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] The present invention provides IL12 receptor agonists and methods of use thereof. [Background technology]

[0002] Interleukin-12 (IL-12 or IL12) is a proinflammatory cytokine that plays an important role in both innate and adaptive immunity. (Hamza et al., 2010, Int. J. Mol. Sci., 11(3):789-806) IL12 functions primarily as a 70-kDa heterodimer composed of disulfide-linked p35 and p40 subunits. (Ibid.) Various immune cells, including B cells, dendritic cells, macrophages, monocytes, and neutrophils, express IL12 upon stimulation (Tugues et al., 2015, Cell Death Differ., 22:237-246), and form an active heterodimer after protein synthesis. Binding of IL-12 to the IL-12 receptor complex on T cells and natural killer (NK) cells results in signal transduction via signal transducer and activator of transcription (STAT4) and signal transducer and activator of transcription 3 (STAT3), followed by the production and secretion of interferon gamma (IFN-γ). Ullrich et al., 2020, EXCLI J., 19:1563-1589. Downstream signaling of IFN-γ involves activation of the T-box transcription factor TBX21 (Tbet), which mediates the transcription of T helper 1 (T helper 1). H 1) Induce inflammatory functions of cells. (Ibid.)

[0003] IL-12 has been investigated as an anticancer therapeutic agent since the early 1990s due to its ability to activate NK cells and cytotoxic T cells (Lasek et al., 2014, Cancer Immunol. Immunother. 63(5):419-435). However, in most patients, repeated administration of IL-12 induced an adaptive response, resulting in a gradual decline in IL-12-induced IFN-γ blood levels (ibid.). Furthermore, the simultaneous induction of other cytokines (e.g., TNF-α) and / or chemokines (IP-10 or MIG) along with IFN-γ resulted in severe toxicity (ibid.). Various dosing and timing protocols have been developed to minimize IFN-γ toxicity and improve IL-12 efficacy (ibid.). These approaches have been largely ineffective, and patient survival rates have not been significantly improved (ibid.).

[0004] Despite the general acceptance of IL12 therapeutics in the field of immunotherapy, such as anti-cancer therapy, IL12 molecules generally have a narrow therapeutic index, requiring highly toxic doses to achieve modest anti-cancer effects.

[0005] Therefore, there is a need in the art for new IL12 therapies with improved therapeutic efficacy and safety profiles. 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 are characterized by an improved therapeutic profile due to 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, such as 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, e.g., 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 in 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, infra, and in numbered embodiments 848-850.

[0009] The present disclosure further provides pharmaceutical compositions comprising the IL12 receptor agonists, IL12 muteins, p35 portions, and p40 portions of the present disclosure. Exemplary pharmaceutical compositions are described in Section 6.10, infra, and in numbered embodiment 851.

[0010] Further provided herein are methods of using the disclosed IL12 receptor agonists, IL12 muteins, p35 moieties, p40 moieties, and pharmaceutical compositions, e.g., to treat cancerous conditions. Exemplary methods are described in Section 6.11, infra, 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-2C are schematic diagrams depicting the structural organization of p35 and p40 moieties of IL12 (FIG. 2A) and various embodiments of monovalent (FIGS. 2B-2G) and bivalent (FIGS. 2H-2O) IL12-Fc fusion proteins that the IL12 receptor agonists of the present disclosure may comprise or consist of. In the embodiments of FIGS. 2B-2P, the IL12 moieties can be attached to either the N-terminus of the Fc (FIGS. 2B, 2C, 2H-2J, and 2N) or the C-terminus of the Fc (FIGS. 2D-2G, 2K-2M, 2O, and 2P). When attached to Fc according to the embodiments of FIGS. 2B-2M, the IL12 moieties can be arranged in the order (N-terminus 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 portion may be provided in the form of a p40 monomer. The asterisk appearing between the p40 and p35 portions indicates an optionally eliminated disulfide bond between the subunits. While only certain IL12 portions are shown with the disulfide bond between the p40 and p35 portions eliminated, any of the IL12-Fc fusion proteins of Figures 2A-2O may 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 proteins of Figures 2A-2O are disclosed, for example, in Section 6.3. The CH2 and CH3 domains shown in Figures 2B-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 of 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), e.g., as described in Section 6.6.1.2 (not shown). [Figure 2B]2A-2C are schematic diagrams depicting the structural organization of p35 and p40 moieties of IL12 (FIG. 2A) and various embodiments of monovalent (FIGS. 2B-2G) and bivalent (FIGS. 2H-2O) IL12-Fc fusion proteins that the IL12 receptor agonists of the present disclosure may comprise or consist of. In the embodiments of FIGS. 2B-2P, the IL12 moieties can be attached to either the N-terminus of the Fc (FIGS. 2B, 2C, 2H-2J, and 2N) or the C-terminus of the Fc (FIGS. 2D-2G, 2K-2M, 2O, and 2P). When attached to Fc according to the embodiments of FIGS. 2B-2M, the IL12 moieties can be arranged in the order (N-terminus 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 portion may be provided in the form of a p40 monomer. The asterisk appearing between the p40 and p35 portions indicates an optionally eliminated disulfide bond between the subunits. While only certain IL12 portions are shown with the disulfide bond between the p40 and p35 portions eliminated, any of the IL12-Fc fusion proteins of Figures 2A-2O may 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 proteins of Figures 2A-2O are disclosed, for example, in Section 6.3. The CH2 and CH3 domains shown in Figures 2B-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 of 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), e.g., as described in Section 6.6.1.2 (not shown). [Figure 2C]2A-2C are schematic diagrams depicting the structural organization of p35 and p40 moieties of IL12 (FIG. 2A) and various embodiments of monovalent (FIGS. 2B-2G) and bivalent (FIGS. 2H-2O) IL12-Fc fusion proteins that the IL12 receptor agonists of the present disclosure may comprise or consist of. In the embodiments of FIGS. 2B-2P, the IL12 moieties can be attached to either the N-terminus of the Fc (FIGS. 2B, 2C, 2H-2J, and 2N) or the C-terminus of the Fc (FIGS. 2D-2G, 2K-2M, 2O, and 2P). When attached to Fc according to the embodiments of FIGS. 2B-2M, the IL12 moieties can be arranged in the order (N-terminus 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 portion may be provided in the form of a p40 monomer. The asterisk appearing between the p40 and p35 portions indicates an optionally eliminated disulfide bond between the subunits. While only certain IL12 portions are shown with the disulfide bond between the p40 and p35 portions eliminated, any of the IL12-Fc fusion proteins of Figures 2A-2O may 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 proteins of Figures 2A-2O are disclosed, for example, in Section 6.3. The CH2 and CH3 domains shown in Figures 2B-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 of 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), e.g., as described in Section 6.6.1.2 (not shown). [Figure 2D]2A-2C are schematic diagrams depicting the structural organization of p35 and p40 moieties of IL12 (FIG. 2A) and various embodiments of monovalent (FIGS. 2B-2G) and bivalent (FIGS. 2H-2O) IL12-Fc fusion proteins that the IL12 receptor agonists of the present disclosure may comprise or consist of. In the embodiments of FIGS. 2B-2P, the IL12 moieties can be attached to either the N-terminus of the Fc (FIGS. 2B, 2C, 2H-2J, and 2N) or the C-terminus of the Fc (FIGS. 2D-2G, 2K-2M, 2O, and 2P). When attached to Fc according to the embodiments of FIGS. 2B-2M, the IL12 moieties can be arranged in the order (N-terminus 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 portion may be provided in the form of a p40 monomer. The asterisk appearing between the p40 and p35 portions indicates an optionally eliminated disulfide bond between the subunits. While only certain IL12 portions are shown with the disulfide bond between the p40 and p35 portions eliminated, any of the IL12-Fc fusion proteins of Figures 2A-2O may 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 proteins of Figures 2A-2O are disclosed, for example, in Section 6.3. The CH2 and CH3 domains shown in Figures 2B-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 