IL12 receptor agonists and methods of use thereof

Abstract

The present disclosure relates to IL12 receptor agonists with improved therapeutic profiles.

Description

[Technical Field]

[0001] 1. CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 63 / 479,839, filed January 13, 2023, the contents of which are incorporated herein by reference in their entirety.

[0002] 2. Sequence Listing This application contains a Sequence Listing that has been submitted electronically and is incorporated herein by reference in its entirety. The copy created on January 11, 2024 is named RGN-034WO_SL.xml and is 191,262 bytes in size. [Background technology]

[0003] Interleukin-12 (IL-12 or IL12) is a pro-inflammatory cytokine that plays an important role in both innate and adaptive immunity. (Non-Patent Document 1). IL12 primarily functions as a 70-kDa heterodimer composed of disulfide-linked p35 and p40 subunits. A variety of different immune cells, including B cells, dendritic cells, macrophages, monocytes, and neutrophils, express IL12 upon stimulation (Non-Patent Document 2), and the active heterodimer is formed after protein synthesis. Binding of IL12 to the IL12 receptor complex on T cells and natural killer (NK) cells leads to signal transduction via signal transducer and activator of transcription 4 (STAT4) and signal transducer and activator of transcription 3 (STAT3), and subsequent production and secretion of interferon gamma (IFN-γ). (Non-Patent Document 3). Downstream signaling of IFN-γ involves activation of the T-box transcription factor TBX21 (Tbet), which activates T helper 1 (T helper 1) receptor. H 1) Induce pro-inflammatory cellular functions. Id. IL-12 has been investigated as an anti-cancer therapeutic agent since the early 1990s due to its ability to activate NK cells and cytotoxic T cells. (Non-Patent Document 4) However, in most patients, repeated administration of IL-12 resulted in a gradual decline in adaptive responses and IL-12-induced IFN-γ blood levels. Furthermore, simultaneous induction of IFN-γ with other cytokines (e.g., TNF-α) and / or chemokines (IP-10 or MIG) resulted in severe toxicity. Different dosing and timing protocols have been developed to minimize IFN-γ toxicity and improve the efficacy of IL-12. These approaches have had minimal effect and have not significantly improved patient survival. Despite general acceptance in the field of IL12 therapy, which is being developed for immunotherapy, including anti-cancer therapy, IL12 molecules generally exhibit a poor therapeutic index, with highly toxic doses required to confer modest anti-cancer efficacy.

[0004] Therefore, there is a need in the art for new IL12 therapies with improved therapeutic efficacy and safety profiles. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Hamza et al.,2010,Int.J.Mol.Sci.,11(3):789-806 [Non-patent document 2] Tugues et al.,2015,Cell Death Differ.,22:237-246 [Non-patent document 3] Ullrich et al.,2020,EXCLI J.,19:1563-1589 [Non-patent document 4] Lasek et al.,2014,Cancer Immunol.Immunother.63(5):419-435 Summary of the Invention

[0006] The present disclosure provides novel IL12 receptor agonists. In certain aspects, the IL12 receptor agonists address the shortcomings of IL12 therapy and feature an improved therapeutic profile due to an improved half-life and / or an improved safety profile. In certain aspects, the IL12 receptor agonists address the aggregation problem associated with conventional IL12 fusion constructs, e.g., fusion proteins comprising p35, p40, and an Fc domain. The IL12 receptor agonists of the present disclosure typically comprise or consist of IL12 muteins that differ from native IL12 by the primary amino acid sequence of p35 and / or p40 and / or by the inclusion of additional domains or moieties not normally present in IL12. Exemplary IL12 receptor agonists are disclosed in Section 6.2, numbered embodiments 3-847.

[0007] The present disclosure further provides variant p35 and p40 moieties that incorporate amino acid substitutions that contribute to improved therapeutic profiles, for example, by attenuating IL12 activity through reduced receptor binding. Exemplary p35 and p40 moieties, including exemplary p35 moieties useful for incorporation into IL12 receptor agonists, are disclosed in Section 6.3 and numbered embodiments 1, 2, 676-719, and 589-674.

[0008] The present disclosure further provides nucleic acids encoding the IL12 receptor agonists, IL12 muteins, p35 portions, and p40 portions of the present disclosure. Nucleic acids encoding IL12 receptor agonists and IL12 muteins composed of two or more polypeptide chains can be a single nucleic acid (e.g., a vector encoding all polypeptide chains) or multiple nucleic acids (e.g., two or more vectors encoding different polypeptide chains). The present disclosure further provides host cells and cell lines engineered to express the nucleic acids and IL12 receptor agonists, IL12 muteins, p35 portions, and p40 portions of the present disclosure. The present disclosure further provides methods for producing the IL12 receptor agonists, IL12 muteins, p35 portions, and p40 portions of the present disclosure. Exemplary nucleic acids, host cells, cell lines, and methods for producing the IL12 receptor agonists, IL12 muteins, p35 portions, and p40 portions of the present disclosure are described in Section 6.9 below and in numbered embodiments 848-850.

[0009] The present disclosure further provides pharmaceutical compositions comprising an IL12 receptor agonist, an IL12 mutein, a p35 portion, and a p40 portion of the present disclosure. Exemplary pharmaceutical compositions are described in Section 6.10 below and in numbered Example 851.

[0010] Further provided herein are methods of using the disclosed IL12 receptor agonists, IL12 muteins, p35 portions, p40 portions, and pharmaceutical compositions, for example, to treat cancerous conditions. Exemplary methods are described in Section 6.11, below, and in numbered embodiments 852-860. [Brief explanation of the drawings]

[0011] [Figure 1] Diagram depicting the structure of IL12 (left), the IL12 receptor complex (center), and IL12 signaling (right). [Figure 2A]2A is a schematic diagram depicting the structural organization of the p35 and p40 moieties of IL12. While only certain IL12 moieties are shown with the disulfide bond between the p40 and p35 moieties eliminated, the IL12-Fc fusion protein of FIG. 2A may have the disulfide bond between the p40 and p35 moieties eliminated. Examples of suitable p35 and p40 moieties that can be incorporated into the IL12 fusion protein of FIG. 2A are disclosed, for example, in Section 6.3. [Figure 2B] 2A-2C are schematic diagrams depicting embodiments of monovalent IL12-Fc fusion proteins that the IL12 receptor agonists of the present disclosure may comprise or consist of. In the embodiment of FIG. 2B, the IL12 moiety can be attached to the N-terminus of the Fc. When attached to the Fc according to the embodiment of FIG. 2B, the IL12 moiety can be arranged in the order (N- to C-terminus) of p40 moiety-p35 moiety (FIGS. 2F, 2H, 2I, 2K, and 2M) or p35 moiety-p40 moiety (FIGS. 2G, 2J, and 2L). Alternatively, the p40 moiety can be provided in the form of a p40 monomer. The asterisk between the p40 moiety and the p35 moiety indicates an optionally removed disulfide bond between the subunits. While only certain IL12 moieties are shown with the disulfide bond between the p40 and p35 moieties eliminated, the IL12-Fc fusion protein of Figure 2B can have the disulfide bond between the p40 and p35 moieties eliminated. Examples of suitable p35 and p40 moieties that can be incorporated into the IL12 fusion protein of Figure 2B are disclosed, for example, in Section 6.3. The CH2 and CH3 domains shown in Figure 2B form an Fc domain, which is a type of multimerization moiety. Fc domains and other multimerization moieties that can be used in IL12 receptor agonists are described in Section 6.6. The Fc domain in a heterodimeric IL12-Fc fusion protein (e.g., as shown in Figures 2B-2G and 2P) can incorporate any combination of mutations suitable for heterodimerization or selective purification (e.g., knob-in-hole and / or star mutations), as described, for example, in Section 6.6.1.2 (not shown). [Figure 2C] 2C is a schematic diagram depicting an embodiment of a monovalent IL12-Fc fusion protein that an IL12 receptor agonist of the present disclosure can comprise or consist of. In the embodiment of FIG. 2C, the IL12 moiety can be attached to the N-terminus of the Fc. When attached to the Fc according to the embodiment of FIG. 2C, the IL12 moiety can be arranged in the order (N- to C-terminus) of p40 moiety-p35 moiety (FIGS. 2F, 2H, 2I, 2K, and 2M) or p35 moiety-p40 moiety (FIGS. 2G, 2J, and 2L). Alternatively, the p40 moiety can be provided in the form of a p40 monomer. The asterisk between the p40 moiety and the p35 moiety indicates an optionally removed disulfide bond between the subunits. While only certain IL12 moieties are shown with the disulfide bond between the p40 and p35 moieties eliminated, the IL12-Fc fusion protein of Figure 2C allows for the disulfide bond between the p40 and p35 moieties to be eliminated. Examples of suitable p35 and p40 moieties that can be incorporated into the IL12 fusion protein of Figure 2C are disclosed, for example, in Section 6.3. The CH2 and CH3 domains shown in Figure 2C form an Fc domain, which is a type of multimerization moiety. Fc domains and other multimerization moieties that can be used in IL12 receptor agonists are described in Section 6.6. The Fc domain in a heterodimeric IL12-Fc fusion protein (e.g., as shown in Figures 2B-2G and 2P) can incorporate any combination of mutations suitable for heterodimerization or selective purification (e.g., knob-in-hole and / or star mutations), as described, for example, in Section 6.6.1.2 (not shown). [Figure 2D]2D are schematic diagrams depicting embodiments of monovalent IL12-Fc fusion proteins that the IL12 receptor agonists of the present disclosure can comprise or consist of. In the embodiment of FIG. 2D, the IL12 moiety can be attached to the C-terminus of the Fc. When attached to the Fc according to the embodiment of FIG. 2D, the IL12 moiety can be arranged in the order (N- to C-terminus) of p40 moiety-p35 moiety (FIGS. 2F, 2H, 2I, 2K, and 2M) or p35 moiety-p40 moiety (FIGS. 2G, 2J, and 2L). Alternatively, the p40 moiety can be provided in the form of a p40 monomer. The asterisk between the p40 moiety and the p35 moiety indicates an optionally removed disulfide bond between the subunits. While only certain IL12 moieties are shown with the disulfide bond between the p40 and p35 moieties eliminated, the IL12-Fc fusion protein of Figure 2D can have the disulfide bond between the p40 and p35 moieties eliminated. Examples of suitable p35 and p40 moieties that can be incorporated into the IL12 fusion protein of Figure 2D are disclosed, for example, in Section 6.3. The CH2 and CH3 domains shown in Figure 2D form an Fc domain, which is a type of multimerization moiety. Fc domains and other multimerization moieties that can be used in IL12 receptor agonists are described in Section 6.6. The Fc domain in a heterodimeric IL12-Fc fusion protein (e.g., as shown in Figures 2B-2G and 2P) can incorporate any combination of mutations suitable for heterodimerization or selective purification (e.g., knob-in-hole and / or star mutations), as described, for example, in Section 6.6.1.2 (not shown). [Figure 2E]2E are schematic diagrams depicting embodiments of monovalent IL12-Fc fusion proteins that the IL12 receptor agonists of the present disclosure may comprise or consist of. In the embodiment of FIG. 2E, the IL12 moiety can be attached to the C-terminus of the Fc. When attached to the Fc according to the embodiment of FIG. 2E, the IL12 moiety can be arranged in the order (N- to C-terminus) of p40 moiety-p35 moiety (FIGS. 2F, 2H, 2I, 2K, and 2M) or p35 moiety-p40 moiety (FIGS. 2G, 2J, and 2L). Alternatively, the p40 moiety can be provided in the form of a p40 monomer. The asterisk between the p40 moiety and the p35 moiety indicates an optionally removed disulfide bond between the subunits. While only certain IL12 moieties are shown with the disulfide bond between the p40 and p35 moieties eliminated, the IL12-Fc fusion protein of Figure 2E can have the disulfide bond between the p40 and p35 moieties eliminated. Examples of suitable p35 and p40 moieties that can be incorporated into the IL12 fusion protein of Figure 2E are disclosed, for example, in Section 6.3. The CH2 and CH3 domains shown in Figure 2E form an Fc domain, which is a type of multimerization moiety. Fc domains and other multimerization moieties that can be used in IL12 receptor agonists are described in Section 6.6. The Fc domain in a heterodimeric IL12-Fc fusion protein (e.g., as shown in Figures 2B-2G and 2P) can incorporate any combination of mutations suitable for heterodimerization or selective purification (e.g., knob-in-hole and / or star mutations), as described, for example, in Section 6.6.1.2 (not shown). [Figure 2F]2F is a schematic diagram depicting an embodiment of a monovalent IL12-Fc fusion protein that an IL12 receptor agonist of the present disclosure can comprise or consist of. In the embodiment of FIG. 2F, the IL12 moiety can be attached to the C-terminus of the Fc. When attached to the Fc according to the embodiment of FIG. 2F, the IL12 moiety can be arranged in the order p40 moiety-p35 moiety (N- to C-terminus). Alternatively, the p40 moiety can be provided in the form of a p40 monomer. The asterisk between the p40 and p35 moieties indicates an optionally eliminated disulfide bond between the subunits. While only certain IL12 moieties are shown having an eliminated disulfide bond between the p40 and p35 moieties, the IL12-Fc fusion protein of FIG. 2F can have the disulfide bond between the p40 and p35 moieties eliminated. Examples of suitable p35 and p40 moieties that can be incorporated into the IL12 fusion protein of FIG. 2F are disclosed, for example, in Section 6.3. The CH2 and CH3 domains shown in Figure 2F form an Fc domain, a type of multimerization moiety. Fc domains and other multimerization moieties that can be used in IL12 receptor agonists are described in Section 6.6. The Fc domain in a heterodimeric IL12-Fc fusion protein (e.g., as shown in Figures 2B-2G and 2P) can incorporate any combination of mutations suitable for heterodimerization or selective purification (e.g., knob-in-hole and / or star mutations), as described in Section 6.6.1.2 (not shown). [Figure 2G]2G is a schematic diagram depicting an embodiment of a monovalent IL12-Fc fusion protein that an IL12 receptor agonist of the present disclosure can comprise or consist of. In the embodiment of FIG. 2G, the IL12 moiety can be attached to the C-terminus of the Fc. When attached to the Fc according to the embodiment of FIG. 2G, the IL12 moiety can be arranged in the order p35 moiety-p40 moiety (N- to C-terminus). Alternatively, the p40 moiety can be provided in the form of a p40 monomer. The asterisk between the p40 moiety and the p35 moiety indicates an optionally eliminated disulfide bond between the subunits. While only certain IL12 moieties are shown to have an eliminated disulfide bond between the p40 moiety and the p35 moiety, the IL12-Fc fusion protein of FIG. 2G can have the disulfide bond between the p40 moiety and the p35 moiety eliminated. Examples of suitable p35 and p40 moieties that can be incorporated into the IL12 fusion protein of FIG. 2G are disclosed, for example, in Section 6.3. The CH2 and CH3 domains shown in Figure 2G form an Fc domain, a type of multimerization moiety. Fc domains and other multimerization moieties that can be used in IL12 receptor agonists are described in Section 6.6. The Fc domain in a heterodimeric IL12-Fc fusion protein (e.g., as shown in Figures 2B-2G and 2P) can incorporate any combination of mutations suitable for heterodimerization or selective purification (e.g., knob-in-hole and / or star mutations), as described in Section 6.6.1.2 (not shown). [Figure 2H]2H is a schematic diagram depicting an embodiment of a bivalent IL12-Fc fusion protein that an IL12 receptor agonist of the present disclosure can comprise or consist of. In the embodiment of FIG. 2H, the IL12 moiety can be attached to the N-terminus of the Fc. When attached to the Fc according to the embodiment of FIG. 2H, the IL12 moieties can be arranged in the order p40 moiety-p35 moiety (N- to C-terminus). Alternatively, the p40 moiety can be provided in the form of a p40 monomer. The asterisk between the p40 and p35 moieties indicates an optionally eliminated disulfide bond between the subunits. While only certain IL12 moieties are shown having an eliminated disulfide bond between the p40 and p35 moieties, the IL12-Fc fusion protein of FIG. 2H can have the disulfide bond between the p40 and p35 moieties eliminated. Examples of suitable p35 and p40 moieties that can be incorporated into the IL12 fusion protein of FIG. 2H are disclosed, for example, in Section 6.3. The CH2 and CH3 domains shown in Figure 2H form an Fc domain, a type of multimerization moiety. Fc domains and other multimerization moieties that can be used in IL12 receptor agonists are described in Section 6.6. The Fc domain in a heterodimeric IL12-Fc fusion protein (e.g., as shown in Figures 2B-2G and 2P) can incorporate any combination of mutations suitable for heterodimerization or selective purification (e.g., knob-in-hole and / or star mutations), as described in Section 6.6.1.2 (not shown). [Figure 2I]

