / 558137) for 1 hour. Cells were washed twice before data was acquired using a Cytek Aurora flow cytometer. %pSTAT4 positive or pSTAT4 gMFI (geometric mean fluorescence intensity) is plotted.8.1.5. In vitro Target Cell Killing Assay

[0380] Human peripheral blood mononuclear cells (PBMCs) were isolated from healthy donor leukocyte packs using the EasySep™ Direct Human PBMC Isolation Kit and following the manufacturers recommended protocol. Subsequently, CD3+ T-cells were isolated using an EasySepTM Human CD3+ T Cell Isolation Kit from StemCell Technologies and following the manufacturer’s recommended instructions. Media used for maintaining CD3+ T-cells and conducting experiments consisted of X-VIVO 15 cell culture media supplemented with 10% FBS, HEPES, NaPyr, NEAA, and 0.01 mM BME. CD3+ T-cells were pre-activated with CD3 / CD28 Dynabeads™ (Thermo / 11132D) and human IL-2 (proleukin) for 3 days. Target cells for this assay were HEK293 cells engineered to co-express hPSMA and hMUC16, HEK293 cells which only express MUC16, LNCaP tumor cells, or OVCAR3 tumor cells. All target cells also express 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 will be reduced. Activated T-cells and target cells were incubated together with 6pM constant dose of MUC16xCD3 (for HEK293 cells), 750pM constant dose of STEAP1xCD3 (for LNCaP cells), a constant dose of MSLNxCD3 (for OVCAR3 cells treated with MUC 16- targeted I L12R|31 and I L12R|32 agonists), or a constant dose of MUC16xCD3 (for OVCAR3 cells treated with MSLN- targeted I L12Rp1 and IL12R 2 agonists), along with a serial dilution (either 9 point, 5-foldtitration starting at 20nM or a 10-point, 5-fold titration starting at 100nM) of hPSMA, hMUC16, or hMSLN-targeted I L12R|31 and I L12Rf32 agonists alone or in equal molar combination. The lowest point on the curve contains no titrated IL12R or Isotype control molecules. Assay was read out after 3 days at 37C 5% CO2. Prior to addition of One-Gio to sample wells for detecting luminescence, supernatant was collected for assessing IFNy release. 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 either a 9 point or 10- point dose response curve using GraphPad Prism software. For assessing IFNy release, commercially available immunoassays were used.8.2. Example 1 : Activation of STAT3-Signaling by the Combination of IL12 Moiety-Comprising Tumor-targeted IL12R 1 and ll_12R|32 Agonists

[0381] IL12 moiety comprising tumor-targeted IL12Rpi and IL12R 2 agonists IL12R[31-Agonist 1 (IL12RP1-Ag1), IL12R[32-Agonist 1 (IL12R[32-Ag1), and IL12R[32-Agonist 2 (IL12R[32-Ag2) were designed and produced as described in Section 8.1.1. The tumor targeting moieties and IL12R binding moieties of these tumor-targeted I L12Rp1 and IL12Rp2 agonist constructs used in this example are set forth in Table E2. The ability of IL12 moiety-comprising tumor-targeted I L12Rp 1 and IL12R 2 agonists to activate STAT3-signaling was assessed in a STAT3-reporter cell-based assay as described in Section 8.1.2.

[0382] The constructs IL12Rp1-Ag1 , IL12R 2-Ag1, and IL12Rp2-Ag2 alone or combinations comprising IL12Rpi-Ag1 + IL12Rp2-Ag1 or IL12Rp1-Ag1 + IL12Rp2-Ag2 did not trigger STAT signaling activation in the absence of PSMA-expressing cells (FIGS. 4A-4B).