of 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), e.g., as described in Section 6.6.1.2 (not shown). [Figure 2E]2A-2C are schematic diagrams depicting the structural organization of p35 and p40 moieties of IL12 (FIG. 2A) and various embodiments of monovalent (FIGS. 2B-2G) and bivalent (FIGS. 2H-2O) IL12-Fc fusion proteins that the IL12 receptor agonists of the present disclosure may comprise or consist of. In the embodiments of FIGS. 2B-2P, the IL12 moieties can be attached to either the N-terminus of the Fc (FIGS. 2B, 2C, 2H-2J, and 2N) or the C-terminus of the Fc (FIGS. 2D-2G, 2K-2M, 2O, and 2P). When attached to Fc according to the embodiments of FIGS. 2B-2M, the IL12 moieties can be arranged in the order (N-terminus 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 portion may be provided in the form of a p40 monomer. The asterisk appearing between the p40 and p35 portions indicates an optionally eliminated disulfide bond between the subunits. While only certain IL12 portions are shown with the disulfide bond between the p40 and p35 portions eliminated, any of the IL12-Fc fusion proteins of Figures 2A-2O may 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 proteins of Figures 2A-2O are disclosed, for example, in Section 6.3. The CH2 and CH3 domains shown in Figures 2B-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 of 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), e.g., as described in Section 6.6.1.2 (not shown). [Figure 2F]2A-2C are schematic diagrams depicting the structural organization of p35 and p40 moieties of IL12 (FIG. 2A) and various embodiments of monovalent (FIGS. 2B-2G) and bivalent (FIGS. 2H-2O) IL12-Fc fusion proteins that the IL12 receptor agonists of the present disclosure may comprise or consist of. In the embodiments of FIGS. 2B-2P, the IL12 moieties can be attached to either the N-terminus of the Fc (FIGS. 2B, 2C, 2H-2J, and 2N) or the C-terminus of the Fc (FIGS. 2D-2G, 2K-2M, 2O, and 2P). When attached to Fc according to the embodiments of FIGS. 2B-2M, the IL12 moieties can be arranged in the order (N-terminus 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 portion may be provided in the form of a p40 monomer. The asterisk appearing between the p40 and p35 portions indicates an optionally eliminated disulfide bond between the subunits. While only certain IL12 portions are shown with the disulfide bond between the p40 and p35 portions eliminated, any of the IL12-Fc fusion proteins of Figures 2A-2O may 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 proteins of Figures 2A-2O are disclosed, for example, in Section 6.3. The CH2 and CH3 domains shown in Figures 2B-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 of 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), e.g., as described in Section 6.6.1.2 (not shown). [Figure 2G]2A-2C are schematic diagrams depicting the structural organization of p35 and p40 moieties of IL12 (FIG. 2A) and various embodiments of monovalent (FIGS. 2B-2G) and bivalent (FIGS. 2H-2O) IL12-Fc fusion proteins that the IL12 receptor agonists of the present disclosure may comprise or consist of. In the embodiments of FIGS. 2B-2P, the IL12 moieties can be attached to either the N-terminus of the Fc (FIGS. 2B, 2C, 2H-2J, and 2N) or the C-terminus of the Fc (FIGS. 2D-2G, 2K-2M, 2O, and 2P). When attached to Fc according to the embodiments of FIGS. 2B-2M, the IL12 moieties can be arranged in the order (N-terminus 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 portion may be provided in the form of a p40 monomer. The asterisk appearing between the p40 and p35 portions indicates an optionally eliminated disulfide bond between the subunits. While only certain IL12 portions are shown with the disulfide bond between the p40 and p35 portions eliminated, any of the IL12-Fc fusion proteins of Figures 2A-2O may 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 proteins of Figures 2A-2O are disclosed, for example, in Section 6.3. The CH2 and CH3 domains shown in Figures 2B-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 of 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), e.g., as described in Section 6.6.1.2 (not shown). [Figure 2H]2A-2C are schematic diagrams depicting the structural organization of p35 and p40 moieties of IL12 (FIG. 2A) and various embodiments of monovalent (FIGS. 2B-2G) and bivalent (FIGS. 2H-2O) IL12-Fc fusion proteins that the IL12 receptor agonists of the present disclosure may comprise or consist of. In the embodiments of FIGS. 2B-2P, the IL12 moieties can be attached to either the N-terminus of the Fc (FIGS. 2B, 2C, 2H-2J, and 2N) or the C-terminus of the Fc (FIGS. 2D-2G, 2K-2M, 2O, and 2P). When attached to Fc according to the embodiments of FIGS. 2B-2M, the IL12 moieties can be arranged in the order (N-terminus 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 portion may be provided in the form of a p40 monomer. The asterisk appearing between the p40 and p35 portions indicates an optionally eliminated disulfide bond between the subunits. While only certain IL12 portions are shown with the disulfide bond between the p40 and p35 portions eliminated, any of the IL12-Fc fusion proteins of Figures 2A-2O may 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 proteins of Figures 2A-2O are disclosed, for example, in Section 6.3. The CH2 and CH3 domains shown in Figures 2B-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 of 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), e.g., as described in Section 6.6.1.2 (not shown). [Figure 2I]2A-2C are schematic diagrams depicting the structural organization of p35 and p40 moieties of IL12 (FIG. 2A) and various embodiments of monovalent (FIGS. 2B-2G) and bivalent (FIGS. 2H-2O) IL12-Fc fusion proteins that the IL12 receptor agonists of the present disclosure may comprise or consist of. In the embodiments of FIGS. 2B-2P, the IL12 moieties can be attached to either the N-terminus of the Fc (FIGS. 2B, 2C, 2H-2J, and 2N) or the C-terminus of the Fc (FIGS. 2D-2G, 2K-2M, 2O, and 2P). When attached to Fc according to the embodiments of FIGS. 2B-2M, the IL12 moieties can be arranged in the order (N-terminus 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 portion may be provided in the form of a p40 monomer. The asterisk appearing between the p40 and p35 portions indicates an optionally eliminated disulfide bond between the subunits. While only certain IL12 portions are shown with the disulfide bond between the p40 and p35 portions eliminated, any of the IL12-Fc fusion proteins of Figures 2A-2O may 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 proteins of Figures 2A-2O are disclosed, for example, in Section 6.3. The CH2 and CH3 domains shown in Figures 2B-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 of 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), e.g., as described in Section 6.6.1.2 (not shown). [Figure 2J]2A-2C are schematic diagrams depicting the structural organization of p35 and p40 moieties of IL12 (FIG. 2A) and various embodiments of monovalent (FIGS. 2B-2G) and bivalent (FIGS. 2H-2O) IL12-Fc fusion proteins that the IL12 receptor agonists of the present disclosure may comprise or consist of. In the embodiments of FIGS. 2B-2P, the IL12 moieties can be attached to either the N-terminus of the Fc (FIGS. 2B, 2C, 2H-2J, and 2N) or the C-terminus of the Fc (FIGS. 2D-2G, 2K-2M, 2O, and 2P). When attached to Fc according to the embodiments of FIGS. 2B-2M, the IL12 moieties can be arranged in the order (N-terminus 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 portion may be provided in the form of a p40 monomer. The asterisk appearing between the p40 and p35 portions indicates an optionally eliminated disulfide bond between the subunits. While only certain IL12 portions are shown with the disulfide bond between the p40 and p35 portions eliminated, any of the IL12-Fc fusion proteins of Figures 2A-2O may 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 proteins of Figures 2A-2O are disclosed, for example, in Section 6.3. The CH2 and CH3 domains shown in Figures 2B-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 of 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), e.g., as described in Section 6.6.1.2 (not shown). [Figure 2K]2A-2C are schematic diagrams depicting the structural organization of p35 and p40 moieties of IL12 (FIG. 2A) and various embodiments of monovalent (FIGS. 2B-2G) and bivalent (FIGS. 2H-2O) IL12-Fc fusion proteins that the IL12 receptor agonists of the present disclosure may comprise or consist of. In the embodiments of FIGS. 2B-2P, the IL12 moieties can be attached to either the N-terminus of the Fc (FIGS. 2B, 2C, 2H-2J, and 2N) or the C-terminus of the Fc (FIGS. 2D-2G, 2K-2M, 2O, and 2P). When attached to Fc according to the embodiments of FIGS. 2B-2M, the IL12 moieties can be arranged in the order (N-terminus 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 portion may be provided in the form of a p40 monomer. The asterisk appearing between the p40 and p35 portions indicates an optionally eliminated disulfide bond between the subunits. While only certain IL12 portions are shown with the disulfide bond between the p40 and p35 portions eliminated, any of the IL12-Fc fusion proteins of Figures 2A-2O may 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 proteins of Figures 2A-2O are disclosed, for example, in Section 6.3. The CH2 and CH3 domains shown in Figures 2B-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 of 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), e.g., as described in Section 6.6.1.2 (not shown). [Figure 2L]2A-2C are schematic diagrams depicting the structural organization of p35 and p40 moieties of IL12 (FIG. 2A) and various embodiments of monovalent (FIGS. 2B-2G) and bivalent (FIGS. 2H-2O) IL12-Fc fusion proteins that the IL12 receptor agonists of the present disclosure may comprise or consist of. In the embodiments of FIGS. 2B-2P, the IL12 moieties can be attached to either the N-terminus of the Fc (FIGS. 2B, 2C, 2H-2J, and 2N) or the C-terminus of the Fc (FIGS. 2D-2G, 2K-2M, 2O, and 2P). When attached to Fc according to the embodiments of FIGS. 2B-2M, the IL12 moieties can be arranged in the order (N-terminus 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 portion may be provided in the form of a p40 monomer. The asterisk appearing between the p40 and p35 portions indicates an optionally eliminated disulfide bond between the subunits. While only certain IL12 portions are shown with the disulfide bond between the p40 and p35 portions eliminated, any of the IL12-Fc fusion proteins of Figures 2A-2O may 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 proteins of Figures 2A-2O are disclosed, for example, in Section 6.3. The CH2 and CH3 domains shown in Figures 2B-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 of 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), e.g., as described in Section 6.6.1.2 (not shown). [Figure 2M]2A-2C are schematic diagrams depicting the structural organization of p35 and p40 moieties of IL12 (FIG. 2A) and various embodiments of monovalent (FIGS. 2B-2G) and bivalent (FIGS. 2H-2O) IL12-Fc fusion proteins that the IL12 receptor agonists of the present disclosure may comprise or consist of. In the embodiments of FIGS. 2B-2P, the IL12 moieties can be attached to either the N-terminus of the Fc (FIGS. 2B, 2C, 2H-2J, and 2N) or the C-terminus of the Fc (FIGS. 2D-2G, 2K-2M, 2O, and 2P). When attached to Fc according to the embodiments of FIGS. 2B-2M, the IL12 moieties can be arranged in the order (N-terminus 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 portion may be provided in the form of a p40 monomer. The asterisk appearing between the p40 and p35 portions indicates an optionally eliminated disulfide bond between the subunits. While only certain IL12 portions are shown with the disulfide bond between the p40 and p35 portions eliminated, any of the IL12-Fc fusion proteins of Figures 2A-2O may 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 proteins of Figures 2A-2O are disclosed, for example, in Section 6.3. The CH2 and CH3 domains shown in Figures 2B-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 of 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), e.g., as described in Section 6.6.1.2 (not shown). [Figure 2N]2A-2C are schematic diagrams depicting the structural organization of p35 and p40 moieties of IL12 (FIG. 2A) and various embodiments of monovalent (FIGS. 2B-2G) and bivalent (FIGS. 2H-2O) IL12-Fc fusion proteins that the IL12 receptor agonists of the present disclosure may comprise or consist of. In the embodiments of FIGS. 2B-2P, the IL12 moieties can be attached to either the N-terminus of the Fc (FIGS. 2B, 2C, 2H-2J, and 2N) or the C-terminus of the Fc (FIGS. 2D-2G, 2K-2M, 2O, and 2P). When attached to Fc according to the embodiments of FIGS. 2B-2M, the IL12 moieties can be arranged in the order (N-terminus 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 portion may be provided in the form of a p40 monomer. The asterisk appearing between the p40 and p35 portions indicates an optionally eliminated disulfide bond between the subunits. While only certain IL12 portions are shown with the disulfide bond between the p40 and p35 portions eliminated, any of the IL12-Fc fusion proteins of Figures 2A-2O may 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 proteins of Figures 2A-2O are disclosed, for example, in Section 6.3. The CH2 and CH3 domains shown in Figures 2B-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 of 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), e.g., as described in Section 6.6.1.2 (not shown). [Figure 2O]2A-2C are schematic diagrams depicting the structural organization of p35 and p40 moieties of IL12 (FIG. 2A) and various embodiments of monovalent (FIGS. 2B-2G) and bivalent (FIGS. 2H-2O) IL12-Fc fusion proteins that the IL12 receptor agonists of the present disclosure may comprise or consist of. In the embodiments of FIGS. 2B-2P, the IL12 moieties can be attached to either the N-terminus of the Fc (FIGS. 2B, 2C, 2H-2J, and 2N) or the C-terminus of the Fc (FIGS. 2D-2G, 2K-2M, 2O, and 2P). When attached to Fc according to the embodiments of FIGS. 2B-2M, the IL12 moieties can be arranged in the order (N-terminus 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 portion may be provided in the form of a p40 monomer. The asterisk appearing between the p40 and p35 portions indicates an optionally eliminated disulfide bond between the subunits. While only certain IL12 portions are shown with the disulfide bond between the p40 and p35 portions eliminated, any of the IL12-Fc fusion proteins of Figures 2A-2O may 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 proteins of Figures 2A-2O are disclosed, for example, in Section 6.3. The CH2 and CH3 domains shown in Figures 2B-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 of 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), e.g., as described in Section 6.6.1.2 (not shown). [Figure 2P]2A-2C are schematic diagrams depicting the structural organization of p35 and p40 moieties of IL12 (FIG. 2A) and various embodiments of monovalent (FIGS. 2B-2G) and bivalent (FIGS. 2H-2O) IL12-Fc fusion proteins that the IL12 receptor agonists of the present disclosure may comprise or consist of. In the embodiments of FIGS. 2B-2P, the IL12 moieties can be attached to either the N-terminus of the Fc (FIGS. 2B, 2C, 2H-2J, and 2N) or the C-terminus of the Fc (FIGS. 2D-2G, 2K-2M, 2O, and 2P). When attached to Fc according to the embodiments of FIGS. 2B-2M, the IL12 moieties can be arranged in the order (N-terminus 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 portion may be provided in the form of a p40 monomer. The asterisk appearing between the p40 and p35 portions indicates an optionally eliminated disulfide bond between the subunits. While only certain IL12 portions are shown with the disulfide bond between the p40 and p35 portions eliminated, any of the IL12-Fc fusion proteins of Figures 2A-2O may 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 proteins of Figures 2A-2O are disclosed, for example, in Section 6.3. The CH2 and CH3 domains shown in Figures 2B-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 of 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), e.g., as described in Section 6.6.1.2 (not shown). [Figure 3A]3A-3I are schematic diagrams depicting p35 and p40 moieties having the structural orientation of IL12 and various embodiments of IL12-Fc fusion proteins (FIGS. 3B-3I) that can be combined with each other to form a bivalent IL12-Fc fusion protein that an IL12 receptor agonist of the present disclosure may comprise or consist of. 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 proteins of FIGS. 3A-3I are disclosed, for example, in Section 6.3. An asterisk appearing 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. The CH2 and CH3 domains shown in FIGS. 3B-3I form an Fc domain, which is a type of multimerization moiety. Fc domains and other multimerizing moieties that can be used in IL12 receptor agonists are described in Section 6.6. [Figure 3B] 3A-3I are schematic diagrams depicting p35 and p40 moieties having the structural orientation of IL12 and various embodiments of IL12-Fc fusion proteins (FIGS. 3B-3I) that can be combined with each other to form a bivalent IL12-Fc fusion protein that an IL12 receptor agonist of the present disclosure may comprise or consist of. 