[0023] Figure 2I is a schematic diagram depicting an embodiment of a bivalent IL12-Fc fusion protein that the IL12 receptor agonist of the present disclosure can comprise or consist of. In the embodiment of Figure 2I, the IL12 moiety can be attached to the N-terminus of the Fc. When attached to the Fc according to the embodiment of Figure 2I, the IL12 moieties can be arranged in the order p40 moiety-p35 moiety (N- to C-terminus). Alternatively, the p40 moiety can be provided in the form of a p40 monomer. The asterisk between the p40 and p35 moieties indicates an optionally eliminated disulfide bond between the subunits. While only certain IL12 moieties are shown having an eliminated disulfide bond between the p40 and p35 moieties, the IL12-Fc fusion protein of Figure I can have the disulfide bond between the p40 and p35 moieties eliminated. Examples of suitable p35 and p40 moieties that can be incorporated into the IL12 fusion protein of Figure 2I are disclosed, for example, in Section 6.3. The CH2 and CH3 domains shown in Figure 2I form an Fc domain, a type of multimerization moiety. Fc domains and other multimerization moieties that can be used in IL12 receptor agonists are described in Section 6.6. The Fc domain in a heterodimeric IL12-Fc fusion protein (e.g., as shown in Figures 2B-2G and 2P) can incorporate any combination of mutations suitable for heterodimerization or selective purification (e.g., knob-in-hole and / or star mutations), as described in Section 6.6.1.2 (not shown). [Figure 2J]2J is a schematic diagram depicting an embodiment of a bivalent IL12-Fc fusion protein that an IL12 receptor agonist of the present disclosure can comprise or consist of. In the embodiment of FIG. 2J, the IL12 moiety can be attached to the N-terminus of the Fc. When attached to the Fc according to the embodiment of FIG. 2J, the IL12 moieties can be arranged in the order p35 moiety-p40 moiety (N-terminus to C-terminus). Alternatively, the p40 moiety can be provided in the form of a p40 monomer. The asterisk between the p40 moiety and the p35 moiety indicates an optionally eliminated disulfide bond between the subunits. While only certain IL12 moieties are shown having an eliminated disulfide bond between the p40 moiety and the p35 moiety, the IL12-Fc fusion protein of FIG. 2J can have the disulfide bond between the p40 moiety and the p35 moiety eliminated. Examples of suitable p35 and p40 moieties that can be incorporated into the IL12 fusion protein of FIG. 2J are disclosed, for example, in Section 6.3. The CH2 and CH3 domains shown in Figure 2J form an Fc domain, a type of multimerization moiety. Fc domains and other multimerization moieties that can be used in IL12 receptor agonists are described in Section 6.6. The Fc domain in a heterodimeric IL12-Fc fusion protein (e.g., as shown in Figures 2B-2G and 2P) can incorporate any combination of mutations suitable for heterodimerization or selective purification (e.g., knob-in-hole and / or star mutations), as described in Section 6.6.1.2 (not shown). [Figure 2K]2K is a schematic diagram illustrating an embodiment of a bivalent IL12-Fc fusion protein that an IL12 receptor agonist of the present disclosure can comprise or consist of. In the embodiment of FIG. 2K, the IL12 moiety can be attached to the C-terminus of the Fc. When attached to the Fc according to the embodiment of FIG. 2K, the IL12 moieties can be arranged in the order p40 moiety-p35 moiety (N- to C-terminus). Alternatively, the p40 moiety can be provided in the form of a p40 monomer. The asterisk between the p40 and p35 moieties indicates an optionally eliminated disulfide bond between the subunits. While only certain IL12 moieties are shown to have an eliminated disulfide bond between the p40 and p35 moieties, the IL12-Fc fusion protein of FIG. 2K can have the disulfide bond between the p40 and p35 moieties eliminated. Examples of suitable p35 and p40 moieties that can be incorporated into the IL12 fusion protein of FIG. 2K are disclosed, for example, in Section 6.3. The CH2 and CH3 domains shown in Figure 2K form an Fc domain, a type of multimerization moiety. Fc domains and other multimerization moieties that can be used in IL12 receptor agonists are described in Section 6.6. The Fc domain in a heterodimeric IL12-Fc fusion protein (e.g., as shown in Figures 2B-2G and 2P) can incorporate any combination of mutations suitable for heterodimerization or selective purification (e.g., knob-in-hole and / or star mutations), as described in Section 6.6.1.2 (not shown). [Figure 2L]2L is a schematic diagram depicting an embodiment of a bivalent IL12-Fc fusion protein that an IL12 receptor agonist of the present disclosure can comprise or consist of. In the embodiment of FIG. 2L, the IL12 moiety can be attached to the C-terminus of the Fc. When attached to the Fc according to the embodiment of FIG. 2L, the IL12 moieties can be arranged in the order p35 moiety-p40 moiety (N- to C-terminus). Alternatively, the p40 moiety can be provided in the form of a p40 monomer. The asterisk between the p40 moiety and the p35 moiety indicates an optionally eliminated disulfide bond between the subunits. While only certain IL12 moieties are shown having an eliminated disulfide bond between the p40 moiety and the p35 moiety, the IL12-Fc fusion protein of FIG. 2L can have the disulfide bond between the p40 moiety and the p35 moiety eliminated. Examples of suitable p35 and p40 moieties that can be incorporated into the IL12 fusion protein of FIG. 2L are disclosed, for example, in Section 6.3. The CH2 and CH3 domains shown in Figure 2L form an Fc domain, a type of multimerization moiety. Fc domains and other multimerization moieties that can be used in IL12 receptor agonists are described in Section 6.6. The Fc domain in a heterodimeric IL12-Fc fusion protein (e.g., as shown in Figures 2B-2G and 2P) can incorporate any combination of mutations suitable for heterodimerization or selective purification (e.g., knob-in-hole and / or star mutations), as described in Section 6.6.1.2 (not shown). [Figure 2M]2M is a schematic diagram depicting an embodiment of a bivalent IL12-Fc fusion protein that the IL12 receptor agonist of the present disclosure can comprise or consist of. In the embodiment of FIG. 2M, the IL12 moiety can be attached to the C-terminus of the Fc. When attached to the Fc according to the embodiment of FIG. 2M, the IL12 moieties can be arranged in the order p40 moiety-p35 moiety (N- to C-terminus). Alternatively, the p40 moiety can be provided in the form of a p40 monomer. The asterisk between the p40 and p35 moieties indicates an optionally eliminated disulfide bond between the subunits. While only certain IL12 moieties are shown having an eliminated disulfide bond between the p40 and p35 moieties, the IL12-Fc fusion protein of FIG. 2M can have the disulfide bond between the p40 and p35 moieties eliminated. Examples of suitable p35 and p40 moieties that can be incorporated into the IL12 fusion protein of FIG. 2M are disclosed, for example, in Section 6.3. The CH2 and CH3 domains shown in Figure 2M form an Fc domain, a type of multimerization moiety. Fc domains and other multimerization moieties that can be used in IL12 receptor agonists are described in Section 6.6. The Fc domain in a heterodimeric IL12-Fc fusion protein (e.g., as shown in Figures 2B-2G and 2P) can incorporate any combination of mutations suitable for heterodimerization or selective purification (e.g., knob-in-hole and / or star mutations), as described in Section 6.6.1.2 (not shown). [Figure 2N]2N is a schematic diagram illustrating an embodiment of a bivalent IL12-Fc fusion protein that the IL12 receptor agonist of the present disclosure can comprise or consist of. In the embodiment of FIG. 2N, the IL12 portion can be attached to the N-terminus of the Fc. The p40 portion can be provided in the form of a p40 monomer. The asterisk between the p40 and p35 portions indicates an optionally eliminated disulfide bond between the subunits. While only certain IL12 portions are shown with an eliminated disulfide bond between the p40 and p35 portions, the IL12-Fc fusion protein of FIG. 2N can have the disulfide bond between the p40 and p35 portions eliminated. Examples of suitable p35 and p40 portions that can be incorporated into the IL12 fusion protein of FIG. 2N are disclosed, for example, in Section 6.3. The CH2 and CH3 domains shown in FIG. 2N form an Fc domain, which is a type of multimerization moiety. Fc domains and other multimerization moieties that can be used in IL12 receptor agonists are described in Section 6.6. The Fc domain in a heterodimeric IL12-Fc fusion protein (e.g., as shown in Figures 2B-2G and 2P) can incorporate any combination of mutations suitable for heterodimerization or selective purification (e.g., knob-in-hole and / or star mutations), for example, as described in Section 6.6.1.2 (not shown). [Figure 2O]2O is a schematic diagram illustrating an embodiment of a bivalent IL12-Fc fusion protein that the IL12 receptor agonist of the present disclosure can comprise or consist of. In the embodiment of FIG. 2O, the IL12 portion can be attached to the C-terminus of the Fc. The p40 portion can be provided in the form of a p40 monomer. The asterisk between the p40 and p35 portions indicates an optionally eliminated disulfide bond between the subunits. While only certain IL12 portions are shown with an eliminated disulfide bond between the p40 and p35 portions, the IL12-Fc fusion protein of FIG. 2O can have the disulfide bond between the p40 and p35 portions eliminated. Examples of suitable p35 and p40 portions that can be incorporated into the IL12 fusion protein of FIG. 2O are disclosed, for example, in Section 6.3. The CH2 and CH3 domains shown in FIG. 2O form an Fc domain, which is a type of multimerization moiety. Fc domains and other multimerization moieties that can be used in IL12 receptor agonists are described in Section 6.6. The Fc domain in a heterodimeric IL12-Fc fusion protein (e.g., as shown in Figures 2B-2G and 2P) can incorporate any combination of mutations suitable for heterodimerization or selective purification (e.g., knob-in-hole and / or star mutations), for example, as described in Section 6.6.1.2 (not shown). [Figure 2P]2P are schematic diagrams depicting various embodiments of monovalent IL12-Fc fusion proteins (FIGS. 2B-2G) and bivalent IL12-Fc fusion proteins (FIGS. 2H-2O) that can be comprised or consist of by the IL12 receptor agonists of the present disclosure. In the embodiment of FIG. 2P, the IL12 moiety can be attached to the C-terminus of the Fc. The p40 moiety can be provided in the form of a p40 monomer. The asterisk between the p40 and p35 moieties indicates an optionally eliminated disulfide bond between the subunits. While only certain IL12 moieties are shown to have an eliminated disulfide bond between the p40 and p35 moieties, the CH2 and CH3 domains shown in FIG. 2P form an Fc domain, which is a type of multimerization moiety. Fc domains and other multimerization moieties that can be used in IL12 receptor agonists are described in Section 6.6. The Fc domain in the heterodimeric IL12-Fc fusion protein (e.g., as shown in Figures 2B-2G and 2P) can incorporate any combination of mutations suitable for heterodimerization or selective purification (e.g., knob-in-hole and / or star mutations), e.g., as described in section 6.6.1.2 (not shown). [Figure 3A] 3A-3C are schematic diagrams depicting p35 and p40 moieties with the structural orientation of IL12. The p40 moiety can include the D1, D2, and D3 domains (FIGS. 3B-3F), or only the D2 and D3 domains (FIGS. 3G-3I). Examples of suitable p35 and p40 moieties that can be incorporated into the IL12 fusion protein of FIG. 3A are disclosed, for example, in Section 6.3. An asterisk occurring in either the p40 or p35 moiety indicates the optional presence of a mutation in the IL12 moiety, for example, one or more of the mutations described in Section 6.3. Fc domains and other multimerization moieties that can be used in IL12 receptor agonists are described in Section 6.6. [Figure 3B]3A-3C are schematic diagrams depicting embodiments of IL12-Fc fusion proteins that can be combined with each other to form a bivalent IL12-Fc fusion protein that can be comprised by or consist of an IL12 receptor agonist of the present disclosure. The p40 portion can include the D1, D2, and D3 domains (FIGS. 3B-3F), or only the D2 and D3 domains (FIGS. 3G-3I). Examples of suitable p35 and p40 moieties that can be incorporated into the IL12 fusion protein of FIG. 3B are disclosed, for example, in Section 6.3. An asterisk occurring in either the p40 or p35 portion indicates the optional presence of a mutation in the IL12 portion, for example, one or more of the mutations described in Section 6.3. The CH2 and CH3 domains shown in FIG. 3B form an Fc domain, which is a type of multimerization moiety. Fc domains and other multimerization moieties that can be used in IL12 receptor agonists are described in Section 6.6. [Figure 3C] 3C are schematic diagrams depicting embodiments of IL12-Fc fusion proteins that can be combined with each other to form a bivalent IL12-Fc fusion protein that can comprise or consist of an IL12 receptor agonist of the present disclosure. The p40 portion can comprise the D1, D2, and D3 domains (FIGS. 3B-3F), or only the D2 and D3 domains (FIGS. 3G-3I). Examples of suitable p35 and p40 moieties that can be incorporated into the IL12 fusion protein of FIG. 3C are disclosed, for example, in Section 6.3. An asterisk occurring in either the p40 or p35 portion indicates the optional presence of a mutation in the IL12 portion, for example, one or more of the mutations described in Section 6.3. The CH2 and CH3 domains shown in FIG. 3C form an Fc domain, which is a type of multimerization moiety. Fc domains and other multimerization moieties that can be used in IL12 receptor agonists are described in Section 6.6. [Figure 3D]3D are schematic diagrams depicting embodiments of IL12-Fc fusion proteins that can be combined with each other to form a bivalent IL12-Fc fusion protein that can be comprised by or consist of an IL12 receptor agonist of the present disclosure. The p40 portion can include the D1, D2, and D3 domains (FIGS. 3B-3F), or only the D2 and D3 domains (FIGS. 3G-3I). Examples of suitable p35 and p40 moieties that can be incorporated into the IL12 fusion protein of FIG. 3D are disclosed, for example, in Section 6.3. An asterisk occurring in either the p40 or p35 portion indicates the optional presence of a mutation in the IL12 portion, for example, one or more of the mutations described in Section 6.3. The CH2 and CH3 domains shown in FIG. 3D form an Fc domain, which is a type of multimerization moiety. Fc domains and other multimerization moieties that can be used in IL12 receptor agonists are described in Section 6.6. [Figure 3E] 3E are schematic diagrams depicting embodiments of IL12-Fc fusion proteins that can be combined with each other to form a bivalent IL12-Fc fusion protein that can be comprised by or consist of an IL12 receptor agonist of the present disclosure. The p40 portion can include the D1, D2, and D3 domains (FIGS. 3B-3F), or only the D2 and D3 domains (FIGS. 3G-3I). Examples of suitable p35 and p40 moieties that can be incorporated into the IL12 fusion protein of FIG. 3E are disclosed, for example, in Section 6.3. An asterisk occurring in either the p40 or p35 portion indicates the optional presence of a mutation in the IL12 portion, for example, one or more of the mutations described in Section 6.3. The CH2 and CH3 domains shown in FIG. 3E form an Fc domain, which is a type of multimerization moiety. Fc domains and other multimerization moieties that can be used in IL12 receptor agonists are described in Section 6.6. [Figure 3F]3A-3C are schematic diagrams depicting embodiments of IL12-Fc fusion proteins that can be combined with each other to form a bivalent IL12-Fc fusion protein that can be comprised by or consist of an IL12 receptor agonist of the present disclosure. The p40 portion can include the D1, D2, and D3 domains (FIGS. 3B-3F), or only the D2 and D3 domains (FIGS. 3G-3I). Examples of suitable p35 and p40 moieties that can be incorporated into the IL12 fusion protein of FIG. 3F are disclosed, for example, in Section 6.3. An asterisk occurring in either the p40 or p35 portion indicates the optional presence of a mutation in the IL12 portion, for example, one or more of the mutations described in Section 6.3. The CH2 and CH3 domains shown in FIG. 3F form an Fc domain, which is a type of multimerization moiety. Fc domains and other multimerization moieties that can be used in IL12 receptor agonists are described in Section 6.6. [Figure 3G] 3A-3C are schematic diagrams depicting embodiments of IL12-Fc fusion proteins that can be combined with each other to form a bivalent IL12-Fc fusion protein that can be comprised by or consist of an IL12 receptor agonist of the present disclosure. The p40 portion can include the D1, D2, and D3 domains (FIGS. 3B-3F), or only the D2 and D3 domains (FIGS. 3G-3I). Examples of suitable p35 and p40 moieties that can be incorporated into the IL12 fusion protein of FIG. 3G are disclosed, for example, in Section 6.3. An asterisk occurring in either the p40 or p35 portion indicates the optional presence of a mutation in the IL12 portion, for example, one or more of the mutations described in Section 6.3. The CH2 and CH3 domains shown in FIG. 3G form an Fc domain, which is a type of multimerization moiety. Fc domains and other multimerization moieties that can be used in IL12 receptor agonists are described in Section 6.6. [Figure 3H]3H are schematic diagrams depicting embodiments of IL12-Fc fusion proteins that can be combined with each other to form a bivalent IL12-Fc fusion protein that can be comprised by or consist of an IL12 receptor agonist of the present disclosure. The p40 portion can include the D1, D2, and D3 domains (FIGS. 3B-3F), or only the D2 and D3 domains (FIGS. 3G-3I). Examples of suitable p35 and p40 moieties that can be incorporated into the IL12 fusion protein of FIG. 3H are disclosed, for example, in Section 6.3. An asterisk occurring in either the p40 or p35 portion indicates the optional presence of a mutation in the IL12 portion, for example, one or more of the mutations described in Section 6.3. The CH2 and CH3 domains shown in FIG. 3H form an Fc domain, which is a type of multimerization moiety. Fc domains and other multimerization moieties that can be used in IL12 receptor agonists are described in Section 6.6. [Figure 3I] 3A-3C are schematic diagrams depicting embodiments of IL12-Fc fusion proteins that can be combined with each other to form a bivalent IL12-Fc fusion protein that can be comprised by or consist of an IL12 receptor agonist of the present disclosure. The p40 portion can include the D1, D2, and D3 domains (FIGS. 3B-3F), or only the D2 and D3 domains (FIGS. 3G-3I). Examples of suitable p35 and p40 moieties that can be incorporated into the IL12 fusion protein of FIG. 3I are disclosed, for example, in Section 6.3. An asterisk occurring in either the p40 or p35 portion indicates the optional presence of a mutation in the IL12 portion, for example, one or more of the mutations described in Section 6.3. The CH2 and CH3 domains shown in FIG. 3I form an Fc domain, which is a type of multimerization moiety. Fc domains and other multimerization moieties that can be used in IL12 receptor agonists are described in Section 6.6. [Figure 4A]4A and 4B are schematic diagrams depicting the structural orientation of additional embodiments of IL12-Fc fusion proteins that the IL12 receptor agonists of the present disclosure can comprise or consist of. Examples of suitable p35 and p40 moieties that can be incorporated into the IL12 fusion protein of FIG. 4A are disclosed, e.g., in Section 6.3. Examples of suitable IL12 receptor moieties are disclosed, e.g., in Sections 6.4.1 and 6.4.1. Examples of suitable IL12 antibody-based masking moieties are disclosed, e.g., in Section 6.4.3. [Figure 4B] 4B is a schematic diagram depicting the structural orientation of additional embodiments of IL12-Fc fusion proteins that can be comprised or consist of by the IL12 receptor agonists of the present disclosure, incorporating a masking moiety in the form of an IL12 receptor (IL12R) β1 receptor moiety or an IL12Rβ2 receptor moiety. Examples of suitable p35 and p40 moieties that can be incorporated into the IL12 fusion protein of FIG. 4B are disclosed, e.g., in Section 6.3. Examples of suitable IL12 receptor moieties are disclosed, e.g., in Sections 6.4.1 and 6.4.1. Examples of suitable IL12 antibody-based masking moieties are disclosed, e.g., in Section 6.4.3. The CH2 and CH3 domains shown in FIG. 4B form an Fc domain, which is a type of multimerization moiety. Fc domains and other multimerization moieties that can be used in IL12 receptor agonists are described in Section 6.6. The Fc domain in the heterodimeric IL12-Fc fusion protein (e.g., as shown in Figure 4F-4W) can incorporate any combination of mutations suitable for heterodimerization or selective purification (e.g., knob-in-hole and / or star mutations), e.g., as described in section 6.6.1.2 (not shown). [Figure 4C]4C is a schematic diagram depicting the structural orientation of additional embodiments of IL12-Fc fusion proteins that can be comprised or consist of by the IL12 receptor agonists of the present disclosure, incorporating a masking moiety in the form of an IL12 receptor (IL12R) β1 receptor moiety or an IL12Rβ2 receptor moiety. Examples of suitable p35 and p40 moieties that can be incorporated into the IL12 fusion protein of FIG. 4C are disclosed, e.g., in Section 6.3. Examples of suitable IL12 receptor moieties are disclosed, e.g., in Sections 6.4.1 and 6.4.1. Examples of suitable IL12 antibody-based masking moieties are disclosed, e.g., in Section 6.4.3. The CH2 and CH3 domains shown in FIG. 4C form an Fc domain, which is a type of multimerization moiety. Fc domains and other multimerization moieties that can be used in IL12 receptor agonists are described in Section 6.6. The Fc domain in the heterodimeric IL12-Fc fusion protein (e.g., as shown in Figure 4F-4W) can incorporate any combination of mutations suitable for heterodimerization or selective purification (e.g., knob-in-hole and / or star mutations), e.g., as described in section 6.6.1.2 (not shown). [Figure 4D]4D is a schematic diagram depicting the structural orientation of additional embodiments of IL12-Fc fusion proteins that can be comprised or consist of by the IL12 receptor agonists of the present disclosure, incorporating a masking moiety in the form of an IL12 receptor (IL12R) β1 receptor moiety or an IL12Rβ2 receptor moiety. Examples of suitable p35 and p40 moieties that can be incorporated into the IL12 fusion protein of FIG. 4D are disclosed, e.g., in Section 6.3. Examples of suitable IL12 receptor moieties are disclosed, e.g., in Sections 6.4.1 and 6.4.1. Examples of suitable IL12 antibody-based masking moieties are disclosed, e.g., in Section 6.4.3. The CH2 and CH3 domains shown in FIG. 4D form an Fc domain, which is a type of multimerization moiety. Fc domains and other multimerization moieties that can be used in IL12 receptor agonists are described in Section 6.6. The Fc domain in the heterodimeric IL12-Fc fusion protein (e.g., as shown in Figure 4F-4W) can incorporate any combination of mutations suitable for heterodimerization or selective purification (e.g., knob-in-hole and / or star mutations), e.g., as described in section 6.6.1.2 (not shown). [Figure 4E]4E is a schematic diagram depicting the structural orientation of additional embodiments of IL12-Fc fusion proteins that can be comprised or consist of by the IL12 receptor agonists of the present disclosure, incorporating a masking moiety in the form of an IL12 receptor (IL12R) β1 receptor moiety or an IL12Rβ2 receptor moiety. Examples of suitable p35 and p40 moieties that can be incorporated into the IL12 fusion protein of FIG. 4E are disclosed, e.g., in Section 6.3. Examples of suitable IL12 receptor moieties are disclosed, e.g., in Sections 6.4.1 and 6.4.1. Examples of suitable IL12 antibody-based masking moieties are disclosed, e.g., in Section 6.4.3. The CH2 and CH3 domains shown in FIG. 4E form an Fc domain, which is a type of multimerization moiety. Fc domains and other multimerization moieties that can be used in IL12 receptor agonists are described in Section 6.6. The Fc domain in the heterodimeric IL12-Fc fusion protein (e.g., as shown in Figure 4F-4W) can incorporate any combination of mutations suitable for heterodimerization or selective purification (e.g., knob-in-hole and / or star mutations), e.g., as described in section 6.6.1.2 (not shown). [Figure 4F]4F is a schematic diagram depicting the structural orientation of additional embodiments of IL12-Fc fusion proteins that can be comprised or consist of by the IL12 receptor agonists of the present disclosure, incorporating a masking moiety in the form of an IL12 receptor (IL12R) β1 receptor moiety or an IL12Rβ2 receptor moiety. Examples of suitable p35 and p40 moieties that can be incorporated into the IL12 fusion protein of FIG. 4F are disclosed, e.g., in Section 6.3. Examples of suitable IL12 receptor moieties are disclosed, e.g., in Sections 6.4.1 and 6.4.1. Examples of suitable IL12 antibody-based masking moieties are disclosed, e.g., in Section 6.4.3. The CH2 and CH3 domains shown in FIG. 4F form an Fc domain, which is a type of multimerization moiety. Fc domains and other multimerization moieties that can be used in IL12 receptor agonists are described in Section 6.6. The Fc domain in the heterodimeric IL12-Fc fusion protein (e.g., as shown in Figure 4F-4W) can incorporate any combination of mutations suitable for heterodimerization or selective purification (e.g., knob-in-hole and / or star mutations), e.g., as described in section 6.6.1.2 (not shown). [Figure 4G]4G is a schematic diagram depicting the structural orientation of additional embodiments of IL12-Fc fusion proteins that can be comprised or consist of by the IL12 receptor agonists of the present disclosure, incorporating a masking moiety in the form of an IL12 receptor (IL12R) β1 receptor moiety or an IL12Rβ2 receptor moiety. Examples of suitable p35 and p40 moieties that can be incorporated into the IL12 fusion protein of FIG. 4G are disclosed, e.g., in Section 6.3. Examples of suitable IL12 receptor moieties are disclosed, e.g., in Sections 6.4.1 and 6.4.1. Examples of suitable IL12 antibody-based masking moieties are disclosed, e.g., in Section 6.4.3. The CH2 and CH3 domains shown in FIG. 4G form an Fc domain, which is a type of multimerization moiety. Fc domains and other multimerization moieties that can be used in IL12 receptor agonists are described in Section 6.6. The Fc domain in the heterodimeric IL12-Fc fusion protein (e.g., as shown in Figure 4F-4W) can incorporate any combination of mutations suitable for heterodimerization or selective purification (e.g., knob-in-hole and / or star mutations), e.g., as described in section 6.6.1.2 (not shown). [Figure 4H]4H is a schematic diagram depicting the structural orientation of additional embodiments of IL12-Fc fusion proteins that can be comprised or consist of by the IL12 receptor agonists of the present disclosure, incorporating a masking moiety in the form of an IL12 receptor (IL12R) β1 receptor moiety or an IL12Rβ2 receptor moiety. Examples of suitable p35 and p40 moieties that can be incorporated into the IL12 fusion protein of Figure 4H are disclosed, e.g., in Section 6.3. Examples of suitable IL12 receptor moieties are disclosed, e.g., in Sections 6.4.1 and 6.4.1. Examples of suitable IL12 antibody-based masking moieties are disclosed, e.g., in Section 6.4.3. The CH2 and CH3 domains shown in Figure 4H form an Fc domain, which is a type of multimerization moiety. Fc domains and other multimerization moieties that can be used in IL12 receptor agonists are described in Section 6.6. The Fc domain in the heterodimeric IL12-Fc fusion protein (e.g., as shown in Figure 4F-4W) can incorporate any combination of mutations suitable for heterodimerization or selective purification (e.g., knob-in-hole and / or star mutations), e.g., as described in section 6.6.1.2 (not shown). [Figure 4I]4I is a schematic diagram depicting the structural orientation of additional embodiments of IL12-Fc fusion proteins that can be comprised or consist of by the IL12 receptor agonists of the present disclosure, incorporating a masking moiety in the form of an IL12 receptor (IL12R) β1 receptor moiety or an IL12Rβ2 receptor moiety. Examples of suitable p35 and p40 moieties that can be incorporated into the IL12 fusion protein of FIG. 4I are disclosed, e.g., in Section 6.3. Examples of suitable IL12 receptor moieties are disclosed, e.g., in Sections 6.4.1 and 6.4.1. Examples of suitable IL12 antibody-based masking moieties are disclosed, e.g., in Section 6.4.3. The CH2 and CH3 domains shown in FIG. 4I form an Fc domain, which is a type of multimerization moiety. Fc domains and other multimerization moieties that can be used in IL12 receptor agonists are described in Section 6.6. The Fc domain in the heterodimeric IL12-Fc fusion protein (e.g., as shown in Figure 4F-4W) can incorporate any combination of mutations suitable for heterodimerization or selective purification (e.g., knob-in-hole and / or star mutations), e.g., as described in section 6.6.1.2 (not shown). [Figure 4J]4J is a schematic diagram depicting the structural orientation of additional embodiments of IL12-Fc fusion proteins that can be comprised or consist of by the IL12 receptor agonists of the present disclosure, incorporating a masking moiety in the form of an IL12 receptor (IL12R) β1 receptor moiety or an IL12Rβ2 receptor moiety. Examples of suitable p35 and p40 moieties that can be incorporated into the IL12 fusion protein of Figure 4J are disclosed, e.g., in Section 6.3. Examples of suitable IL12 receptor moieties are disclosed, e.g., in Sections 6.4.1 and 6.4.1. Examples of suitable IL12 antibody-based masking moieties are disclosed, e.g., in Section 6.4.3. The CH2 and CH3 domains shown in Figure 4J form an Fc domain, which is a type of multimerization moiety. Fc domains and other multimerization moieties that can be used in IL12 receptor agonists are described in Section 6.6. The Fc domain in the heterodimeric IL12-Fc fusion protein (e.g., as shown in Figure 4F-4W) can incorporate any combination of mutations suitable for heterodimerization or selective purification (e.g., knob-in-hole and / or star mutations), e.g., as described in section 6.6.1.2 (not shown). [Figure 4K]4K is a schematic diagram depicting the structural orientation of additional embodiments of IL12-Fc fusion proteins that can be comprised or consist of by the IL12 receptor agonists of the present disclosure, incorporating a masking moiety in the form of an IL12 receptor (IL12R) β1 receptor moiety or an IL12Rβ2 receptor moiety. Examples of suitable p35 and p40 moieties that can be incorporated into the IL12 fusion protein of Figure 4K are disclosed, e.g., in Section 6.3. Examples of suitable IL12 receptor moieties are disclosed, e.g., in Sections 6.4.1 and 6.4.1. Examples of suitable IL12 antibody-based masking moieties are disclosed, e.g., in Section 6.4.3. The CH2 and CH3 domains shown in Figure 4K form an Fc domain, which is a type of multimerization moiety. Fc domains and other multimerization moieties that can be used in IL12 receptor agonists are described in Section 6.6. The Fc domain in the heterodimeric IL12-Fc fusion protein (e.g., as shown in Figure 4F-4W) can incorporate any combination of mutations suitable for heterodimerization or selective purification (e.g., knob-in-hole and / or star mutations), e.g., as described in section 6.6.1.2 (not shown). [Figure 4L]4L is a schematic diagram depicting the structural orientation of additional embodiments of IL12-Fc fusion proteins that can be comprised or consist of by the IL12 receptor agonists of the present disclosure, incorporating a masking moiety in the form of an IL12 receptor (IL12R) β1 receptor moiety or an IL12Rβ2 receptor moiety. Examples of suitable p35 and p40 moieties that can be incorporated into the IL12 fusion protein of FIG. 4L are disclosed, e.g., in Section 6.3. Examples of suitable IL12 receptor moieties are disclosed, e.g., in Sections 6.4.1 and 6.4.1. Examples of suitable IL12 antibody-based masking moieties are disclosed, e.g., in Section 6.4.3. The CH2 and CH3 domains shown in FIG. 4L form an Fc domain, which is a type of multimerization moiety. Fc domains and other multimerization moieties that can be used in IL12 receptor agonists are described in Section 6.6. The Fc domain in the heterodimeric IL12-Fc fusion protein (e.g., as shown in Figure 4F-4W) can incorporate any combination of mutations suitable for heterodimerization or selective purification (e.g., knob-in-hole and / or star mutations), e.g., as described in section 6.6.1.2 (not shown). [Figure 4M]4M is a schematic diagram depicting the structural orientation of additional embodiments of IL12-Fc fusion proteins that can be comprised or consist of by the IL12 receptor agonists of the present disclosure, incorporating a masking moiety in the form of an IL12 receptor (IL12R) β1 receptor moiety or an IL12Rβ2 receptor moiety. Examples of suitable p35 and p40 moieties that can be incorporated into the IL12 fusion protein of FIG. 4M are disclosed, e.g., in Section 6.3. Examples of suitable IL12 receptor moieties are disclosed, e.g., in Sections 6.4.1 and 6.4.1. Examples of suitable IL12 antibody-based masking moieties are disclosed, e.g., in Section 6.4.3. The CH2 and CH3 domains shown in FIG. 4M form an Fc domain, which is a type of multimerization moiety. Fc domains and other multimerization moieties that can be used in IL12 receptor agonists are described in Section 6.6. The Fc domain in the heterodimeric IL12-Fc fusion protein (e.g., as shown in Figure 4F-4W) can incorporate any combination of mutations suitable for heterodimerization or selective purification (e.g., knob-in-hole and / or star mutations), e.g., as described in section 6.6.1.2 (not shown). [Figure 4N]4N is a schematic diagram depicting the structural orientation of additional embodiments of IL12-Fc fusion proteins that can be comprised or consist of by the IL12 receptor agonists of the present disclosure, incorporating a masking moiety in the form of an IL12 receptor (IL12R) β1 receptor moiety or an IL12Rβ2 receptor moiety. Examples of suitable p35 and p40 moieties that can be incorporated into the IL12 fusion protein of FIG. 4N are disclosed, e.g., in Section 6.3. Examples of suitable IL12 receptor moieties are disclosed, e.g., in Sections 6.4.1 and 6.4.1. Examples of suitable IL12 antibody-based masking moieties are disclosed, e.g., in Section 6.4.3. The CH2 and CH3 domains shown in FIG. 4N form an Fc domain, which is a type of multimerization moiety. Fc domains and other multimerization moieties that can be used in IL12 receptor agonists are described in Section 6.6. The Fc domain in the heterodimeric IL12-Fc fusion protein (e.g., as shown in Figure 4F-4W) can incorporate any combination of mutations suitable for heterodimerization or selective purification (e.g., knob-in-hole and / or star mutations), e.g., as described in section 6.6.1.2 (not shown). [Figure 4O]4O is a schematic diagram depicting the structural orientation of additional embodiments of IL12-Fc fusion proteins that can be comprised or consist of by the IL12 receptor agonists of the present disclosure, incorporating a masking moiety in the form of an anti-IL12 antibody fragment. Examples of suitable p35 and p40 moieties that can be incorporated into the IL12 fusion protein of FIG. 4O are disclosed, e.g., in Section 6.3. Examples of suitable IL12 receptor moieties are disclosed, e.g., in Sections 6.4.1 and 6.4.1. Examples of suitable IL12 antibody-based masking moieties are disclosed, e.g., in Section 6.4.3. The CH2 and CH3 domains shown in FIG. 4O form an Fc domain, which is a type of multimerization moiety. Fc domains and other multimerization moieties that can be used in IL12 receptor agonists are described in Section 6.6. The Fc domain in the heterodimeric IL12-Fc fusion protein (e.g., as shown in Figure 4F-4W) can incorporate any combination of mutations suitable for heterodimerization or selective purification (e.g., knob-in-hole and / or star mutations), e.g., as described in section 6.6.1.2 (not shown). [Figure 4P]4P is a schematic diagram depicting the structural orientation of an additional embodiment of an IL12-Fc fusion protein that can be comprised or consist of an IL12 receptor agonist of the present disclosure, incorporating a masking moiety in the form of an anti-IL12 antibody fragment. Examples of suitable p35 and p40 moieties that can be incorporated into the IL12 fusion protein of FIG. 4P are disclosed, e.g., in Section 6.3. Examples of suitable IL12 receptor moieties are disclosed, e.g., in Sections 6.4.1 and 6.4.1. Examples of suitable IL12 antibody-based masking moieties are disclosed, e.g., in Section 6.4.3. The CH2 and CH3 domains shown in FIG. 4P form an Fc domain, which is a type of multimerization moiety. Fc domains and other multimerization moieties that can be used in IL12 receptor agonists are described in Section 6.6. The Fc domain in the heterodimeric IL12-Fc fusion protein (e.g., as shown in Figure 4F-4W) can incorporate any combination of mutations suitable for heterodimerization or selective purification (e.g., knob-in-hole and / or star mutations), e.g., as described in section 6.6.1.2 (not shown). [Figure 4Q]4Q is a schematic diagram depicting the structural orientation of additional embodiments of IL12-Fc fusion proteins that can be comprised or consist of in the IL12 receptor agonists of the present disclosure, incorporating a masking moiety in the form of an anti-IL12 antibody fragment. Examples of suitable p35 and p40 moieties that can be incorporated into the IL12 fusion protein of Figure 4Q are disclosed, e.g., in Section 6.3. Examples of suitable IL12 receptor moieties are disclosed, e.g., in Sections 6.4.1 and 6.4.1. Examples of suitable IL12 antibody-based masking moieties are disclosed, e.g., in Section 6.4.3. The CH2 and CH3 domains shown in Figure 4Q form an Fc domain, which is a type of multimerization moiety. Fc domains and other multimerization moieties that can be used in IL12 receptor agonists are described in Section 6.6. The Fc domain in the heterodimeric IL12-Fc fusion protein (e.g., as shown in Figure 4F-4W) can incorporate any combination of mutations suitable for heterodimerization or selective purification (e.g., knob-in-hole and / or star mutations), e.g., as described in section 6.6.1.2 (not shown). [Figure 4R]4R is a schematic diagram depicting the structural orientation of additional embodiments of IL12-Fc fusion proteins that can be comprised or consist of in the IL12 receptor agonists of the present disclosure, incorporating a masking moiety in the form of an anti-IL12 antibody fragment. Examples of suitable p35 and p40 moieties that can be incorporated into the IL12 fusion protein of FIG. 4R are disclosed, e.g., in Section 6.3. Examples of suitable IL12 receptor moieties are disclosed, e.g., in Sections 6.4.1 and 6.4.1. Examples of suitable IL12 antibody-based masking moieties are disclosed, e.g., in Section 6.4.3. The CH2 and CH3 domains shown in FIG. 4R form an Fc domain, which is a type of multimerization moiety. Fc domains and other multimerization moieties that can be used in IL12 receptor agonists are described in Section 6.6. The Fc domain in the heterodimeric IL12-Fc fusion protein (e.g., as shown in Figure 4F-4W) can incorporate any combination of mutations suitable for heterodimerization or selective purification (e.g., knob-in-hole and / or star mutations), e.g., as described in section 6.6.1.2 (not shown). [Figure 4S]4S is a schematic diagram depicting the structural orientation of additional embodiments of IL12-Fc fusion proteins that can be comprised or consist of by the IL12 receptor agonists of the present disclosure, incorporating a masking moiety in the form of an IL12 receptor (IL12R) β1 receptor moiety or an IL12Rβ2 receptor moiety. Examples of suitable p35 and p40 moieties that can be incorporated into the IL12 fusion protein of FIG. 4S are disclosed, e.g., in Section 6.3. Examples of suitable IL12 receptor moieties are disclosed, e.g., in Sections 6.4.1 and 6.4.1. Examples of suitable IL12 antibody-based masking moieties are disclosed, e.g., in Section 6.4.3. The CH2 and CH3 domains shown in FIG. 4S form an Fc domain, which is a type of multimerization moiety. Fc domains and other multimerization moieties that can be used in IL12 receptor agonists are described in Section 6.6. The Fc domain in the heterodimeric IL12-Fc fusion protein (e.g., as shown in Figure 4F-4W) can incorporate any combination of mutations suitable for heterodimerization or selective purification (e.g., knob-in-hole and / or star mutations), e.g., as described in section 6.6.1.2 (not shown). [Figure 4T]4T is a schematic diagram depicting the structural orientation of additional embodiments of IL12-Fc fusion proteins that can be comprised or consist of by the IL12 receptor agonists of the present disclosure, incorporating a masking moiety in the form of an IL12 receptor (IL12R) β1 receptor moiety or an IL12Rβ2 receptor moiety. Examples of suitable p35 and p40 moieties that can be incorporated into the IL12 fusion protein of FIG. 4T are disclosed, e.g., in Section 6.3. Examples of suitable IL12 receptor moieties are disclosed, e.g., in Sections 6.4.1 and 6.4.1. Examples of suitable IL12 antibody-based masking moieties are disclosed, e.g., in Section 6.4.3. The CH2 and CH3 domains shown in FIG. 4T form an Fc domain, which is a type of multimerization moiety. Fc domains and other multimerization moieties that can be used in IL12 receptor agonists are described in Section 6.6. The Fc domain in the heterodimeric IL12-Fc fusion protein (e.g., as shown in Figure 4F-4W) can incorporate any combination of mutations suitable for heterodimerization or selective purification (e.g., knob-in-hole and / or star mutations), e.g., as described in section 6.6.1.2 (not shown). [Figure 4U]4U is a schematic diagram depicting the structural orientation of additional embodiments of IL12-Fc fusion proteins that can be comprised or consist of by the IL12 receptor agonists of the present disclosure, incorporating a masking moiety in the form of an IL12 receptor (IL12R) β1 receptor moiety or an IL12Rβ2 receptor moiety. Examples of suitable p35 and p40 moieties that can be incorporated into the IL12 fusion protein of FIG. 4U are disclosed, e.g., in Section 6.3. Examples of suitable IL12 receptor moieties are disclosed, e.g., in Sections 6.4.1 and 6.4.1. Examples of suitable IL12 antibody-based masking moieties are disclosed, e.g., in Section 6.4.3. The CH2 and CH3 domains shown in FIG. 4U form an Fc domain, which is a type of multimerization moiety. Fc domains and other multimerization moieties that can be used in IL12 receptor agonists are described in Section 6.6. The Fc domain in the heterodimeric IL12-Fc fusion protein (e.g., as shown in Figure 4F-4W) can incorporate any combination of mutations suitable for heterodimerization or selective purification (e.g., knob-in-hole and / or star mutations), e.g., as described in section 6.6.1.2 (not shown). [Figure 4V]4V is a schematic diagram depicting the structural orientation of additional embodiments of IL12-Fc fusion proteins that can be comprised or consist of by the IL12 receptor agonists of the present disclosure, incorporating a masking moiety in the form of an IL12 receptor (IL12R) β1 receptor moiety or an IL12Rβ2 receptor moiety. Examples of suitable p35 and p40 moieties that can be incorporated into the IL12 fusion protein of FIG. 4V are disclosed, e.g., in Section 6.3. Examples of suitable IL12 receptor moieties are disclosed, e.g., in Sections 6.4.1 and 6.4.1. Examples of suitable IL12 antibody-based masking moieties are disclosed, e.g., in Section 6.4.3. The CH2 and CH3 domains shown in FIG. 4V form an Fc domain, which is a type of multimerization moiety. Fc domains and other multimerization moieties that can be used in IL12 receptor agonists are described in Section 6.6. The Fc domain in the heterodimeric IL12-Fc fusion protein (e.g., as shown in Figure 4F-4W) can incorporate any combination of mutations suitable for heterodimerization or selective purification (e.g., knob-in-hole and / or star mutations), e.g., as described in section 6.6.1.2 (not shown). [Figure 4W]4W is a schematic diagram depicting the structural orientation of additional embodiments of IL12-Fc fusion proteins that can be comprised or consist of by the IL12 receptor agonists of the present disclosure, incorporating a masking moiety in the form of an IL12 receptor (IL12R) β1 receptor moiety or an IL12Rβ2 receptor moiety. Examples of suitable p35 and p40 moieties that can be incorporated into the IL12 fusion protein of FIG. 4W are disclosed, e.g., in Section 6.3. Examples of suitable IL12 receptor moieties are disclosed, e.g., in Sections 6.4.1 and 6.4.1. Examples of suitable IL12 antibody-based masking moieties are disclosed, e.g., in Section 6.4.3. The CH2 and CH3 domains shown in FIG. 4W form an Fc domain, which is a type of multimerization moiety. Fc domains and other multimerization moieties that can be used in IL12 receptor agonists are described in Section 6.6. The Fc domain in the heterodimeric IL12-Fc fusion protein (e.g., as shown in Figure 4F-4W) can incorporate any combination of mutations suitable for heterodimerization or selective purification (e.g., knob-in-hole and / or star mutations), e.g., as described in section 6.6.1.2 (not shown). [Figure 5A] 5A is a schematic diagram depicting the structural orientation of an additional embodiment of an IL12-Fc fusion protein that can be comprised or consist of an IL12 receptor agonist of the present disclosure, incorporating the Fab domain of an antibody (e.g., an anti-PD1 (αPD1) antibody) as a targeting moiety. Examples of suitable p35 and p40 moieties that can be incorporated into the IL12 fusion protein of FIG. 5A are disclosed, e.g., in Section 6.3. Examples of suitable targeting moieties are disclosed, e.g., in Section 6.5. [Figure 5B]5B is a schematic diagram depicting the structural orientation of an additional embodiment of an IL12-Fc fusion protein that can be comprised or consist of an IL12 receptor agonist of the present disclosure, incorporating the Fab domain of an antibody (e.g., an anti-PD1 (αPD1) antibody) as a targeting moiety. Examples of suitable p35 and p40 moieties that can be incorporated into the IL12 fusion protein of FIG. 5B are disclosed, e.g., in Section 6.3. Examples of suitable targeting moieties are disclosed, e.g., in Section 6.5. The CH2 and CH3 domains shown in FIG. 5B form an Fc domain, which is a type of multimerization moiety. Fc domains and other multimerization moieties that can be used in IL12 receptor agonists are described in Section 6.6. The Fc domain in the heterodimeric IL12-Fc fusion protein (e.g., as shown in Figures 5B, 5C, 5E, 5G-5S, and 5V-5X) can incorporate any combination of mutations suitable for heterodimerization or selective purification (e.g., knob-in-hole and / or star mutations), e.g., as described in section 6.6.1.2 (not shown). [Figure 5C]5C is a schematic diagram depicting the structural orientation of an additional embodiment of an IL12-Fc fusion protein that can be comprised or consist of an IL12 receptor agonist of the present disclosure, incorporating the Fab domain of an antibody (e.g., an anti-PD1 (αPD1) antibody) as a targeting moiety. Examples of suitable p35 and p40 moieties that can be incorporated into the IL12 fusion protein of FIG. 5C are disclosed, e.g., in Section 6.3. Examples of suitable targeting moieties are disclosed, e.g., in Section 6.5. The CH2 and CH3 domains shown in FIG. 5C form an Fc domain, which is a type of multimerization moiety. Fc domains and other multimerization moieties that can be used in IL12 receptor agonists are described in Section 6.6. The Fc domain in the heterodimeric IL12-Fc fusion protein (e.g., as shown in Figures 5B, 5C, 5E, 5G-5S, and 5V-5X) can incorporate any combination of mutations suitable for heterodimerization or selective purification (e.g., knob-in-hole and / or star mutations), e.g., as described in section 6.6.1.2 (not shown). [Figure 5D]5D is a schematic diagram depicting the structural orientation of an additional embodiment of an IL12-Fc fusion protein that can be comprised or consist of an IL12 receptor agonist of the present disclosure, incorporating the Fab domain of an antibody (e.g., an anti-PD1 (αPD1) antibody) as a targeting moiety. Examples of suitable p35 and p40 moieties that can be incorporated into the IL12 fusion protein of FIG. 5D are disclosed, e.g., in Section 6.3. Examples of suitable targeting moieties are disclosed, e.g., in Section 6.5. The CH2 and CH3 domains shown in FIG. 5D form an Fc domain, which is a type of multimerization moiety. Fc domains and other multimerization moieties that can be used in IL12 receptor agonists are described in Section 6.6. The Fc domain in the heterodimeric IL12-Fc fusion protein (e.g., as shown in Figures 5B, 5C, 5E, 5G-5S, and 5V-5X) can incorporate any combination of mutations suitable for heterodimerization or selective purification (e.g., knob-in-hole and / or star mutations), e.g., as described in section 6.6.1.2 (not shown). [Figure 5E]5E is a schematic diagram depicting the structural orientation of an additional embodiment of an IL12-Fc fusion protein that can be comprised or consist of an IL12 receptor agonist of the present disclosure, incorporating the Fab domain of an antibody (e.g., an anti-PD1 (αPD1) antibody) as a targeting moiety. Examples of suitable p35 and p40 moieties that can be incorporated into the IL12 fusion protein of FIG. 5E are disclosed, e.g., in Section 6.3. Examples of suitable targeting moieties are disclosed, e.g., in Section 6.5. The CH2 and CH3 domains shown in FIG. 5E form an Fc domain, which is a type of multimerization moiety. Fc domains and other multimerization moieties that can be used in IL12 receptor agonists are described in Section 6.6. The Fc domain in the heterodimeric IL12-Fc fusion protein (e.g., as shown in Figures 5B, 5C, 5E, 5G-5S, and 5V-5X) can incorporate any combination of mutations suitable for heterodimerization or selective purification (e.g., knob-in-hole and / or star mutations), e.g., as described in section 6.6.1.2 (not shown). [Figure 5F]5F is a schematic diagram depicting the structural orientation of an additional embodiment of an IL12-Fc fusion protein that can be comprised or consist of an IL12 receptor agonist of the present disclosure, incorporating the Fab domain of an antibody (e.g., an anti-PD1 (αPD1) antibody) as a targeting moiety. Examples of suitable p35 and p40 moieties that can be incorporated into the IL12 fusion protein of FIG. 5F are disclosed, e.g., in Section 6.3. Examples of suitable targeting moieties are disclosed, e.g., in Section 6.5. The CH2 and CH3 domains shown in FIG. 5F form an Fc domain, which is a type of multimerization moiety. Fc domains and other multimerization moieties that can be used in IL12 receptor agonists are described in Section 6.6. The Fc domain in the heterodimeric IL12-Fc fusion protein (e.g., as shown in Figures 5B, 5C, 5E, 5G-5S, and 5V-5X) can incorporate any combination of mutations suitable for heterodimerization or selective purification (e.g., knob-in-hole and / or star mutations), e.g., as described in section 6.6.1.2 (not shown). [Figure 5G]5G is a schematic diagram depicting the structural orientation of an additional embodiment of an IL12-Fc fusion protein that can be comprised or consist of an IL12 receptor agonist of the present disclosure, incorporating the Fab domain of an antibody (e.g., an anti-PD1 (αPD1) antibody) as a targeting moiety. Examples of suitable p35 and p40 moieties that can be incorporated into the IL12 fusion protein of FIG. 5G are disclosed, e.g., in Section 6.3. Examples of suitable targeting moieties are disclosed, e.g., in Section 6.5. The CH2 and CH3 domains shown in FIG. 5G form an Fc domain, which is a type of multimerization moiety. Fc domains and other multimerization moieties that can be used in IL12 receptor agonists are described in Section 6.6. The Fc domain in the heterodimeric IL12-Fc fusion protein (e.g., as shown in Figures 5B, 5C, 5E, 5G-5S, and 5V-5X) can incorporate any combination of mutations suitable for heterodimerization or selective purification (e.g., knob-in-hole and / or star mutations), e.g., as described in section 6.6.1.2 (not shown). [Figure 5H]5H is a schematic diagram depicting the structural orientation of an additional embodiment of an IL12-Fc fusion protein that can be comprised or consist of an IL12 receptor agonist of the present disclosure, incorporating the Fab domain of an antibody (e.g., an anti-PD1 (αPD1) antibody) as a targeting moiety. Examples of suitable p35 and p40 moieties that can be incorporated into the IL12 fusion protein of FIG. 5H are disclosed, e.g., in Section 6.3. Examples of suitable targeting moieties are disclosed, e.g., in Section 6.5. The CH2 and CH3 domains shown in FIG. 5H form an Fc domain, which is a type of multimerization moiety. Fc domains and other multimerization moieties that can be used in IL12 receptor agonists are described in Section 6.6. The Fc domain in a heterodimeric IL12-Fc fusion protein (e.g., as shown in Figures 5B, 5C, 5E, 5G-5S, and 5V-5X) can incorporate any combination of mutations suitable for heterodimerization or selective purification (e.g., knob-in-hole and / or star mutations), e.g., as described in Section 6.6.1.2 (not shown). In some embodiments, the IL12-Fc fusion protein is masked, for example, by a receptor (as shown in Figures 5H-5K, 5O, 5R, 5S, and 5V-5X) or an antibody (as shown in Figures 5L-5N). While the antibody in Figures 5L-5N is depicted as an Fv, particularly an scFv, the scFv can be replaced with a Fab, as shown in Figures 39A-39B. [Figure 5I]5I is a schematic diagram depicting the structural orientation of an additional embodiment of an IL12-Fc fusion protein that can be comprised or consist of an IL12 receptor agonist of the present disclosure, incorporating the Fab domain of an antibody (e.g., an anti-PD1 (αPD1) antibody) as a targeting moiety. Examples of suitable p35 and p40 moieties that can be incorporated into the IL12 fusion protein of FIG. 5I are disclosed, e.g., in Section 6.3. Examples of suitable targeting moieties are disclosed, e.g., in Section 6.5. The CH2 and CH3 domains shown in FIG. 5I form an Fc domain, which is a type of multimerization moiety. Fc domains and other multimerization moieties that can be used in IL12 receptor agonists are described in Section 6.6. The Fc domain in a heterodimeric IL12-Fc fusion protein (e.g., as shown in Figures 5B, 5C, 5E, 5G-5S, and 5V-5X) can incorporate any combination of mutations suitable for heterodimerization or selective purification (e.g., knob-in-hole and / or star mutations), e.g., as described in Section 6.6.1.2 (not shown). In some embodiments, the IL12-Fc fusion protein is masked, for example, by a receptor (as shown in Figures 5H-5K, 5O, 5R, 5S, and 5V-5X) or an antibody (as shown in Figures 5L-5N). While the antibody in Figures 5L-5N is depicted as an Fv, particularly an scFv, the scFv can be replaced with a Fab, as shown in Figures 39A-39B. [Figure 5J]5J is a schematic diagram depicting the structural orientation of an additional embodiment of an IL12-Fc fusion protein that can be comprised or consist of an IL12 receptor agonist of the present disclosure, incorporating the Fab domain of an antibody (e.g., an anti-PD1 (αPD1) antibody) as a targeting moiety. Examples of suitable p35 and p40 moieties that can be incorporated into the IL12 fusion protein of FIG. 5J are disclosed, e.g., in Section 6.3. Examples of suitable targeting moieties are disclosed, e.g., in Section 6.5. The CH2 and CH3 domains shown in FIG. 5J form an Fc domain, which is a type of multimerization moiety. Fc domains and other multimerization moieties that can be used in IL12 receptor agonists are described in Section 6.6. The Fc domain in a heterodimeric IL12-Fc fusion protein (e.g., as shown in Figures 5B, 5C, 5E, 5G-5S, and 5V-5X) can incorporate any combination of mutations suitable for heterodimerization or selective purification (e.g., knob-in-hole and / or star mutations), e.g., as described in Section 6.6.1.2 (not shown). In some embodiments, the IL12-Fc fusion protein is masked, for example, by a receptor (as shown in Figures 5H-5K, 5O, 5R, 5S, and 5V-5X) or an antibody (as shown in Figures 5L-5N). While the antibody in Figures 5L-5N is depicted as an Fv, particularly an scFv, the scFv can be replaced with a Fab, as shown in Figures 39A-39B. [Figure 5K]5K is a schematic diagram depicting the structural orientation of an additional embodiment of an IL12-Fc fusion protein that can be comprised or consist of an IL12 receptor agonist of the present disclosure, incorporating the Fab domain of an antibody (e.g., an anti-PD1 (αPD1) antibody) as a targeting moiety. Examples of suitable p35 and p40 moieties that can be incorporated into the IL12 fusion protein of FIG. 5K are disclosed, e.g., in Section 6.3. Examples of suitable targeting moieties are disclosed, e.g., in Section 6.5. The CH2 and CH3 domains shown in FIG. 5K form an Fc domain, which is a type of multimerization moiety. Fc domains and other multimerization moieties that can be used in IL12 receptor agonists are described in Section 6.6. The Fc domain in a heterodimeric IL12-Fc fusion protein (e.g., as shown in Figures 5B, 5C, 5E, 5G-5S, and 5V-5X) can incorporate any combination of mutations suitable for heterodimerization or selective purification (e.g., knob-in-hole and / or star mutations), e.g., as described in Section 6.6.1.2 (not shown). In some embodiments, the IL12-Fc fusion protein is masked, for example, by a receptor (as shown in Figures 5H-5K, 5O, 5R, 5S, and 5V-5X) or an antibody (as shown in Figures 5L-5N). While the antibody in Figures 5L-5N is depicted as an Fv, particularly an scFv, the scFv can be replaced with a Fab, as shown in Figures 39A-39B. [Figure 5L]5L is a schematic diagram depicting the structural orientation of an additional embodiment of an IL12-Fc fusion protein that can be comprised or consist of an IL12 receptor agonist of the present disclosure, incorporating the Fab domain of an antibody (e.g., an anti-PD1 (αPD1) antibody) as a targeting moiety. Examples of suitable p35 and p40 moieties that can be incorporated into the IL12 fusion protein of FIG. 5L are disclosed, e.g., in Section 6.3. Examples of suitable targeting moieties are disclosed, e.g., in Section 6.5. The CH2 and CH3 domains shown in FIG. 5L form an Fc domain, which is a type of multimerization moiety. Fc domains and other multimerization moieties that can be used in IL12 receptor agonists are described in Section 6.6. The Fc domain in a heterodimeric IL12-Fc fusion protein (e.g., as shown in Figures 5B, 5C, 5E, 5G-5S, and 5V-5X) can incorporate any combination of mutations suitable for heterodimerization or selective purification (e.g., knob-in-hole and / or star mutations), e.g., as described in Section 6.6.1.2 (not shown). In some embodiments, the IL12-Fc fusion protein is masked, for example, by a receptor (as shown in Figures 5H-5K, 5O, 5R, 5S, and 5V-5X) or an antibody (as shown in Figures 5L-5N). While the antibody in Figures 5L-5N is depicted as an Fv, particularly an scFv, the scFv can be replaced with a Fab, as shown in Figures 39A-39B. [Figure 5M]5M is a schematic diagram depicting the structural orientation of an additional embodiment of an IL12-Fc fusion protein that can be comprised or consist of an IL12 receptor agonist of the present disclosure, incorporating the Fab domain of an antibody (e.g., an anti-PD1 (αPD1) antibody) as a targeting moiety. Examples of suitable p35 and p40 moieties that can be incorporated into the IL12 fusion protein of FIG. 5M are disclosed, e.g., in Section 6.3. Examples of suitable targeting moieties are disclosed, e.g., in Section 6.5. The CH2 and CH3 domains shown in FIG. 5M form an Fc domain, which is a type of multimerization moiety. Fc domains and other multimerization moieties that can be used in IL12 receptor agonists are described in Section 6.6. The Fc domain in a heterodimeric IL12-Fc fusion protein (e.g., as shown in Figures 5B, 5C, 5E, 5G-5S, and 5V-5X) can incorporate any combination of mutations suitable for heterodimerization or selective purification (e.g., knob-in-hole and / or star mutations), e.g., as described in Section 6.6.1.2 (not shown). In some embodiments, the IL12-Fc fusion protein is masked, for example, by a receptor (as shown in Figures 5H-5K, 5O, 5R, 5S, and 5V-5X) or an antibody (as shown in Figures 5L-5N). While the antibody in Figures 5L-5N is depicted as an Fv, particularly an scFv, the scFv can be replaced with a Fab, as shown in Figures 39A-39B. [Figure 5N]5N is a schematic diagram depicting the structural orientation of an additional embodiment of an IL12-Fc fusion protein that can be comprised or consist of an IL12 receptor agonist of the present disclosure, incorporating the Fab domain of an antibody (e.g., an anti-PD1 (αPD1) antibody) as a targeting moiety. Examples of suitable p35 and p40 moieties that can be incorporated into the IL12 fusion protein of FIG. 5N are disclosed, e.g., in Section 6.3. Examples of suitable targeting moieties are disclosed, e.g., in Section 6.5. The CH2 and CH3 domains shown in FIG. 5N form an Fc domain, which is a type of multimerization moiety. Fc domains and other multimerization moieties that can be used in IL12 receptor agonists are described in Section 6.6. The Fc domain in a heterodimeric IL12-Fc fusion protein (e.g., as shown in Figures 5B, 5C, 5E, 5G-5S, and 5V-5X) can incorporate any combination of mutations suitable for heterodimerization or selective purification (e.g., knob-in-hole and / or star mutations), e.g., as described in Section 6.6.1.2 (not shown). In some embodiments, the IL12-Fc fusion protein is masked, for example, by a receptor (as shown in Figures 5H-5K, 5O, 5R, 5S, and 5V-5X) or an antibody (as shown in Figures 5L-5N). While the antibody in Figures 5L-5N is depicted as an Fv, particularly an scFv, the scFv can be replaced with a Fab, as shown in Figures 39A-39B. [Figure 5O]5O is a schematic diagram depicting the structural orientation of an additional embodiment of an IL12-Fc fusion protein that can be comprised or consist of an IL12 receptor agonist of the present disclosure, incorporating the Fab domain of an antibody (e.g., an anti-PD1 (αPD1) antibody) as a targeting moiety. Examples of suitable p35 and p40 moieties that can be incorporated into the IL12 fusion protein of FIG. 5O are disclosed, e.g., in Section 6.3. Examples of suitable targeting moieties are disclosed, e.g., in Section 6.5. The CH2 and CH3 domains shown in FIG. 5O form an Fc domain, which is a type of multimerization moiety. Fc domains and other multimerization moieties that can be used in IL12 receptor agonists are described in Section 6.6. The Fc domain in a heterodimeric IL12-Fc fusion protein (e.g., as shown in Figures 5B, 5C, 5E, 5G-5S, and 5V-5X) can incorporate any combination of mutations suitable for heterodimerization or selective purification (e.g., knob-in-hole and / or star mutations), e.g., as described in Section 6.6.1.2 (not shown). In some embodiments, the IL12-Fc fusion protein is masked, for example, by a receptor (as shown in Figures 5H-5K, 5O, 5R, 5S, and 5V-5X) or an antibody (as shown in Figures 5L-5N). While the antibody in Figures 5L-5N is depicted as an Fv, particularly an scFv, the scFv can be replaced with a Fab, as shown in Figures 39A-39B. [Figure 5P]5P is a schematic diagram depicting the structural orientation of an additional embodiment of an IL12-Fc fusion protein that can be comprised or consist of an IL12 receptor agonist of the present disclosure, incorporating the Fab domain of an antibody (e.g., an anti-PD1 (αPD1) antibody) as a targeting moiety. Examples of suitable p35 and p40 moieties that can be incorporated into the IL12 fusion protein of FIG. 5P are disclosed, e.g., in Section 6.3. Examples of suitable targeting moieties are disclosed, e.g., in Section 6.5. The CH2 and CH3 domains shown in FIG. 5P form an Fc domain, which is a type of multimerization moiety. Fc domains and other multimerization moieties that can be used in IL12 receptor agonists are described in Section 6.6. The Fc domain in a heterodimeric IL12-Fc fusion protein (e.g., as shown in Figures 5B, 5C, 5E, 5G-5S, and 5V-5X) can incorporate any combination of mutations suitable for heterodimerization or selective purification (e.g., knob-in-hole and / or star mutations), e.g., as described in Section 6.6.1.2 (not shown). In some embodiments, the IL12-Fc fusion protein is masked, for example, by a receptor (as shown in Figures 5H-5K, 5O, 5R, 5S, and 5V-5X) or an antibody (as shown in Figures 5L-5N). While the antibody in Figures 5L-5N is depicted as an Fv, particularly an scFv, the scFv can be replaced with a Fab, as shown in Figures 39A-39B. [Figure 5Q]5Q is a schematic diagram depicting the structural orientation of an additional embodiment of an IL12-Fc fusion protein that can be comprised or consist of an IL12 receptor agonist of the present disclosure, incorporating the Fab domain of an antibody (e.g., an anti-PD1 (αPD1) antibody) as a targeting moiety. Examples of suitable p35 and p40 moieties that can be incorporated into the IL12 fusion protein of FIG. 5Q are disclosed, e.g., in Section 6.3. Examples of suitable targeting moieties are disclosed, e.g., in Section 6.5. The CH2 and CH3 domains shown in FIG. 5Q form an Fc domain, which is a type of multimerization moiety. Fc domains and other multimerization moieties that can be used in IL12 receptor agonists are described in Section 6.6. The Fc domain in a heterodimeric IL12-Fc fusion protein (e.g., as shown in Figures 5B, 5C, 5E, 5G-5S, and 5V-5X) can incorporate any combination of mutations suitable for heterodimerization or selective purification (e.g., knob-in-hole and / or star mutations), e.g., as described in Section 6.6.1.2 (not shown). In some embodiments, the IL12-Fc fusion protein is masked, for example, by a receptor (as shown in Figures 5H-5K, 5O, 5R, 5S, and 5V-5X) or an antibody (as shown in Figures 5L-5N). While the antibody in Figures 5L-5N is depicted as an Fv, particularly an scFv, the scFv can be replaced with a Fab, as shown in Figures 39A-39B. [Figure 5R]5R is a schematic diagram depicting the structural orientation of an additional embodiment of an IL12-Fc fusion protein that can be comprised or consist of an IL12 receptor agonist of the present disclosure, incorporating the Fab domain of an antibody (e.g., an anti-PD1 (αPD1) antibody) as a targeting moiety. Examples of suitable p35 and p40 moieties that can be incorporated into the IL12 fusion protein of FIG. 5R are disclosed, e.g., in Section 6.3. Examples of suitable targeting moieties are disclosed, e.g., in Section 6.5. The CH2 and CH3 domains shown in FIG. 5R form an Fc domain, which is a type of multimerization moiety. Fc domains and other multimerization moieties that can be used in IL12 receptor agonists are described in Section 6.6. The Fc domain in a heterodimeric IL12-Fc fusion protein (e.g., as shown in Figures 5B, 5C, 5E, 5G-5S, and 5V-5X) can incorporate any combination of mutations suitable for heterodimerization or selective purification (e.g., knob-in-hole and / or star mutations), e.g., as described in Section 6.6.1.2 (not shown). In some embodiments, the IL12-Fc fusion protein is masked, for example, by a receptor (as shown in Figures 5H-5K, 5O, 5R, 5S, and 5V-5X) or an antibody (as shown in Figures 5L-5N). While the antibody in Figures 5L-5N is depicted as an Fv, particularly an scFv, the scFv can be replaced with a Fab, as shown in Figures 39A-39B. [Figure 5S]5S is a schematic diagram depicting the structural orientation of an additional embodiment of an IL12-Fc fusion protein that can be comprised or consist of an IL12 receptor agonist of the present disclosure, incorporating the Fab domain of an antibody (e.g., an anti-PD1 (αPD1) antibody) as a targeting moiety. Examples of suitable p35 and p40 moieties that can be incorporated into the IL12 fusion protein of FIG. 5S are disclosed, e.g., in Section 6.3. Examples of suitable targeting moieties are disclosed, e.g., in Section 6.5. The CH2 and CH3 domains shown in FIG. 5S form an Fc domain, which is a type of multimerization moiety. Fc domains and other multimerization moieties that can be used in IL12 receptor agonists are described in Section 6.6. The Fc domain in a heterodimeric IL12-Fc fusion protein (e.g., as shown in Figures 5B, 5C, 5E, 5G-5S, and 5V-5X) can incorporate any combination of mutations suitable for heterodimerization or selective purification (e.g., knob-in-hole and / or star mutations), e.g., as described in Section 6.6.1.2 (not shown). In some embodiments, the IL12-Fc fusion protein is masked, for example, by a receptor (as shown in Figures 5H-5K, 5O, 5R, 5S, and 5V-5X) or an antibody (as shown in Figures 5L-5N). While the antibody in Figures 5L-5N is depicted as an Fv, particularly an scFv, the scFv can be replaced with a Fab, as shown in Figures 39A-39B. [Figure 5T] FIG. 5 is a schematic diagram depicting an exemplary mechanism of action of the targeted IL12-Fc fusion proteins disclosed herein, eg, in FIGS. 5B-5S and 5V-5X. [Figure 5U] FIG. 5 is a schematic diagram depicting an exemplary mechanism of action of the targeted IL12-Fc fusion proteins disclosed herein, eg, in FIGS. 5B-5S and 5V-5X. [Figure 5V]5V is a schematic diagram depicting the structural orientation of an additional embodiment of an IL12-Fc fusion protein that can be comprised or consist of an IL12 receptor agonist of the present disclosure, incorporating the Fab domain of an antibody (e.g., an anti-PD1 (αPD1) antibody) as a targeting moiety. Examples of suitable p35 and p40 moieties that can be incorporated into the IL12 fusion protein of FIG. 5V are disclosed, e.g., in Section 6.3. Examples of suitable targeting moieties are disclosed, e.g., in Section 6.5. The CH2 and CH3 domains shown in FIG. 5V form an Fc domain, which is a type of multimerization moiety. Fc domains and other multimerization moieties that can be used in IL12 receptor agonists are described in Section 6.6. The Fc domain in a heterodimeric IL12-Fc fusion protein (e.g., as shown in Figures 5B, 5C, 5E, 5G-5S, and 5V-5X) can incorporate any combination of mutations suitable for heterodimerization or selective purification (e.g., knob-in-hole and / or star mutations), e.g., as described in Section 6.6.1.2 (not shown). In some embodiments, the IL12-Fc fusion protein is masked, for example, by a receptor (as shown in Figures 5H-5K, 5O, 5R, 5S, and 5V-5X) or an antibody (as shown in Figures 5L-5N). While the antibody in Figures 5L-5N is depicted as an Fv, particularly an scFv, the scFv can be replaced with a Fab, as shown in Figures 39A-39B. [Figure 5W]5W is a schematic diagram depicting the structural orientation of an additional embodiment of an IL12-Fc fusion protein that can be comprised or consist of an IL12 receptor agonist of the present disclosure, incorporating the Fab domain of an antibody (e.g., an anti-PD1 (αPD1) antibody) as a targeting moiety. Examples of suitable p35 and p40 moieties that can be incorporated into the IL12 fusion protein of FIG. 5W are disclosed, e.g., in Section 6.3. Examples of suitable targeting moieties are disclosed, e.g., in Section 6.5. The CH2 and CH3 domains shown in FIG. 5W form an Fc domain, which is a type of multimerization moiety. Fc domains and other multimerization moieties that can be used in IL12 receptor agonists are described in Section 6.6. The Fc domain in a heterodimeric IL12-Fc fusion protein (e.g., as shown in Figures 5B, 5C, 5E, 5G-5S, and 5V-5X) can incorporate any combination of mutations suitable for heterodimerization or selective purification (e.g., knob-in-hole and / or star mutations), e.g., as described in Section 6.6.1.2 (not shown). In some embodiments, the IL12-Fc fusion protein is masked, for example, by a receptor (as shown in Figures 5H-5K, 5O, 5R, 5S, and 5V-5X) or an antibody (as shown in Figures 5L-5N). While the antibody in Figures 5L-5N is depicted as an Fv, particularly an scFv, the scFv can be replaced with a Fab, as shown in Figures 39A-39B. [Figure 5X]5X is a schematic diagram depicting the structural orientation of an additional embodiment of an IL12-Fc fusion protein that can be comprised or consist of an IL12 receptor agonist of the present disclosure, incorporating the Fab domain of an antibody (e.g., an anti-PD1 (αPD1) antibody) as a targeting moiety. Examples of suitable p35 and p40 moieties that can be incorporated into the IL12 fusion protein of FIG. 5X are disclosed, e.g., in Section 6.3. Examples of suitable targeting moieties are disclosed, e.g., in Section 6.5. The CH2 and CH3 domains shown in FIG. 5X form an Fc domain, which is a type of multimerization moiety. Fc domains and other multimerization moieties that can be used in IL12 receptor agonists are described in Section 6.6. The Fc domain in a heterodimeric IL12-Fc fusion protein (e.g., as shown in Figures 5B, 5C, 5E, 5G-5S, and 5V-5X) can incorporate any combination of mutations suitable for heterodimerization or selective purification (e.g., knob-in-hole and / or star mutations), e.g., as described in Section 6.6.1.2 (not shown). In some embodiments, the IL12-Fc fusion protein is masked, for example, by a receptor (as shown in Figures 5H-5K, 5O, 5R, 5S, and 5V-5X) or an antibody (as shown in Figures 5L-5N). While the antibody in Figures 5L-5N is depicted as an Fv, particularly an scFv, the scFv can be replaced with a Fab, as shown in Figures 39A-39B. [Figure 6] 6 depicts an alignment of mouse and human IL12 p35, with arrows depicting examples of the location of representative mutant proteins. FIG. 6 discloses, in order of appearance, SEQ ID NOs: 126 and 6, respectively. [Figure 7] 7 depicts an alignment of mouse and human IL12 p40, with arrows depicting examples of the location of representative mutant proteins. FIG. 7 discloses, in order of appearance, SEQ ID NOs: 127 and 5, respectively. [Figure 8]8 depicts a sequence alignment of human IL12 p35 with other representative IL6 family cytokines. Arrows depict the locations of representative amino acid substitutions. FIG. 8 discloses SEQ ID NOS: 6, 128-132, respectively, in order of appearance. [Figure 9] The three-dimensional structure of IL12 (p35 and p40) is depicted, highlighting potential residues involved in p35 interaction with IL12Rβ2, residues at the p35 / p40 heterodimer interface, and surface-exposed residues located at the D1 or D1-D2 junction of p40 potentially involved in the interaction with IL12Rβ1. [Figure 10A] Photograph of an SDS-PAGE gel depicting the size of IL12-Fc fusion proteins: Lane 1) Monovalent: IL12(p35×p40)-Fc; Lane 2) Monovalent: Fc-IL12(p35×p40); Lane 3) Monovalent: IL12*(p35*×p40*)-Fc. [Figure 10B] Photograph of an SDS-PAGE gel depicting the size of IL12-Fc fusion proteins: Lane 4) Bivalent: IL12(p40-p35)-Fc; Lane 5) Bivalent: IL12(p35-p40)-Fc; Lane 6) Bivalent: Fc-IL12(p40-p35); Lane 7) Bivalent: Fc-IL12(p35-p40); Lane 10) Bivalent: IL12*(p40*-p35*)-Fc; Lane 11) Bivalent: IL12*(p35*-p40*)-Fc; Lane 12) Bivalent: Fc-IL12*(p40*-p35*); Lane 13) Bivalent: Fc-IL12*(p35*-p40*). [Figure 11]A trace from size-exclusion ultra-performance liquid chromatography (SEC) coupled with multi-angle light scattering (MALS) (SEC-MALS) is depicted, showing the size and location of the monovalent IL12(p35 × p40)-Fc. The predicted molecular weight of the fusion protein is 110.5 kDa. The fusion protein has six predicted glycosylation sites, resulting in an estimated MW of 122.5 kDa with glycosylation. The fusion protein exhibited a 125.6 kDa monomeric protein with approximately 75% peak area, and two HMW species (peak 2, MW approximately 258 kDa, 18.1% peak area) and (peak 1, 6.0% peak area). [Figure 12] A trace from SEC-MALS is depicted, showing the size and arrangement of Fc-monovalent:Fc-IL12(p35×p40). The predicted molecular weight of the fusion protein is 110.5 kDa. The fusion protein has six predicted glycosylation sites, resulting in an estimated MW of 122.5 kDa with glycosylation. The fusion protein was primarily represented as a potential dimeric protein of 244.3 kDa, with a total peak area of ​​approximately 50%. Putative monomeric and trimeric oligomers were also detected (peaks 1 and 3, respectively). [Figure 13] A trace from SEC-MALS is depicted, showing the size and arrangement of monovalent IL12*(p35*×p40*)-Fc. The predicted molecular weight of the fusion protein is 110.5 kDa. The fusion protein has six predicted glycosylation sites, resulting in an estimated MW of 122.5 kDa with glycosylation. The fusion protein exhibited a peak area of ​​approximately 72% for a 128.6 kDa monomeric protein, with putative dimeric and tetrameric oligomers detected (peaks 2 and 3). [Figure 14]A trace from SEC-MALS is depicted, showing the size and arrangement of the bivalent IL12(p35-p40)-Fc. The predicted molecular weight of the fusion protein is 170.0 kDa. The fusion protein has 10 predicted glycosylation sites, resulting in an estimated MW of 190.0 kDa with glycosylation. The fusion protein exhibited mostly aggregated protein, with the high molecular weight species accounting for approximately 45% of the total peak area and an apparent molar mass of 1.7 MDa. [Figure 15] A trace from SEC-MALS is depicted, showing the size and arrangement of the bivalent IL12(p40-p35)-Fc. The predicted molecular weight of the fusion protein is 171.2 kDa. The fusion protein has 12 predicted glycosylation sites, resulting in an estimated MW of 195.2 kDa with glycosylation. The fusion protein exhibited a 195.2 kDa monomeric protein with a total peak area of ​​approximately 70%. A putative dimer was also detected (peak 2). [Figure 16] A trace from SEC-MALS is depicted, showing the size and arrangement of Fc-bivalent:Fc-IL12(p35-p40). The predicted molecular weight of the fusion protein is 171.2 kDa. The fusion protein has 12 predicted glycosylation sites, resulting in an estimated MW of 195.2 kDa with glycosylation. The fusion protein showed mostly aggregated protein, with the predominant species being approximately 450 kDa with a peak area of ​​47.7%. [Figure 17] A trace from SEC-MALS is depicted, showing the size and arrangement of the bivalent Fc-IL12 (p40-p35). The predicted molecular weight of the fusion protein is 170 kDa. The fusion protein has 10 predicted glycosylation sites, resulting in an estimated MW of 190.0 kDa with glycosylation. The fusion protein exhibited a total peak area of ​​approximately 82% of a 198.9 kDa monomeric protein. A putative dimer was also detected (peak 2). [Figure 18]A trace from SEC-MALS is depicted, showing the size and arrangement of the bivalent IL12*(p40*-p35*)-Fc. The predicted molecular weight of the fusion protein is 171.2 kDa. The fusion protein has 12 predicted glycosylation sites, resulting in an estimated MW of 195.2 kDa with glycosylation. The fusion protein consisted primarily of a monomeric species (approximately 60% of the total peak area) with an apparent molar mass of 201.0 kDa. [Figure 19] Curves showing the bioactivity of the indicated control or IL12-Fc fusion proteins on CTLL2 / STAT3-Luc cells are presented. [Figure 20A] Curves showing the biological activity of the indicated control or IL12-Fc fusion proteins or mutant proteins on CTLL2 / STAT3-Luc cells are presented. [Figure 20B] Curves showing the biological activity of the indicated control or IL12-Fc fusion proteins or mutant proteins on CTLL2 / STAT3-Luc cells are presented. [Figure 21] FIG. 1 is a schematic diagram depicting the experimental protocol for implanting MC38 cancer cells in C57BL / 6 mice followed by dosing with test fusion proteins. [Figure 22] 1 is a graph depicting the effect of the indicated control or fusion proteins on tumor volume in the MC38 tumor model. [Figure 23A] 1 is a graph depicting the effect of the indicated control or fusion proteins on individual tumor growth in the MC38 tumor model. [Figure 23B] 1 is a graph depicting the effect of the indicated control or fusion proteins on individual tumor growth in the MC38 tumor model. [Figure 23C] 1 is a graph depicting the effect of the indicated control or fusion proteins on individual tumor growth in the MC38 tumor model. [Figure 23D] 1 is a graph depicting the effect of the indicated control or fusion proteins on individual tumor growth in the MC38 tumor model. [Figure 23E] 1 is a graph depicting the effect of the indicated control or fusion proteins on individual tumor growth in the MC38 tumor model. [Figure 23F] 1 is a graph depicting the effect of the indicated control or fusion proteins on individual tumor growth in the MC38 tumor model. [Figure 24] 1 is a graph depicting the effect of the indicated control or fusion proteins on mouse weight change in the MC38 tumor model. [Figure 25A] 1 depicts the effect of the indicated control or fusion or mutant protein fusion proteins on tumor volume and body weight change in the MC38 tumor model. [Figure 25B] 1 depicts the effect of the indicated control or fusion or mutant protein fusion proteins on tumor volume and body weight change in the MC38 tumor model. [Figure 26A] Traces from binding assays are depicted showing binding of the indicated IL12-Fc fusion proteins to primary murine T cells. [Figure 26B] Traces from binding assays are depicted showing binding of the indicated IL12-Fc fusion proteins to primary murine T cells. [Figure 27A] 1 depicts traces from a pSTAT4-based bioassay showing the effect of the indicated IL12-Fc fusion proteins on pSTAT4 activity in primary murine T cells. [Figure 27B] 1 depicts traces from a pSTAT4-based bioassay showing the effect of the indicated IL12-Fc fusion proteins on pSTAT4 activity in primary murine T cells. [Figure 28] 1 depicts traces from a STAT3-based bioassay showing the effect of the indicated IL12-Fc fusion proteins on STAT3 activity in NK92 cells (NK92 / STAT3-Luc cl.7F7). [Figure 29] Traces from a STAT3-based bioassay are depicted showing the effect of the indicated IL12-Fc fusion proteins on STAT3 activity in NK92 cells (NK92 / STAT3-Luc cl.7F7). [Figure 30] 1 depicts traces from a pSTAT4-based bioassay showing the effect of the indicated IL12-Fc fusion proteins on pSTAT4 activity in primary murine T cells. [Figure 31] 1 depicts traces from a STAT3-based bioassay showing the effect of the indicated IL12-Fc fusion proteins on STAT3 activity in NK92 cells (NK92 / STAT3-Luc cl.7F7). [Figure 32] 1 depicts traces from a STAT3-based bioassay showing the effect of the indicated IL12-Fc fusion proteins on STAT3 activity in target-expressing cells compared to a non-targeting control construct. [Figure 33] 1 depicts traces from a STAT3-based bioassay showing the effect of the indicated IL12-Fc fusion proteins on STAT3 activity in human NK92 cells (NK92 / STAT3-Luc cl.7F7). [Figure 34] 1 depicts traces from a STAT3-based bioassay showing the effect of the indicated IL12-Fc fusion proteins on STAT3 activity in murine HT-2 cells. [Figure 35A] 1 depicts that receptor-masked Fc-IL12 reduces toxicity and retains levels of anti-tumor activity in vivo. [Figure 35B] 1 depicts that receptor-masked Fc-IL12 reduces toxicity and retains levels of anti-tumor activity in vivo. [Figure 35C] 1 depicts that receptor-masked Fc-IL12 reduces toxicity and retains levels of anti-tumor activity in vivo. [Figure 36A]Depicts that PD1-targeted receptor-masked IL12 has targeted antitumor efficacy without weight loss and reduction in systemic IFNγ. [Figure 36B] Depicts that PD1-targeted receptor-masked IL12 has targeted antitumor efficacy without weight loss and reduction in systemic IFNγ. [Figure 36C] Depicts that PD1-targeted receptor-masked IL12 has targeted antitumor efficacy without weight loss and reduction in systemic IFNγ. [Figure 36D] Depicts that PD1-targeted receptor-masked IL12 has targeted antitumor efficacy without weight loss and reduction in systemic IFNγ. [Figure 36E] Depicts that PD1-targeted receptor-masked IL12 has targeted antitumor efficacy without weight loss and reduction in systemic IFNγ. [Figure 37A] 1 depicts that PD1-targeted receptor-masked IL12 has superior antitumor efficacy than PD-1 blockade or the combination of non-targeted receptor-masked IL12 and PD-1 blockade. [Figure 37B] 1 depicts that PD1-targeted receptor-masked IL12 has superior antitumor efficacy than PD-1 blockade or the combination of non-targeted receptor-masked IL12 and PD-1 blockade. [Figure 38A] Depicts that PD1-targeted receptor-masked IL12 has targeted antitumor efficacy without weight loss and minimal systemic IFNγ. [Figure 38B-1] Depicts that PD1-targeted receptor-masked IL12 has targeted antitumor efficacy without weight loss and minimal systemic IFNγ. [Figure 38B-2] Depicts that PD1-targeted receptor-masked IL12 has targeted antitumor efficacy without weight loss and minimal systemic IFNγ. [Figure 38C-1] Depicts that PD1-targeted receptor-masked IL12 has targeted antitumor efficacy without weight loss and minimal systemic IFNγ. [Figure 38C-2]Depicts that PD1-targeted receptor-masked IL12 has targeted antitumor efficacy without weight loss and minimal systemic IFNγ. [Figure 38C-3] Depicts that PD1-targeted receptor-masked IL12 has targeted antitumor efficacy without weight loss and minimal systemic IFNγ. [Figure 38C-4] Depicts that PD1-targeted receptor-masked IL12 has targeted antitumor efficacy without weight loss and minimal systemic IFNγ. [Figure 38C-5] Depicts that PD1-targeted receptor-masked IL12 has targeted antitumor efficacy without weight loss and minimal systemic IFNγ. [Figure 39A] 39A-39D are schematic diagrams depicting exemplary antibody-masked IL12 / Fc fusion constructs. The masking antibody is depicted as a Fab, although the Fab can be replaced with an Fv, as shown in Figures 5L-5N. The construct depicted in Figure 39A can further comprise a targeting moiety. Thus, embodiments described herein encompassing the formats of Figures 39A and 39B (including the numbered embodiments and claims in Section 6.2, Groups A and B) can further comprise a targeting moiety, e.g., a targeting moiety described in Section 6.5.2, using the Fab format illustrated in Figures 39A and 39D. [Figure 39B] 39A-39D are schematic diagrams depicting exemplary antibody-masked IL12 / Fc fusion constructs. The masking antibody is depicted as a Fab, although the Fab can be replaced with an Fv, as shown in Figures 5L-5N. The construct depicted in Figure 39B can further comprise a targeting moiety. Thus, embodiments described herein encompassing the formats of Figures 39A and 39B (including those in the numbered embodiments and claims of Section 6.2, Groups A and B) can further comprise a targeting moiety, e.g., a targeting moiety described in Section 6.5.2, using the Fab format illustrated in Figures 39A and 39D. [Figure 39C]39A-39D are schematic diagrams depicting exemplary antibody-masked IL12 / Fc fusion constructs. The masking antibody is depicted as a Fab, although the Fab can be replaced with an Fv, as shown in Figures 5L-5N. Figure 39C depicts an embodiment with a targeting moiety at the N-terminus of the constructs in Figures 39A and 39B, respectively. In Figure 39C, the targeting moiety is depicted as a Fab, although other formats can be used. Thus, embodiments described herein encompassing the formats of Figures 39A and 39B (including those in the numbered embodiments and claims of Section 6.2, Groups A and B) may further include a targeting moiety, e.g., a targeting moiety described in Section 6.5.2, using the Fab format illustrated in Figures 39A and 39D. [Figure 39D] 39A-39D are schematic diagrams depicting exemplary antibody-masked IL12 / Fc fusion constructs. The masking antibody is depicted as a Fab, although the Fab can be replaced with an Fv, as shown in Figures 5L-5N. Figure 39D depicts an embodiment with a targeting moiety at the N-terminus of the constructs in Figures 39A and 39B, respectively. In Figure 39D, the targeting moiety is depicted as a Fab, although other formats can be used. Thus, embodiments described herein encompassing the formats of Figures 39A and 39B (including those in the numbered embodiments and claims of Section 6.2, Groups A and B) may further include a targeting moiety, e.g., a targeting moiety described in Section 6.5.2, using the Fab format illustrated in Figures 39A and 39D. [Figure 40A] 1 depicts traces from a STAT3-based bioassay in NK92 cells for PD1-targeted mIL12 with an R1 mask or an scFv mask. [Figure 40A-1] Same as above. [Figure 40B] 1 depicts traces from a STAT3-based bioassay in NK92 cells for PD1-targeted mIL12 with an R1 mask or an scFv mask. [Figure 41A]1 depicts that PD1-targeting antibody masks IL12 has tumor growth inhibition without weight loss and minimal systemic IFNγ. [Figure 41B] 1 depicts that PD1-targeting antibody masks IL12 has tumor growth inhibition without weight loss and minimal systemic IFNγ. [Figure 41C] 1 depicts that PD1-targeting antibody masks IL12 has tumor growth inhibition without weight loss and minimal systemic IFNγ. [Figure 41D] 1 depicts that PD1-targeting antibody masks IL12 has tumor growth inhibition without weight loss and minimal systemic IFNγ. [Figure 42] 1 illustrates that the combination of a receptor mask and a p40 mutein for one IL12 subunit further attenuates activity compared to the receptor mask alone. [Figure 43] 1 illustrates that the combination of a receptor mask and a p35 mutein for one IL12 subunit further attenuates activity compared to the receptor mask alone. [Figure 44] 1 depicts that a "three-chain" format protein construct with a receptor mask attenuates IL12 bioactivity. [Figure 45] 1 depicts an exemplary format of receptor-masked IL12 with target-enhanced bioactivity. [Figure 46A] 1 depicts a protocol for in vivo administration of a receptor-masked "three-chain" format protein construct. [Figure 46B] The activity obtained against tumor growth is depicted. [Figure 46C] The activity obtained on weight loss is depicted. [Figure 46D] The activity obtained on IFNγ production is depicted. DETAILED DESCRIPTION OF THE INVENTION