[0383] When NK92 / STAT3-Luc cells were co-cultured with hPSMA-expressing Raji cells, both combinations were associated with STAT3-Luc activity, in which combination of I L12R i -Ag1 + IL12R[32-Ag2 produced a stronger activation that that produced by the combination of I L12Rp 1 - Ag1 + IL12R|32-Ag1 (FIGS. 4C-4D).8.3. Example 2: Activation of STAT3-Signaling and IFNy Release by the Combination of Tumor-targeted IL12RP1 and IL12RP2 Agonists

[0384] Tumor-targeted IL12Rp1 and IL12F?[32 agonists comprising Fab arms were designed and produced as described in Section 8.1.1. The tumor targeting moieties and IL12R binding moieties of these tumor-targeted IL12R|31 and IL12Rp2 agonist constructs used in this example are set forth in Table E3. The ability of tumor-targeted I L12R|31 and 11_12R|32 agonists to activate STAT3-signaling was assessed in a STAT3-reporter cell-based assay as described in Section 8.1.2. The ability of tumor-targeted IL12Rp1 and IL12Rp2 agonists to induce the release of IFNy from T-cells was assessed as described in Section 8.1.3.

[0385] First, the tumor-targeted IL12R 1 and IL12R 2 agonists were evaluated alone or in combinations using NK92 / STAT3-Luc cells that were co-cultured with Raji cells that do not express PSMA. Only control bispecific antibodies comprising an anti-l L12p1 Fab arm and an anti-l L12|32 Fab arm were associated with STAT3-Luc activity (FIG. 5A). Next, the tumor- targeted I L12Rp1 and IL12R 2 agonists were evaluated alone or in combinations using NK92 / STAT3-Luc cells that were co-cultured with hPSMA-expressing Raji cells. In this case, the four tumor-targeted IL12R 1 and I L12Rp2 agonist combinations resulted in STAT3-Luc activity, and the strongest STAT3-Luc activation among the four combinations was observed with IL12Rpi-Ag4 + IL12R[32-Ag3. These results suggest that the tumor-targeted I L12R|31 and I L12Rp2 agonist combinations induce STAT signaling activation only in the presence of PSMA- expressing tumor cells.

[0386] Next, the combinations of tumor-targeted IL12R|31 and IL12R|32 agonists to induce the release of IFNy from T-cells was evaluated in the presence of either Raji cells that do notexpress PSMA or in the presence of PSMA-expressing Raji cells. The combination of I L12R01- Ag3 + IL12R[32-Ag6 was not associated with IFNy release in the presence of Raji cells that do not express PSMA (FIG. 6A), but induced IFNy release in the presence of hPSMA-expressing Raji cells (FIG. 6B). Similarly, the combination of I L12R|31 -Ag4 + IL12R02-Ag3 was not associated with IFNy release in the presence of Raji cells that do not express PSMA (FIG. 6C), but induced IFNy release in the presence of hPSMA-expressing Raji cells (FIG. 6D). These results suggest that the combinations of tumor-targeted IL12Rp1 and IL12R02 agonists induce the release of IFNy from T-cells only in the presence of PSMA-expressing cells.8.4. Example 3: Activation of pSTAT4 Signaling, IFNy Release and Target Cell Killing by Tumor-targeted IL12R01 and IL12R02 Agonist Combinations

[0387] The tumor-targeted I L12R|31 and IL12R|32 agonists generated and used in Section 8.3 were evaluated for their ability to activate pSTAT4 signaling as described in Section 8.1.4, for their ability to induce IFNy release as described in Section 8.1.3, and for their ability to induce target cell death as described in Section 8.1.5.

[0388] The combinations of tumor-targeted IL12R|31 and IL12Rp2 agonists to activate pSTAT4 signaling in activated T-cells was evaluated in the presence and absence of PSMA-expressing C4-2 tumor cells. In the absence of tumor cells, tumor-targeted I L12R|31 and I L12R|32 agonists, alone or in combination, did not activate pSTAT4 signaling (FIG. 7A). In the presence of PSMA- expressing cells, both combinations that were evaluated (IL12R|31-Ag3 + IL12R[32-Ag6 and IL12R01-Ag5 + IL12R02-Ag4) activated pSTAT4 signaling in CD25+T-cells (FIG. 7B). None of the tumor-targeted I L12R|31 and I L12R|32 agonist constructs were associated with pSTAT4 activations when tested alone (FIG. 7B).