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 proteins of FIGS. 3A-3I are disclosed, for example, in Section 6.3. An asterisk appearing 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. The CH2 and CH3 domains shown in FIGS. 3B-3I form an Fc domain, which is a type of multimerization moiety. Fc domains and other multimerizing moieties that can be used in IL12 receptor agonists are described in Section 6.6. [Figure 3C]3A-3I are schematic diagrams depicting p35 and p40 moieties having the structural orientation of IL12 and various embodiments of IL12-Fc fusion proteins (FIGS. 3B-3I) that can be combined with each other to form a bivalent IL12-Fc fusion protein that an IL12 receptor agonist of the present disclosure may comprise or consist of. 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 proteins of FIGS. 3A-3I are disclosed, for example, in Section 6.3. An asterisk appearing 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. The CH2 and CH3 domains shown in FIGS. 3B-3I form an Fc domain, which is a type of multimerization moiety. Fc domains and other multimerizing moieties that can be used in IL12 receptor agonists are described in Section 6.6. [Figure 3D] 3A-3I are schematic diagrams depicting p35 and p40 moieties having the structural orientation of IL12 and various embodiments of IL12-Fc fusion proteins (FIGS. 3B-3I) that can be combined with each other to form a bivalent IL12-Fc fusion protein that an IL12 receptor agonist of the present disclosure may comprise or consist of. 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 proteins of FIGS. 3A-3I are disclosed, for example, in Section 6.3. An asterisk appearing 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. The CH2 and CH3 domains shown in FIGS. 3B-3I form an Fc domain, which is a type of multimerization moiety. Fc domains and other multimerizing moieties that can be used in IL12 receptor agonists are described in Section 6.6. [Figure 3E]3A-3I are schematic diagrams depicting p35 and p40 moieties having the structural orientation of IL12 and various embodiments of IL12-Fc fusion proteins (FIGS. 3B-3I) that can be combined with each other to form a bivalent IL12-Fc fusion protein that an IL12 receptor agonist of the present disclosure may comprise or consist of. 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 proteins of FIGS. 3A-3I are disclosed, for example, in Section 6.3. An asterisk appearing 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. The CH2 and CH3 domains shown in FIGS. 3B-3I form an Fc domain, which is a type of multimerization moiety. Fc domains and other multimerizing moieties that can be used in IL12 receptor agonists are described in Section 6.6. [Figure 3F] 3A-3I are schematic diagrams depicting p35 and p40 moieties having the structural orientation of IL12 and various embodiments of IL12-Fc fusion proteins (FIGS. 3B-3I) that can be combined with each other to form a bivalent IL12-Fc fusion protein that an IL12 receptor agonist of the present disclosure may comprise or consist of. 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 proteins of FIGS. 3A-3I are disclosed, for example, in Section 6.3. An asterisk appearing 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. The CH2 and CH3 domains shown in FIGS. 3B-3I form an Fc domain, which is a type of multimerization moiety. Fc domains and other multimerizing moieties that can be used in IL12 receptor agonists are described in Section 6.6. [Figure 3G]3A-3I are schematic diagrams depicting p35 and p40 moieties having the structural orientation of IL12 and various embodiments of IL12-Fc fusion proteins (FIGS. 3B-3I) that can be combined with each other to form a bivalent IL12-Fc fusion protein that an IL12 receptor agonist of the present disclosure may comprise or consist of. 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 proteins of FIGS. 3A-3I are disclosed, for example, in Section 6.3. An asterisk appearing 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. The CH2 and CH3 domains shown in FIGS. 3B-3I form an Fc domain, which is a type of multimerization moiety. Fc domains and other multimerizing moieties that can be used in IL12 receptor agonists are described in Section 6.6. [Figure 3H] 3A-3I are schematic diagrams depicting p35 and p40 moieties having the structural orientation of IL12 and various embodiments of IL12-Fc fusion proteins (FIGS. 3B-3I) that can be combined with each other to form a bivalent IL12-Fc fusion protein that an IL12 receptor agonist of the present disclosure may comprise or consist of. 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 proteins of FIGS. 3A-3I are disclosed, for example, in Section 6.3. An asterisk appearing 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. The CH2 and CH3 domains shown in FIGS. 3B-3I form an Fc domain, which is a type of multimerization moiety. Fc domains and other multimerizing moieties that can be used in IL12 receptor agonists are described in Section 6.6. [Figure 3I]3A-3I are schematic diagrams depicting p35 and p40 moieties having the structural orientation of IL12 and various embodiments of IL12-Fc fusion proteins (FIGS. 3B-3I) that can be combined with each other to form a bivalent IL12-Fc fusion protein that an IL12 receptor agonist of the present disclosure may comprise or consist of. 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 proteins of FIGS. 3A-3I are disclosed, for example, in Section 6.3. An asterisk appearing 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. The CH2 and CH3 domains shown in FIGS. 3B-3I form an Fc domain, which is a type of multimerization moiety. Fc domains and other multimerizing moieties that can be used in IL12 receptor agonists are described in Section 6.6. [Figure 4A]4A-4W , which are schematic diagrams showing the structural orientation of additional embodiments of IL12-Fc fusion proteins that the IL12 receptor agonists of the present disclosure may comprise or consist of, incorporating a masking moiety in the form of an IL12 receptor (IL12R) β1 receptor moiety or an IL12Rβ2 receptor moiety (FIGS. 4B-4N and 4S-4W) or an anti-IL12 antibody fragment (FIGS. 4O-4R). Examples of suitable p35 and p40 moieties that may be incorporated into the IL12 fusion proteins of FIGS. 4A-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 6.4.2. Examples of suitable IL12 antibody-based masking moieties are disclosed, e.g., in Section 6.4.3. The CH2 and CH3 domains shown in FIGS. 4B-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 of a heterodimeric IL12-Fc fusion protein (e.g., as shown in Figures 4F-4W) can incorporate any combination of mutations suitable for heterodimerization or selective purification (e.g., knobs-in-holes and / or star mutations), e.g., as described in Section 6.6.1.2 (not shown). [Figure 4B]4A-4W , which are schematic diagrams showing the structural orientation of additional embodiments of IL12-Fc fusion proteins that the IL12 receptor agonists of the present disclosure may comprise or consist of, incorporating a masking moiety in the form of an IL12 receptor (IL12R) β1 receptor moiety or an IL12Rβ2 receptor moiety (FIGS. 4B-4N and 4S-4W) or an anti-IL12 antibody fragment (FIGS. 4O-4R). Examples of suitable p35 and p40 moieties that may be incorporated into the IL12 fusion proteins of FIGS. 4A-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 6.4.2. Examples of suitable IL12 antibody-based masking moieties are disclosed, e.g., in Section 6.4.3. The CH2 and CH3 domains shown in FIGS. 4B-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 of a heterodimeric IL12-Fc fusion protein (e.g., as shown in Figures 4F-4W) can incorporate any combination of mutations suitable for heterodimerization or selective purification (e.g., knobs-in-holes and / or star mutations), e.g., as described in Section 6.6.1.2 (not shown). [Figure 4C]4A-4W , which are schematic diagrams showing the structural orientation of additional embodiments of IL12-Fc fusion proteins that the IL12 receptor agonists of the present disclosure may comprise or consist of, incorporating a masking moiety in the form of an IL12 receptor (IL12R) β1 receptor moiety or an IL12Rβ2 receptor moiety (FIGS. 4B-4N and 4S-4W) or an anti-IL12 antibody fragment (FIGS. 4O-4R). Examples of suitable p35 and p40 moieties that may be incorporated into the IL12 fusion proteins of FIGS. 4A-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 6.4.2. Examples of suitable IL12 antibody-based masking moieties are disclosed, e.g., in Section 6.4.3. The CH2 and CH3 domains shown in FIGS. 4B-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 of a heterodimeric IL12-Fc fusion protein (e.g., as