[0012] 6.1.Definition About, Approximately: The terms "about," "approximately," and the like are used throughout the specification before a numerical value to indicate that the numerical value is not necessarily exact (e.g., to account for fractions, variations in measurement precision and / or accuracy, timing, etc.). A disclosure of "about X" or "approximately X," where X is a numerical value, should be understood to also be a disclosure of "X." Thus, for example, disclosure of embodiments in which a sequence has "about X% sequence identity" to another sequence is also a disclosure of embodiments in which the sequence has "X% sequence identity" to the other sequence.

[0013] And / Or: Unless otherwise indicated, the conjunction "or" is intended to be used in its proper sense as a Boolean logic operator, encompassing both the selection of features in an alternative (selection of A is mutually exclusive from B, A OR B) and the selection of conjunctive features (selection of both A and B, A OR B). In several places in the text, the term "and / or" is used interchangeably and should not be construed to mean that "or" is used in reference to mutually exclusive alternatives.

[0014] Antigen Binding Domain or ABD: As used herein, the term "antigen binding domain" or "ABD" refers to the portion of a targeting moiety that is capable of specifically, non-covalently, and reversibly binding to a target molecule.

[0015] Associated: The term "associated" in the context of an IL12 receptor agonist or a component thereof (e.g., an IL12 p40 moiety, an IL12 p35 moiety, a targeting moiety such as an antibody) refers to a functional relationship between two or more polypeptide chains. In particular, the term "associated" means that two or more polypeptides are associated with each other, for example, non-covalently through molecular interactions or covalently through one or more disulfide or chemical crosslinks, to produce a functional IL12 receptor agonist. Examples of associations that may be present in the IL12 receptor agonists of the present disclosure include (but are not limited to) an association between an IL12 p40 moiety and an IL12 p35 moiety, an association between homodimeric or heterodimeric Fc domains within an Fc region, an association between a VH region and a VL region within a Fab or scFv, an association between a CH1 and a CL within a Fab, and an association between a CH3 and a CH3 within a domain-substituted Fab.

[0016] Bivalent: As used herein, the term "bivalent" with respect to IL12 and / or a targeting moiety in an IL12 receptor agonist refers to an IL12 receptor agonist having two IL12 heterodimers (i.e., two p40 x p35 heterodimers) and / or a targeting moiety, respectively. Typically, an IL12 receptor agonist that is bivalent with respect to an IL12 moiety and / or a targeting moiety is a dimer (either a homodimer or a heterodimer).

[0017] Cancer: The term "cancer" refers to a disease characterized by the uncontrolled (and often rapid) growth of abnormal cells. Cancer cells can spread locally or to other parts of the body via the bloodstream and lymphatic system. Examples of various cancers are described herein, including, but not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, adrenal gland cancer, autonomic ganglion cancer, biliary tract cancer, bone cancer, endometrial cancer, eye cancer, fallopian tube cancer, reproductive tract cancer, colon cancer, meningeal cancer, esophageal cancer, peritoneal cancer, pituitary cancer, penile cancer, placental cancer, pleural cancer, salivary gland cancer, small intestine cancer, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, upper aerodigestive tract cancer, urinary tract cancer, vaginal cancer, vulvar cancer, lymphoma, leukemia, lung cancer, and the like.

[0018] Complementarity determining region or CDR: The term "complementarity determining region" or "CDR" as used herein refers to the sequence of amino acids in an antibody variable region that confers antigen specificity and binding affinity. Generally, each heavy chain variable region has three CDRs (CDR-H1, CDR-H2, CDR-H3), and each light chain variable region has three CDRs (CDR1-L1, CDR-L2, CDR-L3). Exemplary rules that can be used to identify the boundaries of CDRs include, for example, 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 antibodies.