[0389] The combinations of tumor-targeted IL12R|31 and IL12Rp2 agonists to promote target cell killing and IFNy release by human T-cells was evaluated with target HEK293 cells that express only hMUC16 or both hMUC16 and hPSMA. In the presence of a MUC16xCD3 antibody, tumor-targeted I L12R01 and I L12R|32 agonist combinations IL12R|31-Ag3 + I L12R|32- Ag6 and I L12R|31 -Ag5 + IL12R02-Ag4 promoted cell killing only if the target cells expressed PSMA (FIGS. 8A-8B). Similarly, these combinations were associated with IFNy release only when HEK293 cells expressed hPSMA (FIGS. 8C-8D).8.5. Example 4: Activation of STAT3-Signaling, pSTAT4 Signaling, IFNy Release and Target Cell Killing by PSMA-targeted IL12R01 and IL12R02 Agonist Combinations

[0390] The tumor-targeted IL12R01 and IL12R02 agonists generated and used in Section 8.3 were evaluated for their ability to activate STAT3-signaling as described in Section 8.1.2, toactivate pSTAT4 signaling as described in Section 8.1.4, to induce IFNy release as described in Section 8.1.3, and to induce target cell death as described in Section 8.1.5.

[0391] The PSMA-targeted IL12R|31 and IL12R|32 agonists did not trigger STAT3 signaling, either alone or in combination, in Jurkat cells which did not express PSMA, either for the first combination of IL12R01-Ag3 + I L12R|31 -Ag6 (FIG. 9A) or for the second combination of IL12R01-Ag4 + IL12R01-Ag3 (FIG. 9C). IL12R01-Ag3 + IL12R01-Ag6 in combination triggered STAT3 activation in Jurkat / PSMA cells, but not when administered alone (FIG. 9B). Similarly, IL12R01-Ag4 + IL12R|31-Ag3 in combination triggered STAT3 activation in Jurkat / PSMA cells, but not when administered alone (FIG. 9D).

[0392] The PSMA-targeted I L12R|31 and IL12R02 agonists were evaluated for their ability to induce cell killing and IFNy release in LNCaP tumor cells, which express PSMA at high levels. The combination of IL12R|31-Ag3 + IL12R02-Ag6 (together with STEAP1xCD3 antibody) promoted cell killing (FIG. 10A) and IFNy release (FIG. 10B). Similarly, the combination of IL12R01-Ag4 + IL12R02-Ag3 (together with STEAP1xCD3 antibody) promoted cell killing (FIG. 10C) and IFNy release (FIG. 10D). No cell killing or IFNy release was observed with administration of IL12R01-Ag3, I L12R02-Ag6, IL12R[32-Ag3 or IL12R01-Ag4 alone.

[0393] Finally, the PSMA-targeted I L12R01 and I L12Rp2 agonists were evaluated for their ability to activate pSTAT4 signaling in activated T-cells in the presence of PSMA-positive C4-2 tumor cells. Both combinations, IL12R[31-Ag3 + IL12R02-Ag6 (FIG. 11A) and I L12R|31 -Ag4 + IL12R02-Ag3 (FIG. 11 B) activated STAT5 signaling in CD25+T-cells.8.6. Example 5: Activation of pSTAT4 Signaling and Target Cell Killing by MUC16- and MSLN-targeted IL12R01 and IL12R 2 Agonist Combinations

[0394] Tumor-targeted IL12R01 and IL12R02 agonists comprising Fab arms were designed and produced as described in Section 8.1.1. The tumor targeting moieties and IL12R binding moieties of these tumor-targeted IL12R01 and IL12R02 agonist constructs used in this example are set forth in Table E4. The ability of tumor-targeted IL12R01 and IL12R02 agonists to activate pSTAT4 signaling as described in Section 8.1.4 and to induce target cell death as described in Section 8.1.5 was evaluated in OVCAR3 cells, which express MUC16 and MSLN at high levels.