shown in Figures 4F-4W) can incorporate any combination of mutations suitable for heterodimerization or selective purification (e.g., knobs-in-holes and / or star mutations), e.g., as described in Section 6.6.1.2 (not shown). [Figure 4D]4A-4W , which are schematic diagrams showing the structural orientation of additional embodiments of IL12-Fc fusion proteins that the IL12 receptor agonists of the present disclosure may comprise or consist of, incorporating a masking moiety in the form of an IL12 receptor (IL12R) β1 receptor moiety or an IL12Rβ2 receptor moiety (FIGS. 4B-4N and 4S-4W) or an anti-IL12 antibody fragment (FIGS. 4O-4R). Examples of suitable p35 and p40 moieties that may be incorporated into the IL12 fusion proteins of FIGS. 4A-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 6.4.2. Examples of suitable IL12 antibody-based masking moieties are disclosed, e.g., in Section 6.4.3. The CH2 and CH3 domains shown in FIGS. 4B-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 of a heterodimeric IL12-Fc fusion protein (e.g., as shown in Figures 4F-4W) can incorporate any combination of mutations suitable for heterodimerization or selective purification (e.g., knobs-in-holes and / or star mutations), e.g., as described in Section 6.6.1.2 (not shown). [Figure 4E]4A-4W , which are schematic diagrams showing the structural orientation of additional embodiments of IL12-Fc fusion proteins that the IL12 receptor agonists of the present disclosure may comprise or consist of, incorporating a masking moiety in the form of an IL12 receptor (IL12R) β1 receptor moiety or an IL12Rβ2 receptor moiety (FIGS. 4B-4N and 4S-4W) or an anti-IL12 antibody fragment (FIGS. 4O-4R). Examples of suitable p35 and p40 moieties that may be incorporated into the IL12 fusion proteins of FIGS. 4A-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 6.4.2. Examples of suitable IL12 antibody-based masking moieties are disclosed, e.g., in Section 6.4.3. The CH2 and CH3 domains shown in FIGS. 4B-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 of a heterodimeric IL12-Fc fusion protein (e.g., as shown in Figures 4F-4W) can incorporate any combination of mutations suitable for heterodimerization or selective purification (e.g., knobs-in-holes and / or star mutations), e.g., as described in Section 6.6.1.2 (not shown). [Figure 4F]4A-4W , which are schematic diagrams showing the structural orientation of additional embodiments of IL12-Fc fusion proteins that the IL12 receptor agonists of the present disclosure may comprise or consist of, incorporating a masking moiety in the form of an IL12 receptor (IL12R) β1 receptor moiety or an IL12Rβ2 receptor moiety (FIGS. 4B-4N and 4S-4W) or an anti-IL12 antibody fragment (FIGS. 4O-4R). Examples of suitable p35 and p40 moieties that may be incorporated into the IL12 fusion proteins of FIGS. 4A-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 6.4.2. Examples of suitable IL12 antibody-based masking moieties are disclosed, e.g., in Section 6.4.3. The CH2 and CH3 domains shown in FIGS. 4B-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 of a heterodimeric IL12-Fc fusion protein (e.g., as shown in Figures 4F-4W) can incorporate any combination of mutations suitable for heterodimerization or selective purification (e.g., knobs-in-holes and / or star mutations), e.g., as described in Section 6.6.1.2 (not shown). [Figure 4G]4A-4W , which are schematic diagrams showing the structural orientation of additional embodiments of IL12-Fc fusion proteins that the IL12 receptor agonists of the present disclosure may comprise or consist of, incorporating a masking moiety in the form of an IL12 receptor (IL12R) β1 receptor moiety or an IL12Rβ2 receptor moiety (FIGS. 4B-4N and 4S-4W) or an anti-IL12 antibody fragment (FIGS. 4O-4R). Examples of suitable p35 and p40 moieties that may be incorporated into the IL12 fusion proteins of FIGS. 4A-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 6.4.2. Examples of suitable IL12 antibody-based masking moieties are disclosed, e.g., in Section 6.4.3. The CH2 and CH3 domains shown in FIGS. 4B-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 of a heterodimeric IL12-Fc fusion protein (e.g., as shown in Figures 4F-4W) can incorporate any combination of mutations suitable for heterodimerization or selective purification (e.g., knobs-in-holes and / or star mutations), e.g., as described in Section 6.6.1.2 (not shown). [Figure 4H]4A-4W , which are schematic diagrams showing the structural orientation of additional embodiments of IL12-Fc fusion proteins that the IL12 receptor agonists of the present disclosure may comprise or consist of, incorporating a masking moiety in the form of an IL12 receptor (IL12R) β1 receptor moiety or an IL12Rβ2 receptor moiety (FIGS. 4B-4N and 4S-4W) or an anti-IL12 antibody fragment (FIGS. 4O-4R). Examples of suitable p35 and p40 moieties that may be incorporated into the IL12 fusion proteins of FIGS. 4A-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 6.4.2. Examples of suitable IL12 antibody-based masking moieties are disclosed, e.g., in Section 6.4.3. The CH2 and CH3 domains shown in FIGS. 4B-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 of a heterodimeric IL12-Fc fusion protein (e.g., as shown in Figures 4F-4W) can incorporate any combination of mutations suitable for heterodimerization or selective purification (e.g., knobs-in-holes and / or star mutations), e.g., as described in Section 6.6.1.2 (not shown). [Figure 4I]4A-4W , which are schematic diagrams showing the structural orientation of additional embodiments of IL12-Fc fusion proteins that the IL12 receptor agonists of the present disclosure may comprise or consist of, incorporating a masking moiety in the form of an IL12 receptor (IL12R) β1 receptor moiety or an IL12Rβ2 receptor moiety (FIGS. 4B-4N and 4S-4W) or an anti-IL12 antibody fragment (FIGS. 4O-4R). Examples of suitable p35 and p40 moieties that may be incorporated into the IL12 fusion proteins of FIGS. 4A-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 6.4.2. Examples of suitable IL12 antibody-based masking moieties are disclosed, e.g., in Section 6.4.3. The CH2 and CH3 domains shown in FIGS. 4B-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 of a heterodimeric IL12-Fc fusion protein (e.g., as shown in Figures 4F-4W) can incorporate any combination of mutations suitable for heterodimerization or selective purification (e.g., knobs-in-holes and / or star mutations), e.g., as described in Section 6.6.1.2 (not shown). [Figure 4J]4A-4W , which are schematic diagrams showing the structural orientation of additional embodiments of IL12-Fc fusion proteins that the IL12 receptor agonists of the present disclosure may comprise or consist of, incorporating a masking moiety in the form of an IL12 receptor (IL12R) β1 receptor moiety or an IL12Rβ2 receptor moiety (FIGS. 4B-4N and 4S-4W) or an anti-IL12 antibody fragment (FIGS. 4O-4R). Examples of suitable p35 and p40 moieties that may be incorporated into the IL12 fusion proteins of FIGS. 4A-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 6.4.2. Examples of suitable IL12 antibody-based masking moieties are disclosed, e.g., in Section 6.4.3. The CH2 and CH3 domains shown in FIGS. 4B-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 of a heterodimeric IL12-Fc fusion protein (e.g., as shown in Figures 4F-4W) can incorporate any combination of mutations suitable for heterodimerization or selective purification (e.g., knobs-in-holes and / or star mutations), e.g., as described in Section 6.6.1.2 (not shown). [Figure 4K]4A-4W , which are schematic diagrams showing the structural orientation of additional embodiments of IL12-Fc fusion proteins that the IL12 receptor agonists of the present disclosure may comprise or consist of, incorporating a masking moiety in the form of an IL12 receptor (IL12R) β1 receptor moiety or an IL12Rβ2 receptor moiety (FIGS. 4B-4N and 4S-4W) or an anti-IL12 antibody fragment (FIGS. 4O-4R). Examples of suitable p35 and p40 moieties that may be incorporated into the IL12 fusion proteins of FIGS. 4A-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 6.4.2. Examples of suitable IL12 antibody-based masking moieties are disclosed, e.g., in Section 6.4.3. The CH2 and CH3 domains shown in FIGS. 4B-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 of a heterodimeric IL12-Fc fusion protein (e.g., as shown in Figures 4F-4W) can incorporate any combination of mutations suitable for heterodimerization or selective purification (e.g., knobs-in-holes and / or star mutations), e.g., as described in Section 6.6.1.2 (not shown). [Figure 4L]4A-4W , which are schematic diagrams showing the structural orientation of additional