[0019] EC50: The term "EC50" refers to the half maximal effective concentration of a molecule (such as an IL12 receptor agonist) that induces a response midway between baseline and maximum after a specified exposure time. EC50 essentially represents the concentration of an antibody or IL12 receptor agonist at which 50% of its maximal effect is observed. In certain embodiments, the EC50 value is equal to the concentration of an IL12 receptor agonist that confers half-maximal STAT3 activation in the assay described in Section 8.1.2.

[0020] Epitope: An epitope, or antigenic determinant, is the portion of an antigen (e.g., a target molecule) that is recognized by an antibody or other antigen-binding moiety described herein. Epitopes can be linear or conformational.

[0021] Fab: The term "Fab" in the context of the targeting moiety of the present disclosure refers to a pair of polypeptide chains, where the first polypeptide chain comprises an antibody N-terminal variable heavy (VH) domain through a first constant domain (referred to herein as C1), and the second polypeptide chain comprises an antibody N-terminal variable light (VL) domain through a second constant domain (referred to herein as C2) that can pair 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 (CL) of the light chain. The Fabs of the present disclosure can be oriented according to their natural orientation or can include domain substitutions or swaps that promote correct VH and VL pairing. For example, the CH1 and CL domain pair in a Fab can be replaced with a CH3 domain pair to promote correct engineered Fab-chain pairing in a heterodimeric molecule. It is also possible to reverse the CH1 and CL so that CH1 is attached to VL and CL is attached to VH, a configuration commonly known as a crossmab.

[0022] Fc domain and Fc region: The term "Fc domain" refers to the portion of a heavy chain that pairs with the corresponding portion of another heavy chain. The term "Fc region" refers to the region of an antibody-based binding molecule formed by the association of two heavy chain Fc domains. The two Fc domains within an Fc region can be identical or different from each other. In natural antibodies, the Fc domains are typically identical, but one or both Fc domains can be advantageously modified to enable heterodimerization, for example, via knob-in-hole interactions.

[0023] Host cell: As used herein, the term "host cell" refers to a cell into which a nucleic acid of the present 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 the progeny or potential progeny of such a cell. Because certain modifications may occur later, either due to mutation or environmental influences, such progeny may not actually be identical to the parent cell, but still fall 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, e.g., vertebrate cells such as mouse, rat, monkey, or human cell lines, e.g., HKB11 cells, PER.C6 cells, HEK cells, or CHO cells.

[0024] IL12 agonist or IL12 receptor agonist: The terms "IL12 agonist" and "IL12 receptor agonist" are used interchangeably herein to refer to a molecule that comprises or consists of an IL12 mutein and has IL12 activity. The IL12 activity can be greater than, less than, or equal to the activity of wild-type or recombinant IL12 (e.g., human or murine IL12) in one or more in vitro or in vivo biological assays, such as the STAT3-driven luciferase-based reporter assay described in Section 8.1.2 or the MC38 synergistic tumor model described in Section 8.1.3. In various embodiments, the IL12 agonist has activity in the range of 5% to 90%, 5% to 85%, 5% to 80%, 10% to 80%, 15% to 80%, 20% to 80%, 25% to 80%, 30% to 80%, 35% to 80%, 45% to 80%, 50% to 80%, 5% to 70%, 10% to 70%, 15% to 70%, 20% to 70%, 25% to 70%, 30% to 70%, 35% to 70%, 45% to 70%, or 50% to 70% compared to recombinant IL12.

[0025] IL12 moiety: The term "IL12 moiety" refers to a p35 moiety or a p40 moiety. Accordingly, the related term "IL12 inter-moiety linker" refers to a linker connecting two IL12 moieties, e.g., a p35 moiety and a p40 moiety.

[0026] IL12 Mutein: An "IL12 mutein" is a variant IL12 molecule composed of one or more polypeptide chains (e.g., one, two, three, or four polypeptide chains) comprising an IL12 p35 (referred to as "p35") portion and an IL12 p40 ("p40") portion associated with each other, which differs from native IL12 by (a) primary amino acid sequence and / or (b) association with an additional domain not naturally associated with IL12, e.g., (i) a multimerization moiety (e.g., a dimerization domain such as an Fc domain), and / or (ii) a targeting moiety, and / or (iii) a stabilizing moiety, and / or (iv) an IL12βR portion.

[0027] In some embodiments, the term mutein refers to a structure (a) with or without a targeting moiety, and / or (b) with or without a stabilizing moiety, and / or (c) with or without a multimerizing moiety. In the context of the IL12 agonists of the present disclosure, the term "IL12 mutein" can refer to the core components of a variant IL12 molecule, i.e., the p35 and p40 moieties, and can also refer to multimerizing moieties, such as the Fc domain and any / or associated linker moieties, and it should be understood that the term "IL12 mutein" also extends to IL12 molecules that include additional features, such as one or more targeting moieties, one or more stabilizing moieties, one or more multimerizing moieties, one or more IL12R moieties, one or more linker moieties, and any combination of the foregoing, unless the context dictates otherwise.

[0028] Thus, an IL12 mutein can include a p35 and / or p40 portion that has one or more amino acid substitutions, deletions, and / or insertions compared to wild-type p35 and / or p40.

[0029] As disclosed herein, the p35 portion can include an IL12Rβ2 portion, and the p40 portion can include an IL12Rβ1 portion. The p35 portion and the IL12Rβ2 portion can be on the same or different polypeptide chains. The p40 portion and the IL12Rβ1 portion can be on the same or different polypeptide chains. The IL12Rβ1 and IL12Rβ2 portions generally function as masking moieties and, therefore, when present, are typically configured to interact with the p40 and p35 portions, respectively.

[0030] In some embodiments, an IL12 mutein has one or more mutations in its p35 subunit or its p40 subunit, or one or more mutations in both its p35 subunit and its p40 subunit. Exemplary mutations, e.g., substitutions, are disclosed, inter alia, in Section 6.3 and its subsections, Tables 1 and 2, and numbered embodiments 1, 2, 676-719, and 589-674. The p35 and p40 subunits of an IL12 mutein can be included on the same polypeptide chain or on different polypeptide chains. Exemplary configurations of the IL12 muteins and agonists of the present disclosure are disclosed, inter alia, in Figures 2A-5X, Section 6.2, and numbered embodiments 3-847.

[0031] In some embodiments, the IL12 mutein comprises a masking moiety, among other exemplary masking moieties of the present disclosure as disclosed in the numbered embodiments disclosed in Figures 4B-4E, 4G-4W, 5H-5O, 5R-5S, 5V-5X, and 39A-39D, and in Section 6.4 and in Section 7 below, which reference exemplary monomers of these figures and / or their configurations.

[0032] In some embodiments, the IL12 mutein comprises a receptor-based masking moiety. In other embodiments, the IL12 mutein comprises an antibody-based masking moiety. Exemplary antibody-based masking moieties and IL12 receptor agonists comprising them are disclosed, inter alia, in Figures 4O-4R, 5L-5N, and 39A-39D, Sections 6.2 and 6.4, and in the numbered embodiments disclosed in Section 7 below with reference to these figures and / or exemplary monomers of those compositions. Exemplary receptor-based masking moieties and IL12 receptor agonists comprising them are disclosed, inter alia, in Figures 4B-4E, 4G-4N, 4S-4W, 5H-5K, 5O, 5R, 5S, and 5V-5X, Sections 6.2 and 6.4, and in the numbered embodiments disclosed in Section 7 below with reference to these figures and / or exemplary monomers of those compositions.

[0033] An IL12 mutein can be monovalent with respect to p35 and p40 (i.e., has a single p35 moiety and a single p40 moiety), or multivalent with respect to p35 and p40 (i.e., has multiple p35 moieties and p40 moieties). In some embodiments, an IL12 mutein is bivalent with respect to p35 and p40 (i.e., has two p35 moieties and two p40 moieties). When an IL12 mutein is multivalent with respect to p35 and p40, the multiple p35 moieties can be the same or different from each other, and / or the multiple p40 moieties can be the same or different from each other.

[0034] An IL12 mutein can have altered function (eg, receptor binding, affinity, cytokine activity) and / or altered pharmacokinetics compared to wild-type IL12.

[0035] IL12 p35 moiety or p35 moiety: An IL12 p35 moiety or p35 moiety is an amino acid sequence that comprises 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 IL12Rβ2-binding portion of mammalian, e.g., human or mouse, p35 (sometimes referred to as the alpha subunit of IL12, or IL12α), and optionally has one or more amino acid substitutions as defined in Section 6.3.2, below. The sequence of human p35 has the Uniprot identifier P29459 (uniprot.org / uniprot / P29459). The sequence of mouse p35 has the Uniprot identifier P43431 (uniprot.org / uniprot / P43431).

[0036] p35 contains a signal sequence (amino acids 1-22 of human p35). In native IL12, p35 has four conserved cysteine ​​residues that form two interchain disulfide bonds, bridging C64 and C96, and C85 and C123 of human p35. p35 also contains a cysteine ​​(C74 of human p35) that forms an interchain bond with p40 (at amino acid C177 of human p40).

[0037] The p35 portion 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 mammalian p35, e.g., human or mouse p35 (corresponding to amino acids 23-219 of human p35), and optionally having one or more amino acid substitutions as defined in Section 6.3.2 below.

[0038] In various embodiments, the p35 portion of the IL12 muteins of the present disclosure retains (a) neither, either, or both interchain disulfide bonds, and / or (b) any combination of cysteines that form interchain bonds with p40.

[0039] IL12 p40 moiety or p40 moiety: An IL12 p40 moiety or p40 moiety is an amino acid sequence that comprises 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 IL12Rβ1-binding portion of mammalian, e.g., human or mouse, p40 (sometimes referred to as the beta subunit of IL12, or IL12β), and optionally has one or more amino acid substitutions as defined in Section 6.3.1 below. The sequence of human p40 has Uniprot identifier P29460 (uniprot.org / uniprot / P29460). The sequence of mouse p40 has Uniprot identifier P43432 (uniprot.org / uniprot / P43432). p40 contains a signal sequence (at amino acids 1-22 of human p40), an Ig-like C2-type domain designated D1 (at amino acids 23-106 of human p40), a first fibronectin type III domain designated D2 (at amino acids 107-236 of human p40), and a second fibronectin type III domain designated D3 (at amino acids 237-328 of human p40). In native IL12, the D2 domain of p40 has four conserved cysteine ​​residues that form two interchain disulfide bonds, bridging C109 and C120, and C148 and C171 in human p40, and the D3 domain also contains an interchain disulfide bond bridging C278 and C305 in human p40. D2 also contains a cysteine ​​(C177 in human p40) that forms an interchain bond with p35 (at amino acid C74 in human p35). D3 also contains the highly conserved WSXWS motif (SEQ ID NO: 3) (WSEWAS in human p40 (SEQ ID NO: 4)).

[0040] The p40 portion preferably comprises the D2 and D3 domains of mammalian, e.g., human or mouse, p40 (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), optionally with one or more amino acid substitutions as defined in Section 6.3.1 below.

[0041] The p40 portion can also include a D1 domain or 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 mammalian, e.g., human or mouse, p40, optionally with one or more amino acid substitutions as defined in Section 6.3.1 below.

[0042] In various embodiments, the p40 portion of the IL12 muteins of the present disclosure retains (a) none, any one, any two, or all three interchain disulfide bonds, and / or (b) cysteines that form interchain bonds with p35, and / or (c) any combination of the conserved WSXWS motif (SEQ ID NO: 3).

[0043] IL12Rβ1 portion: An IL12Rβ1 portion is an amino acid sequence of up to 350 amino acids that comprises 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 IL12 p40-binding portion of a mammalian, e.g., human or mouse, IL12 receptor subunit beta-1 (IL12Rβ1). The sequence of human IL12Rβ1 has Uniprot identifier P42701 (uniprot.org / uniprot / P42701), with amino acids 24-545 constituting the extracellular domain. The sequence of mouse IL12Rβ1 has Uniprot identifier Q60837 (uniprot.org / uniprot / Q60837), with amino acids 20-565 constituting the extracellular domain. IL12Rβ1 contains a signal sequence (at amino acids 1-23 of human IL12Rβ1), an extracellular p40-binding domain (at amino acids 24-545 of human IL12Rβ1), a helical transmembrane domain (at amino acids 546-570 of human IL12Rβ1), and a cytoplasmic domain (at amino acids 571-662 of human IL12Rβ1). The extracellular domain of IL12Rβ1 contains five fibronectin type III domains: D1 (corresponding to amino acids 46-136 of full-length human IL12Rβ1), D2 (corresponding to amino acids 142-234 of full-length human IL12Rβ1), D3 (corresponding to amino acids 237-337 of full-length human IL12Rβ1), D4 (corresponding to amino acids 338-444 of full-length human IL12Rβ1), and D5 (corresponding to amino acids 448-542 of full-length human IL12Rβ1). The "IL12 p40-binding portion" of IL12Rβ1 refers to the portion of the extracellular domain of IL12Rβ1 that is capable of binding to IL12 p40.Thus, in some embodiments, the IL12Rβ1 portion is a portion of the extracellular domain of IL12Rβ1 (e.g., a portion including or consisting of D1, D2, D3, both D1 and D2, or all of D1, D2, and D3 of IL12Rβ1) or a polypeptide having 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 such a portion of the extracellular domain of IL12Rβ1.

[0044] In various aspects of the numbered embodiments and claims described in Section 7 below, the term IL12Rβ1 moiety refers to: (a) an amino acid sequence of 90-100, 90-110, or 90-120 amino acids in length that has at least 90% sequence identity to amino acids 46-136 of human IL12Rβ1, optionally wherein the amino acid sequence has at least 95%, at least 97%, or 100% sequence identity to amino acids 46-136 of human IL12Rβ1; (b) an amino acid sequence of 110-120, 110-130, or 110-140 amino acids in length that has at least 90% sequence identity to amino acids 24-136 of human IL12Rβ1, optionally wherein the amino acid sequence has at least 95%, at least 97%, or 100% sequence identity to amino acids 24-136 of human IL12Rβ1; (c) an amino acid sequence of 185-195, 185-205, or 185-215 amino acids in length that has at least 90% sequence identity to amino acids 46-234 of human IL12Rβ1, optionally wherein the amino acid sequence has at least 95%, at least 97%, or 100% sequence identity to amino acids 46-234 of human IL12Rβ1; (d) an amino acid sequence of 210-220, 210-230, or 210-240 amino acids in length that has at least 90% sequence identity to amino acids 24-234 of human IL12Rβ1, optionally wherein the amino acid sequence has at least 95%, at least 97%, or 100% sequence identity to amino acids 24-234 of human IL12Rβ1; (e) an amino acid sequence of 285-300, 285-310, or 285-320 amino acids in length that has at least 90% sequence identity to amino acids 46-337 of human IL12Rβ1, optionally wherein the amino acid sequence has at least 95%, at least 97%, or 100% sequence identity to amino acids 46-337 of human IL12Rβ1; (f) an amino acid sequence of 310-320, 310-330, or 310-340 amino acids in length that has at least 90% sequence identity to amino acids 24-337 of human IL12Rβ1, optionally wherein the amino acid sequence has at least 95%, at least 97%, or 100% sequence identity to amino acids 24-337 of human IL12Rβ1; (g) (i) an amino acid sequence consisting of amino acids 24 to 136 of human IL12Rβ1 with up to 5 or up to 10 contiguous amino acids at its N-terminus and / or C-terminus, or (ii) an amino acid sequence having at least 95% or at least 97% sequence identity to the sequence defined in (i); (h) (i) an amino acid sequence consisting of amino acids 46 to 136 of human IL12Rβ1 with up to 5 or up to 10 contiguous amino acids at its N-terminus and / or C-terminus, or (ii) an amino acid sequence having at least 95% or at least 97% sequence identity to the sequence defined in (i); (i) (i) an amino acid sequence consisting of amino acids 24 to 234 of human IL12Rβ1 with up to 5 or up to 10 contiguous amino acids at its N-terminus and / or C-terminus, or (ii) an amino acid sequence having at least 95% or at least 97% sequence identity to the sequence defined in (i), (j) (i) an amino acid sequence consisting of amino acids 46 to 234 of human IL12Rβ1 with up to 5 or up to 10 contiguous amino acids at its N-terminus and / or C-terminus, or (ii) an amino acid sequence having at least 95% or at least 97% sequence identity to the sequence defined in (i); (k) (i) an amino acid sequence consisting of amino acids 24 to 337 of human IL12Rβ1 with up to 5 or up to 10 contiguous amino acids at its N-terminus and / or C-terminus, or (ii) an amino acid sequence having at least 95% or at least 97% sequence identity to the sequence defined in (i); or (l) (i) an amino acid sequence consisting of amino acids 46 to 337 of human IL12Rβ1 with up to 5 or up to 10 contiguous amino acids at its N-terminus and / or C-terminus, or (ii) an amino acid sequence having at least 95% or at least 97% sequence identity to the sequence defined in (i).

[0045] IL12Rβ moiety: As used herein, the term IL12Rβ moiety refers to an IL12Rβ1 or IL12Rβ2 moiety. IL12Rβ2 portion: An IL12Rβ2 portion is an amino acid sequence of up to 450 amino acids that contains 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 IL12 p35-binding portion of a mammalian, e.g., human or mouse, IL12 receptor subunit beta-2 (IL12Rβ2). The sequence of human IL12Rβ2 has Uniprot identifier Q99665 (uniprot.org / uniprot / Q99665), with amino acids 24-622 constituting the extracellular domain. The sequence of mouse IL12Rβ2 has Uniprot identifier P97378 (uniprot.org / uniprot / Q60837), with amino acids 24-637 constituting the extracellular domain. IL12Rβ2 contains a signal sequence (at amino acids 1-23 of human IL12Rβ2), an extracellular p40-binding domain (at amino acids 24-622 of human IL12Rβ2), a helical transmembrane domain (at amino acids 623-643 of human IL12Rβ2), and a cytoplasmic domain (at amino acids 644-862 of human IL12Rβ2). The extracellular domain of IL12Rβ2 contains five fibronectin type III domains: D1 (corresponding to amino acids 126-221 of full-length human IL12Rβ2), D2 (corresponding to amino acids 226-319 of full-length human IL12Rβ2), D3 (corresponding to amino acids 320-419 of full-length human IL12Rβ2), D4 (corresponding to amino acids 423-520 of full-length human IL12Rβ2), and D5 (corresponding to amino acids 521-620 of full-length human IL12Rβ2). The "IL12 p40-binding portion" of IL12Rβ2 refers to the portion of the extracellular domain of IL12Rβ2 that can bind to IL12 p35.Thus, in some embodiments, the IL12Rβ1 portion is a portion of the extracellular domain of IL12Rβ2 (e.g., a portion including or consisting of D1, D2, D3, both D1 and D2, or all of D1, D2, and D3 of IL12Rβ2) or a polypeptide having 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 such a portion of the extracellular domain of IL12Rβ2.

[0046] In various aspects of the numbered embodiments and claims described in Section 7 below, the term IL12Rβ2 moiety refers to: (a) an amino acid sequence of 90-100, 90-110, or 90-120 amino acids in length that has at least 90% sequence identity to amino acids 126-221 of human IL12Rβ2, optionally wherein the amino acid sequence has at least 95%, at least 97%, or 100% sequence identity to amino acids 126-221 of human IL12Rβ2; (b) an amino acid sequence of 190-220, 190-230, or 190-240 amino acids in length that has at least 90% sequence identity to amino acids 24-221 of human IL12Rβ2, optionally wherein the amino acid sequence has at least 95%, at least 97%, or 100% sequence identity to amino acids 24-221 of human IL12Rβ2; (c) an amino acid sequence of 185-195, 185-205, or 185-215 amino acids in length that has at least 90% sequence identity to amino acids 126-319 of human IL12Rβ2, optionally wherein the amino acid sequence has at least 95%, at least 97%, or 100% sequence identity to amino acids 126-319 of human IL12Rβ2; (d) an amino acid sequence of 290-310, 290-320, or 290-330 amino acids in length that has at least 90% sequence identity to amino acids 24-319 of human IL12Rβ2, optionally wherein the amino acid sequence has at least 95%, at least 97%, or 100% sequence identity to amino acids 24-319 of human IL12Rβ2; (e) an amino acid sequence of 290-310, 290-320, or 290-330 amino acids in length that has at least 90% sequence identity to amino acids 126-419 of human IL12Rβ2, optionally wherein the amino acid sequence has at least 95%, at least 97%, or 100% sequence identity to amino acids 126-419 of human IL12Rβ2; (f) an amino acid sequence of 390-410, 390-420, or 390-430 amino acids in length that has at least 90% sequence identity to amino acids 24-419 of human IL12Rβ2, optionally wherein the amino acid sequence has at least 95%, at least 97%, or 100% sequence identity to amino acids 24-419 of human IL12Rβ2; (g) (i) an amino acid sequence consisting of amino acids 24 to 221 of human IL12Rβ2 with up to 5 or up to 10 contiguous amino acids at its N-terminus and / or C-terminus, or (ii) an amino acid sequence having at least 95% or at least 97% sequence identity to the sequence defined in (i); (h) (i) an amino acid sequence consisting of amino acids 126 to 221 of human IL12Rβ2 with up to 5 or up to 10 contiguous amino acids at its N-terminus and / or C-terminus, or (ii) an amino acid sequence having at least 95% or at least 97% sequence identity to the sequence defined in (i); (i) (i) an amino acid sequence consisting of amino acids 24 to 319 of human IL12Rβ2 with up to 5 or up to 10 contiguous amino acids at its N-terminus and / or C-terminus, or (ii) an amino acid sequence having at least 95% or at least 97% sequence identity to the sequence defined in (i), (j) (i) an amino acid sequence consisting of amino acids 126 to 319 of human IL12Rβ2 with up to 5 or up to 10 contiguous amino acids at its N-terminus and / or C-terminus, or (ii) an amino acid sequence having at least 95% or at least 97% sequence identity to the sequence defined in (i); (k) (i) an amino acid sequence consisting of amino acids 24 to 419 of human IL12Rβ2 with up to 5 or up to 10 contiguous amino acids at its N-terminus and / or C-terminus, or (ii) an amino acid sequence having at least 95% or at least 97% sequence identity to the sequence defined in (i); or (l) (i) an amino acid sequence consisting of amino acids 126 to 419 of human IL12Rβ2 with up to 5 or up to 10 contiguous amino acids at its N-terminus and / or C-terminus, or (ii) an amino acid sequence having at least 95% or at least 97% sequence identity to the sequence defined in (i).

[0047] Major histocompatibility complex and MHC: These terms refer to naturally occurring MHC molecules, individual chains of MHC molecules (e.g., MHC class I α (heavy) chain, β2 microglobulin, MHC class II α chain, and MHC class II β chain), individual subunits of such chains of MHC molecules (e.g., α1, α2, and / or α3 subunits of the MHC class I α chain, α1-α2 subunits of the MHC class II α chain, and β1-β2 subunits of the MHC class II β chain), as well as portions (e.g., peptide-binding portions, e.g., peptide-binding grooves), variants, and various derivatives (including fusion proteins) thereof, which retain the ability to display antigenic peptides for recognition by T cell receptors (TCRs), e.g., antigen-specific TCRs. MHC class I molecules contain a peptide-binding groove formed by the α1 and α2 domains of the heavy α chain, which can accommodate peptides of approximately 8-10 amino acids. Despite the fact that both classes of MHC bind to a core of approximately nine amino acids (e.g., 5-17 amino acids) within a peptide, the open-ended nature of the MHC class II peptide-binding groove (the α1 domain of a class II MHC a polypeptide associated with the β1 domain of a class II MHC β polypeptide) allows for a wider range of peptide lengths. Peptides that bind to MHC class II typically vary in length between 13-17 amino acids, although shorter or longer lengths are not uncommon. As a result, peptides can shift within the MHC class II peptide-binding groove, altering which 9-mer is directly positioned within the groove at any given time. Conventional identification of specific MHC variants is used herein. This term encompasses "human leukocyte antigen" or "HLA."

[0048] Masking moiety or IL12 masking moiety: The terms "masking moiety" (with respect to IL12) and "IL12 masking moiety" refer to a moiety that can reversibly bind to a p35 moiety and / or a p40 moiety. In some embodiments, the masking moiety is an IL12Rβ moiety (e.g., an IL12Rβ1 or IL12Rβ2 moiety). In other embodiments, the masking moiety is an anti-IL12 (e.g., an anti-p35 or anti-p40) antibody fragment.

[0049] Monomer and IL12 Monomer: As used herein, the terms "monomer" and "IL12 monomer" refer to a molecule comprising a first polypeptide chain that (a) comprises a p35 portion and a p40 portion and is capable of associating with a second polypeptide chain, (b) comprises a p35 portion and is capable of associating with a p40 portion on a second polypeptide chain, (c) comprises a p40 portion and is capable of associating with a p35 portion on a second polypeptide chain, (d) comprises a multimerization portion (e.g., an Fc domain) and is capable of associating with a corresponding multimerization portion (e.g., another Fc domain) on a second polypeptide chain, or (e) is any combination of (a), (b), (c), and (d) above. Thus, a monomer can associate with other monomers through the pairing of the p35 / p40 portion and / or the pairing of the multimerization portion (e.g., an Fc domain). In some embodiments, one or more of the associations between the monomers is stabilized, for example, at the p35 / p40 interface, through interchain disulfide bridges, or through the hinge sequence or other portions of the Fc domain. Thus, a monomer of the present disclosure can associate with another monomer to form a dimer. The dimer can be a homodimer, in which each constituent monomer is identical, or a heterodimer, in which each constituent monomer is different. As used herein, reference to a "monomer" does not exclude the presence of a second polypeptide chain that does not contain p35, p40, or a multimerization moiety, such as a light chain of a Fab domain. Thus, a "dimer" of two monomers can contain more than two polypeptide chains, for example, three or four polypeptide chains.

[0050] Monomeric p40 or monomeric p40 polypeptide chain: The terms "monomeric p40," "monomeric p40 polypeptide chain," and the like refer to a polypeptide chain that includes an IL12-p40 portion that does not have a dimerization moiety, e.g., does not have an Fc domain. The monomeric p40 polypeptide chain can optionally include a p40 masking moiety (e.g., a p40-binding portion of IL12Rβ1 or an anti-p40 antibody-based masking moiety). Such polypeptide chains are sometimes referred to herein as "masked monomeric p40."

[0051] Monovalent: As used herein, the term "monovalent" with respect to IL12 and / or a targeting moiety in an IL12 receptor agonist refers to an IL12 receptor agonist having only a single IL12 heterodimer (i.e., one p40 x p35 heterodimer) and / or a targeting moiety, respectively.

[0052] Operably linked: As used herein, the term "operably linked" refers to a functional relationship between two or more regions of a polypeptide chain, where the two or more regions are joined 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.

[0053] Peptide-MHC complex, pMHC complex, peptide-in-groove: "Peptide-MHC complex," "pMHC complex," and "in-groove peptide" refer to (i) an MHC domain (e.g., a human MHC molecule or portion thereof (e.g., its peptide-binding groove and, e.g., its extracellular portion), (ii) an antigenic peptide, and, optionally, (iii) a β2 microglobulin domain (e.g., human β2 microglobulin or portion thereof), wherein the MHC domain, antigenic peptide, and optional β2 microglobulin domain are complexed in a manner that allows specific binding to a T cell receptor. In some embodiments, the pMHC complex comprises at least the extracellular domain of a human HLA class I / human β2 microglobulin molecule and / or a human HLA class II molecule.

[0054] Single-chain Fv or scFv: As used herein, the term "single-chain Fv" or "scFv" refers to a polypeptide chain comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain.

[0055] Specific (or selective) binding: As used herein, the term "specifically (or selectively) binding" means that the targeting moiety, e.g., an antibody or antigen-binding domain ("ABD"), forms a complex with the target molecule that is relatively stable under physiological conditions. Specific binding occurs when the target molecule binds to a target molecule with a specific binding affinity of about 5×10 -2 M or less (e.g., 5 × 10 -2 Under M, 10 -2 Less than M, 5 x 10 -3 Under M, 10 -3 Less than M, 5 x 10 -4 Under M, 10 -4 Less than M, 5 x 10 -5 Under M, 10 -5 Less than M, 5 x 10 -6 Under M, 10 -6 Less than M, 5 x 10 -7 Under M, 10 -7 Less than M, 5 x 10 -8 Under M, 10 -8 Less than M, 5 x 10-9 Under M, 10 -9 Less than M or 10 -10 K (less than M) D Methods for determining the binding affinity of an antibody or antibody fragment, e.g., an 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., a Biacore assay), fluorescence-activated cell sorting (FACS) binding assays, and the like. An IL12 receptor agonist of the present disclosure that includes a targeting moiety or its ABD that specifically binds to a target molecule from one species may, however, have cross-reactivity to target molecules from one or more other species.

[0056] 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, dogs, cows, chickens, amphibians, and reptiles. Except where noted, the terms "patient" and "subject" are used interchangeably herein.

[0057] Target molecule: As used herein, the term "target molecule" refers to any biological molecule (e.g., a 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 an IL12 receptor agonist of the present disclosure.

[0058] Targeting moiety: As used herein, the term "targeting moiety" refers to any molecule or binding portion thereof (e.g., an immunoglobulin or antigen-binding fragment) that can bind to a cell surface or extracellular matrix molecule at the site where the IL12 receptor agonist of the present disclosure is localized, e.g., on a tumor cell or lymphocyte in the tumor microenvironment. A targeting moiety can also have functional activity in addition to localizing the IL12 receptor agonist to a specific site. For example, a targeting moiety that is an anti-PD1 antibody or an antigen-binding portion thereof can also exhibit anti-tumor activity or enhance the anti-tumor activity of an IL12 mutein by inhibiting PD1 signaling.

[0059] Treat, Treatment, Treating: As used herein, the terms "treat," "treatment," and "treating" refer to a reduction or amelioration of the progression, severity, and / or duration of a proliferative disorder, or an amelioration of one or more symptoms (preferably one or more discernible symptoms) of a proliferative disorder resulting from the administration of one or more IL12 receptor agonists of the present disclosure. In certain embodiments, the terms "treat," "treatment," and "treating" refer to an improvement in at least one measurable physical parameter of a proliferative disorder, such as tumor growth, not necessarily discernible by the patient. In other embodiments, the terms "treat," "treatment," and "treating" refer to an inhibition of the progression of a proliferative disorder, either physical, e.g., by stabilization of a discernible symptom, physiological, e.g., by stabilization of a physical parameter, or both. In other embodiments, the terms "treat," "treatment," and "treating" refer to a reduction or stabilization of tumor size or cancerous cell number.

[0060] Tumor: The term "tumor" is used interchangeably herein with the term "cancer," e.g., both terms encompass solid and liquid, e.g., diffuse or circulating, tumors. As used herein, the term "cancer" or "tumor" includes precancerous and malignant cancers and tumors.

[0061] 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 cancer cells, either in whole or as fragments (e.g., MHC / peptides), and is useful for preferential targeting of pharmacological agents to cancer cells. In some embodiments, the TAA is a marker expressed by both normal and cancer cells, e.g., a lineage marker, e.g., CD19 on B cells. In some embodiments, the TAA is a cell surface molecule that is overexpressed in cancer cells compared to normal cells, e.g., by 1-fold overexpression, 2-fold overexpression, 3-fold overexpression, or more compared to normal cells. In some embodiments, the TAA is a cell surface molecule that is inappropriately synthesized in cancer cells, e.g., a molecule that contains a deletion, addition, or mutation compared to the molecule expressed on normal cells. In some embodiments, the TAA is expressed exclusively on the cell surface of cancer cells, either in whole or as fragments (e.g., MHC / peptides), and is not synthesized or expressed on the surface of normal cells. Thus, the term "TAA" encompasses antigens specific to cancer cells, sometimes known in the art as tumor-specific antigens ("TSAs").

[0062] Universal light chain: As used herein in connection with a targeting moiety, the term "universal light chain" refers to a light chain polypeptide that can pair with the heavy chain region of the targeting moiety and can also pair with other heavy chain regions. A universal light chain is also known as a "common light chain."

[0063] 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 Fab. VL: The term "VL" refers to the variable region of an immunoglobulin light chain, including the light chain of an scFv or Fab.

[0064] 6.2. IL-12 receptor agonists The present disclosure provides an IL12 receptor agonist comprising or consisting of an IL12 mutein, which comprises a p35 portion and a p40 portion and differs from wild-type IL12 by (a) primary amino acid sequence (e.g., amino acid insertion, deletion, or substitution, or any combination of the foregoing, compared to p35 and / or p40), and / or (b) additional domains not naturally associated with IL12, for example, (i) a multimerization moiety (e.g., a dimerization domain such as an Fc domain), and / or (ii) a targeting moiety, and / or (iii) a stabilizing moiety, and / or (iv) association with the IL12β receptor (IL12βR1 and / or IL12βR2) sequence.

[0065] The IL12 receptor agonists and / or IL12 muteins therein of the present disclosure can have amino acid modifications that result in reduced binding affinity to the IL12 receptor complex (e.g., a receptor complex including IL12Rβ1 and IL12Rβ2) compared to wild-type IL12. Overall, the IL12 receptor agonists and / or IL12 muteins therein of the present disclosure can have normal or attenuated binding (i.e., reduced affinity) to the IL12 receptor complex (e.g., up to 10-fold, up to 50-fold, up to 100-fold, up to 200-fold, up to 500-fold, up to 1,000-fold, up to 2,000-fold, or up to 5,000-fold). In some embodiments, binding is attenuated by 100-5,000-fold, 200-2,000-fold, 500-2,000-fold, or 500-1,000-fold. Binding can be attenuated through one or more amino acid substitutions in the p35 and / or p40 sequences and / or the inclusion of one or more IL12Rβ moieties in the IL12 receptor agonist.

[0066] In certain embodiments, the IL12 receptor agonists and IL12 muteins of the present disclosure have one or more amino acid substitutions in the IL12 p40 portion, the IL12 p35 portion, or both the IL12 p40 and p35 portions that reduce binding to the IL12 receptor complex, e.g., as disclosed in Section 6.3 and its subsections. For example, in some embodiments, the IL12 mutein can have binding to the human IL12 receptor complex that is attenuated by up to 100- to 1,000-fold compared to wild-type human IL12. Exemplary amino acid substitutions are disclosed in Sections 6.3.1 and 6.3.2 and include a substitution at W37 of full-length human or mouse p40 that reduces binding to IL12Rβ1, e.g., the substitution W37A.

[0067] Certain aspects of the present disclosure relate to IL12 receptor agonists comprising: (a) a first polypeptide chain comprising, in N- to C-terminal orientation, a first targeting moiety or targeting moiety component, a first Fc domain, and a p35 moiety; (b) a second polypeptide chain comprising, in N- to C-terminal orientation, a second targeting moiety or targeting moiety component, and a second Fc domain; (c) a p40 moiety between the first Fc domain and the p35 moiety, or in the form of monomeric p40; and (d) an IL12Rβ moiety or an IL12 antibody fragment configured to mask the p35 moiety or p40 moiety. The p40 moiety and / or the p35 moiety may have attenuating substitutions, e.g., as described in Section 6.3. In some embodiments, the p40 portion has an amino acid substitution at a position corresponding to amino acid W37 of full-length human p40 or amino acid W37 of full-length mouse p40, eg, the substitution W37A.

[0068] A further aspect of the present disclosure relates to an IL12 receptor agonist comprising an IL12 mutein, wherein the IL12 receptor agonist has at least a 500-fold attenuation compared to wild-type IL12, and the IL12 receptor agonist comprises: (a) a first polypeptide chain and a second polypeptide chain dimerized through a first Fc domain and a second Fc domain; (b) an optional first targeting moiety or targeting moiety component on the first polypeptide chain, and an optional second targeting moiety or targeting moiety component on the second polypeptide chain; (c) a p35 moiety and a p40 moiety; and (d) an IL12Rβ moiety or an IL12 antibody fragment configured to mask the p35 moiety or the p40 moiety.