[0395] The pair of MUC16-targeted constructs (IL12Rp1-Ag11 + IL12Rp2-Ag11), when administered in combination, induced pSTAT4 signaling in activated T-cells in the presence of OVCAR3 tumor cells (FIG. 12A). The pair of MUC16-targeted constructs also induced cell killing of OVCAR3 tumor cells when administered in combination to a significantly higher degree than either construct alone (FIG. 12B).

[0396] The pair of MSLN-targeted constructs (IL12Rp1-Ag21 + IL12Rp2-Ag21 ), when administered in combination, induced pSTAT4 signaling in activated T-cells in the presence of OVCAR3 tumor cells (FIG. 13A). The pair of MSLN-targeted constructs also induced cell killing of OVCAR3 tumor cells when administered in combination, while no cell killing was observed with administration of either construct alone (FIG. 13B).8.7. Example 6: Activation of STAT3 Signaling by Additional PSMA-targeted IL12RP1 and IL12RP2 Agonist Combinations

[0397] PSMA-targeted IL12R 1 and IL12RP2 agonists comprising Fab arms were designed and produced as described in Section 8.1.1. The tumor targeting moieties and IL12R binding moieties of these PSMA-targeted IL12R 1 and IL12R 2 agonist constructs used in this example are set forth in Table E5. The ability of PSMA-targeted IL12R 1 and IL12RP2 agonists to activate STAT3-signaling was assessed in a STAT3-reporter cell-based assay as described in Section 6.1.2.

[0398] The combinations of PSMA-targeted IL12R01 and IL12R02 agonist constructs triggered minimal STAT3 signaling activation in NK92 / STAT3-Luc cells in the absence of PSMA- expressing cells (FIG. 14A).

[0399] When NK92 / STAT3-Luc cells were co-cultured with hPSMA-expressing Raji cells, all combinations were associated with STAT3-Luc activity, in which combinations of IL12R01-Ag6 + IL12R02-Ag7 and IL12R01-Ag7 + IL12R02-Ag8 produced a stronger activation than that produced by the combinations of IL12R01-Ag4 + IL12R02-Ag3 and IL12R01-Ag3 + IL12R02- Ag6 (FIG. 14B).8.8. Example 7: Activation of pSTAT4 Signaling by Combinations of PSMA- targeted IL12R01 and IL12R02 Agonists with Different Formats

[0400] PSMA-targeted IL12R01 and IL12R02 agonists comprising scFv arms were designed and produced similarly to the protocol described in Section 8.1.1. The PSMA-targeting moieties and IL12R binding moieties of the PSMA-targeted IL12R01 and IL12R02 agonist constructs used in this example are set forth in Table E6. The ability of PSMA-targeted IL12R01 and IL12R02 agonists to activate pSTAT4 signaling was evaluated as described in Section 8.1.4 in the absence and presence of C4-2 tumor cells.

[0401] STAT4 signaling of combinations PSMA-targeted IL12R01 and IL12R02 agonists withFab or scFv arms was evaluated. The results of this assessment showed that both combinations were associated with minimal STAT4 signaling in the absence of C4-2 tumor cells(FIG. 15A) but displayed increased levels of STAT4 phosphorylation in the presence of C4-2 tumor cells (FIG. 15B).9. SEQUENCE LISTING

[0402] Exemplary sequences of the present disclosure are provided in Table S below (with the column “SEQ” indicating the SEQ ID NO:).

Claims

WHAT IS CLAIMED IS:

1. A combination comprising:(a) a tumor-targeted IL12Rp1 agonist comprising:(i) a first tumor-targeting moiety; and(ii) an IL12Rp1 binding moiety; and(b) a tumor-targeted IL12R|32 agonist comprising:(iii) a second tumor-targeting moiety; and(iv) an IL12RP2 binding moiety.

2. The combination of claim 1 , wherein the I L12R|31 binding moiety and the IL12R|32 binding moiety each comprises or consists of an antigen binding domain of an antibody.

3. The combination of claim 2, wherein the I L12Rp1 binding moiety and the I L12R|32 binding moiety are Fabs, scFvs, or sdAbs.