embodiments of IL12-Fc fusion proteins that the IL12 receptor agonists of the present disclosure may comprise or consist of, incorporating a masking moiety in the form of an IL12 receptor (IL12R) β1 receptor moiety or an IL12Rβ2 receptor moiety (FIGS. 4B-4N and 4S-4W) or an anti-IL12 antibody fragment (FIGS. 4O-4R). Examples of suitable p35 and p40 moieties that may be incorporated into the IL12 fusion proteins of FIGS. 4A-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 6.4.2. Examples of suitable IL12 antibody-based masking moieties are disclosed, e.g., in Section 6.4.3. The CH2 and CH3 domains shown in FIGS. 4B-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 of a heterodimeric IL12-Fc fusion protein (e.g., as shown in Figures 4F-4W) can incorporate any combination of mutations suitable for heterodimerization or selective purification (e.g., knobs-in-holes and / or star mutations), e.g., as described in Section 6.6.1.2 (not shown). [Figure 4M]4A-4W , which are schematic diagrams showing the structural orientation of additional embodiments of IL12-Fc fusion proteins that the IL12 receptor agonists of the present disclosure may comprise or consist of, incorporating a masking moiety in the form of an IL12 receptor (IL12R) β1 receptor moiety or an IL12Rβ2 receptor moiety (FIGS. 4B-4N and 4S-4W) or an anti-IL12 antibody fragment (FIGS. 4O-4R). Examples of suitable p35 and p40 moieties that may be incorporated into the IL12 fusion proteins of FIGS. 4A-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 6.4.2. Examples of suitable IL12 antibody-based masking moieties are disclosed, e.g., in Section 6.4.3. The CH2 and CH3 domains shown in FIGS. 4B-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 of a heterodimeric IL12-Fc fusion protein (e.g., as shown in Figures 4F-4W) can incorporate any combination of mutations suitable for heterodimerization or selective purification (e.g., knobs-in-holes and / or star mutations), e.g., as described in Section 6.6.1.2 (not shown). [Figure 4N]4A-4W , which are schematic diagrams showing the structural orientation of additional embodiments of IL12-Fc fusion proteins that the IL12 receptor agonists of the present disclosure may comprise or consist of, incorporating a masking moiety in the form of an IL12 receptor (IL12R) β1 receptor moiety or an IL12Rβ2 receptor moiety (FIGS. 4B-4N and 4S-4W) or an anti-IL12 antibody fragment (FIGS. 4O-4R). Examples of suitable p35 and p40 moieties that may be incorporated into the IL12 fusion proteins of FIGS. 4A-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 6.4.2. Examples of suitable IL12 antibody-based masking moieties are disclosed, e.g., in Section 6.4.3. The CH2 and CH3 domains shown in FIGS. 4B-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 of a heterodimeric IL12-Fc fusion protein (e.g., as shown in Figures 4F-4W) can incorporate any combination of mutations suitable for heterodimerization or selective purification (e.g., knobs-in-holes and / or star mutations), e.g., as described in Section 6.6.1.2 (not shown). [Figure 4O]4A-4W , which are schematic diagrams showing the structural orientation of additional embodiments of IL12-Fc fusion proteins that the IL12 receptor agonists of the present disclosure may comprise or consist of, incorporating a masking moiety in the form of an IL12 receptor (IL12R) β1 receptor moiety or an IL12Rβ2 receptor moiety (FIGS. 4B-4N and 4S-4W) or an anti-IL12 antibody fragment (FIGS. 4O-4R). Examples of suitable p35 and p40 moieties that may be incorporated into the IL12 fusion proteins of FIGS. 4A-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 6.4.2. Examples of suitable IL12 antibody-based masking moieties are disclosed, e.g., in Section 6.4.3. The CH2 and CH3 domains shown in FIGS. 4B-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 of a heterodimeric IL12-Fc fusion protein (e.g., as shown in Figures 4F-4W) can incorporate any combination of mutations suitable for heterodimerization or selective purification (e.g., knobs-in-holes and / or star mutations), e.g., as described in Section 6.6.1.2 (not shown). [Figure 4P]4A-4W , which are schematic diagrams showing the structural orientation of additional embodiments of IL12-Fc fusion proteins that the IL12 receptor agonists of the present disclosure may comprise or consist of, incorporating a masking moiety in the form of an IL12 receptor (IL12R) β1 receptor moiety or an IL12Rβ2 receptor moiety (FIGS. 4B-4N and 4S-4W) or an anti-IL12 antibody fragment (FIGS. 4O-4R). Examples of suitable p35 and p40 moieties that may be incorporated into the IL12 fusion proteins of FIGS. 4A-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 6.4.2. Examples of suitable IL12 antibody-based masking moieties are disclosed, e.g., in Section 6.4.3. The CH2 and CH3 domains shown in FIGS. 4B-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 of a heterodimeric IL12-Fc fusion protein (e.g., as shown in Figures 4F-4W) can incorporate any combination of mutations suitable for heterodimerization or selective purification (e.g., knobs-in-holes and / or star mutations), e.g., as described in Section 6.6.1.2 (not shown). [Figure 4Q]4A-4W , which are schematic diagrams showing the structural orientation of additional embodiments of IL12-Fc fusion proteins that the IL12 receptor agonists of the present disclosure may comprise or consist of, incorporating a masking moiety in the form of an IL12 receptor (IL12R) β1 receptor moiety or an IL12Rβ2 receptor moiety (FIGS. 4B-4N and 4S-4W) or an anti-IL12 antibody fragment (FIGS. 4O-4R). Examples of suitable p35 and p40 moieties that may be incorporated into the IL12 fusion proteins of FIGS. 4A-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 6.4.2. Examples of suitable IL12 antibody-based masking moieties are disclosed, e.g., in Section 6.4.3. The CH2 and CH3 domains shown in FIGS. 4B-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 of a heterodimeric IL12-Fc fusion protein (e.g., as shown in Figures 4F-4W) can incorporate any combination of mutations suitable for heterodimerization or selective purification (e.g., knobs-in-holes and / or star mutations), e.g., as described in Section 6.6.1.2 (not shown). [Figure 4R]4A-4W , which are schematic diagrams showing the structural orientation of additional embodiments of IL12-Fc fusion proteins that the IL12 receptor agonists of the present disclosure may comprise or consist of, incorporating a masking moiety in the form of an IL12 receptor (IL12R) β1 receptor moiety or an IL12Rβ2 receptor moiety (FIGS. 4B-4N and 4S-4W) or an anti-IL12 antibody fragment (FIGS. 4O-4R). Examples of suitable p35 and p40 moieties that may be incorporated into the IL12 fusion proteins of FIGS. 4A-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 6.4.2. Examples of suitable IL12 antibody-based masking moieties are disclosed, e.g., in Section 6.4.3. The CH2 and CH3 domains shown in FIGS. 4B-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 of a heterodimeric IL12-Fc fusion protein (e.g., as shown in Figures 4F-4W) can incorporate any combination of mutations suitable for heterodimerization or selective purification (e.g., knobs-in-holes and / or star mutations), e.g., as described in Section 6.6.1.2 (not shown). [Figure 4S]4A-4W , which are schematic diagrams showing the structural orientation of additional embodiments of IL12-Fc fusion proteins that the IL12 receptor agonists of the present disclosure may comprise or consist of, incorporating a masking moiety in the form of an IL12 receptor (IL12R) β1 receptor moiety or an IL12Rβ2 receptor moiety (FIGS. 4B-4N and 4S-4W) or an anti-IL12 antibody fragment (FIGS. 4O-4R). Examples of suitable p35 and p40 moieties that may be incorporated into the IL12 fusion proteins of FIGS. 4A-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 6.4.2. Examples of suitable IL12 antibody-based masking moieties are disclosed, e.g., in Section 6.4.3. The CH2 and CH3 domains shown in FIGS. 4B-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 of a heterodimeric IL12-Fc fusion protein (e.g., as shown in Figures 4F-4W) can incorporate any combination of mutations suitable for heterodimerization or selective purification (e.g., knobs-in-holes and / or star mutations), e.g., as described in Section 6.6.1.2 (not shown). [Figure 4T]4A-4W , which are schematic diagrams showing the structural orientation of additional embodiments of IL12-Fc fusion proteins that the IL12 receptor agonists of the present disclosure may comprise or consist of, incorporating a masking moiety in the form of an IL12 receptor (IL12R) β1 receptor moiety or an IL12Rβ2 receptor moiety (FIGS. 4B-4N and 4S-4W) or an anti-IL12 antibody fragment (FIGS. 4O-4R). Examples