[0069] A still further aspect of the present disclosure is an IL12 mutein comprising: (a) an optional first targeting moiety and an optional second targeting moiety; and (b) a p35 portion and a p40 portion, on first and second polypeptide chains dimerized through a first and second Fc domain, wherein: (i) the p35 portion comprises an attenuating amino acid substitution, optionally, the attenuating amino acid substitution is at (A) amino acid Y189 of full-length human p35 or amino acid Y185 of full-length mouse p35, and the substitution is optionally (B) at amino acid 1193 of full-length human p35 or amino acid M189 of full-length mouse p35, optionally with a substitution of A, V, or E; (C) at amino acid R211 of full-length human p35 or amino acid R207 of full-length mouse p35, optionally with a substitution of A or K; or (D) any combination of (A)-(C); and / or (ii) the p40 portion comprises an attenuating amino acid substitution, optionally with an attenuating amino acid substitution, (B) at amino acid W37 of full-length human p40 or at amino acid W37 of full-length mouse p40, optionally with a substitution of A; (C) at amino acid D115 of full-length human p40 or at amino acid E115 of full-length mouse p40, optionally with a substitution of A; (D) at amino acid K118 of full-length human p40 or at amino acid K118 of full-length mouse p40, optionally with a substitution of (E) at amino acid K126 of full-length human p40 or at amino acid K126 of full-length mouse p40, optionally with a substitution of A; (F) at amino acid Y268 of full-length human p40 or at amino acid Y265 of full-length mouse p40, optionally with a substitution of V or F; (G) at amino acid Y314 of full-length human p40 or at amino acid Y318 of full-length mouse p40, optionally with a substitution of F; or (H) any combination of (A)-(G).(c) an IL12Rβ portion or an IL12 antibody fragment configured to mask the p35 portion or p40 portion.

[0070] The binding affinity of p40 to IL12Rβ1 and p35 to IL12Rβ2 can be assayed by surface plasmon resonance (SPR) techniques (analyzed on a Biacore instrument) (Liljeblad et al., 2000, Glyco J 17:323-329).

[0071] In some embodiments, the IL12 receptor agonists and IL12 muteins of the present disclosure may comprise an IL12 receptor sequence, e.g., an IL12Rβ1 and / or IL12Rβ2 sequence, as described in Section 6.4 and its subsections, which is believed to attenuate the side effects of IL12 receptor agonist treatment.

[0072] An IL12 receptor agonist or IL12 mutein can be composed of one or more polypeptides. In some embodiments, the IL12 receptor agonist is composed of multiple (e.g., two) monomers that include a p40 and / or p35 portion, and in some embodiments, also include a multimerization portion.

[0073] The IL12 receptor agonist or IL12 mutein may further comprise one or more targeting moieties, one or more stabilizing moieties, and / or one or more IL12βR moieties. Exemplary multimerization moieties are described in Section 6.6 and include an Fc domain, which confers homodimerization or heterodimerization capabilities to the IL12 receptor agonist. Free IL12 has poor pharmacokinetics (serum half-life of about 5 hours to about 20 hours), and without being bound by theory, it is believed that the inclusion of a multimerization domain, such as an Fc domain, improves the serum stability and pharmacokinetic profile of the IL12 receptor agonist. Thus, the Fc domain can be a dual-purpose domain, conferring the stabilizing properties of the stabilizing moiety, as described in Section 6.7.

[0074] Exemplary targeting moieties are described in Section 6.5 and include antigen-binding domains (e.g., scFv or Fab) that bind to tumor-associated antigens, tumor microenvironment antigens, or tumor lymphocytes, and peptide-MHC complexes that recognize tumor lymphocytes.

[0075] In some embodiments, the IL12 receptor agonist comprises one or more masking moieties. In some embodiments, the IL12 receptor agonist comprises one or more IL12Rβ-based masking moieties, e.g., an IL12Rβ1 moiety, an IL12Rβ2 moiety, or both an IL12Rβ1 moiety and an IL12Rβ1 moiety. Exemplary IL12Rβ1 moieties are described in Section 6.4.1. Exemplary IL12Rβ2 moieties are described in Section 6.4.2.

[0076] In other embodiments, the IL12 receptor agonist comprises one or more antibody-based masking moieties, such as an anti-p35 antibody-based masking moiety or an anti-p40 antibody-based masking moiety. In some embodiments, the antibody-based masking moiety is an Fv (e.g., an scFv). In other embodiments, the antibody-based masking moiety is a Fab.

[0077] In some embodiments, the IL12 agonist of the present disclosure is composed of two monomers, optionally associated with one or more additional polypeptide chains (e.g., associated with a polypeptide chain comprising the light chain of a Fab targeting moiety). The monomers can be identical, thereby forming a homodimer, or different, thereby forming a heterodimer. The multimerization moieties of each monomer of the IL12 receptor agonist can be configured to dimerize together. Exemplary multimerization moieties are described in Section 6.6 and include Fc domains.

[0078] It has been discovered that incorporation of a p40 moiety and a p35 moiety at the C-terminus of the Fc domain, with the p40 moiety N-terminal to the p35 moiety, improves expression yields, reduces aggregation, and results in higher quality preparations of the IL12 receptor agonists of the present disclosure. When the p35 moiety is immediately C-terminal to the Fc domain (with or without a linker separating the p35 moiety and the Fc domain), expression and aggregation can be improved by providing the p40 moiety as a monomeric p40 moiety on a separate polypeptide chain lacking the Fc domain. Thus, in some embodiments, the IL12 agonists of the present disclosure further comprise a monomeric p40 polypeptide chain associated with the p35 moiety in one of the two monomers.

[0079] In some embodiments, an IL12 mutein or IL12 agonist comprises various components of one or more of its polypeptide chains, for example, (1) the p35 and p40 portions of IL12 when present on the same polypeptide chain, (2) the p35 portion and a multimerization domain (e.g., Fc domain), (3) the p40 portion and a multimerization domain (e.g., Fc domain), (4) the p35 portion and its targeting portion or component (e.g., the heavy chain of an scFv or Fab), (5) the p40 portion and and a targeting moiety or component thereof (e.g., the heavy chain of an scFv or Fab), (6) a multimerization domain (e.g., an Fc domain) and a targeting moiety or component thereof (e.g., the heavy chain of an scFv or Fab), (7) a p35 moiety, a p40 moiety, a multimerization domain, or a targeting moiety or component thereof, and an IL12βR moiety, e.g., an IL12βR1 or IL12βR2 moiety, or (8) one or more linker sequences connecting any combination of the foregoing. Exemplary linkers are described in Section 6.8.

[0080] Most IL12 muteins and IL12 agonists are multimeric, e.g., dimeric, due to the association of p35 and p40 moieties present on different polypeptide chains and / or due to the association of multimerization moieties (e.g., Fc domains) configured to associate with each other. The present disclosure generally refers to a polypeptide chain containing a p35 moiety, a p40 moiety, and / or a multimerization moiety (e.g., a first Fc domain) that can associate with another polypeptide chain containing a p40 moiety, a p35 moiety, and / or a corresponding multimerization moiety (e.g., a second Fc domain) as a "monomer" or "IL12 monomer," respectively. Below are some illustrative examples of IL12 monomers of the present disclosure, described in the N- to C-terminal orientation. The individual elements of each monomer are described in detail herein, e.g., in the subsections and numbered embodiments below.

[0081] (1) Exemplary Monomer 1: IL12 p35 moiety-optional linker-multimerization moiety (see, e.g., Figure 2B, left monomer; Figure 2C, left monomer; and Figure 2N (both monomers)).

[0082] (2) Exemplary Monomer 2: IL12 p40 moiety-optional linker-multimerization moiety (see, e.g., Figure 2B, right monomer, and Figure 2C, right monomer). (3) Exemplary Monomer 3: Multimerization Moiety-Optional Linker-IL12 p35 Moiety (see, e.g., Figure 2D (left monomer), Figure 2E (left monomer), Figure 2O (both monomers), Figure 2P (left monomer), Figure 4M (left monomer), Figure 4U (left monomer), Figure 4V (left monomer), and Figure 4W (left monomer)).

[0083] (4) Exemplary Monomer 4: Multimerization Moiety-Optional Linker-IL12 p40 Moiety (see, e.g., Figure 2D (right monomer), Figure 2E (right monomer), and Figure 4L (right monomer)).

[0084] (5) Exemplary Monomer 5: IL12 p40 portion-optional linker-IL12 p35 portion-optional linker-multimerization portion (see, e.g., Figure 2H (both monomers), Figure 2I (both monomers), Figure 3B, Figure 3D, Figure 3G, Figure 5E (right monomer), Figure 5P (left monomer), and Figure 5Q (left monomer)).

[0085] (6) Exemplary Monomer 6: IL12 p35 moiety-optional linker-IL12 p40 moiety-optional linker-multimerization moiety (see, e.g., Figure 2J, both monomers).

[0086] (7) Exemplary Monomer 7: multimerization moiety-optional linker-IL12 p35 moiety-optional linker-IL12 p40 moiety (see, e.g., Figure 2G (left monomer), Figure 2L (both monomers), Figure 3I).

[0087] (8) Exemplary Monomer 8: multimerization moiety-optional linker-IL12 p40 moiety-optional linker-IL12 p35 moiety (see, e.g., Figure 2F (left monomer), Figure 2K (both monomers), Figure 2M (both monomers), Figure 3C, Figure 3E, Figure 3F, Figure 3H, Figure 4F (left monomer), Figure 4J (left monomer), Figure 4K (left monomer), Figure 4S (left monomer), Figure 39A (left monomer), and Figure 39B (left monomer)).

[0088] (9) Exemplary Monomer 9: multimerization moiety-optional linker-IL12Rβ moiety (e.g., an IL12Rβ1 moiety or an IL12Rβ2 moiety)-optional linker-IL12 p40 moiety-optional linker-IL12 p35 moiety (see, e.g., Figure 4C (both monomers), Figure 4E (both monomers), Figure 41 (left monomer), Figure 4N (left monomer), and Figure 4T (left monomer)). In some embodiments, the IL12Rβ moiety is an IL12Rβ1 moiety. In other embodiments, the IL12Rβ moiety is an IL12Rβ2 moiety.

[0089] (10) Exemplary Monomer 10: multimerization moiety-optional linker-IL12Rβ moiety (e.g., IL12Rβ1 moiety or IL12Rβ2 moiety)-optional linker-IL12 p35 moiety-optional linker-IL12 p40 moiety. In some embodiments, the IL12Rβ moiety is an IL12Rβ1 moiety. In other embodiments, the IL12Rβ moiety is an IL12Rβ2 moiety.

[0090] (11) Exemplary Monomer 11: Multimerization moiety-Optional linker-IL12 p40 moiety-Optional linker-IL12 p35 moiety-Optional linker-IL12Rβ moiety (e.g., IL12Rβ1 moiety or IL12Rβ2 moiety) (see, e.g., Figure 4B (both monomers), Figure 4D (both monomers), Figure 4G (left monomer), and Figure 4H (left monomer)). In some embodiments, the IL12Rβ moiety is an IL12Rβ1 moiety. In other embodiments, the IL12Rβ moiety is an IL12Rβ2 moiety.

[0091] (12) Exemplary Monomer 12: multimerization moiety-optional linker-IL12 p35 moiety-optional linker-IL12 p40 moiety-optional linker-IL12Rβ moiety (e.g., IL12Rβ1 moiety or IL12Rβ2 moiety).

[0092] (13) Exemplary Monomer 13: IL12 p40 portion-optional linker-IL12 p35 portion-optional linker-IL12Rβ portion (e.g., IL12Rβ1 portion or IL12Rβ2 portion)-optional linker-multimerization portion. In some embodiments, the IL12Rβ portion is an IL12Rβ1 portion. In other embodiments, the IL12Rβ portion is an IL12Rβ2 portion.

[0093] (14) Exemplary Monomer 14: IL12 p35 portion-optional linker-IL12 p40 portion-optional linker-IL12Rβ portion (e.g., IL12Rβ1 portion or IL12Rβ2 portion)-optional linker-multimerization portion. In some embodiments, the IL12Rβ portion is an IL12Rβ1 portion. In other embodiments, the IL12Rβ portion is an IL12Rβ2 portion.

[0094] (15) Exemplary Monomer 15: IL12Rβ portion (e.g., IL12Rβ1 portion or IL12Rβ2 portion)-optional linker-IL12 p40 portion-optional linker-IL12 p35 portion-optional linker-multimerization portion (see, e.g., Figure 5O (left monomer)). In some embodiments, the IL12Rβ portion is an IL12Rβ1 portion. In other embodiments, the IL12Rβ portion is an IL12Rβ2 portion.

[0095] (16) Exemplary Monomer 16: IL12Rβ portion (e.g., IL12Rβ1 portion or IL12Rβ2 portion)-optional linker-IL12 p35 portion-optional linker-IL12 p40 portion-optional linker-multimerization portion. In some embodiments, the IL12Rβ portion is an IL12Rβ1 portion. In other embodiments, the IL12Rβ portion is an IL12Rβ2 portion.

[0096] (17) Exemplary Monomer 17: IL12 p35 moiety-optional linker-multimerization moiety-optional linker-targeting moiety (see, e.g., Figure 5G, left monomer).

[0097] (18) Exemplary Monomer 18: IL12 p40 moiety-optional linker-multimerization moiety-optional linker-targeting moiety (see, e.g., Figure 5G, right monomer).

[0098] (19) Exemplary Monomer 19: targeting moiety-optional linker-multimerization moiety-optional linker-IL12 p35 moiety (see, e.g., Figure 5B (left monomer), Figure 5V (left monomer), Figure 5W (left monomer), and Figure 5X (left monomer)).

[0099] (20) Exemplary monomer 20: targeting moiety-optional linker-multimerization moiety-optional linker-IL12 p40 moiety (see, e.g., Figure 5B, right monomer).

[0100] (21) Exemplary Monomer 21: targeting moiety-optional linker-IL12 p35 moiety-optional linker-multimerization moiety. (22) Exemplary Monomer 22: targeting moiety-optional linker-IL12 p40 moiety-optional linker-multimerization moiety.

[0101] (23) Exemplary Monomer 23: multimerization moiety-optional linker-IL12 p35 moiety-optional linker-targeting moiety. (24) Exemplary Monomer 24: multimerization moiety-optional linker-IL12 p40 moiety-optional linker-targeting moiety.

[0102] (25) Exemplary Monomer 25: IL12 p40 moiety-optional linker-IL12 p35 moiety-optional linker-multimerization moiety-optional linker-targeting moiety (see, e.g., Figure 5F, both monomers).

[0103] (26) Exemplary Monomer 26: IL12 p35 moiety-optional linker-IL12 p40 moiety-optional linker-multimerization moiety-optional linker-targeting moiety.

[0104] (27) Exemplary Monomer 27: targeting moiety-optional linker-multimerization moiety-optional linker-IL12 p35 moiety-optional linker-IL12 p40 moiety.

[0105] (28) Exemplary Monomer 28: targeting moiety-optional linker-multimerization moiety-optional linker-IL12 p40 moiety-optional linker-IL12 p35 moiety (see, e.g., Figure 5C (left monomer), Figure 5D (both monomers), Figure 5I (left monomer), Figure 5J (left monomer), Figure 5M (left monomer), Figure 5N (left monomer), Figure 39C (left monomer), and Figure 39D (left monomer)).

[0106] (29) Exemplary Monomer 29: targeting moiety-optional linker-IL12 p40 moiety-optional linker-IL12 p35 moiety-optional linker-multimerization moiety.

[0107] (30) Exemplary monomer 30: targeting moiety-optional linker-IL12 p35 moiety-optional linker-IL12 p40 moiety-optional linker-multimerization moiety.

[0108] (31) Exemplary Monomer 31: multimerization moiety-optional linker-IL12 p35 moiety-optional linker-IL12 p40 moiety-optional linker-targeting moiety.

[0109] (32) Exemplary Monomer 32: multimerization moiety-optional linker-IL12 p40 moiety-optional linker-IL12 p35 moiety-optional linker-targeting moiety.

[0110] (33) Exemplary Monomer 33: Targeting Moiety—Optional Linker—Multimerization Moiety (see, e.g., Figure 5C (Right Monomer), Figure 5E (Left Monomer), Figure 5H (Right Monomer), Figure 5L (Right Monomer), Figure 5O (Right Monomer), Figure 5P (Right Monomer), Figure 5Q (Right Monomer), Figure 5R (Right Monomer), and Figure 5V (Right Monomer)).

[0111] (34) Exemplary Monomer 34: Multimerization Moiety—Optional Linker—Targeting Moiety. (35) Exemplary Monomer 35: targeting moiety-optional linker-multimerization moiety-optional linker-IL12Rβ moiety (e.g., IL12Rβ1 moiety or IL12Rβ2 moiety)-optional linker-IL12 p40 moiety-optional linker-IL12 p35 moiety (see, e.g., Figure 5H (left monomer), Figure 5K (left monomer), and Figure 5R (left monomer)). In some embodiments, the IL12Rβ moiety is an IL12Rβ1 moiety. In other embodiments, the IL12Rβ moiety is an IL12Rβ2 moiety.

[0112] (36) Exemplary Monomer 36: targeting moiety-optional linker-multimerization moiety-optional linker-IL12Rβ moiety (e.g., an IL12Rβ1 moiety or an IL12Rβ2 moiety)-optional linker-IL12 p35 moiety-optional linker-IL12 p40 moiety. In some embodiments, the IL12Rβ moiety is an IL12Rβ1 moiety. In other embodiments, the IL12Rβ moiety is an IL12Rβ2 moiety.

[0113] (37) Exemplary Monomer 37: targeting moiety-optional linker-multimerization moiety-optional linker-IL12 p40 moiety-optional linker-IL12 p35 moiety-optional linker-IL12Rβ moiety (e.g., IL12Rβ1 moiety or IL12Rβ2 moiety). In some embodiments, the IL12Rβ moiety is an IL12Rβ1 moiety. In other embodiments, the IL12Rβ moiety is an IL12Rβ2 moiety.

[0114] (38) Exemplary Monomer 38: targeting moiety-optional linker-multimerization moiety-optional linker-IL12 p35 moiety-optional linker-IL12 p40 moiety-optional linker-IL12Rβ moiety (e.g., IL12Rβ1 moiety or IL12Rβ2 moiety). In some embodiments, the IL12Rβ moiety is an IL12Rβ1 moiety. In other embodiments, the IL12Rβ moiety is an IL12Rβ2 moiety.

[0115] (39) Exemplary Monomer 39: multimerization moiety-optional linker-IL12Rβ moiety (e.g., IL12Rβ1 moiety or IL12Rβ2 moiety)-optional linker-IL12 p40 moiety-optional linker-IL12 p35 moiety-optional linker-targeting moiety. In some embodiments, the IL12Rβ moiety is an IL12Rβ1 moiety. In other embodiments, the IL12Rβ moiety is an IL12Rβ2 moiety.

[0116] (40) Exemplary monomer 40: multimerization moiety-optional linker-IL12Rβ moiety (e.g., IL12Rβ1 moiety or IL12Rβ2 moiety)-optional linker-IL12 p35 moiety-optional linker-IL12 p40 moiety-optional linker-targeting moiety. In some embodiments, the IL12Rβ moiety is an IL12Rβ1 moiety. In other embodiments, the IL12Rβ moiety is an IL12Rβ2 moiety.

[0117] (41) Exemplary Monomer 41: multimerization moiety-optional linker-IL12 p40 moiety-optional linker-IL12 p35 moiety-optional linker-IL12Rβ moiety (e.g., IL12Rβ1 moiety or IL12Rβ2 moiety)-optional linker-targeting moiety. In some embodiments, the IL12Rβ moiety is an IL12Rβ1 moiety. In other embodiments, the IL12Rβ moiety is an IL12Rβ2 moiety.

[0118] (42) Exemplary Monomer 42: multimerization moiety-optional linker-IL12 p35 moiety-optional linker-IL12 p40 moiety-optional linker-IL12Rβ moiety (e.g., IL12Rβ1 moiety or IL12Rβ2 moiety)-optional linker-targeting moiety. In some embodiments, the IL12Rβ moiety is an IL12Rβ1 moiety. In other embodiments, the IL12Rβ moiety is an IL12Rβ2 moiety.

[0119] (43) Exemplary Monomer 43: IL12 p40 portion-optional linker-IL12 p35 portion-optional linker-IL12Rβ portion (e.g., IL12Rβ1 portion or IL12Rβ2 portion)-optional linker-multimerization portion-optional linker-targeting portion. In some embodiments, the IL12Rβ portion is an IL12Rβ1 portion. In other embodiments, the IL12Rβ portion is an IL12Rβ2 portion.

[0120] (44) Exemplary Monomer 44: IL12 p35 portion-optional linker-IL12 p40 portion-optional linker-IL12Rβ portion (e.g., IL12Rβ1 portion or IL12Rβ2 portion)-optional linker-multimerization portion-optional linker-targeting portion. In some embodiments, the IL12Rβ portion is an IL12Rβ1 portion. In other embodiments, the IL12Rβ portion is an IL12Rβ2 portion.

[0121] (45) Exemplary Monomer 45: IL12Rβ portion (e.g., IL12Rβ1 portion or IL12Rβ2 portion)-optional linker-IL12 p40 portion-optional linker-IL12 p35 portion-optional linker-multimerization portion-optional linker-targeting portion. In some embodiments, the IL12Rβ portion is an IL12Rβ1 portion. In other embodiments, the IL12Rβ portion is an IL12Rβ2 portion.

[0122] (46) Exemplary Monomer 46: IL12Rβ portion (e.g., IL12Rβ1 portion or IL12Rβ2 portion)-optional linker-IL12 p35 portion-optional linker-IL12 p40 portion-optional linker-multimerization portion-optional linker-targeting portion. In some embodiments, the IL12Rβ portion is an IL12Rβ1 portion. In other embodiments, the IL12Rβ portion is an IL12Rβ2 portion.

[0123] (47) Exemplary Monomer 47: targeting moiety-optional linker-IL12 p40 moiety-optional linker-IL12 p35 moiety-optional linker-IL12Rβ moiety (e.g., an IL12Rβ1 moiety or an IL12Rβ2 moiety)-optional linker-multimerization moiety-optional linker-targeting moiety. In some embodiments, the IL12Rβ moiety is an IL12Rβ1 moiety. In other embodiments, the IL12Rβ moiety is an IL12Rβ2 moiety.

[0124] (48) Exemplary Monomer 48: targeting moiety-optional linker-IL12 p35 moiety-optional linker-IL12 p40 moiety-optional linker-IL12Rβ moiety (e.g., an IL12Rβ1 moiety or an IL12Rβ2 moiety)-optional linker-multimerization moiety-optional linker-targeting moiety. In some embodiments, the IL12Rβ moiety is an IL12Rβ1 moiety. In other embodiments, the IL12Rβ moiety is an IL12Rβ2 moiety.

[0125] (49) Exemplary Monomer 49: targeting moiety-optional linker-IL12Rβ moiety (e.g., an IL12Rβ1 moiety or an IL12Rβ2 moiety)-optional linker-IL12 p40 moiety-optional linker-IL12 p35 moiety-optional linker-multimerization moiety-optional linker-targeting moiety. In some embodiments, the IL12Rβ moiety is an IL12Rβ1 moiety. In other embodiments, the IL12Rβ moiety is an IL12Rβ2 moiety.

[0126] (50) Exemplary monomer 50: targeting moiety-optional linker-IL12Rβ moiety (e.g., an IL12Rβ1 moiety or an IL12Rβ2 moiety)-optional linker-IL12 p35 moiety-optional linker-IL12 p40 moiety-optional linker-multimerization moiety-optional linker-targeting moiety. In some embodiments, the IL12Rβ moiety is an IL12Rβ1 moiety. In other embodiments, the IL12Rβ moiety is an IL12Rβ2 moiety.

[0127] (51) Exemplary Monomer 51: Multimerization Moiety-Optional Linker-IL12Rβ Moiety (e.g., IL12Rβ1 Moiety or IL12Rβ2 Moiety) (see, e.g., Figure 4J (Right Monomer), Figure 4K (Right Monomer), Figure 4N (Right Monomer), Figure 4S (Right Monomer), Figure 4V (Right Monomer), and Figure 4W (Right Monomer)). In some embodiments, the IL12Rβ moiety is an IL12Rβ1 moiety. In other embodiments, the IL12Rβ moiety is an IL12Rβ2 moiety.

[0128] (52) Exemplary monomer 52: IL12Rβ portion (e.g., IL12Rβ1 portion or IL12Rβ2 portion)-optional linker-multimerization portion. (53) Exemplary Monomer 53: Multimerization Moiety-Optional Linker-IL12 p35 Moiety-Optional Linker-IL12Rβ Moiety (e.g., IL12Rβ1 Moiety or IL12Rβ2 Moiety) (See, e.g., Figure 4L (Left Monomer)). In some embodiments, the IL12Rβ Moiety is an IL12Rβ1 Moiety. In other embodiments, the IL12Rβ Moiety is an IL12Rβ2 Moiety.

[0129] (54) Exemplary Monomer 54: Multimerization Moiety-Optional Linker-IL12 p40 Moiety-Optional Linker-IL12Rβ Moiety (e.g., IL12Rβ1 Moiety or IL12Rβ2 Moiety) (See, e.g., Figure 4M (right monomer)). In some embodiments, the IL12Rβ Moiety is an IL12Rβ1 Moiety. In other embodiments, the IL12Rβ Moiety is an IL12Rβ2 Moiety.

[0130] (55) Exemplary monomer 55: multimerization moiety-optional linker-IL12 p40 moiety-optional linker-IL12 p35 moiety-optional linker-antibody-based masking moiety (see, e.g., Figure 4O (left monomer) and Figure 4P (left monomer)).

[0131] (56) Exemplary monomer 56: multimerization moiety—optional linker—antibody-based masking moiety (see, e.g., Figure 4Q (right monomer), Figure 4R (right monomer), Figure 39A (right monomer), and Figure 39B (right monomer)).

[0132] (57) Exemplary Monomer 57: Targeting Moiety-Optional Linker-Multimerization Moiety-Optional Linker-IL12Rβ Moiety (e.g., IL12Rβ1 Moiety or IL12Rβ2 Moiety) (see, e.g., Figure 5I (Right Monomer), Figure 5J (Right Monomer), Figure 5K (Right Monomer), Figure 5S (Right Monomer), Figure 5W (Right Monomer), and Figure 5X (Right Monomer)). In some embodiments, the IL12Rβ moiety is an IL12Rβ1 moiety. In other embodiments, the IL12Rβ moiety is an IL12Rβ2 moiety.

[0133] (58) Exemplary monomer 58: targeting moiety-optional linker-multimerization moiety-optional linker-IL12 p40 moiety-optional linker-IL12 p35 moiety-optional linker-antibody-based masking moiety (see, e.g., Figure 5L (left monomer)).

[0134] (59) Exemplary monomer 59: targeting moiety-optional linker-multimerization moiety-optional linker-antibody-based masking moiety (see, e.g., Figure 5M (right monomer), Figure 5N (right monomer), Figure 39C (right monomer), and Figure 39D (right monomer)).

[0135] (60) Exemplary monomer 60: multimerization moiety (see, e.g., Figure 2F (right monomer), Figure 2G (right monomer), Figure 4O (right monomer), Figure 4P (right monomer), Figure 4T (right monomer), and Figure 4U (right monomer)).

[0136] (61) Exemplary Monomer 61: IL12Rβ portion (e.g., IL12Rβ1 portion or IL12Rβ2 portion)-optional linker-IL12p40 portion-optional linker-IL12p35 portion-optional linker-multimerization portion. In some embodiments, the IL12Rβ portion is an IL12Rβ1 portion. In other embodiments, the IL12Rβ portion is an IL12Rβ2 portion.

[0137] (62) Exemplary Monomer 62: IL12Rβ portion (e.g., IL12Rβ1 portion or IL12Rβ2 portion) - optional linker - multimerization portion. In some embodiments, the IL12Rβ portion is an IL12Rβ1 portion. In other embodiments, the IL12Rβ portion is an IL12Rβ2 portion.

[0138] (63) Exemplary monomer 63: targeting moiety-optional linker-multimerization moiety-optional linker-IL12 p40 moiety-optional linker-IL12 p35 moiety (see, e.g., Figure 5S (left monomer)).

[0139] In some embodiments, the present disclosure provides an IL12 receptor agonist comprising two monomers according to exemplary monomer 1 (see, eg, FIG. 2N). In some embodiments, the present disclosure provides an IL12 receptor agonist comprising exemplary monomer 1 and exemplary monomer 2 (see, eg, Figures 2B and 2C).

[0140] In some embodiments, the present disclosure provides an IL12 receptor agonist comprising two monomers according to exemplary monomer 3 (see, eg, Figure 2O). In some embodiments, the present disclosure provides IL12 receptor agonists comprising exemplary monomer 3 and exemplary monomer 4 (see, eg, Figures 2D and 2E).

[0141] In some embodiments, the present disclosure provides IL12 receptor agonists including exemplary monomer 3 and exemplary monomer 51, where exemplary monomer 3 is associated with monomeric p40 (see, e.g., Figure 4V (left monomer) and Figure 4W (left monomer)). In some embodiments, the monomeric p40 is masked monomeric p40 (see, e.g., Figure 4U and Figure 4W). The mask can be, for example, an IL12Rβ1-based mask or an anti-p40 antibody-based mask.

[0142] In some embodiments, the present disclosure provides IL12 receptor agonists comprising exemplary monomer 3 and exemplary monomer 54 (see, eg, Figure 4M). In some embodiments, the present disclosure provides IL12 receptor agonists comprising exemplary monomer 3 and exemplary monomer 60, where exemplary monomer 3 is associated with monomeric p40 (see, e.g., Figure 2P (left monomer) and Figure 4U (left monomer)). In some embodiments, the monomeric p40 is masked monomeric p40 (see, e.g., Figure 4U). The mask can be, for example, an IL12Rβ1-based mask or an anti-p40 antibody-based mask.

[0143] In some embodiments, the present disclosure provides IL12 receptor agonists comprising exemplary monomer 4 and exemplary monomer 53 (see, eg, Figure 4L). In some embodiments, the present disclosure provides an IL12 receptor agonist comprising two monomers according to exemplary monomer 5 (see, eg, Figures 2H and 2I).

[0144] In some embodiments, the present disclosure provides IL12 receptor agonists comprising exemplary monomer 5 and exemplary monomer 33 (see, e.g., Figures 5E, 5P, and 5Q).

[0145] In some embodiments, the present disclosure provides an IL12 receptor agonist comprising two monomers according to exemplary monomer 6 (see, eg, Figure 2J). In some embodiments, the present disclosure provides an IL12 receptor agonist comprising two monomers according to exemplary monomer 7 (see, eg, Figure 2L).

[0146] In some embodiments, the present disclosure provides IL12 receptor agonists comprising exemplary monomer 7 and exemplary monomer 60 (see, eg, Figure 2G). In some embodiments, the present disclosure provides an IL12 receptor agonist comprising two monomers according to exemplary monomer 8 (see, eg, Figures 2K and 2M).

[0147] In some embodiments, the present disclosure provides IL12 receptor agonists comprising exemplary monomer 8 and exemplary monomer 60 (see, eg, Figures 2F and 4F).

[0148] In some embodiments, the present disclosure provides IL12 receptor agonists comprising exemplary monomer 8 and exemplary monomer 51 (see, e.g., Figures 4J, 4K, and 4S).

[0149] In some embodiments, the present disclosure provides IL12 receptor agonists comprising exemplary monomer 8 and exemplary monomer 56 (see, e.g., Figures 4Q, 4R, 39A, and 39B).

[0150] In some embodiments, the present disclosure provides an IL12 receptor agonist comprising two monomers according to exemplary monomer 9 (see, e.g., Figures 4C and 4E). The IL12Rβ moieties in a monomer according to exemplary monomer 9 can both be IL12Rβ1 moieties, both can be IL12Rβ2 moieties, or a combination of IL12Rβ1 and IL12Rβ2 moieties.

[0151] In some embodiments, the present disclosure provides IL12 receptor agonists comprising exemplary monomer 9 and exemplary monomer 60 (see, eg, Figures 4I and 4T).

[0152] In some embodiments, the present disclosure provides an IL12 receptor agonist comprising two monomers according to exemplary monomer 11 (see, e.g., Figures 4B and 4D). The IL12Rβ moieties in a monomer according to exemplary monomer 11 can both be IL12Rβ1 moieties, both be IL12Rβ2 moieties, or a combination of IL12Rβ1 and IL12Rβ2 moieties.

[0153] In some embodiments, the present disclosure provides IL12 receptor agonists comprising exemplary monomer 11 and exemplary monomer 60 (see, eg, Figures 4G and 4H).

[0154] In some embodiments, the present disclosure provides IL12 receptor agonists including exemplary monomer 15 and exemplary monomer 33 (see, e.g., Figure 5O). In one embodiment, the IL12Rβ moiety in exemplary monomer 15 is an IL12Rβ1 moiety. In another embodiment, the IL12Rβ moiety in exemplary monomer 15 is an IL12Rβ2 moiety.

[0155] In some embodiments, the present disclosure provides an IL12 receptor agonist comprising exemplary monomer 17 and exemplary monomer 18 (see, eg, Figure 5G). In some embodiments, the present disclosure provides an IL12 receptor agonist comprising exemplary monomer 19 and exemplary monomer 20 (see, eg, Figure 5B).

[0156] In some embodiments, the present disclosure provides IL12 receptor agonists including exemplary monomer 19 and exemplary monomer 33, where exemplary monomer 19 is associated with monomeric p40 (see, e.g., Figure 5V (left monomer)). In some embodiments, the monomeric p40 is masked monomeric p40 (see, e.g., Figure 5V). The mask can be, for example, an IL12Rβ1-based mask or an anti-p40 antibody-based mask.

[0157] In some embodiments, the present disclosure provides IL12 receptor agonists including exemplary monomer 19 and exemplary monomer 57, where exemplary monomer 19 is associated with monomeric p40 (see, e.g., Figure 5W (left monomer) and Figure 5X (left monomer)). In some embodiments, the monomeric p40 is masked monomeric p40 (see, e.g., Figure 5X). The mask can be, for example, an IL12Rβ1-based mask or an anti-p40 antibody-based mask.

[0158] In some embodiments, the present disclosure provides an IL12 receptor agonist comprising two monomers according to exemplary monomer 25 (see, eg, Figure 5F). In some embodiments, the present disclosure provides an IL12 receptor agonist comprising two monomers according to exemplary monomer 28 (see, eg, FIG. 5D).

[0159] In some embodiments, the present disclosure provides IL12 receptor agonists comprising exemplary monomer 28 and exemplary monomer 33 (see, eg, Figure 5C). In some embodiments, the present disclosure provides IL12 receptor agonists comprising exemplary monomer 28 and exemplary monomer 57 (see, e.g., Figures 5I and 5J). In one embodiment, the IL12Rβ moiety in exemplary monomer 57 is an IL12Rβ1 moiety. In another embodiment, the IL12Rβ moiety in exemplary monomer 57 is an IL12Rβ2 moiety.

[0160] In some embodiments, the present disclosure provides IL12 receptor agonists comprising exemplary monomer 28 and exemplary monomer 59 (see, e.g., Figures 5M, 5N, 39C, and 39D).

[0161] In some embodiments, the present disclosure provides IL12 receptor agonists including exemplary monomer 33 and exemplary monomer 35 (see, e.g., Figures 5H and 5R). In one embodiment, the IL12Rβ moiety in exemplary monomer 35 is an IL12Rβ1 moiety. In another embodiment, the IL12Rβ moiety in exemplary monomer 35 is an IL12Rβ2 moiety.

[0162] In some embodiments, the present disclosure provides IL12 receptor agonists comprising exemplary monomer 33 and exemplary monomer 58 (see, eg, Figure 5L). In some embodiments, the present disclosure provides an IL12 receptor agonist comprising exemplary monomer 35 and exemplary monomer 57 (see, e.g., FIG. 5K). The IL12Rβ moieties in exemplary monomer 35 and exemplary monomer 57 can both be IL12Rβ1 moieties, both be IL12Rβ2 moieties, or a combination of IL12Rβ1 and IL12Rβ2 moieties. In one embodiment, the IL12Rβ moiety in exemplary monomer 35 is an IL12Rβ1 moiety, and the IL12Rβ moiety in exemplary monomer 57 is an IL12Rβ2 moiety. In another embodiment, the IL12Rβ moiety in exemplary monomer 35 is an IL12Rβ2 moiety, and the IL12Rβ moiety in exemplary monomer 57 is an IL12Rβ1 moiety.

[0163] In some embodiments, the present disclosure provides IL12 receptor agonists comprising exemplary monomer 55 and exemplary monomer 60 (see, eg, Figures 4O and 4P).

[0164] In some embodiments, the present disclosure provides IL12 receptor agonists comprising exemplary monomer 57 and exemplary monomer 63 (see, eg, Figure 5S). Additional embodiments of exemplary monomer pairings are described in the numbered embodiments disclosed in Section 7.