4. The combination of claim 2 or 3, wherein the I L12R|31 binding moiety binds to the D2 domain of IL12R 1 and the IL12RP2 binding moiety binds to the D1 domain of IL12R|32.

5. The combination of claim 1 , wherein:(a) the I L12R|31 binding moiety is a first IL12 moiety comprising a first p40 moiety associated with a first p35 moiety; and(b) the IL12RP2 binding moiety is a second I L12 moiety comprising a second p35 moiety associated with a second p40 moiety.

6. The combination of claim 5, wherein:(a) the first IL12 moiety has greater selectivity to I L12Rpi than IL12RP2 as compared to wild-type IL12 (e.g., wild-type human IL12); and / or(b) the second I L12 moiety has greater selectivity to I L12Rp2 than I L12Rp 1 as compared to wild-type IL12 (e.g., wild-type human IL12).

7. The combination of claim 5 or 6, wherein the first p35 moiety comprises an amino acid sequence having 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:2.

8. The combination of any one of claims 5 to 7, wherein the first p35 moiety is a variant p35 moiety having reduced binding to I L12F?|32 as compared to a wild-type p35 moiety (e.g., a p35 moiety having the amino acid sequence of SEQ ID NO:2).

9. The combination of claim 8, wherein the variant p35 moiety comprises one or more of the mutations set forth in Table B1 , optionally wherein the one or more mutations comprise or consist of Y189E as compared to wild-type p35 (e.g., wherein the variant p35 moiety comprises the amino acid sequence of SEQ ID NO:40).

10. The combination of claim 5 or 6, wherein the first p35 moiety comprises the amino acid sequence of SEQ ID NO:2.

11. The combination of any one of claims 5 to 10, wherein the first p40 moiety comprises an amino acid sequence having 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 or SEQ ID NO:7.

12. The combination of any one of claims 5 to 11 , wherein the first p40 moiety comprises the amino acid sequence of SEQ ID NO:6 or SEQ ID NO:7.

13. The combination of any one of claims 5 to 12, wherein the second p35 moiety comprises an amino acid sequence having 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:2.

14. The combination of any one of claims 5 to 12, wherein the second p35 moiety comprises the amino acid sequence of SEQ ID NO:2.

15. The combination of any one of claims 5 to 14, wherein the second p40 moiety comprises an amino acid sequence having 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 or SEQ ID NO:7.

16. The combination of claim 15, wherein the second p40 moiety is a variant p40 moiety having reduced binding to I L12R|31 as compared to a wild-type p40 moiety.

17. The combination of claim 16, wherein the variant p40 moiety comprises a D1 domain mutation or a D1 domain deletion.

18. The combination of any one of claims 5 to 17, wherein the second p40 moiety comprises the amino acid sequence of SEQ ID NO:6.

19. The combination of any one of claims 5 to 18, wherein the first p35 moiety and the first p40 moiety are separated by a linker (a first “IL12 moiety linker”) and / or the second p35 moiety and the second p40 moiety are separated by a linker (a second “IL12 moiety linker”).

20. The combination of claim 19, wherein the first IL12 moiety linker and / or the second IL12 moiety linker are each at least 5, at least 10, or at least 15 amino acids in length.

21. The combination of claim 19 or 20, wherein the first IL12 moiety linker and / or the second IL12 moiety linker is a non-cleavable linker.

22. The combination of any one of claims 1 to 21 , wherein the first tumor-targeting moiety binds to a first tumor-associated antigen and the second tumor-targeting moiety binds to a second tumor-associated antigen.

23. The combination of claim 22, wherein the first tumor-associated antigen and the second tumor-associated antigen are expressed on the same tumor cell.

24. The combination of claim 22 or 23, wherein the first tumor-associated antigen and the second tumor-associated antigen are different.

25. The combination of claim 22 or 23, wherein the first tumor-associated antigen and the second tumor-associated antigen are the same.

26. The combination of claim 25, wherein the first tumor-targeting moiety and the second tumor-targeting moiety are the same.