of suitable p35 and p40 moieties that may be incorporated into the IL12 fusion proteins of FIGS. 4A-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 6.4.2. Examples of suitable IL12 antibody-based masking moieties are disclosed, e.g., in Section 6.4.3. The CH2 and CH3 domains shown in FIGS. 4B-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 of a heterodimeric IL12-Fc fusion protein (e.g., as shown in Figures 4F-4W) can incorporate any combination of mutations suitable for heterodimerization or selective purification (e.g., knobs-in-holes and / or star mutations), e.g., as described in Section 6.6.1.2 (not shown). [Figure 4U]4A-4W , which are schematic diagrams showing the structural orientation of additional embodiments of IL12-Fc fusion proteins that the IL12 receptor agonists of the present disclosure may comprise or consist of, incorporating a masking moiety in the form of an IL12 receptor (IL12R) β1 receptor moiety or an IL12Rβ2 receptor moiety (FIGS. 4B-4N and 4S-4W) or an anti-IL12 antibody fragment (FIGS. 4O-4R). Examples of suitable p35 and p40 moieties that may be incorporated into the IL12 fusion proteins of FIGS. 4A-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 6.4.2. Examples of suitable IL12 antibody-based masking moieties are disclosed, e.g., in Section 6.4.3. The CH2 and CH3 domains shown in FIGS. 4B-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 of a heterodimeric IL12-Fc fusion protein (e.g., as shown in Figures 4F-4W) can incorporate any combination of mutations suitable for heterodimerization or selective purification (e.g., knobs-in-holes and / or star mutations), e.g., as described in Section 6.6.1.2 (not shown). [Figure 4V]4A-4W , which are schematic diagrams showing the structural orientation of additional embodiments of IL12-Fc fusion proteins that the IL12 receptor agonists of the present disclosure may comprise or consist of, incorporating a masking moiety in the form of an IL12 receptor (IL12R) β1 receptor moiety or an IL12Rβ2 receptor moiety (FIGS. 4B-4N and 4S-4W) or an anti-IL12 antibody fragment (FIGS. 4O-4R). Examples of suitable p35 and p40 moieties that may be incorporated into the IL12 fusion proteins of FIGS. 4A-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 6.4.2. Examples of suitable IL12 antibody-based masking moieties are disclosed, e.g., in Section 6.4.3. The CH2 and CH3 domains shown in FIGS. 4B-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 of a heterodimeric IL12-Fc fusion protein (e.g., as shown in Figures 4F-4W) can incorporate any combination of mutations suitable for heterodimerization or selective purification (e.g., knobs-in-holes and / or star mutations), e.g., as described in Section 6.6.1.2 (not shown). [Figure 4W]4A-4W , which are schematic diagrams showing the structural orientation of additional embodiments of IL12-Fc fusion proteins that the IL12 receptor agonists of the present disclosure may comprise or consist of, incorporating a masking moiety in the form of an IL12 receptor (IL12R) β1 receptor moiety or an IL12Rβ2 receptor moiety (FIGS. 4B-4N and 4S-4W) or an anti-IL12 antibody fragment (FIGS. 4O-4R). Examples of suitable p35 and p40 moieties that may be incorporated into the IL12 fusion proteins of FIGS. 4A-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 6.4.2. Examples of suitable IL12 antibody-based masking moieties are disclosed, e.g., in Section 6.4.3. The CH2 and CH3 domains shown in FIGS. 4B-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 of a heterodimeric IL12-Fc fusion protein (e.g., as shown in Figures 4F-4W) can incorporate any combination of mutations suitable for heterodimerization or selective purification (e.g., knobs-in-holes and / or star mutations), e.g., as described in Section 6.6.1.2 (not shown). [Figure 5A]5A-5X , which are schematic diagrams showing the structural orientation of additional embodiments of IL12-Fc fusion proteins that may be incorporated into the IL12 receptor agonists 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 may be incorporated into the IL12 fusion proteins of FIGS. 5A-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 FIGS. 5B-5S and 5V-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 of 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 (shown in Figures 5H-5K, 5O, 5R, 5S, and 5V-5X) or an antibody (shown in Figures 5L-5N). While the antibody in Figures 5L-5N is depicted as an Fv, particularly an scFv, the scFv may be substituted with a Fab, as shown in Figures 39A-39B. [Figure 5B]5A-5X , which are schematic diagrams showing the structural orientation of additional embodiments of IL12-Fc fusion proteins that may be incorporated into the IL12 receptor agonists 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 may be incorporated into the IL12 fusion proteins of FIGS. 5A-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 FIGS. 5B-5S and 5V-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 of 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 (shown in Figures 5H-5K, 5O, 5R, 5S, and 5V-5X) or an antibody (shown in Figures 5L-5N). While the antibody in Figures 5L-5N is depicted as an Fv, particularly an scFv, the scFv may be substituted with a Fab, as shown in Figures 39A-39B. [Figure 5C]5A-5X , which are schematic diagrams showing the structural orientation of additional embodiments of IL12-Fc fusion proteins that may be incorporated into the IL12 receptor agonists 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 may be incorporated into the IL12 fusion proteins of FIGS. 5A-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 FIGS. 5B-5S and 5V-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 of 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 (shown in Figures 5H-5K, 5O, 5R, 5S, and 5V-5X) or an antibody (shown in Figures 5L-5N). While the antibody in Figures 5L-5N is depicted as an Fv, particularly an scFv, the scFv may be substituted with a Fab, as shown in Figures 39A-39B. [Figure 5D]5A-5X , which are schematic diagrams showing the structural orientation of additional embodiments of IL12-Fc fusion proteins that may be incorporated into the IL12 receptor agonists 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 may be incorporated into the IL12 fusion proteins of FIGS. 5A-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 FIGS. 5B-5S and 5V-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 of 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 (shown in Figures 5H-5K, 5O, 5R, 5S, and 5V-5X) or an antibody (shown in Figures 5L-5N). While the antibody in Figures 5L-5N is depicted as an Fv, particularly an scFv, the scFv may be substituted with a Fab, as shown in Figures 39A-39B. [Figure 5E]5A-5X , which are schematic diagrams showing the structural orientation of additional embodiments of IL12-Fc fusion proteins that may be incorporated into the IL12 receptor agonists 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 may be incorporated into the IL12 fusion proteins of FIGS. 5A-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 FIGS. 5B-5S and 5V-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 of 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 (shown in Figures 5H-5K, 5O, 5R, 5S, and 5V-5X) or an antibody (shown in Figures 5L-5N). While the antibody in Figures 5L-5N is depicted as an Fv, particularly an scFv, the scFv may be substituted with a Fab, as shown in Figures 39A-39B. [Figure 5F]5A-5X , which are schematic diagrams showing the structural orientation of additional embodiments of IL12-Fc fusion proteins that may be incorporated into the IL12 receptor agonists 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 may be incorporated into the IL12 fusion proteins of FIGS. 5A-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 FIGS. 5B-5S and 5V-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 of 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 (shown in Figures 5H-5K, 5O, 5R, 5S, and 5V-5X) or an antibody (shown in Figures 5L-5N). While the antibody in Figures 5L-5N is depicted as an Fv, particularly an scFv, the scFv may be substituted with a Fab, as shown in Figures 39A-39B. [Figure 5G]5A-5X , which are schematic diagrams showing the structural orientation of additional embodiments of IL12-Fc fusion proteins that may be incorporated into the IL12 receptor agonists 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 may be incorporated into the IL12 fusion proteins of FIGS. 5A-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 FIGS. 5B-5S and 5V-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 of 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 (shown in Figures 5H-5K, 5O, 5R, 5S, and 5V-5X) or an antibody (shown in Figures 