[0165] In the aforementioned IL12 receptor agonist embodiments that include two exemplary monomers having IL12Rβ moieties, the IL12Rβ moieties can both be IL12Rβ1 moieties, both be IL12Rβ2 moieties, or a combination of IL12Rβ1 and IL12Rβ2 moieties.

[0166] In the IL12 receptor agonists of the present disclosure, when the targeting moiety is an antibody antigen-binding domain ("ABD"), each monomer can be composed of two polypeptide chains, one carrying a heavy chain variable region and the other carrying a light chain variable region. The targeting moiety itself can comprise heavy and light chain variable domains on separate polypeptide chains. For example, for an IL12 receptor agonist monomer comprising a targeting moiety, the monomer can be composed of polypeptide A and polypeptide B. Polypeptide A can comprise, for example, from N-terminus to C-terminus, the heavy chain variable domain of the targeting moiety-optional linker-multimerization moiety-optional linker-IL12 p40 moiety-IL12 p35 moiety, and polypeptide B can comprise the light chain variable domain of the targeting moiety.

[0167] Alternatively, an scFv can be used as a targeting moiety, in which the heavy and light chain variable regions of the targeting moiety are fused to each other in a single polypeptide. In certain aspects, the IL12 receptor agonists of the present disclosure have a therapeutic index of greater than 1, and preferably greater than 2, and even more preferably greater than 10. In certain embodiments, the therapeutic index is about 10, about 20, about 100, or about 200.

[0168] In various embodiments, the IL12 receptor agonist does not include (a) a cytokine other than IL12, (b) an anti-IL12 antibody or antibody fragment, (c) an anti-DNA antibody or antibody fragment, (b) a non-binding antibody variable domain, or any combination of two, three, or all four of these.

[0169] Further details of the components of the IL12 receptor agonists of the present disclosure are provided below. 6.2.1. Biochemical Properties of IL-12 Receptor Agonists In vivo, large antibody complexes are rapidly removed by phagocytosis, potentially resulting in reduced antibody efficacy. Large complexes may also increase the immunogenicity of therapeutic antibodies. See, for example, WO2020047067A1. During manufacturing, aggregation is a common problem that compromises the quality, safety, and efficacy of antibodies. IL12 receptor agonists of the present disclosure (e.g., masked IL12 receptor agonists comprising a single receptor domain type (e.g., D1 of IL12Rβ1 or IL12Rβ2)) may be less prone to aggregation, for example, in vivo or ex vivo, compared to receptor agonists with alternative structures (e.g., masked IL12 receptor agonists comprising two receptor domain types (e.g., D1 and D2 of IL12Rβ1 or IL12Rβ2)). Thus, in some embodiments, an IL12 receptor agonist of the present disclosure aggregates at least 50%, at least 60%, at least 70%, at least 80%, at least 95%, or at least 99% less during recombinant production in a mammalian cell line than an IL12 receptor agonist with an alternative structure. The oligomerization state of an IL12 receptor agonist can be determined, for example, by size-exclusion ultra-performance liquid chromatography.

[0170] In some embodiments, the IL12 receptor agonists of the present disclosure are believed to have good thermal stability. High thermal stability and low tendency to aggregate facilitate antibody production and storage and promote a long serum half-life. Carter and Merchant, 1997, Curr Opin Biotechnol, 8(4):449-454. Thermal stability can be measured by methods known in the art, including differential scanning fluorimetry (DSF).

[0171] 6.3.IL12 p40 and p35 moieties The present disclosure provides IL12 receptor agonists having p35 and p40 moieties with wild-type or variant p35 and p40 sequences. The present disclosure further provides p35 and p40 moieties having variant p35 and p40 sequences, respectively. Exemplary p40 moieties are disclosed in Section 6.3.1 and exemplary p35 moieties are disclosed in Section 6.3.2.

[0172] 6.3.1. IL12 p40 moiety Each IL12 p40 moiety of the IL12 receptor agonists of the present disclosure comprises a wild-type or variant IL12 p40 moiety. In some embodiments, the IL12 receptor agonists of the present disclosure comprise a single IL12 p40 moiety (e.g., in embodiments where the IL12 receptor agonist is monovalent with respect to IL12, the IL12 p40 moiety on the first monomer or the second monomer). In some embodiments, the IL12 receptor agonists of the present disclosure comprise two IL12 p40 moieties (e.g., in embodiments where the IL12 agonist is divalent with respect to IL12, the first IL12 p40 moiety on the first monomer and the second IL12 p40 moiety on the second monomer). In such embodiments, the two IL12 p40 moieties can be the same or they can be different.

[0173] In eukaryotic cells, the human IL12 p40 subunit is synthesized as a 328 amino acid precursor polypeptide, from which 22 amino acids are removed to generate mature IL12 p40. In some embodiments, the IL12 p40 portion is an amino acid sequence that contains 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 IL12Rβ1-binding portion of mammalian, e.g., human or mouse, p40 (sometimes referred to as the beta subunit of IL12, or IL12β). In some embodiments, the mammalian p40 is full-length human p40. In other embodiments, the mammalian p40 is mature human p40. The sequence of human p40 has Uniprot identifier P29460 (uniprot.org / uniprot / P29460). In some embodiments, the mammalian p40 is full-length mouse p40. In some embodiments, the mammalian p40 is mature mouse p40. The sequence of mouse p40 has Uniprot identifier P43432 (uniprot.org / uniprot / P43432).

[0174] In some embodiments, the p40 portion excludes the p40 D1 domain and includes the p40 D2 and D3 domains, hi other embodiments, the p40 portion includes the p40 D1, D2, and D3 domains.

[0175] Full-length human IL12 p40 has the following amino acid sequence (signal sequence = underlined; D1 domain = italic; D2 domain = bold; D3 domain = bold and underlined):

[0176] [ka]

[0177] Amino acid 23 of full-length human p40 is amino acid 1 of mature human p40. In certain embodiments, the IL12 p40 moiety comprises one or more amino acid substitutions that reduce binding to IL12Rβ1. For example, in some embodiments, the IL12 p40 moiety can have up to 1,000-fold attenuated binding to human IL12Rβ1 compared to wild-type human IL12 p40. In some embodiments, the IL12 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 IL12Rβ1 compared to wild-type human IL12 p40.

[0178] Other characteristics of useful IL12 p40 variants may include the ability to destabilize dimerization with IL12 p35. 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, where amino acid positions are relative to the mature human IL12 p40 amino acid sequence excluding the 22 amino acid signal sequence unless otherwise stated. The corresponding amino acid positions in the full-length human sequence, the full-length mouse sequence, and the mature mouse sequence are provided in Table 1. Table 1 also provides exemplary substitutions at each listed position.

[0179] [Table 1-1]

[0180] [Table 1-2]

[0181] An exemplary amino acid substitution in mature human K6 is K6A. An exemplary amino acid substitution in mature human W15 is W15A. Exemplary amino acid substitutions in mature human D18 include D18N, D18K, and D18A.

[0182] Exemplary amino acid substitutions in mature human E32 include E32Q and E32A. Exemplary amino acid substitutions in mature human E33 include E33Q and E33A.

[0183] Exemplary amino acid substitutions in mature human D34 include D34N, D34K, and D34A. An exemplary amino acid substitution in mature human Q42 is Q42E.

[0184] Exemplary amino acid substitutions in mature human S43 include S43E and S34K. An exemplary amino acid substitution in mature human E45 is E45Q.

[0185] An exemplary amino acid substitution in mature human Q56 is Q56E. Exemplary amino acid substitutions in mature human E59 include E59K, E59Q, and E59A.

[0186] An exemplary amino acid substitution in mature human F60 is F60A. An exemplary amino acid substitution in mature human D62 is D62N. An exemplary amino acid substitution in mature human E73 is E73Q.

[0187] An exemplary amino acid substitution in mature human K84 is K84A. An exemplary amino acid substitution in mature human D87 is D87N. An exemplary amino acid substitution in mature human D93 is D93A.

[0188] An exemplary amino acid substitution in mature human K96 is E93A. Exemplary amino acid substitutions in mature human K99 include K99E, K99Y, and K99A.

[0189] An exemplary amino acid substitution in mature human E100 is E100Q. Exemplary amino acid substitutions in mature human N103 include N103D and N103Q.

[0190] An exemplary amino acid substitution in mature human K104 is K104A. Exemplary amino acid substitutions in mature human N113 include N113D and N113Q.

[0191] An exemplary amino acid substitution in mature human Q144 is Q144E. An exemplary amino acid substitution in mature human R159 is R159E. An exemplary amino acid substitution in mature human D161 is D161N.

[0192] An exemplary amino acid substitution in mature human K163 is K163E. An exemplary amino acid substitution in mature human E187 is E187Q. Exemplary amino acid substitutions in mature human N200 include N200D and N200Q.

[0193] An exemplary amino acid substitution in mature human N218 is N218Q. An exemplary amino acid substitution in mature human Q229 is Q229E. An exemplary amino acid substitution in mature human E235 is E235Q.

[0194] Exemplary amino acid substitutions in mature human Y246 include Y246V and Y246F. An exemplary amino acid substitution in mature human C252 is C252S.

[0195] An exemplary amino acid substitution in mature human Q256 is Q256N. An exemplary amino acid substitution in mature human K258 is K258E. An exemplary amino acid substitution in mature human K260 is K260E.

[0196] An exemplary amino acid substitution in mature human E262 is E262Q. An exemplary amino acid substitution in mature human K264 is K264E. Exemplary amino acid substitutions in mature human N281 include N281D and N281Q.

[0197] An exemplary amino acid substitution in mature human Y292 is Y292F. An exemplary amino acid substitution in mature human E299 is E299Q. In certain embodiments, amino acid substitutions at mature human Y246 and / or Y292 destabilize the p40 / p35 heterodimer by preventing disulfide bond formation between the two subunits. Exemplary amino acid substitutions at Y246 include Y246V and Y246F. An exemplary amino acid substitution at Y292 is Y292F.

[0198] In some embodiments, the p40 moiety is optionally fused, either directly or indirectly (e.g., as described in Section 6.8), via a linker, to the IL12 p40 binding domain of IL12Rβ1 (i.e., the IL12Rβ1 moiety, e.g., as described in Section 6.4.1). When present, the IL12 p40 binding domain of IL12Rβ1 can be at the N-terminus or C-terminus of the IL12 p40 moiety. When the p40 moiety is "directly" fused to the IL12 p40 binding domain of IL12Rβ1, the p40 moiety and the IL12 p40 binding domain of IL12Rβ1 are arranged adjacent to each other on the same monomer and, when present, are separated only by a linker. When the p40 moiety is "indirectly" fused to the IL12 p40 binding domain of IL12Rβ1, the p40 moiety and the IL12 p40 binding domain of IL12Rβ1 are separated by one or more other domains (e.g., the IL12 p35 moiety) on the same monomer or are located on separate monomers.

[0199] 6.3.2. IL12 p35 moiety Each IL12 p35 moiety of the IL12 receptor agonists of the present disclosure comprises a wild-type or variant IL12 p35 moiety. In some embodiments, the IL12 receptor agonists of the present disclosure comprise a single IL12 p35 moiety (e.g., in embodiments where the IL12 receptor agonist is monovalent with respect to IL12, the IL12 p35 moiety on the first monomer or the second monomer). In some embodiments, the IL12 receptor agonists of the present disclosure comprise two IL12 p35 moieties. In such embodiments, the two IL12 p35 moieties can be identical or they can be different.

[0200] In eukaryotic cells, the human IL12 p35 subunit is synthesized as a 219 amino acid precursor polypeptide from which 22 amino acids are removed to generate mature IL12 p35. In some embodiments, the IL12 p35 portion is an amino acid sequence that contains 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 IL12Rβ2-binding portion of mammalian, e.g., human or mouse, p35 (sometimes referred to as the alpha subunit of IL12 or IL12α). 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 Uniprot identifier P29459 (uniprot.org / uniprot / P29459). In some embodiments, the mammalian p35 is full-length mouse p35. In some embodiments, the mammalian p35 is mature mouse p40. The sequence of mouse p40 has Uniprot identifier P43431 (uniprot.org / uniprot / P43431).

[0201] Full-length human IL12 p35 has the following amino acid sequence (signal sequence = underlined):

[0202] [ka]

[0203] Amino acid 23 of full-length human p35 is amino acid 1 of mature human p35. In certain embodiments, the IL12 p35 moiety comprises one or more amino acid substitutions that reduce binding to IL12Rβ2. For example, in some embodiments, the IL12 p35 moiety can have binding to human IL12Rβ1 that is attenuated by up to 1,000-fold compared to wild-type human IL12 p35. In some embodiments, the IL12 moiety can have binding to human IL12Rβ2 that is attenuated by 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 compared to wild-type human IL12 p35.

[0204] Other characteristics of useful IL12 p35 variants may include the ability to destabilize dimerization with IL12 p40. 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, I171, and R189, where the amino acid positions are relative to the mature human IL12 p35 amino acid sequence excluding the 22-amino acid signal sequence. The corresponding amino acid positions in the full-length human sequence, full-length mouse sequence, and mature mouse sequence are provided in Table 2. Table 2 also provides exemplary substitutions at each listed position.

[0205] [Table 2]

[0206] An exemplary amino acid substitution in mature human N21 is N21D. An exemplary amino acid substitution in mature human Q35 is Q35D. An exemplary amino acid substitution in mature human E38 is E38Q.

[0207] An exemplary amino acid substitution in mature human E45 is E45Q. Exemplary amino acid substitutions in mature human D55 include D55Q and D55K.

[0208] An exemplary amino acid substitution in mature human N71 is N71D. An exemplary amino acid substitution in mature human L75 is L75A. An exemplary amino acid substitution in mature human N76 is N76D.

[0209] An exemplary amino acid substitution in mature human E79 is E79Q. Exemplary amino acid substitutions in mature human N85 include N85D and N85Q.

[0210] An exemplary amino acid substitution in mature human L89 is L89A. An exemplary amino acid substitution in mature human F96 is F96A. An exemplary amino acid substitution in mature human M97 is M97A.

[0211] An exemplary amino acid substitution in mature human L124 is L124A. An exemplary amino acid substitution in mature human M125 is M125A. An exemplary amino acid substitution in mature human Q130 is Q130E.

[0212] An exemplary amino acid substitution in mature human Q135 is Q135E. An exemplary amino acid substitution in mature human N136 is N136D. An exemplary amino acid substitution in mature human E143 is E143Q.

[0213] An exemplary amino acid substitution in mature human Q146 is Q146E. Exemplary amino acid substitutions in mature human Y167 include Y167A, Y167V, Y167R, and Y167E.

[0214] Exemplary amino acid substitutions in mature human I171 include I171A, I171V, and I171E. In certain embodiments, amino acid substitutions in mature human R189 destabilize the p40 / p35 heterodimer by preventing disulfide bond formation between the two subunits. Exemplary amino acid substitutions in mature human R189 include R189A and R189K.

[0215] In some embodiments, the p35 moiety is fused, either directly or indirectly, optionally via a linker (e.g., as described in Section 6.8), to the IL12 p35 binding domain of IL12Rβ2 (i.e., an IL12Rβ2 moiety, e.g., as described in Section 6.4.2). If present, the IL12 p35 binding domain of IL12Rβ2 can be N- or C-terminal to the IL12 p35 moiety. When the p35 moiety is fused "directly" to the IL12 p35 binding domain of IL12Rβ2, the p35 moiety and the IL12 p35 binding domain of IL12Rβ2 are positioned adjacently on the same monomer, separated only by a linker, if present. When the p35 moiety is "indirectly" fused to the IL12 p35 binding domain of IL12Rβ2, the p35 moiety and the IL12 p35 binding domain of IL12Rβ2 are separated by one or more other domains (e.g., an IL12 p40 moiety) on the same monomer or are located on separate monomers.

[0216] 6.4.IL12 Masking Part The present disclosure provides IL12 receptor agonists having one or more IL12 masking moieties capable of binding to IL12 p40 and / or p35 moieties. In some aspects, the IL12 generating moieties described herein bind to the IL12 p40 and / or p35 moieties, thereby attenuating IL12 activity on target cells. In some embodiments, the IL12 masking moiety is an IL12Rβ1 moiety capable of binding to the IL12 p40 moiety. In other embodiments, the IL12 masking moiety is an IL12Rβ2 moiety capable of binding to the IL12 p35 moiety. Exemplary IL12Rβ1 moieties are disclosed in Section 6.4.1, and exemplary IL12Rβ2 moieties are disclosed in Section 6.4.2. In other embodiments, the IL12 masking moiety is an IL12 antibody fragment. Exemplary IL12 antibody fragments are disclosed in Section 6.4.3.

[0217] 6.4.1.IL12Rβ1 part The IL12 receptor agonists of the present disclosure optionally comprise one or more IL12Rβ1 moieties as masking moieties (sometimes referred to herein as "IL12Rβ1 masking moieties"). In some embodiments, the IL12 receptor agonist comprises an IL12Rβ1 moiety in combination with an IL12Rβ2 moiety as an additional masking moiety.

[0218] In certain embodiments, the IL12Rβ1 portion comprises an amino acid sequence having at least 70% sequence identity, e.g., at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, to the p40-binding portion of mammalian, e.g., human or murine, IL12 receptor subunit beta-1 (IL12Rβ1). The sequence of human IL12Rβ1 has Uniprot identifier P42701 (uniprot.org / uniprot / P42701), with amino acids 24-545 comprising the extracellular domain. The sequence of mouse IL12Rβ1 has the Uniprot identifier Q60837 (uniprot.org / uniprot / Q60837), with amino acids 24 to 545 constituting the extracellular domain.

[0219] The extracellular domain of IL12Rβ1 contains five fibronectin type III domains: D1, D2, D3, D4, and D5. The sequences of D1, D2, D3, D4, and D5 of mouse and human IL12Rβ1 are provided below.

[0220] [Table 3]

[0221] In some embodiments, an IL12Rβ1 portion of the present disclosure comprises or consists of D1, D2, D3, D4, and / or D5 of IL12Rβ1, including any combination thereof (e.g., D1 and D2, D1-D3, D1-D4, D1-D5, etc.). In some embodiments, an IL12Rβ1 portion does not include one or more of D1, D2, D3, D4, or D5 of IL12Rβ1. In certain aspects, an IL12Rβ1 portion does not include any of D3, D4, or D5 of IL12Rβ1. In further aspects, an IL12Rβ1 portion does not include any of D2, D3, D4, or D5 of IL12Rβ1.

[0222] Preferably, the IL12Rβ1 portion is capable of binding to IL12p40. In some embodiments, the IL12Rβ1 portion comprises or consists of 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 the amino acid sequence of D1 of IL12Rβ1. The IL12Rβ1 portion may comprise or consist of at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids at the N-terminus of D1 of IL12Rβ1, and / or at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids at the C-terminus of D1 in addition to D1. In some embodiments, the IL12Rβ1 portion comprises or consists of 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 the amino acid sequence of D2 of IL12Rβ1.The IL12Rβ1 portion may comprise or consist of at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids at the N-terminus of D2 of IL12Rβ1, and / or at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids at the C-terminus of D2 in addition to D2.

[0223] In some embodiments, the IL12Rβ1 portion is an amino acid sequence 90-100, 90-110, or 90-120 amino acids in length that has at least 90% sequence identity to amino acids 46-136 of human IL12Rβ1, and optionally, the amino acid sequence has at least 95%, at least 97%, or 100% sequence identity to amino acids 46-136 of human IL12Rβ1.

[0224] In some embodiments, the IL12Rβ1 portion is an amino acid sequence 110-120, 110-130, or 110-140 amino acids in length that has at least 90%, at least 95%, at least 97%, or 100% sequence identity to amino acids 24-136 of human IL12Rβ1.

[0225] In some embodiments, the IL12Rβ1 portion is an amino acid sequence 185-195, 185-205, or 185-215 amino acids in length that has at least 90%, at least 95%, at least 97%, or 100% sequence identity to amino acids 46-234 of human IL12Rβ1.

[0226] In some embodiments, the IL12Rβ1 portion is an amino acid sequence 210-220, 210-230, or 210-240 amino acids in length that has at least 90%, at least 95%, at least 97%, or 100% sequence identity to amino acids 24-234 of human IL12Rβ1.

[0227] In some embodiments, the IL12Rβ1 portion is an amino acid sequence 285-300, 285-310, or 285-320 amino acids in length that has at least 90%, at least 95%, at least 97%, or 100% sequence identity to amino acids 46-337 of human IL12Rβ1.

[0228] In some embodiments, the IL12Rβ1 portion is an amino acid sequence 310-320, 310-330, or 310-340 amino acids in length that has at least 90%, at least 95%, at least 97%, or 100% sequence identity to amino acids 24-337 of human IL12Rβ1.

[0229] In some embodiments, the IL12Rβ1 portion is (i) an amino acid sequence consisting of amino acids 24-136 of human IL12Rβ1 with up to 5 or up to 10 contiguous amino acids at its N-terminus and / or C-terminus, or (ii) an amino acid sequence having at least 95% or at least 97% sequence identity to the sequence defined in (i).

[0230] In some embodiments, the IL12Rβ1 portion is (i) an amino acid sequence consisting of amino acids 46-136 of human IL12Rβ1 with up to 5 or up to 10 contiguous amino acids at its N-terminus and / or C-terminus, or (ii) an amino acid sequence having at least 95% or at least 97% sequence identity to the sequence defined in (i).

[0231] In some embodiments, the IL12Rβ1 portion is (i) an amino acid sequence consisting of amino acids 24-234 of human IL12Rβ1 with up to 5 or up to 10 contiguous amino acids at its N-terminus and / or C-terminus, or (ii) an amino acid sequence having at least 95% or at least 97% sequence identity to the sequence defined in (i).

[0232] In some embodiments, the IL12Rβ1 portion is (j) an amino acid sequence consisting of amino acids 46-234 of human IL12Rβ1 with up to 5 or up to 10 contiguous amino acids at its N-terminus and / or C-terminus, or (ii) an amino acid sequence having at least 95% or at least 97% sequence identity to the sequence defined in (i).

[0233] In some embodiments, the IL12Rβ1 portion is (i) an amino acid sequence consisting of amino acids 24-337 of human IL12Rβ1 with up to 5 or up to 10 contiguous amino acids at its N-terminus and / or C-terminus, or (ii) an amino acid sequence having at least 95% or at least 97% sequence identity to the sequence defined in (i).

[0234] In some embodiments, the IL12Rβ1 portion is (i) an amino acid sequence consisting of amino acids 46-337 of human IL12Rβ1 with up to 5 or up to 10 contiguous amino acids at its N-terminus and / or C-terminus, or (ii) an amino acid sequence having at least 95% or at least 97% sequence identity to the sequence defined in (i).

[0235] In some embodiments, the IL12Rβ1 portion comprises an IL12p40-binding portion of the extracellular domain of mammalian, e.g., human or murine, IL12Rβ1 (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 IL12p40-binding portion of the extracellular domain).

[0236] In some embodiments, the IL12Rβ1 portion comprises or consists of an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to a portion of the extracellular domain of IL12Rβ1 lacking up to 5 amino acids, up to 10 amino acids, up to 15 amino acids, up to 20 amino acids, up to 30 amino acids, up to 40 amino acids, up to 50 amino acids, up to 60 amino acids, up to 70 amino acids, up to 80 amino acids, up to 90 amino acids, up to 100 amino acids, up to 150 amino acids, up to 200 amino acids, up to 250 amino acids, up to 300 amino acids, up to 350 amino acids, or up to 400 amino acids from the C-terminus and / or N-terminus.

[0237] 6.4.2.IL12Rβ2 part The IL12 receptor agonists of the present disclosure optionally include an IL12Rβ2 moiety as a masking moiety (sometimes referred to herein as an "IL12Rβ2 masking moiety"), in some embodiments in combination with an IL12Rβ1 moiety as an additional masking moiety.

[0238] In certain embodiments, the IL12Rβ2 portion comprises an amino acid sequence having at least 70% sequence identity, e.g., at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, to the p35-binding portion of mammalian, e.g., human or murine, IL12 receptor subunit beta-2 (IL12Rβ2). The sequence of human IL12Rβ has Uniprot identifier Q99665 (uniprot.org / uniprot / Q99665), with amino acids 24 to 622 comprising the extracellular domain.

[0239] The extracellular domain of IL12Rβ2 contains five fibronectin type III domains: D1, D2, D3, D4, and D5. The sequences of D1, D2, D3, D4, and D5 of mouse and human IL12Rβ2 are provided below.

[0240] [Table 4]

[0241] In some embodiments, an IL12Rβ2 portion of the present disclosure comprises or consists of D1, D2, and / or D3 of IL12Rβ2, including any combination thereof (e.g., D1 and D2, D1-D3, etc.). In some embodiments, an IL12Rβ2 portion does not comprise one or more of D1, D2, or D3 of IL12Rβ2. In certain aspects, an IL12Rβ2 portion does not comprise any of D3, D4, or D5 of IL12Rβ2. In further aspects, an IL12Rβ2 portion does not comprise any of D2, D3, D4, or D5 of IL12Rβ2.

[0242] In some embodiments, the IL12Rβ2 portion is an amino acid sequence 90-100, 90-110, or 90-120 amino acids in length that has at least 90%, at least 95%, at least 97%, or 100% sequence identity to amino acids 126-221 of human IL12Rβ2.

[0243] In some embodiments, the IL12Rβ2 portion is an amino acid sequence 190-220, 190-230, or 190-240 amino acids in length that has at least 90%, at least 95%, at least 97%, or 100% sequence identity to amino acids 24-221 of human IL12Rβ2; In some embodiments, the IL12Rβ2 portion is an amino acid sequence 185-195, 185-205, or 185-215 amino acids in length that has at least 90%, at least 95%, at least 97%, or 100% sequence identity to amino acids 126-319 of human IL12Rβ2; In some embodiments, the IL12Rβ2 portion is an amino acid sequence of 290-310, 290-320, or 290-330 amino acids in length that has at least 90%, at least 95%, at least 97%, or 100% sequence identity to amino acids 24-319 of human IL12Rβ2; In some embodiments, the IL12Rβ2 portion is an amino acid sequence of 290-310, 290-320, or 290-330 amino acids in length that has at least 90%, at least 95%, at least 97%, or 100% sequence identity to amino acids 126-419 of human IL12Rβ2; In some embodiments, the IL12Rβ2 portion is an amino acid sequence 390-410, 390-420, or 390-430 amino acids in length that has at least 90%, at least 95%, at least 97%, or 100% sequence identity to amino acids 24-419 of human IL12Rβ2; In some embodiments, the IL12Rβ2 portion is (i) an amino acid sequence consisting of amino acids 24-221 of human IL12Rβ2 with up to 5 or up to 10 contiguous amino acids at its N-terminus and / or C-terminus, or (ii) an amino acid sequence having at least 95% or at least 97% sequence identity to the sequence defined in (i); In some embodiments, the IL12Rβ2 portion is (i) an amino acid sequence consisting of amino acids 126-221 of human IL12Rβ2 with up to 5 or up to 10 contiguous amino acids at its N-terminus and / or C-terminus, or (ii) an amino acid sequence having at least 95% or at least 97% sequence identity to the sequence defined in (i); In some embodiments, the IL12Rβ2 portion is (i) an amino acid sequence consisting of amino acids 24-319 of human IL12Rβ2 with up to 5 or up to 10 contiguous amino acids at its N-terminus and / or C-terminus, or (ii) an amino acid sequence having at least 95% or at least 97% sequence identity to the sequence defined in (i); In some embodiments, the IL12Rβ2 portion is (i) an amino acid sequence consisting of amino acids 126-319 of human IL12Rβ2 with up to 5 or up to 10 contiguous amino acids at its N-terminus and / or C-terminus, or (ii) an amino acid sequence having at least 95% or at least 97% sequence identity to the sequence defined in (i); In some embodiments, the IL12Rβ2 portion is (i) an amino acid sequence consisting of amino acids 24-419 of human IL12Rβ2 with up to 5 or up to 10 contiguous amino acids at its N-terminus and / or C-terminus, or (ii) an amino acid sequence having at least 95% or at least 97% sequence identity to the sequence defined in (i); or In some embodiments, the IL12Rβ2 portion is (i) an amino acid sequence consisting of amino acids 126-419 of human IL12Rβ2 with up to 5 or up to 10 contiguous amino acids at its N-terminus and / or C-terminus, or (ii) an amino acid sequence having at least 95% or at least 97% sequence identity to the sequence defined in (i).

[0244] Preferably, the IL12Rβ2 portion is capable of binding to IL12p35. In some embodiments, the IL12Rβ2 portion comprises or consists of 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 the amino acid sequence of D1 of IL12Rβ2. The IL12Rβ2 portion may comprise or consist of at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids N-terminal to D1 of IL12Rβ2, and / or at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids C-terminal to D1 in addition to D1. In some embodiments, the IL12Rβ2 portion comprises or consists of 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 the amino acid sequence of D2 of IL12Rβ2.The IL12Rβ2 portion may comprise or consist of at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids at the N-terminus of D2 of IL12Rβ2, and / or at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids at the C-terminus of D2 in addition to D2.

[0245] In some embodiments, the IL12Rβ2 portion comprises an IL12p35-binding portion of the extracellular domain of mammalian, e.g., human or murine, IL12Rβ2 (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 IL12p35-binding portion of the extracellular domain).

[0246] In some embodiments, the IL12Rβ2 portion comprises or consists of an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to a portion of the extracellular domain of IL12Rβ2 lacking up to 5 amino acids, up to 10 amino acids, up to 15 amino acids, up to 20 amino acids, up to 30 amino acids, up to 40 amino acids, up to 50 amino acids, up to 60 amino acids, up to 70 amino acids, up to 80 amino acids, up to 90 amino acids, up to 100 amino acids, up to 150 amino acids, up to 200 amino acids, up to 250 amino acids, up to 300 amino acids, up to 350 amino acids, or up to 400 amino acids from the C-terminus and / or N-terminus.

[0247] 6.4.3.IL12 antibody fragment In some aspects, the IL12 receptor agonist of the present disclosure comprises an IL12 antibody fragment. In some embodiments, an IL12 monomer comprises a p40 moiety and a p35 moiety connected to the N-terminus or C-terminus of a multimerization moiety (e.g., an Fc domain), and the IL12 antibody fragment is disposed at the N-terminus or C-terminus of the p40 and p35 moieties (see, e.g., Figures 4O and 4P, left monomer). In other embodiments, an IL12 monomer lacking both the p40 and p35 moieties comprises a multimerization moiety (e.g., an Fc domain) and an IL12 antibody fragment connected to its N-terminus or C-terminus. In some embodiments, the IL12 receptor agonist comprises a single IL12 antibody fragment (e.g., one of the two IL12 monomers comprising the IL12 receptor agonist comprises an IL12 antibody fragment; see, e.g., Figures 4O-4R). In other embodiments, the IL12 receptor agonist comprises two IL12 antibody fragments (e.g., both IL12 monomers comprising the IL12 receptor agonist comprise an IL12 antibody fragment; e.g., two monomers shown in the left of Figure 4O, two monomers shown in the left of Figure 4P, or one monomer shown in the left of Figure 4O and one monomer shown in the left of Figure 4P). In such embodiments, the two IL12 antibody fragments can be identical or different. In some embodiments, a first IL12 antibody fragment can target the p40 portion and a second IL12 antibody fragment can target the p35 portion. In other embodiments, both IL12 antibody fragments can target the p40 portion or both IL12 antibody fragments can target the p35 portion.

[0248] In some embodiments, the IL12 antibody fragment comprises the antibody binding domain of any known anti-IL12 antibody, including, but not limited to, ustekinumab, briakinumab, the anti-IL12 antibodies described in WO / 2017 / 172771, the anti-IL12 antibodies described in WO / 2012 / 094623, the anti-IL12 antibodies described in WO / 2006 / 069036, the anti-IL12 antibodies described in WO / 2009 / 068627, clone B-T21 (Diaclone), MAB219 (R&D Systems), MAB1510 (R&D Systems), clone C17.8 (Bio X Cell), clone R1-5D9 (Bio X Cell), AP-MAB0853 (ab80682) (abcam), and ab9992 (abcam). The anti-IL12 antibody can bind to p35 and / or p40 (eg, to p35, p40, or both p35 and p40).

[0249] In some embodiments, the IL12 antibody fragment comprises an antibody domain that binds to the same epitope as, and / or competes for binding to IL12 with, ustekinumab, briakinumab, the anti-IL12 antibodies described in WO / 2017 / 172771, the anti-IL12 antibodies described in WO / 2012 / 094623, the anti-IL12 antibodies described in WO / 2006 / 069036, the anti-IL12 antibodies described in WO / 2009 / 068627, clone B-T21 (Diaclone), MAB219 (R&D Systems), MAB1510 (R&D Systems), clone C17.8 (Bio X Cell), clone R1-5D9 (Bio X Cell), AP-MAB0853 (ab80682) (abcam), and ab9992 (abcam). Assays for measuring antibody competition are known in the art. For example, a sample of IL12 can be bound to a solid support. Next, a first antibody and a second antibody are added. One of the two antibodies is labeled. If the labeled antibody and the unlabeled antibody bind to separate, distinct sites on IL12, the labeled antibody will bind at the same level regardless of whether the unlabeled antibody is present. However, if the interaction sites are identical or overlapping, the unlabeled antibody will compete and reduce the amount of labeled antibody that binds to the antigen. If an excess of unlabeled antibody is present, the labeled antibody will hardly bind, if at all. In some embodiments, a competing antibody is an antibody that reduces the binding of another antibody to IL12 by about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 95%, or about 99%. Details of the procedures for conducting such competitive assays are well known in the art and can be found, for example, in Greenfield, Ed., Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2014. Such assays can be made quantitative by using purified antibodies. A standard curve can be established by titrating one antibody against itself, i.e., using the same antibody as both the label and the competitor.The ability of unlabeled competing antibodies to inhibit the binding of labeled antibodies to the plate is titrated. The results can be plotted and compared to the concentration required to achieve the desired degree of binding inhibition. In some embodiments, competition for binding to target molecules can be determined using, for example, a real-time, label-free biolayer interferometry assay on an Octet HTX biosensor platform (Pall ForteBio Corp.).

[0250] For example, the IL12 antibody fragment can be formatted in any of the formats described in Section 6.5.2 for targeting moieties. For example, the IL12 antibody fragment can be in scFv format as described in Section 6.5.2.1 or Fab format as described in Section 6.5.2.2. Other formats (e.g., nanobodies) can also be suitably used. In some embodiments, the VH of the scFV is N-terminal to the VL. In other embodiments, the VH of the scFv is C-terminal to the VL.

[0251] Antigen-binding fragments of anti-IL12 antibodies can be incorporated into IL12 receptor agonists having any of the configurations described herein. IL12 receptor agonists are typically composed of multiple polypeptide chains, as represented, for example, by the exemplary monomers described in Section 6.2. As described in Section 6.2, IL12 antibody fragments can be incorporated into any one of exemplary monomers 8, 28, 33, and 59 to form exemplary monomers 54, 57, 58, and 55, respectively. Exemplary IL12 receptor agonists incorporating one or more of exemplary monomers 54, 55, 57, and 58 are detailed in Section 6.2.

[0252] 6.5. Targeting part Incorporation of a targeting moiety into the IL12 receptor agonists of the present disclosure allows for the delivery of high concentrations of IL12 to the tumor microenvironment or tumor-reactive lymphocytes (including CART lymphocytes), while simultaneously reducing systemic exposure and causing fewer side effects than those obtained with wild-type IL12.

[0253] Suitable targeting moiety formats are described in Section 6.5.2. The targeting moiety is preferably an antigen-binding portion, e.g., an antibody or an antigen-binding portion of an antibody, e.g., an scFv as described in Section 6.5.2.1, or a Fab as described in Section 6.5.2.2.

[0254] Antibodies and antigen-binding moieties generally bind to a specific antigenic determinant and can direct the IL12 receptor agonist to a target site, e.g., a particular tumor cell type or tumor stroma that bears the antigenic determinant. Exemplary target molecules recognized by targeting moieties of the present disclosure are described in Section 6.5.1.

[0255] In other embodiments, the targeting moiety is a peptide-MHC complex as described in Section 6.5.3, eg, a peptide-MHC complex recognized by tumor lymphocytes.