27. The combination of claim 25, wherein the first tumor-targeting moiety and the second tumor-targeting moiety are different, e.g., bind to different epitopes.

28. The combination of any one of claims 22 to 27, wherein the first tumor-targeting moiety and the second tumor-targeting moiety do not compete for binding to the tumor-associated antigen.

29. The combination of any one of claims 22 to 28, wherein the first tumor-targeting moiety and / or the second tumor-targeting moiety are Fabs, scFvs, or sdAbs.

30. The combination of any one of claims 22 to 29, wherein the first tumor-targeting moiety and / or second tumor-targeting moiety bind(s) to Fibroblast Activation Protein (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, 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 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, [3-catenin and y- catenin, p120ctn, gp100 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, PDGFfBR ([3-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, -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)-I, IGF-II, IGFI receptor, 5T4, ROR1 , Nkp30, NKG2D, tumor stromal antigens, CA166-9, extra domain A (EDA) of fibronectin or the A1 domain of tenascin-C(TnC A1).

31. The combination of any one of claims 1 to 30, wherein the tumor-targeted I L12Rp1 agonist comprises(a) a first polypeptide chain comprising, in N- to C-terminal orientation:(i) the first tumor-targeting moiety or component thereof (or a component thereof, e.g., a VH-CH1 or VL-CL), optionally associated with another component thereof on a separate polypeptide chain (or a component thereof, e.g., a VL-CL or VH-CH1);(ii) optionally, a linker (a “TAA-Fc linker”); and(iii) a first Fc domain; and(b) a second polypeptide chain comprising, in N- to C-terminal orientation:(i) the I L12Rpi binding moiety or component thereof (or a component thereof, e.g., a VH-CH1 or VL-CL), optionally associated with another component thereof on a separate polypeptide chain (or a component thereof, e.g., a VL-CL or VH-CH1);(ii) optionally, a linker (“an IL12-Fc linker”); and(iii) a second Fc domain associated with the first Fc domain.

32. The combination of claim 31 , wherein the first Fc domain and second Fc domain each comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:11 , SEQ ID NO:12, or SEQ ID NO:13.

33. The combination of claim 31 or 32, wherein the first Fc domain and second Fc domain each comprises a chimeric hinge domain.

34. The combination of claim 33, wherein the first Fc domain and second Fc domain each comprises an amino acid sequence having at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:14 or SEQ ID NO:15.

35. The combination of any one of claims 31 to 34, wherein the first Fc domain and second Fc domain each has reduced effector function.

36. The combination of any one of claims 31 to 35, wherein the first Fc domain and secondFc domain form an Fc heterodimer.

37. The combination of any one of claims 1 to 36, wherein the tumor-targeted I L12Rp2 agonist comprises(a) a third polypeptide chain comprising, in N- to C-terminal orientation:(i) the second tumor-targeting moiety or component thereof (or a component thereof, e.g., a VH-CH1 or VL-CL), optionally associated with another component thereof on a separate polypeptide chain (or a component thereof, e.g., a VL-CL or VH-CH1);(ii) optionally, a linker (a “TAA-Fc linker”); and(iii) a third Fc domain; and(b) a third polypeptide chain comprising, in N- to C-terminal orientation:(i) the IL12RP2 binding moiety or component thereof (or a component thereof, e.g., a VH-CH1 or VL-CL), optionally associated with another component thereof on a separate polypeptide chain (or a component thereof, e.g., a VL-CL or VH-CH1);(ii) optionally, a linker (“an IL12-Fc linker”); and(iii) a fourth Fc domain associated with the third Fc domain.

38. The combination of claim 37, wherein the third Fc domain and fourth Fc domain each comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:11 , SEQ ID NO:12, or SEQ ID NO:13.

39. The combination of claim 37 or 38, wherein the third Fc domain and fourth Fc domain each comprises a chimeric hinge domain.

40. The combination of any one of claims 37 to 39, wherein the third Fc domain and fourth Fc domain each has reduced effector function.

41. The combination of any one of claims 37 to 40, wherein the third Fc domain and fourth Fc domain form an Fc heterodimer.