[0256] 6.5.1.Target molecules The target molecules recognized by the targeting moiety of the IL12 receptor agonist of the present disclosure are generally found, for example, on the surface of activated T cells, tumor cells, virus-infected cells, other diseased cells, free in serum, in the extracellular matrix (ECM), or on immune cells present at the target site, such as tumor-reactive lymphocytes. When immune cells (e.g., chimeric antigen receptor ("CAR")-expressing T cells) are exogenously administered, the targeting moiety can recognize the chimeric antigen receptor (CAR) or another molecule found on the surface of the CAR T cells. In various embodiments, the CAR comprises a CDR or VH sequence and a VL sequence (e.g., in the format of an scFv) that specifically recognize a TAA or pMHC complex.

[0257] Exemplary target molecules are fibroblast activation protein (FAP), the A1 domain of tenascin-C (TNC A1), the A2 domain of tenascin-C (TNC A2), fibronectin extra domain B (EDB), melanoma-associated chondroitin sulfate proteoglycan (MCSP), MART-1 / Melan-A, gp100, dipeptidyl peptidase IV (DPPIV), adenosine deaminase-binding protein (ADAbp), cyclophilin b, colorectal-associated antigen (CRC)-C017-1A / GA733, carcinoembryonic antigen (CEA) and its immunogenic epitopes CAP-1 and CAP-2, etv6, aml1, prostate-specific antigen (PSA) and its immunogenic epitopes PSA-1, PSA-2, and PSA-3, prostate-specific membrane antigen (PSMA), T-cell receptor / CD3-zeta chain, MAGE-tumor antigen family (e.g., MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAG MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5), GAGE-tumor antigen family (e.g., GAGE-1, GAGE-2, GAGE- 3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9), BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, tyrosinase, p53, MUC family, HER2 / neu, p21ras, RCAS1, α-fetoprotein, E-cadherin, α-catenin, β-catenin, and γ-catenin, p120ctn, gp100Pmel117, PRAME, NY-ESO-1, cdc27, adenomatous polyposis coli protein (APC), fodrin, connexin 37, Ig-idiotypes, p15, gp75, GM2, and GD2 gangliosides, viral products such as human papillomavirus proteins, the Smad family of tumor antigens, Imp-1, P1A, EBV-encoded nuclear antigen (EBNA)-1, brain glycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1, and CT-7, c-erbB-2, Her2, EGFR, IGF-1R, CD2 (T cell surface antigen), CD3 (T cell receptor-related antigen), and CD22 (B cell receptor), CD23 (low affinity IgE receptor), CD30 (cytokine receptor), CD33 (myeloid cell surface antigen), CD40 (tumor necrosis factor receptor), IL-6R (IL6 receptor), CD20, MCSP, PDGFβR (β-platelet-derived growth factor receptor), ErbB2 epithelial cell adhesion molecule (EpCAM), EGFR variant III (EGFRvIII), CD19, disialoganglioside GD2, ductal epithelial mucin, gp36, TAG-72, glioma-associated antigen, β-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostase, prostase-specific antigen (PSA), PAP, LAGA-1a, p53, prostein, PSMA, survival and telomerase, prostate cancer tumor antigen-1 (PCTA-1), ELF2M, neutrophil elastase, ephrin B2, insulin growth factor I (IGF1)-I, IGF-II, IGF-I receptor, 5T4, ROR1, Nkp30, NKG2D, tumor stromal antigen, CA166-9, fibronectin extra domain A (EDA) and extra domain B (EDB), and tenascin-C A1 domain (TnC A1).

[0258] In some embodiments, the target molecule is CD20. In such embodiments, the targeting moiety comprises the antibody binding domain of any known anti-CD20 antibody. In a non-limiting example, the CD20 targeting moiety comprises the antigen binding domain from one of the following heavy chain variable regions (VH) and light chain variable regions (VL): anti-CD20 VH

[0259] [ka]

[0260] anti-CD20 VL1

[0261] [ka]

[0262] anti-CD20 VL2

[0263] [ka]

[0264] anti-CD20 VL3

[0265] [ka]

[0266] anti-CD20 VL4

[0267] [ka]

[0268] anti-CD20 VL5

[0269] [ka]

[0270] anti-CD20 VL6

[0271] [ka]

[0272] Non-limiting examples of viral antigens include EBV antigens (e.g., Epstein-Barr virus LMP-1), hepatitis C virus antigens (e.g., hepatitis C virus E2 glycoprotein), HIV antigens (e.g., HIV gp160 and HIV gp120), CMV antigens, HPV-specific antigens, or influenza virus antigens (e.g., influenza virus hemagglutinin).

[0273] Non-limiting examples of ECM antigens include syndecans, heparanase, integrins, osteopontin, link, cadherins, laminins, laminin EGF type, lectins, fibronectin, fibronectin extra domain B (ED-B), notch, tenascin, collagens, and matrixins.

[0274] Other target molecules are cell surface molecules of tumor or viral lymphocytes, for example, T cell costimulatory proteins such as CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, and B7-H3.

[0275] In certain embodiments, the target molecule is a checkpoint inhibitor, such as CTLA-4, PD1, PDL1, PDL2, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, or CHK2. In certain embodiments, the target molecule is PD1. In other embodiments, the target molecule is LAG3.

[0276] In some embodiments, the target molecule is PD1. In such embodiments, the targeting moiety comprises the antibody binding domain of any known anti-PD1 antibody. In a non-limiting example, the PD1 targeting moiety comprises the antigen binding domain from the heavy chain variable region (VH) and light chain variable region (VL) of the following: Anti-PD1 VH

[0277] [ka]

[0278] Anti-PD1 VL

[0279] [ka]

[0280] Additional CD20 and PD1 targeting moieties are listed in Table 3 below. In some embodiments, the targeting moiety targets the exemplary target molecules set forth in Table 3 below, along with reference to exemplary antibodies or antibody sequences on which the targeting moiety can be based.

[0281] [Table 5-1]

[0282] [Table 5-2]

[0283] [Table 5-3]

[0284] [Table 5-4]

[0285] [Table 5-5]

[0286] Table 5-6

[0287] Table 5-7

[0288] Table 5-8

[0289] Table 5-9

[0290] Table 5-10

[0291] Table 5-11

[0292] Table 5-12

[0293] Table 5-13

[0294] Table 5-14

[0295] In some aspects, the targeting moiety competes with an antibody described above, including those in Table 3, for binding to a target molecule. In further aspects, the targeting moiety comprises a CDR having the CDR sequence of an antibody described above, including those in Table 3. In some embodiments, the targeting moiety comprises all six CDR sequences of an antibody described above, including an antibody described in Table 3. In other embodiments, the targeting moiety comprises at least the heavy chain CDR sequences (CDR-H1, CDR-H2, CDR-H3) of such an antibody and the light chain CDR sequence of a universal light chain. In further aspects, the targeting moiety comprises a VH comprising the amino acid sequence of the VH of an antibody described above, e.g., in Table 3. In some embodiments, the targeting moiety further comprises a VL comprising the amino acid sequence of the VL of an antibody described above, e.g., in Table 3. In other embodiments, the targeting moiety further comprises a universal light chain VL sequence.

[0296] In some embodiments, the checkpoint inhibitor targeting moiety does not block or has low blocking of ligand-receptor binding. Examples of non-blocking or low-blocking anti-PD1 antibodies include antibodies having the VH / VL amino acid sequences of SEQ ID NOs: 2 / 10 in PCT Publication No. WO2015 / 112800A1, SEQ ID NOs: 16 / 17 in U.S. Patent No. 11,034,765B2, and SEQ ID NOs: 164 / 178, 165 / 179, 166 / 180, 167 / 181, 168 / 182, 169 / 183, 170 / 184, 171 / 185, 172 / 186, 173 / 187, 174 / 188, 175 / 189, 176 / 190, and 177 / 190 in U.S. Patent No. 10,294,299B2. Examples of non-blocking or low-blocking anti-LAG3 antibodies include antibodies having the VH / VL amino acid sequences of SEQ ID NOs: 23 / 24, 3 / 4, and 11 / 12 of US Publication No. 2022 / 0056126A1.

[0297] Additional target molecules that can be targeted by IL12 receptor agonists are disclosed below in Table 6 and are disclosed, for example, in Hafeez et al., 2020, Molecules 25:4764, doi:10.3390 / molecules25204764, particularly Table 1. Table 1 of Hafeez et al. is incorporated herein by reference in its entirety.

[0298] 6.5.2. Targeting Part Format In certain aspects, the 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 immunoglobulin molecule of the IgG class, more particularly an IgG1 or IgG4 immunoglobulin molecule. Antibody fragments include VH (or V H ) fragment, VL (or V L ) fragments, Fab fragments, F(ab')2 fragments, scFv fragments, Fv fragments, minibodies, diabodies, triabodies, and tetrabodies.

[0299] 6.5.2.1.scFv Single-chain Fv or "scFv" antibody fragments comprise the VH and VL domains of an antibody within a single polypeptide chain, can be expressed as single-chain polypeptides, and retain the specificity of the intact antibody from which they are derived. Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for target binding. Examples of linkers suitable for linking the VH and VL chains of an scFv are the linkers identified in Section 6.8.

[0300] As used herein, unless otherwise specified, an scFv can have the VL variable region and the VH variable region in either order, e.g., with respect to the N-terminus and C-terminus of the polypeptide, and can comprise a VL-linker-VH or a VH-linker-VL.

[0301] The scFv can comprise VH and VL sequences from any suitable species, such as murine, human, or humanized VH and VL sequences. To generate nucleic acids encoding scFvs, DNA fragments encoding the VH and VL are operably linked to another fragment encoding a linker, for example, a fragment encoding any of the linkers described in Section 6.8 (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: 16)), such that the VH and VL sequences can be expressed as a contiguous single-chain protein with the VL and VH regions connected by a 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).

[0302] 6.5.2.2.Fab Fab domains have traditionally been produced by proteolytic cleavage of immunoglobulin molecules using enzymes such as papain. In the IL12 receptor agonists of the present disclosure, the Fab domain is typically recombinantly expressed as part of the IL12 receptor agonist.

[0303] The Fab domain can comprise constant domain and variable region sequences from any suitable species, and thus can be murine, chimeric, human, or humanized.

[0304] A Fab domain typically comprises a CH1 domain attached to a VH domain, which pairs with a CL domain attached to a VL domain. In wild-type immunoglobulins, the VH domain pairs with the VL domain to form the Fv region, and the CH1 domain pairs with the CL domain to further stabilize the link module. Disulfide bonds between the two constant domains can further stabilize the Fab domain.

[0305] For the IL12 receptor agonists of the present disclosure, particularly when the light chain is not a common or universal light chain, it is advantageous to use a Fab heterodimerization strategy to enable correct association of Fab domains belonging to the same ABD and minimize aberrant pairing of Fab domains belonging to different ABDs. For example, the Fab heterodimerization strategy shown in Table 4 below can be used:

[0306] [Table 6]

[0307] Thus, in certain embodiments, correct association between the two polypeptides of a Fab is facilitated by exchanging the VL and VH domains of the Fab with one another, or by exchanging the CH1 and CL domains with one another, as described, for example, in WO2009 / 080251.

[0308] 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 by introducing one or more amino acid modifications in the VH domain and one or more amino acid modifications in the VL domain of the Fab. The modified amino acids are typically part of the VH:VL and CH1:CL interfaces such that the Fab components preferentially pair with each other rather than with other Fab components.

[0309] In one embodiment, the one or more amino acid modifications are limited to conserved framework residues of the variable (VH, VL) and constant (CH1, CL) domains, as indicated by the Kabat numbering of the residues. Almagro, 2008, Frontiers In Bioscience 13:1619-1633 provides definitions of framework residues based on the Kabat, Chothia, and IMGT numbering schemes.

[0310] 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 based on steric and hydrophobic contacts, electrostatic / charge interactions, or a combination of various interactions. Complementarity between protein surfaces has been widely described in the literature in terms of lock and key fit, knob into hole, protrusion and cavity, donor and acceptor, etc., all of which suggest the nature of the structural and chemical match between the two interacting surfaces.

[0311] In one embodiment, one or more of the introduced modifications introduce new hydrogen bonds across the interface of the Fab component. In one embodiment, one or more of the introduced modifications introduce new salt bridges across the interface of the Fab component. Exemplary substitutions are described in WO2014 / 150973 and WO2014 / 082179, the contents of which are incorporated herein by reference.

[0312] 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).

[0313] In some embodiments, the Fab domain comprises 143Q and 188V substitutions in the CH1 domain and 113T and 176V substitutions in the CL domain, which serve to exchange hydrophobic and polar contact regions between the CH1 and CL domains (see, e.g., Golay et al., 2016, J Immunol 196:3199-211).

[0314] In some embodiments, the Fab domain can include modifications in some or all of the VH, CH1, VL, and CL domains to introduce an orthogonal Fab interface that promotes correct assembly of the Fab domain (Lewis et al., 2014 Nature Biotechnology 32:191-198). In embodiments, a 39K, 62E modification is introduced in the VH domain, an H172A, F174G modification is introduced in the CH1 domain, a 1R, 38D, (36F) modification is introduced in the VL domain, and an L135Y, S176W modification is 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.

[0315] Fab domains can also be modified to replace the native CH1:CL disulfide bond with an engineered disulfide bond, thereby increasing the efficiency of pairing of the Fab components. For example, an engineered disulfide bond can be introduced by introducing 126C into the CH1 domain and 121C into the CL domain (see, e.g., Mazor et al., 2015, MAbs 7:377-89).

[0316] Fab domains can also be modified by replacing the CH1 and CL domains with alternative domains that promote correct assembly. For example, Wu et al., 2015, MAbs 7:364-76, describe replacing the CH1 domain with a constant domain of a T cell receptor and the CL domain with a b domain of a T cell receptor, and combining these domain replacements with additional charge-charge interactions between the VL and VH domains by introducing a 38D modification in the VL domain and a 39K modification in the VH domain.

[0317] Instead of, or in addition to, using a Fab heterodimerization strategy to promote correct VH-VL pairing, a VL of a common light chain (also referred to as a universal light chain) can be used in each Fab VL region of the IL12 receptor agonist of the present disclosure. In various embodiments, using a common light chain as described herein reduces the number of inappropriate species of the IL12 receptor agonist compared to using the original cognate VL. In various embodiments, the VL domain of the IL12 receptor agonist is identified from a monospecific antibody that includes a common light chain. In various embodiments, the VH region of the IL12 receptor agonist comprises human heavy chain variable gene segments that are rearranged in vivo in mouse B cells that have been previously engineered to express a limited human light chain repertoire or a single human light chain, cognate to the human heavy chain, and in response to exposure to an antigen of interest, generates an antibody repertoire containing one of two possible human VLs or multiple human VHs that are cognate to one of the human heavy chains, and this antibody repertoire is specific for the antigen of interest. The common light chain is derived from a rearranged human Vκ1-39Jκ5 sequence or a rearranged human Vκ3-20Jκ1 sequence, including somatically mutated (e.g., affinity matured) forms. See, e.g., U.S. Patent No. 10,412,940.

[0318] 6.5.3.MHC-peptide fusions The targeting moiety of the IL12 receptor agonist of the present disclosure can be a peptide-MHC complex ("pMHC complex"), e.g., a peptide complexed with an MHC class I domain, or a peptide complexed with an MHC class II domain, in each case optionally complexed with a β2 microglobulin domain.

[0319] The peptide in the pMHC complex can have an amino acid sequence of a peptide that can associate with, e.g., be presented by, an MHC class I molecule. In certain embodiments, the sequence can comprise 6 to 20 contiguous amino acids. In certain embodiments, the peptide sequence can be of a protein fragment, e.g., a portion thereof, derived from a cellular protein, e.g., a protein associated with cancer or a cancer neoantigen, and the peptide can bind to an MHC class I heavy chain.

[0320] In some embodiments, the pMHC complex targeting moiety comprises an antigenic peptide, an MHC polypeptide, or a fragment, variant, or derivative thereof, and optionally a β2 microglobulin polypeptide, or a fragment, variant, or derivative thereof, having the characteristics and / or configurations described in Section 6.4.3 of PCT Publication WO2021 / 127487A2, which section is specifically incorporated herein by reference. In some embodiments, one or more components of the pMHC complex are connected via a pMHC linker as described in Section 6.7.1 of PCT Publication WO2021 / 127487A2, which section is specifically incorporated herein by reference.

[0321] The peptides in the pMHC complexes of the present disclosure are typically at least portions, e.g., antigenic determinants, of proteins of infectious pathogens (e.g., bacteria, viruses, or parasites), allergens, and tumor-associated proteins. Preferably, the pMHC complexes include antigenic determinants of cancer cells. Exemplary antigenic determinants of cancer cells include LCMV-derived peptide gp33-41, APF (126-134), BALF (276-284), CEA (571-579), CMV pp65 (495-503), FLU-M1 (58-66), gp100 (154-162), gp100 (209-217), HBV core (18-27), Her2 / neu (369-377, V2v9), HPV E7 (11-20), HPV E7 (11-19), HPV These include E7(82-90), KLK4(11-19), LMP1(125-133), MAG-A3(112-120), NYES01(157-165, C165A), NYES1(157-165, C165V), p54 WT(264-272), PAP-3(136-143), PSMA(4-12), PSMA(135-145), survivin(96-014), tyrosinase(369-377, 371D), and WT1(126-134). Additional antigenic determinants of cancer cells are described in Section 6.4.3 and Table 3 of PCT Publication No. WO2021 / 127487A2, which section and table are specifically incorporated herein by reference.

[0322] 6.6. Multimerization part Fc Domain In some embodiments, the IL12 agonists and IL12 monomers of the present disclosure comprise one or more multimerization moieties, e.g., one or more multimerization moieties that are or include an Fc domain. In certain embodiments, the IL12 monomers of the present disclosure comprise a single multimerization moiety (e.g., a single Fc domain), and / or the IL12 agonists of the present disclosure comprise two multimerization moieties (e.g., two Fc domains that can associate to form an Fc region).

[0323] The IL12 receptor agonists and IL12 monomers of the present disclosure can comprise an Fc domain, or a pair of Fc domains that associate to form an Fc region, from any suitable species operably linked to an IL12 moiety. In one embodiment, the Fc domain is derived from a human Fc domain. In a preferred embodiment, the IL12 moiety is fused to an IgG Fc molecule.

[0324] The IL12 moiety can be fused to the N-terminus or C-terminus of the IgG Fc domain. As shown in the Examples, IL12 agonists comprising an IL12 moiety fused to the C-terminus of the IgG Fc domain retain IL12 activity to a greater extent than when the IL12 moiety is fused to the N-terminus of the IgG Fc.

[0325] One embodiment of the present disclosure is directed to dimers comprising two Fc fusion polypeptides created by fusing one or more IL12 moieties (e.g., a p35 moiety and a p40 moiety) to the Fc region of an antibody, for example, by fusing both the p35 moiety and the p40 moiety to an Fc domain that can form an IL12 monomer that can homodimerize upon expression, or by fusing a p35 moiety to a first Fc domain and a p40 moiety to a second Fc domain that form two different IL12 monomers that can heterodimerize upon expression. Dimers can be created, for example, by inserting a gene fusion encoding the fusion protein(s) into an appropriate expression vector, expressing the gene fusion(s) in a host cell transformed with the recombinant expression vector, and assembling the expressed fusion protein(s) in a manner similar to antibody molecules, whereupon interchain bonds form between the Fc moieties, resulting in a dimer.

[0326] The Fc domain that can be incorporated into the IL12 monomer can be derived from any suitable class of antibody, including IgA (including subclasses IgA1 and IgA2), IgD, IgE, IgG (including subclasses IgG1, IgG2, IgG3, and IgG4), and IgM. In one embodiment, the Fc domain is derived from IgG1, IgG2, IgG3, or IgG4. In one embodiment, the Fc domain is derived from IgG1. In one embodiment, the Fc domain is derived from IgG4.

[0327] The two Fc domains within an Fc region can be identical or different from one another. In natural antibodies, the Fc domains are typically identical, but for purposes of producing multispecific binding molecules, such as the IL12 receptor agonists of the present disclosure, the Fc domains may advantageously be different to allow heterodimerization, as described in Section 6.6.1.2, below.

[0328] In natural antibodies, the heavy chain Fc domain of IgA, IgD, and IgG consists of two heavy chain constant domains (CH2 and CH3), while the domain of IgE and IgM consists of three heavy chain constant domains (CH2, CH3, and CH4), which dimerize to create the Fc region.

[0329] In the IL12 receptor agonists of the present disclosure, the Fc region, and / or Fc domains therein, can comprise heavy chain constant domains from antibodies of one or more different classes, for example, from one, two, or three different classes.

[0330] In one embodiment, the Fc region comprises a CH2 domain and a CH3 domain derived from IgG1. In one embodiment, the Fc region comprises a CH2 domain and a CH3 domain derived from IgG2.

[0331] In one embodiment, the Fc region comprises a CH2 domain and a CH3 domain derived from IgG3. In one embodiment, the Fc region comprises a CH2 domain and a CH3 domain derived from IgG4.

[0332] In one embodiment, the Fc region comprises a CH4 domain from IgM. The IgM CH4 domain is typically located C-terminal to the CH3 domain. In one embodiment, the Fc region comprises a CH2 domain and a CH3 domain derived from an IgG and a CH4 domain derived from an IgM.

[0333] It will be understood that heavy chain constant domains for use in producing Fc regions for the IL12 receptor agonists of the present disclosure can include variants of the above-described naturally occurring constant domains. Such variants can include one or more amino acid mutations compared to the wild-type constant domain. In one example, the Fc region of the present disclosure includes at least one constant domain that differs in sequence from the wild-type constant domain. It will be understood that the variant constant domain can be longer or shorter than the wild-type constant domain. Preferably, the variant constant domain is at least 60% identical or similar to the wild-type constant domain. In another example, the variant constant domain is at least 70% identical or similar. In another example, the variant constant domain is at least 80% identical or similar. In another example, the variant constant domain is at least 90% identical or similar. In another example, the variant constant domain is at least 95% identical or similar.

[0334] IgM and IgA naturally occur in humans as covalently linked multimers of a common H2L2 antibody unit. IgM exists as a pentamer when a J chain is incorporated and as a hexamer when the J chain is absent. IgA exists in both monomeric and dimeric forms. The heavy chains of IgM and IgA have an 18-amino acid extension to the C-terminal constant domain known as the tail. The tail contains cysteine ​​residues that form disulfide bonds between heavy chains within the polymer and is thought to play an important role in polymerization. The tail also contains glycosylation sites. In certain embodiments, the IL12 receptor agonist of the present disclosure does not contain a tail.

[0335] The Fc domain incorporated into the IL12 receptor agonists of the present disclosure may contain one or more modifications that alter the functional properties of the protein, for example, binding to an Fc receptor such as FcRn or a leukocyte receptor, binding to complement, modified disulfide bond structures, or altered glycosylation patterns. Exemplary Fc modifications that alter effector function are described in Section 6.6.1.1. The Fc domain can also be altered to include modifications that improve the manufacturability of asymmetric IL12 receptor agonists, for example, by allowing heterodimerization, the preferential pairing of non-identical Fc domains with identical Fc domains. Heterodimerization allows for the production of IL12 receptor agonists in which different polypeptide components are connected to each other by Fc regions containing Fc domains that differ in sequence. Examples of heterodimerization strategies are illustrated in Section 6.6.1.2.

[0336] It will be appreciated that any of the above modifications 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 IL12 receptor agonist.

[0337] 6.6.1.1. Fc Domains with Altered Effector Function In some embodiments, the Fc domain comprises one or more amino acid substitutions that reduce binding to Fc receptors and / or effector functions.

[0338] In a specific embodiment, the Fc receptor is an Fcγ 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 Fcγ receptor, more specifically, human FcγRIIIa, FcγRI, or FcγRIIa, most specifically, human FcγRIIIa. In one embodiment, the effector function is one or more selected from the group consisting of complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and cytokine secretion. In a specific embodiment, the effector function is ADCC.

[0339] In one embodiment, the Fc domain (e.g., the Fc domain of an IL12 monomer) or Fc region (e.g., one or both Fc domains of an IL12 receptor 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 (numbering according to the Kabat EU index). In a more particular embodiment, the Fc domain or Fc region comprises an amino acid substitution at a position selected from the group of L234, L235, and P329 (numbering according to the Kabat EU index). In some embodiments, the Fc domain or region comprises the amino acid substitutions L234A and L235A (numbering according to the Kabat EU index). In one such embodiment, the Fc domain or region is an Igd Fc domain or region, particularly a human Igd Fc domain or region. In one embodiment, the Fc domain or Fc region comprises an amino acid substitution at position P329. In a more particular embodiment, the amino acid substitution is P329A or P329G, particularly P329G (numbering according to the Kabat EU index). In one embodiment, the Fc domain or 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 (numbering according to the Kabat EU index). In a more particular embodiment, the further amino acid substitution is E233P, L234A, L235A, L235E, N297A, N297D or P331S. In a particular embodiment, the Fc domain or Fc region comprises amino acid substitutions at positions P329, L234, and L235 (numbering according to the Kabat EU index). In a more specific embodiment, the Fc domain comprises the amino acid mutations L234A, L235A and P329G (“P329G LALA,” “PGLALA,” or “LALAPG”).

[0340] Typically, the same one or more amino acid substitutions are present in each of the two Fc domains of the Fc region. Thus, in certain embodiments, 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 second Fc domains within 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 with a glycine residue (P329G) (Kabat EU index numbering).

[0341] In one embodiment, the Fc domain is an IgG1 Fc domain, particularly a human IgG1 Fc domain. In some embodiments, the IgG1 Fc domain is a variant IgG1 containing D265A, N297A mutations (EU numbering) to reduce effector function.

[0342] In another embodiment, the Fc domain is an IgG4 Fc domain with reduced binding to Fc receptors. Exemplary IgG4 Fc domains with reduced binding to Fc receptors may comprise an amino acid sequence selected from Table 5 below. In some embodiments, the Fc domain comprises only the bolded portion of the sequence shown below:

[0343] [Table 7-1]

[0344] [Table 7-2]

[0345] [Table 7-3]

[0346] [Table 7-4]

[0347] [Table 7-5]

[0348] In certain embodiments, the IgG4 with reduced effector function comprises the bolded portion of the amino acid sequence of SEQ ID NO: 31 of WO2014 / 121087 (SEQ ID NO: 20), and is sometimes referred to herein as IgG4s or hIgG4s.

[0349] For heterodimeric Fc regions, it is possible to incorporate combinations of the variant IgG4 Fc sequences described above, for example an Fc region comprising an Fc domain comprising the amino acid sequence of SEQ ID NO: 30 of WO2014 / 121087 (SEQ ID NO: 19) (or a bolded portion thereof) and an Fc domain comprising the amino acid sequence of SEQ ID NO: 37 of WO2014 / 121087 (SEQ ID NO: 21) (or a bolded portion thereof), or an Fc region comprising an Fc domain comprising the amino acid sequence of SEQ ID NO: 31 of WO2014 / 121087 (SEQ ID NO: 20) (or a bolded portion thereof) and an Fc domain comprising the amino acid sequence of SEQ ID NO: 38 of WO2014 / 121087 (SEQ ID NO: 22) (or a bolded portion thereof).

[0350] Fc Heterodimerization Variants Certain IL12 receptor agonists, unlike natural immunoglobulins, involve dimerization between two Fc domains operably linked at non-identical N-terminal regions, e.g., one Fc domain connected to the Fab and the other Fc domain connected to the IL12 moiety. Inefficient heterodimerization of the two Fc domains to form an Fc region can be an obstacle to increasing the yield of the desired heterodimeric molecule and presents a purification challenge. Various approaches available in the art can be used to enhance dimerization of Fc domains that may be present in the IL12 receptor agonists of the present disclosure, as disclosed, for example, in EP 1870459 A1, U.S. Pat. No. 5,582,996, U.S. Pat. No. 5,731,168, U.S. Pat. No. 5,910,573, U.S. Pat. No. 5,932,448, U.S. Pat. No. 6,833,441, U.S. Pat. No. 7,183,076, U.S. Patent Application Publication No. 2006 / 204493 A1, and PCT Publication No. WO 2009 / 089004 A1.

[0351] The present disclosure provides IL12 receptor agonists comprising Fc heterodimers, i.e., Fc regions comprising heterologous, non-identical Fc domains. Typically, each Fc domain in the Fc heterodimer comprises an antibody CH3 domain. The CH3 domain is derived from the constant region of an antibody of any isotype, class, or subclass, preferably of the IgG (lgG1, lgG2, lgG3, and lgG4) class, as described in the preceding section.

[0352] Heterodimerization of two different heavy chains at the CH3 domains generates the desired IL12 receptor agonist, whereas homodimerization of the same heavy chain reduces the yield of the desired IL12 receptor agonist. Thus, in preferred embodiments, polypeptides that assemble to form the IL12 receptor agonists of the present disclosure contain a CH3 domain with modifications that favor heterodimeric association compared to an unmodified Fc domain.

[0353] In certain embodiments, the modification that promotes Fc heterodimer formation is a so-called "knob-into-hole" or "knob-in-hole" modification, which comprises a "knob" modification in one of the Fc domains and a "hole" modification in the other Fc domain. Knob-into-hole technology is described, for example, in U.S. Pat. No. 5,731,168, U.S. Pat. No. 7,695,936, Ridgway et al., 1996, Prot Eng 9:617-621, and Carter, 2001, Immunol Meth 248:7-15. Generally, this method involves introducing a protrusion ("knob") at the interface of a first polypeptide and a corresponding cavity ("hole") at the interface of a second polypeptide, allowing the protrusion to be positioned within the cavity, to promote heterodimer formation and impede homodimer formation. The protrusions 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 the same or similar size as the protrusions are created in the interface of the second polypeptide by replacing the large amino acid side chains with smaller amino acid side chains (e.g., alanine or threonine).

[0354] Thus, in some embodiments, amino acid residues in the CH3 domain of a first subunit of an Fc domain are replaced with amino acid residues having a larger side chain volume, thereby creating a protrusion in the CH3 domain of the first subunit that can be positioned within a cavity in the CH3 domain of a second subunit, and amino acid residues in the CH3 domain of a second subunit of an Fc domain are replaced with amino acid residues having a smaller side chain volume, thereby creating a cavity in the CH3 domain of the second subunit into which the protrusion in the CH3 domain of the first subunit can be positioned. Preferably, the amino acid residues having a larger side chain volume are selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W). Preferably, the amino acid residues having a smaller side chain volume are selected from the group consisting of alanine (A), serine (S), threonine (T), and valine (V). The protrusion and cavity can be created by altering the nucleic acid encoding the polypeptide, for example, by site-directed mutagenesis or by peptide synthesis. An exemplary substitution is Y470T.

[0355] In particular such embodiments, 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 the Kabat EU index). In further embodiments, the first Fc domain additionally has a serine residue at position 354 replaced with a cysteine ​​residue (S354C) or a glutamic acid residue at position 356 replaced with a cysteine ​​residue (E356C) (particularly, the serine residue at position 354 is replaced with a cysteine ​​residue). The second Fc domain additionally has the tyrosine residue at position 349 replaced with a cysteine ​​residue (Y349C) (Kabat EU index numbering). In a specific embodiment, the first Fc domain comprises amino acid substitutions S354C and T366W, and the second Fc domain comprises amino acid substitutions Y349C, T366S, L368A, and Y407V (Kabat EU index numbering).

[0356] 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 association of the first and second Fc domains of the Fc region.

[0357] Alternatively, or in addition to using an Fc domain modified to promote heterodimerization, the Fc domain can be modified to enable a purification strategy that allows for the selection of Fc heterodimers. In one such embodiment, one polypeptide contains a modified Fc domain that abrogates its binding to protein A, thus enabling a purification method that results in a heterodimeric protein. See, e.g., U.S. Patent No. 8,586,713. Thus, an IL12 receptor agonist comprises a first CH3 domain and a second Ig CH3 domain, wherein the first and second Ig CH3 domains differ from each other by at least one amino acid, and the at least one amino acid difference reduces binding of the IL12 receptor agonist to protein A compared to a corresponding IL12 receptor agonist lacking the amino acid difference. In one embodiment, the first CH3 domain binds to protein A and the second CH3 domain contains a mutation / modification that reduces or eliminates protein A binding, e.g., an H95R modification (according to IMGT exon numbering; H435R by EU numbering). The second CH3 may further comprise a Y96F modification (according to IMGT; Y436F by EU). This class of modifications is referred to herein as a "star" mutation.

[0358] In some embodiments, the Fc may comprise one or more mutations to promote heterodimerization (e.g., knob and hole mutations) and a star mutation to facilitate purification.

[0359] 6.7. Stabilization part The IL12 receptor agonists of the present disclosure can include a stabilizing moiety that can extend the serum half-life of the molecule in vivo. Serum half-life is often divided into an alpha phase and a beta phase. Either or both phases can be significantly improved by the addition of an appropriate stabilizing moiety. For example, a stabilizing moiety can increase the serum half-life of an IL12 receptor agonist by more than 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, 200, 400, 600, 800, 1000%, or more, compared to a corresponding IL12 receptor agonist that does not contain a stabilizing moiety. For purposes of this disclosure, serum half-life can refer to half-life in humans or other mammals (e.g., mice or non-human primates).

[0360] Wild-type IL12 has a serum half-life of less than 10 minutes. The IL12 receptor agonist of the present disclosure preferably has a serum half-life in humans and / or mice of at least about 2 hours, at least about 4 hours, at least about 6 hours, or at least about 8 hours. In some embodiments, the IL12 receptor agonist of the present disclosure has a serum half-life of at least 10 hours, at least 12 hours, at least 15 hours, at least 18 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 60 hours, or at least 72 hours.

[0361] Stabilizing moieties include polyoxyalkylene moieties (e.g., polyethylene glycol), sugars (e.g., sialic acid), and well-tolerated protein moieties (e.g., Fc and its fragments and variants, transferrin, or serum albumin).

[0362] Other stabilizing moieties that can be used in the IL12 receptor agonists of the present disclosure include those described in Kontermann et al., 2011, Current Opinion in Biotechnology 22:868-76. Such stabilizing moieties include, but are not limited to, human serum albumin fusions, human serum albumin conjugates, human serum albumin binders (e.g., Adnectin PKE, AlbudAb, ABD), XTEN fusions, PAS fusions (i.e., recombinant PEG mimetics based on the three amino acids proline, alanine, and serine), carbohydrate conjugates (e.g., hydroxyethyl starch (HES)), glycosylation, polysialic acid conjugates, and fatty acid conjugates.

[0363] Thus, in some embodiments, the present disclosure provides an IL12 receptor agonist that includes a stabilizing moiety that is a polymeric sugar. Serum albumin can also contribute to half-life extension through modules capable of non-covalently interacting with albumin. Therefore, the IL12 receptor agonist of the present disclosure can include an albumin-binding protein as a stabilizing moiety. The albumin-binding protein can be either conjugated or genetically fused to one or more other components of the IL12 receptor agonist of the present disclosure. Proteins with albumin-binding activity are known from certain bacteria. For example, streptococcal protein G contains several small albumin-binding domains consisting of approximately 50 amino acid residues (6 kDa). Additional examples of serum albumin-binding proteins include those described in U.S. Publication Nos. 2007 / 0178082 and 2007 / 0269422. Fusing an albumin-binding domain to a protein results in a significantly extended half-life (see Kontermann et al., 2011, Current Opinion in Biotechnology 22:868-76).

[0364] In other embodiments, the stabilizing moiety is human serum albumin, hi other embodiments, the stabilizing moiety is transferrin. In some embodiments, the stabilizing moiety is an Fc domain, such as any of the Fc domains described in Section 6.6.1 and its subsections, which are incorporated herein by reference. The Fc domains described in Section 6.6.1 are generally capable of dimerization. However, for stabilization purposes, the Fc domain can be a soluble monomeric Fc domain with reduced ability to self-associate. See, for example, Helm et al., 1996, J. Biol. Chem. 271:7494-7500, and Ying et al., 2012, J. Biol. Chem. 287(23):19399-19408. An example of a soluble monomeric Fc domain contains amino acid substitutions at positions corresponding to T366 and / or Y407 in CH3, as described in U.S. Patent Publication No. 2019 / 0367611. The monomeric Fc domain may be of any Ig subtype and may contain additional substitutions that reduce effector function, as described in Section 6.6.1 and its subsections.

[0365] In still other embodiments, the stabilizing moiety is a polyethylene glycol moiety or another polymer, as described in Section 6.7 below. The stabilizing moiety can be connected to one or more other components of the IL12 receptor agonist of the present disclosure via a linker, for example, as described in Section 6.8 below.