42. The combination of any one of claims 1 to 41 , wherein the tumor-targeted I L12Rp1 agonist is monovalent for the first tumor-targeting moiety.

43. The combination of any one of claims 1 to 42, wherein the tumor-targeted I L12Rp1 agonist is monovalent for the IL12R|31 binding moiety.

44. The combination of any one of claims 1 to 43, wherein the tumor-targeted I L12Rp2 agonist is monovalent for the second tumor-targeting moiety.

45. The combination of any one of claims 1 to 44, wherein the tumor-targeted I L12Rf32 agonist is monovalent for the IL12R 2 binding moiety.

46. The combination of any one of claims 1 to 45, wherein the tumor-targeted I L12Rp1 agonist and the tumor-targeted IL12R 2 agonist are both in the form of a pharmaceutical composition comprising the agonist and an excipient.

47. The combination of claim 46, wherein the tumor-targeted IL12Rp1 agonist and the tumor-targeted IL12RP2 agonist are in the same pharmaceutical composition.

48. The combination of claim 46, wherein the tumor-targeted IL12Rp1 agonist and the tumor-targeted I L12RP2 agonist are in different pharmaceutical compositions.

49. The combination of any one of claims 1 to 48, wherein tumor-targeted I L12Rpi agonist is configured as illustrated in FIG. 1A, 1 B, 1 C, 1 D, 1 E, 1 F, 1G, 1 H, 11, 1 J, 1 K, or 1 L.

50. The combination of any one of claims 1 to 48, wherein tumor-targeted IL12RP2 agonist is configured as illustrated in FIG. 2A, 2B, 2C, 2D, 2E, 2F, 2G, 2H, 2I, 2J, 2K, or 2L.

51. The combination of any one of claims 1 to 50, wherein the combination further comprises a multispecific T-cell engager.

52. The combination of claim 51 , wherein the multispecific T-cell engager is a bispecific T- cell engager.

53. The combination of claim 51 or 52, wherein the multispecific T-cell engager comprises a TAA targeting moiety and a CD3 targeting moiety.

54. The combination of any one of claims 1 to 53, for use as a medicament.

55. The combination for use of claim 54, for use in a method for the treatment of cancer.

56. A tumor-targeted I L12Rp 1 agonist for use in a method for the treatment of cancer, wherein(a) the IL12R 1 agonist is defined as in any one of claims 1 to 53, and(b) the method comprises administering to a subject in need thereof the tumor-targeted I L12R 1 agonist and a tumor-targeted IL12RP2 agonist as defined in any one of claims 1 to 53.

57. A tumor-targeted I L12R|32 agonist for use in a method for the treatment of cancer, wherein(a) the IL12RP2 agonist is defined as in any one of claims 1 to 53, and(b) the method comprises administering to a subject in need thereof the tumor-targeted I L12R 2 agonist and a tumor-targeted I L12R 1 agonist as defined in any one of claims 1 to 53.

58. A method comprising administering to a subject a combination as defined in any one of claims 1 to 53.

59. The method of claim 58, wherein the method is a method of combination therapy for the treatment of cancer.

60. The combination for use of claim 55, the tumor-targeted I L12R|31 agonist for use of claim 56, the tumor-targeted 11_12R|32 agonist for use of claim 57, of the method of claims 58 or 59, 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 clustering of I L12Rp1 and I L12R|32 receptor subunits in a lymphocyte,(d) by eliciting signaling through the IL12 receptor in a lymphocyte,(e) by inducing tumor cytolysis,(f) by inducing anti-tumor cytotoxicity,(g) by stimulating an immune response against a tumor, or(h) a combination of any two or more of the foregoing uses.

61. The combination for use of any one of claims 55 to 56, the tumor-targetedI L12R|31 agonist for use of claims 56 or 60, the tumor-targeted I L12R|32 agonist for use of claim 57 or 60, or the method of claims 58, 59 or 60, wherein, the method comprises administering a multispecific T-cell engager (e.g., simultaneously, sequentially or separately).

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