[0366] In some embodiments, the IL12 receptor agonist comprises polyethylene glycol (PEG) or another hydrophilic polymer as a stabilizing moiety, such as ethylene glycol / propylene glycol copolymer, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymer, polyamino acids (either homopolymers or random copolymers), dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propropylene glycol homopolymer, prolypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyol (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. The polymer may be of any molecular weight and may be branched or unbranched.

[0367] Linker In certain embodiments, the present disclosure provides IL12 receptor agonists in which two or more components of the IL12 receptor agonist are connected to each other by a peptide linker. By way of example and not limitation, a linker can be used to connect (a) an IL12 portion and a multimerization portion, (b) an IL12 portion and a targeting portion, (c) a targeting portion and a multimerization portion (e.g., an Fab domain and an Fc domain), (d) different domains within an IL12 portion (e.g., an IL12 domain and an IL-Rα domain), or (e) different domains within a targeting portion (e.g., different components of a peptide-MHC complex or a VH domain and a VL domain within an scFv).

[0368] Peptide linkers can range from 2 amino acids to 60 amino acids or more, and in certain embodiments, peptide linkers can range in length from 3 amino acids to 50 amino acids, 4 to 30 amino acids, 5 to 25 amino acids, 10 to 25 amino acids, 10 to 60 amino acids, 12 to 20 amino acids, 20 to 50 amino acids, or 25 to 35 amino acids.

[0369] In certain embodiments, the peptide linker is at least 5 amino acids, at least 6 amino acids, or at least 7 amino acids in length, and optionally up to 30 amino acids, up to 40 amino acids, up to 50 amino acids, or up to 60 amino acids in length.

[0370] In some of the foregoing embodiments, the linker is between 5 amino acids and 50 amino acids in length, e.g., between 5 and 50, 5 and 45, 5 and 40, 5 and 35, 5 and 30, 5 and 25, or 5 and 20 amino acids in length. In other of the foregoing embodiments, the linker is between 6 amino acids and 50 amino acids in length, e.g., between 6 and 50, 6 and 45, 6 and 40, 6 and 35, 6 and 30, 6 and 25, or 6 and 20 amino acids in length. In still other of the foregoing embodiments, the linker is between 7 amino acids and 50 amino acids in length, e.g., between 7 and 50, 7 and 45, 7 and 40, 7 and 35, 7 and 30, 7 and 25, or 7 and 20 amino acids in length.

[0371] Charged (eg, charged hydrophilic linkers) and / or flexible linkers are particularly preferred. Examples of flexible linkers that can be used in the IL12 receptor agonists of the present 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 include repeats of glycine and serine, e.g., G n S (SEQ ID NO: 23) or SG n 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 a repeat of G4S (SEQ ID NO: 25), e.g., (GGGGS) n (SEQ ID NO: 26).

[0372] Polyglycine linkers are suitable for use in the IL12 receptor agonists of the present disclosure. In some embodiments, the 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: 32).

[0373] 6.8.1. pMHC Linker For pMHC complexes, suitable linkers can range from 1 amino acid (e.g., Gly) to 20 amino acids, 2 to 15 amino acids, 3 to 12 amino acids, including 4 to 10 amino acids, 5 to 9 amino acids, 6 to 8 amino acids, or 7 to 8 amino acids, and can be 1, 2, 3, 4, 5, 6, or 7 amino acids. In addition to the above linkers, pMHC linkers include glycine polymers (G)n, glycine-serine polymers (e.g., (GS)n, (GSGGS)n (SEQ ID NO: 33), and (GGGS)n, where n is at least one integer (SEQ ID NO: 34)), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers can be used. Both Gly and Ser are relatively unstructured and can function as neutral tethers between components. Glycine polymers can be used. Glycine has access to much more phi-psi space than alanine and is much less restricted than residues with longer side chains (see Scheraga, 1992, Rev. Computational Chem. 1 1173-142, the entire contents of which are incorporated herein by reference). Exemplary linkers can include, but are not limited to, amino acid sequences such as GGSG (SEQ ID NO: 35), GGSGG (SEQ ID NO: 36), GSGSG (SEQ ID NO: 37), GSGGG (SEQ ID NO: 38), GGGSG (SEQ ID NO: 39), GSSSG (SEQ ID NO: 40), GCGASGGGGSGGGGS (SEQ ID NO: 41), GGGGSGGGGS (SEQ ID NO: 42), GGGASGGGGSGGGGS (SEQ ID NO: 43), GGGGSGGGGSGGGGGS (SEQ ID NO: 44), GGGASGGGGS (SEQ ID NO: 45), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 46), GCGGS (SEQ ID NO: 47), etc. In some embodiments, the linker polypeptide comprises a cysteine ​​residue that can form a disulfide bond with a cysteine ​​residue present in another part of the pMHC complex.In certain embodiments, the linker comprises the amino acid sequence GCGGS (SEQ ID NO: 47). Substitution of glycine in the G4S linker (SEQ ID NO: 25) with a cysteine ​​can result in the formation of a disulfide bond, e.g., an MHC targeting moiety with the corresponding cysteine ​​substitution in HLA.A2, which stabilizes the MHC peptide within the MHC complex.

[0374] 6.8.2. Hinge arrangement In other embodiments, the IL12 receptor agonist of the present disclosure includes a linker that is a hinge region. In particular, when the IL12 receptor agonist contains an immunoglobulin-based targeting moiety, a 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 natural hinge region or a modified hinge region. The hinge region is typically found at the N-terminus of the Fc region. The term "hinge region," unless otherwise indicated by context, refers to a naturally occurring or non-natural hinge sequence, which can be a monomeric hinge domain in the context of a single or monomeric polypeptide chain, or two associated hinge sequences on separate polypeptide chains in the context of a dimeric polypeptide (e.g., a homodimeric or heterodimeric IL12 receptor agonist formed by the association of two Fc domains).

[0375] A native hinge region is typically the hinge region found between the Fab and Fc domains of naturally occurring antibodies. 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. Other modified hinge regions can include a complete hinge region derived from an antibody of a different class or subclass than that of the heavy chain Fc domain or Fc region. Alternatively, the modified hinge region can include a portion of a native hinge or repeat unit, with each repeat unit derived from a native hinge region. In a further alternative, the native hinge region can be altered by converting one or more cysteine ​​or other residues to neutral residues such as serine or alanine, or by converting appropriately positioned residues to cysteine ​​residues. The number of cysteine ​​residues in the hinge region can be increased or decreased by such means. Other modified hinge regions may be entirely synthetic and may be designed to have desired properties such as length, cysteine ​​composition, and flexibility.

[0376] Several modified hinge regions have been previously described, for example, in U.S. Pat. No. 5,677,425, WO99 / 15549, WO2005 / 003170, WO2005 / 003169, WO2005 / 003170, WO98 / 25971, and WO2005 / 003171, which are incorporated herein by reference.

[0377] In one embodiment, an IL12 receptor agonist of the present disclosure comprises an Fc region in which one or both Fc domains have an intact hinge region at their N-terminus. In various embodiments, positions 233-236 in the hinge region can be G, G, G, and empty; G, G, empty, and empty; G, empty, empty, and empty; or all empty, and the positions are numbered according to EU numbering.

[0378] In some embodiments, the IL12 receptor agonists of the present disclosure comprise a modified hinge region that reduces binding affinity to an Fcγ receptor compared to a wild-type hinge region of the same isotype (e.g., human IgG1 or human IgG4).

[0379] In one embodiment, the IL12 receptor agonist of the present disclosure comprises an Fc region, each Fc domain having an intact hinge region at its N-terminus, wherein each Fc domain and hinge region is derived from IgG4, and each hinge region contains the modified sequence CPPC (SEQ ID NO: 48). The core hinge region of human IgG4 contains the sequence CPSC (SEQ ID NO: 49), compared to IgG1, which contains the sequence CPPC (SEQ ID NO: 48). The serine residues present in the IgG4 sequence provide increased flexibility in this region, and therefore a proportion of the molecules form disulfide bonds within the same protein chain (intrachain disulfides) rather than cross-linking to other heavy chains within an IgG molecule to form interchain disulfides. (Angel et al., 1993, Mol Immunol 30(1):105-108). Changing the serine residues to prolines to obtain the same core sequence as IgG1 allows for complete formation of interchain disulfides within the IgG4 hinge region, thus reducing heterogeneity in the purified product. This altered isotype is called IgG4P.

[0380] 6.8.2.1. Chimeric Hinge Sequences The hinge region can be a chimeric hinge region. For example, a chimeric hinge may comprise an "upper hinge" sequence derived from a human IgG1, human IgG2, or human IgG4 hinge region combined with a "lower hinge" sequence derived from a human IgG1, human IgG2, or human IgG4 hinge region.

[0381] In certain embodiments, the chimeric hinge region comprises the amino acid sequence EPKSCDKTHTCPPCPAPPVA (SEQ ID NO: 50) (previously disclosed as SEQ ID NO: 8 of WO2014 / 121087, incorporated herein by reference in its entirety) or ESKYGPPCPPCPAPPVA (SEQ ID NO: 51) (previously disclosed as SEQ ID NO: 9 of WO2014 / 121087). Such chimeric hinge sequences may be suitably linked to an IgG4 CH2 region (e.g., by incorporation into an IgG4 Fc domain, e.g., a human or mouse Fc domain, which may be further modified in the CH2 and / or CH3 domains to reduce effector function, e.g., as described in Section 6.6.1.1).

[0382] 6.8.2.2. Hinge sequences with reduced effector function In further embodiments, the hinge region can be modified to reduce effector function, for example, as described in WO2016 / 161010A2, which is incorporated herein by reference in its entirety. In various embodiments, positions 233-236 of the modified hinge region can be G, G, G, and empty; G, G, empty, and empty; G, empty, empty, and empty; or all empty, with positions numbered according to EU numbering (as shown in Figure 1 of WO2016161010A2). These segments can be represented as GGG-, GG--, G---, or ----, where "-" represents an empty position.

[0383] Position 236 is vacant in canonical human IgG2 but occupied in other canonical human IgG isotypes. Positions 233-235 are occupied by residues other than G in all four human isotypes (as shown in Figure 1 of WO2016 / 161010A2).

[0384] Hinge modifications within positions 233-236 can be combined with position 228 being occupied by P. Position 228 is naturally occupied by P in human IgG1 and IgG2, but is occupied by S in human IgG4 and by R in human IgG3. The S228P mutation in IgG4 antibodies is advantageous for stabilizing IgG4 antibodies and reducing heavy-light chain pair exchange between exogenous and endogenous antibodies. Preferably, positions 226-229 are occupied by C, P, P, and C, respectively.

[0385] Exemplary hinge regions have residues 226-236, sometimes referred to as the middle (or core) and lower hinge, occupied by 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:52) (previously disclosed as SEQ ID NO:1 in WO2016 / 161010A2), CPPCPAPGG--GPSVF (SEQ ID NO:53) (previously disclosed as SEQ ID NO:2 in WO2016 / 161010A2), CPPCPAPG---GPSVF (SEQ ID NO:54) (previously disclosed as SEQ ID NO:3 in WO2016 / 161010A2), or CPPCPAP----GPSVF (SEQ ID NO:55) (previously disclosed as SEQ ID NO:4 in WO2016 / 161010A2).

[0386] The above-described modified hinge regions can be incorporated into heavy chain constant regions, which typically include a CH2 domain and a CH3 domain and may have additional hinge segments (e.g., upper hinges) adjacent to the designated regions. The additional constant region segments present are typically of the same isotype, preferably a human isotype, but can be hybrids of different isotypes. The isotype of such additional human constant region segments is preferably human IgG4, but can also be human IgG1, IgG2, or IgG3, or hybrids thereof, in which the domains are of different isotypes. Exemplary sequences of human IgG1, IgG2, and IgG4 are shown in Figures 2 to 4 of WO2016 / 161010A2.

[0387] In certain embodiments, a modified hinge sequence can be linked to an IgG4 CH2 region (e.g., by incorporation into an IgG4 Fc domain, e.g., a human or mouse Fc domain, which can be further modified in the CH2 and / or CH3 domains to reduce effector function, e.g., as described in Section 6.6.1.1).

[0388] 6.9. Nucleic Acids and Host Cells In another aspect, the present disclosure provides nucleic acids encoding the IL12 receptor agonists of the present disclosure. In some embodiments, the IL12 receptor agonist is encoded by a single nucleic acid. In other embodiments, for example, in the case of heterodimeric molecules or molecules comprising a targeting moiety composed of two or more polypeptide chains, the IL12 receptor agonist can be encoded by multiple (e.g., two, three, four, or more) nucleic acids.

[0389] A single nucleic acid can encode an IL12 receptor agonist comprising a single polypeptide chain, an IL12 receptor agonist comprising two or more polypeptide chains, or a portion of an IL12 receptor agonist comprising three or more polypeptide chains (e.g., a single nucleic acid can encode two polypeptide chains of an IL12 receptor agonist comprising three, four, or more polypeptide chains, or three polypeptide chains of an IL12 receptor agonist comprising four or more polypeptide chains). To separately control expression, open reading frames encoding two or more polypeptide chains can be placed under the control of separate transcriptional regulatory elements (e.g., promoters and / or enhancers). Open reading frames encoding two or more polypeptides can also be controlled by the same transcriptional regulatory element and separated by an internal ribosome entry site (IRES) sequence, allowing translation into separate polypeptides.

[0390] In some embodiments, an IL12 receptor agonist comprising two or more polypeptide chains is encoded by two or more nucleic acids. The number of nucleic acids encoding the IL12 receptor agonist can be equal to or less than the number of polypeptide chains in the IL12 receptor agonist (e.g., when two or more polypeptide chains are encoded by a single nucleic acid).

[0391] The nucleic acids of the present disclosure can be DNA or RNA (eg, mRNA). In another aspect, the disclosure provides host cells and vectors containing the nucleic acids of the disclosure. The nucleic acids can be present in a single vector or can be present in separate vectors that are present in the same host cell or in separate host cells, as described in more detail herein below.

[0392] Vectors The present disclosure provides vectors comprising nucleotide sequences encoding one or two polypeptide chains of an IL12 receptor agonist or IL12 receptor agonist component described herein, e.g., a half antibody. Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phage, or yeast artificial chromosomes (YACs).

[0393] Numerous vector systems can be used. For example, one class of vectors utilizes DNA elements derived from animal viruses such as bovine papillomavirus, polyomavirus, adenovirus, vaccinia virus, baculovirus, retrovirus (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.

[0394] Additionally, cells that have stably integrated the DNA into their chromosomes can be selected by introducing one or more markers that allow for the selection of transfected host cells. Markers can provide, for example, prototropy to auxotrophic hosts, biocide resistance (e.g., antibiotics), or resistance to heavy metals such as copper. The selectable marker gene can be either directly linked to the DNA sequence to be expressed or introduced into the same cell by cotransformation. Additional elements may also be required for optimal mRNA synthesis. These elements may include splice signals, as well as transcription promoters, enhancers, and termination signals.

[0395] After the DNA sequence containing expression vector or construct is prepared for expression, expression vector can be transfected or introduced into suitable host cell.To achieve this, various techniques can be used, such as protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, lipid-based transfection or other conventional techniques.The method and conditions for culturing the obtained transfected cells and recovering expressed polypeptide are known to those skilled in the art, and can be changed or optimized according to the specific expression vector and mammalian host cell used based on this specification.

[0396] 6.9.2.Cells The present disclosure also provides a host cell comprising a nucleic acid of the present disclosure. In one embodiment, the host cell is genetically engineered to contain one or more nucleic acids described herein.

[0397] In one embodiment, the host cell is genetically engineered using an expression cassette. The term "expression cassette" refers to a nucleotide sequence that can affect the expression of a gene in a host compatible with such a sequence. Such a cassette can include a promoter, an open reading frame with or without introns, and a termination signal. Additional factors necessary or useful for effecting expression, such as an inducible promoter, can also be used.

[0398] The present disclosure also provides host cells comprising the vectors described herein. 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.

[0399] Pharmaceutical Compositions 6.10.1. Pharmaceutical Compositions Comprising IL12 Receptor Agonist Polypeptides The IL12 receptor agonist of the present disclosure may be in the form of a composition comprising the IL12 receptor agonist and one or more carriers, excipients, and / or diluents. The composition may be formulated for a particular use, such as veterinary use or pharmaceutical use in humans. The form of the composition (e.g., dry powder, liquid formulation, etc.) and the excipients, diluents, and / or carriers used will depend on the intended use of the IL12 receptor agonist and, in the case of therapeutic applications, the mode of administration.

[0400] For therapeutic use, the composition may be supplied as part of a sterile pharmaceutical composition containing a pharmaceutically acceptable carrier. The composition may be in any suitable form (depending on the desired method of administration to the patient). The pharmaceutical composition may be administered to the patient by a variety of routes, including oral, transdermal, subcutaneous, intranasal, intravenous, intramuscular, intratumoral, intrathecal, local, or topical. 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, as well as the subject's physical condition. Typically, the pharmaceutical composition will be administered intravenously or subcutaneously.

[0401] The pharmaceutical composition can be conveniently presented in a unit dosage form containing a predetermined amount of the IL12 receptor agonist of the present disclosure per dose. The amount of IL12 receptor agonist contained in the unit dose will depend on the disease being treated and other factors well known in the art. Such unit dosages can be in the form of a lyophilized dry powder containing an amount of IL12 receptor agonist suitable for a single administration, or in liquid form. The dry powder unit dosage form can be packaged in a kit with a syringe, a suitable amount of diluent, and / or other components useful for administration. The liquid unit dosage can be conveniently supplied in the form of a syringe pre-filled with an amount of IL12 receptor agonist suitable for a single administration.

[0402] The pharmaceutical compositions may also be supplied in bulk, as they contain an amount of IL12 receptor agonist suitable for multiple administration. Pharmaceutical compositions can be prepared for storage as lyophilized formulations or aqueous solutions by mixing an IL12 receptor agonist having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients, or stabilizers (all of which are referred to herein as "carriers") typically used in the art, i.e., buffers, stabilizers, preservatives, tonicity agents, non-ionic detergents, antioxidants, and various other additives. See Remington's Pharmaceutical Sciences, 16th edition (Osol, ed. 1980). Such additives should be nontoxic to recipients at the dosages and concentrations employed.

[0403] Buffering agents help maintain pH in a range close to physiological conditions. They can be present in a wide variety of concentrations, but will typically be present at concentrations ranging from about 2 mM to about 50 mM. Suitable buffering agents for use in the present disclosure include both organic and inorganic acids and their salts, such as citrate buffers (e.g., monosodium citrate-disodium citrate mixtures, citric acid-trisodium citrate mixtures, citric acid-monosodium citrate mixtures, etc.), succinate buffers (e.g., succinic acid-monosodium succinate mixtures, succinic acid-sodium hydroxide mixtures, succinic acid-disodium succinate mixtures, etc.), tartrate buffers (e.g., tartaric acid-sodium tartrate mixtures, tartaric acid-po...

Claims

1. IL12 receptor agonists, (a) a first polypeptide chain comprising, in N-terminal to C-terminal orientation, a first targeting moiety or targeting moiety component, a first Fc domain, and a p35 moiety; (b) a second polypeptide chain comprising, in N-terminal to C-terminal orientation, a second targeting moiety or targeting moiety component, and a second Fc domain; (c) a p40 portion between the first Fc domain and the p35 portion, or in the form of a monomeric p40, optionally wherein the p40 portion has an attenuating substitution at a position corresponding to amino acid W37 of full-length human p40 or amino acid W37 of full-length mouse p40; (d) an IL12 receptor agonist comprising an IL12Rβ portion configured to mask said p35 portion or said p40 portion.

2. 1. An IL12 receptor agonist comprising an IL12 mutein, wherein the IL12 receptor agonist has at least a 500-fold attenuation compared to wild-type IL12, and wherein the IL12 receptor agonist comprises: (a) a first polypeptide chain and a second polypeptide chain dimerized through a first Fc domain and a second Fc domain; (b) an optional first targeting moiety or targeting moiety component on the first polypeptide chain, and an optional second targeting moiety or targeting moiety component on the second polypeptide chain; (c) a p35 portion and a p40 portion; (d) an IL12Rβ portion configured to mask the p35 portion or the p40 portion.

3. 1. An IL12 receptor agonist, comprising: on a first polypeptide chain and a second polypeptide chain dimerized through a first Fc domain and a second Fc domain, (a) an optional first targeting moiety and an optional second targeting moiety; (b) an IL12 mutein comprising a p35 portion and a p40 portion, (iv) the p35 portion comprises an attenuating amino acid substitution, optionally the attenuating amino acid substitution being: (A) at amino acid Y189 of full-length human p35, or at amino acid Y185 of full-length mouse p35, which is optionally A, V, R, or E; (B) at amino acid I193 of full-length human p35, or at amino acid M189 of full-length mouse p35, which is optionally A, V, or E; (C) at amino acid R211 of full-length human p35, or at amino acid R207 of full-length mouse p35, which is optionally A or K; or (D) any combination of (A)-(C); and / or (v) the p40 portion comprises an attenuating amino acid substitution, optionally the attenuating amino acid substitution being: (A) at amino acid K28 of full-length human p40 or at amino acid K28 of full-length mouse p40, wherein the substitution is optionally A; (B) at amino acid W37 of full-length human p40 or at amino acid W37 of full-length mouse p40, wherein the substitution is optionally A; (C) at amino acid D115 of full-length human p40 or at amino acid E115 of full-length mouse p40, wherein the substitution is optionally A; or (D) at amino acid K118 of full-length human p40 or at amino acid K119 of full-length mouse p40. (E) at amino acid K126 of full-length human p40 or at amino acid K126 of full-length mouse p40, wherein the substitution is optionally A; (F) at amino acid Y268 of full-length human p40 or at amino acid Y265 of full-length mouse p40, wherein the substitution is optionally V or F; (G) at amino acid Y314 of full-length human p40 or at amino acid Y318 of full-length mouse p40, wherein the substitution is optionally F; or (H) any combination of (A)-(G). (c) an IL12 receptor agonist comprising an IL12Rβ portion configured to mask said p35 portion or said p40 portion.

4. 4. The IL12 receptor agonist of any one of claims 1 to 3, wherein the IL12Rβ portion is an amino acid sequence 90-100, 90-110, or 90-120 amino acids in length that has at least 90% sequence identity to amino acids 46-136 of human IL12Rβ1, and optionally, the amino acid sequence has at least 95%, at least 97%, or 100% sequence identity to amino acids 46-136 of human IL12Rβ1.

5. 4. The IL12 receptor agonist of any one of claims 1 to 3, wherein the IL12Rβ portion is an amino acid sequence 110-120, 110-130, or 110-140 amino acids in length that has at least 90% sequence identity to amino acids 24-136 of human IL12Rβ1, and optionally, the amino acid sequence has at least 95%, at least 97%, or 100% sequence identity to amino acids 24-136 of human IL12Rβ1.

6. 4. The IL12 receptor agonist of any one of claims 1 to 3, wherein the IL12Rβ portion is an amino acid sequence of 185-195, 185-205, or 185-215 amino acids in length that has at least 90% sequence identity to amino acids 46-234 of human IL12Rβ1, and optionally, the amino acid sequence has at least 95%, at least 97%, or 100% sequence identity to amino acids 46-234 of human IL12Rβ1.

7. 4. The IL12 receptor agonist of any one of claims 1 to 3, wherein the IL12Rβ portion is an amino acid sequence 210-220, 210-230, or 210-240 amino acids in length that has at least 90% sequence identity to amino acids 24-234 of human IL12Rβ1, and optionally, the amino acid sequence has at least 95%, at least 97%, or 100% sequence identity to amino acids 24-234 of human IL12Rβ1.

8. 4. The IL12 receptor agonist of any one of claims 1 to 3, wherein the IL12Rβ portion is an amino acid sequence of 285-300, 285-310, or 285-320 amino acids in length that has at least 90% sequence identity to amino acids 46-337 of human IL12Rβ1, and optionally, the amino acid sequence has at least 95%, at least 97%, or 100% sequence identity to amino acids 46-337 of human IL12Rβ1.

9. 4. The IL12 receptor agonist of any one of claims 1 to 3, wherein the IL12Rβ portion is an amino acid sequence 310-320, 310-330, or 310-340 amino acids in length that has at least 90% sequence identity to amino acids 24-337 of human IL12Rβ1, and optionally, the amino acid sequence has at least 95%, at least 97%, or 100% sequence identity to amino acids 24-337 of human IL12Rβ1.

10. 4. The IL12 receptor agonist of claim 1, wherein the IL12Rβ portion is (i) an amino acid sequence consisting of amino acids 24 to 136 of human IL12Rβ1 with up to 5 or up to 10 contiguous amino acids at its N-terminus and / or C-terminus, or (ii) an amino acid sequence having at least 95% or at least 97% sequence identity to the sequence defined in (i).

11. 4. The IL12 receptor agonist of claim 1, wherein the IL12Rβ portion is (i) an amino acid sequence consisting of amino acids 46 to 136 of human IL12Rβ1 with up to 5 or up to 10 contiguous amino acids at its N-terminus and / or C-terminus, or (ii) an amino acid sequence having at least 95% or at least 97% sequence identity to the sequence defined in (i).

12. 4. The IL12 receptor agonist of claim 1, wherein the IL12Rβ portion is (i) an amino acid sequence consisting of amino acids 24 to 234 of human IL12Rβ1 with up to 5 or up to 10 contiguous amino acids at its N-terminus and / or C-terminus, or (ii) an amino acid sequence having at least 95% or at least 97% sequence identity to the sequence defined in (i).

13. 4. The IL12 receptor agonist of claim 1, wherein the IL12Rβ portion is (j) an amino acid sequence consisting of amino acids 46 to 234 of human IL12Rβ1 with up to 5 or up to 10 contiguous amino acids at its N-terminus and / or C-terminus, or (ii) an amino acid sequence having at least 95% or at least 97% sequence identity to the sequence defined in (i).

14. 4. The IL12 receptor agonist of claim 1, wherein the IL12Rβ portion is (i) an amino acid sequence consisting of amino acids 24 to 337 of human IL12Rβ1 with up to 5 or up to 10 contiguous amino acids at its N-terminus and / or C-terminus, or (ii) an amino acid sequence having at least 95% or at least 97% sequence identity to the sequence defined in (i).

15. 4. The IL12 receptor agonist of claim 1, wherein the IL12Rβ portion is (i) an amino acid sequence consisting of amino acids 46 to 337 of human IL12Rβ1 with up to 5 or up to 10 contiguous amino acids at its N-terminus and / or C-terminus, or (ii) an amino acid sequence having at least 95% or at least 97% sequence identity to the sequence defined in (i).

16. 4. The IL12 receptor agonist of any one of claims 1 to 3, wherein the IL12Rβ portion is an amino acid sequence 90-100, 90-110, or 90-120 amino acids in length that has at least 90% sequence identity to amino acids 126-221 of human IL12Rβ2, and optionally, the amino acid sequence has at least 95%, at least 97%, or 100% sequence identity to amino acids 126-221 of human IL12Rβ2.

17. 4. The IL12 receptor agonist of any one of claims 1 to 3, wherein the IL12Rβ portion is an amino acid sequence 190-220, 190-230, or 190-240 amino acids in length that has at least 90% sequence identity to amino acids 24-221 of human IL12Rβ2, and optionally, the amino acid sequence has at least 95%, at least 97%, or 100% sequence identity to amino acids 24-221 of human IL12Rβ2.

18. 4. The IL12 receptor agonist of any one of claims 1 to 3, wherein the IL12Rβ portion is an amino acid sequence of 185-195, 185-205, or 185-215 amino acids in length that has at least 90% sequence identity to amino acids 126-319 of human IL12Rβ2, and optionally, the amino acid sequence has at least 95%, at least 97%, or 100% sequence identity to amino acids 126-319 of human IL12Rβ2.

19. 4. The IL12 receptor agonist of any one of claims 1 to 3, wherein the IL12Rβ portion is an amino acid sequence of 290-310, 290-320, or 290-330 amino acids in length that has at least 90% sequence identity to amino acids 24-319 of human IL12Rβ2, and optionally, the amino acid sequence has at least 95%, at least 97%, or 100% sequence identity to amino acids 24-319 of human IL12Rβ2.

20. 4. The IL12 receptor agonist of any one of claims 1 to 3, wherein the IL12Rβ portion is an amino acid sequence of 290-310, 290-320, or 290-330 amino acids in length that has at least 90% sequence identity to amino acids 126-419 of human IL12Rβ2, and optionally, the amino acid sequence has at least 95%, at least 97%, or 100% sequence identity to amino acids 126-419 of human IL12Rβ2.

21. 4. The IL12 receptor agonist of any one of claims 1 to 3, wherein the IL12Rβ portion is an amino acid sequence of 390-410, 390-420, or 390-430 amino acids in length that has at least 90% sequence identity to amino acids 24-419 of human IL12Rβ2, and optionally, the amino acid sequence has at least 95%, at least 97%, or 100% sequence identity to amino acids 24-419 of human IL12Rβ2.

22. 4. The IL12 receptor agonist of claim 1, wherein the IL12Rβ portion is (i) an amino acid sequence consisting of amino acids 24 to 221 of human IL12Rβ2 with up to 5 or up to 10 contiguous amino acids at its N-terminus and / or C-terminus, or (ii) an amino acid sequence having at least 95% or at least 97% sequence identity to the sequence defined in (i).

23. 4. The IL12 receptor agonist of claim 1, wherein the IL12Rβ portion is (i) an amino acid sequence consisting of amino acids 126-221 of human IL12Rβ2 with up to 5 or up to 10 contiguous amino acids at its N-terminus and / or C-terminus, or (ii) an amino acid sequence having at least 95% or at least 97% sequence identity to the sequence defined in (i).

24. 4. The IL12 receptor agonist of claim 1, wherein the IL12Rβ portion is (i) an amino acid sequence consisting of amino acids 24 to 319 of human IL12Rβ2 with up to 5 or up to 10 contiguous amino acids at its N-terminus and / or C-terminus, or (ii) an amino acid sequence having at least 95% or at least 97% sequence identity to the sequence defined in (i).

25. 4. The IL12 receptor agonist of claim 1, wherein the IL12Rβ portion is (i) an amino acid sequence consisting of amino acids 126 to 319 of human IL12Rβ2 with up to 5 or up to 10 contiguous amino acids at its N-terminus and / or C-terminus, or (ii) an amino acid sequence having at least 95% or at least 97% sequence identity to the sequence defined in (i).

26. 4. The IL12 receptor agonist of claim 1, wherein the IL12Rβ portion is (i) an amino acid sequence consisting of amino acids 24 to 419 of human IL12Rβ2 with up to 5 or up to 10 contiguous amino acids at its N-terminus and / or C-terminus, or (ii) an amino acid sequence having at least 95% or at least 97% sequence identity to the sequence defined in (i).

27. 4. The IL12 receptor agonist of claim 1, wherein the IL12Rβ portion is (i) an amino acid sequence consisting of amino acids 126 to 419 of human IL12Rβ2 with up to 5 or up to 10 contiguous amino acids at its N-terminus and / or C-terminus, or (ii) an amino acid sequence having at least 95% or at least 97% sequence identity to the sequence defined in (i).

28. 28. The IL12 receptor agonist of any one of claims 1 to 27, wherein the p40 portion comprises an amino acid sequence having at least 90%, at least 95%, or at least 97% sequence identity to the receptor binding domain of mature human or mature mouse p40.

29. 29. The IL12 receptor agonist of any one of claims 1 to 28, wherein the p35 portion has at least 90%, at least 95%, or at least 97% sequence identity to the receptor binding domain of mature human or mature mouse p35.

30. 30. The IL12 receptor agonist of any one of claims 1 to 29, which is monovalent with respect to said p35 portion and said p40 portion.

31. Optionally, an IL12 receptor agonist, which is an IL12 receptor according to any one of claims 1 to 30, comprising a first IL12 monomer and a second IL12 monomer associated with a monomeric p40 (optionally a masked monomeric p40); (a) the first monomer has the structure of Exemplary Monomer 19 and the second monomer has the structure of Exemplary Monomer 33; or (b) the first monomer has the structure of Exemplary Monomer 19 and the second monomer has the structure of Exemplary Monomer 57; or (c) the first monomer has the configuration of Exemplary Monomer 28 and the second monomer has the configuration of Exemplary Monomer 57; or (d) the first monomer has the structure of Exemplary Monomer 33 and the second monomer has the structure of Exemplary Monomer 35; or (e) the first monomer has the configuration of Exemplary Monomer 35 and the second monomer has the configuration of Exemplary Monomer 57; or (f) An IL12 receptor agonist, wherein the first monomer has the configuration of exemplary monomer 57 and the second monomer has the configuration of exemplary monomer 57.

32. 32. The IL12 receptor agonist of claim 31, comprising an IL12Rβ1 portion configured to mask (a) the p40 portion, (b) the p35 portion, or (c) both the p40 portion and the p35 portion.

33. Optionally, an IL12 receptor agonist, which is an IL12 receptor according to any one of claims 1 to 30, comprising a first IL12 monomer and a second IL12 monomer; (a) the first monomer has the structure of exemplary monomer 33 and the second IL12 monomer has the structure of exemplary monomer 58; or (b) an IL12 receptor agonist, wherein the first IL12 monomer has the configuration of exemplary monomer 28 and the second IL12 monomer has the configuration of exemplary monomer 59.

34. 34. The IL12 receptor agonist of any one of claims 1 to 33, wherein the p40 portion comprises the p40 D2 and p40 D3 domains, and optionally the p40 D1 domain.

35. the first targeting moiety and / or the second targeting moiety are: (a) binds to a tumor-associated antigen; (b) binds to a tumor microenvironment antigen; (c) binding to a cell surface molecule of a tumor-reactive lymphocyte; (d) binds to a checkpoint inhibitor; (e) binding to a peptide-MHC complex; (f) a peptide-MHC complex; (g) binds to an antigen associated with or targeted by an autoimmune response, or (h) An IL12 receptor agonist according to any one of claims 1 to 34, independently selected from (a) to (g) above.

36. 36. The IL12 receptor agonist of claim 35, wherein the first targeting moiety and / or the second targeting moiety is an antibody or an antigen-binding fragment thereof, and optionally, the targeting moiety is a Fab or scFv.

37. 37. The IL12 receptor agonist of claim 35 or 36, wherein the first targeting moiety and / or the second targeting moiety binds to a tumor-associated antigen.

38. 37. The IL12 receptor agonist of claim 35 or 36, wherein the first targeting moiety and / or the second targeting moiety bind to a tumor microenvironment antigen.

39. 37. The IL12 receptor agonist of claim 35 or 36, wherein the first targeting moiety and / or the second targeting moiety bind to a cell surface molecule of a tumor lymphocyte, and optionally the cell surface molecule is CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, LAG3, TIM3, or B7-H3.

40. 37. The IL12 receptor agonist of claim 35 or 36, wherein the first targeting moiety and / or second targeting moiety bind to a checkpoint inhibitor, and optionally the checkpoint inhibitor is CTLA-4, PD1, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, VISTA, PSGL1, or CHK2.

41. 37. The IL12 receptor agonist of claim 35 or 36, wherein the first targeting moiety and / or the second targeting moiety bind to an MHC-peptide complex.

42. 37. The IL12 receptor agonist of claim 35 or 36, wherein the first targeting moiety and / or the second targeting moiety is a peptide-MHC complex.

43. A nucleic acid or nucleic acids encoding an IL12 receptor agonist according to any one of claims 1 to 42.

44. A host cell engineered to express an IL12 receptor agonist according to any one of claims 1 to 42 or a nucleic acid(s) according to claim 43.

45. 43. A method of producing an IL12 receptor agonist according to any one of claims 1 to 42, comprising culturing the host cell of claim 44 and recovering the IL12 receptor agonist or p40 portion expressed thereby.

46. A pharmaceutical composition comprising an IL12 receptor agonist according to any one of claims 1 to 42 and an excipient.

47. (a) a method of treating cancer; (b) a method for targeted cancer treatment; (c) a method for localized delivery of IL12 protein; (d) a method of administering IL12 therapy with reduced systemic exposure and / or reduced systemic toxicity; or (e) A method for locally inducing an immune response in a target tissue, comprising: The method comprises administering to a subject in need thereof an IL12 receptor agonist of any one of claims 1 to 42, or a pharmaceutical composition of claim 46, optionally wherein the administration is (i) subcutaneous or (ii) systemic.

48. 48. The method of claim 47, further comprising administering to the subject an anti-PD1 antibody, optionally wherein the anti-PD1 antibody is MDX-1106 (nivolumab), MK-3475 (pembrolizumab), MEDI-0680 (AMP-514), PDR001, or BGB-108.

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