5L-5N). While the antibody in Figures 5L-5N is depicted as an Fv, particularly an scFv, the scFv may be substituted with a Fab, as shown in Figures 39A-39B. [Figure 5H]5A-5X , which are schematic diagrams showing the structural orientation of additional embodiments of IL12-Fc fusion proteins that may be incorporated into the IL12 receptor agonists 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 may be incorporated into the IL12 fusion proteins of FIGS. 5A-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 FIGS. 5B-5S and 5V-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 of 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 (shown in Figures 5H-5K, 5O, 5R, 5S, and 5V-5X) or an antibody (shown in Figures 5L-5N). While the antibody in Figures 5L-5N is depicted as an Fv, particularly an scFv, the scFv may be substituted with a Fab, as shown in Figures 39A-39B. [Figure 5I]5A-5X , which are schematic diagrams showing the structural orientation of additional embodiments of IL12-Fc fusion proteins that may be incorporated into the IL12 receptor agonists 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 may be incorporated into the IL12 fusion proteins of FIGS. 5A-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 FIGS. 5B-5S and 5V-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 of 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 (shown in Figures 5H-5K, 5O, 5R, 5S, and 5V-5X) or an antibody (shown in Figures 5L-5N). While the antibody in Figures 5L-5N is depicted as an Fv, particularly an scFv, the scFv may be substituted with a Fab, as shown in Figures 39A-39B. [Figure 5J]5A-5X , which are schematic diagrams showing the structural orientation of additional embodiments of IL12-Fc fusion proteins that may be incorporated into the IL12 receptor agonists 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 may be incorporated into the IL12 fusion proteins of FIGS. 5A-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 FIGS. 5B-5S and 5V-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 of 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 (shown in Figures 5H-5K, 5O, 5R, 5S, and 5V-5X) or an antibody (shown in Figures 5L-5N). While the antibody in Figures 5L-5N is depicted as an Fv, particularly an scFv, the scFv may be substituted with a Fab, as shown in Figures 39A-39B. [Figure 5K]5A-5X , which are schematic diagrams showing the structural orientation of additional embodiments of IL12-Fc fusion proteins that may be incorporated into the IL12 receptor agonists 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 may be incorporated into the IL12 fusion proteins of FIGS. 5A-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 FIGS. 5B-5S and 5V-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 of 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 (shown in Figures 5H-5K, 5O, 5R, 5S, and 5V-5X) or an antibody (shown in Figures 5L-5N). While the antibody in Figures 5L-5N is depicted as an Fv, particularly an scFv, the scFv may be substituted with a Fab, as shown in Figures 39A-39B. [Figure 5L]5A-5X , which are schematic diagrams showing the structural orientation of additional embodiments of IL12-Fc fusion proteins that may be incorporated into the IL12 receptor agonists 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 may be incorporated into the IL12 fusion proteins of FIGS. 5A-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 FIGS. 5B-5S and 5V-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 of 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 (shown in Figures 5H-5K, 5O, 5R, 5S, and 5V-5X) or an antibody (shown in Figures 5L-5N). While the antibody in Figures 5L-5N is depicted as an Fv, particularly an scFv, the scFv may be substituted with a Fab, as shown in Figures 39A-39B. [Figure 5M]5A-5X , which are schematic diagrams showing the structural orientation of additional embodiments of IL12-Fc fusion proteins that may be incorporated into the IL12 receptor agonists 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 may be incorporated into the IL12 fusion proteins of FIGS. 5A-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 FIGS. 5B-5S and 5V-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 of 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 (shown in Figures 5H-5K, 5O, 5R, 5S, and 5V-5X) or an antibody (shown in Figures 5L-5N). While the antibody in Figures 5L-5N is depicted as an Fv, particularly an scFv, the scFv may be substituted with a Fab, as shown in Figures 39A-39B. [Figure 5N]5A-5X , which are schematic diagrams showing the structural orientation of additional embodiments of IL12-Fc fusion proteins that may be incorporated into the IL12 receptor agonists 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 may be incorporated into the IL12 fusion proteins of FIGS. 5A-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 FIGS. 5B-5S and 5V-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 of 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 (shown in Figures 5H-5K, 5O, 5R, 5S, and 5V-5X) or an antibody (shown in Figures 5L-5N). While the antibody in Figures 5L-5N is depicted as an Fv, particularly an scFv, the scFv may be substituted with a Fab, as shown in Figures 39A-39B. [Figure 5O]5A-5X , which are schematic diagrams showing the structural orientation of additional embodiments of IL12-Fc fusion proteins that may be incorporated into the IL12 receptor agonists 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 may be incorporated into the IL12 fusion proteins of FIGS. 5A-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 FIGS. 5B-5S and 5V-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 of 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 (shown in Figures 5H-5K, 5O, 5R, 5S, and 5V-5X) or an antibody (shown in Figures 5L-5N). While the antibody in Figures 5L-5N is depicted as an Fv, particularly an scFv, the scFv may be substituted with a Fab, as shown in Figures 39A-39B. [Figure 5P]5A-5X , which are schematic diagrams showing the structural orientation of additional embodiments of IL12-Fc fusion proteins that may be incorporated into the IL12 receptor agonists 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 may be incorporated into the IL12 fusion proteins of FIGS. 5A-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 FIGS. 5B-5S and 5V-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 of 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 (shown in Figures 5H-5K, 5O, 5R, 5S, and 5V-5X) or an antibody (shown in Figures 5L-5N). While the antibody in Figures 5L-5N is depicted as an Fv, particularly an scFv, the scFv may be substituted with a Fab, as shown in Figures 39A-39B. [Figure 5Q]5A-5X , which are schematic diagrams showing the structural orientation of additional embodiments of IL12-Fc fusion proteins that may be incorporated into the IL12 receptor agonists 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 may be incorporated into the IL12 fusion proteins of FIGS. 5A-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 FIGS. 5B-5S and 5V-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 of 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 (shown in Figures 5H-5K, 5O, 5R, 5S, and 5V-5X) or an antibody (shown in Figures 5L-5N). While the antibody in Figures 5L-5N is depicted as an Fv, particularly an scFv, the scFv may be substituted with a Fab, as shown in Figures 39A-39B. [Figure 5R]5A-5X , which are schematic diagrams showing the structural orientation of additional embodiments of IL12-Fc fusion proteins that may be incorporated into the IL12 receptor agonists 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 may be incorporated into the IL12 fusion proteins of FIGS. 5A-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 FIGS. 5B-5S and 5V-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 of 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 (shown in Figures 5H-5K, 5O, 5R, 5S, and 5V-5X) or an antibody (shown in Figures 5L-5N). While the antibody in Figures 5L-5N is depicted as an Fv, particularly an scFv, the scFv may be substituted with a Fab, as shown in Figures 39A-39B. [Figure 5S]5A-5X , which are schematic diagrams showing the structural orientation of additional embodiments of IL12-Fc fusion proteins that may be incorporated into the IL12 receptor agonists 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 may be incorporated into the IL12 fusion proteins of FIGS. 5A-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 FIGS. 5B-5S and 5V-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 of 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 (shown in Figures 5H-5K, 5O, 5R, 5S, and 5V-5X) or an antibody (shown in Figures 5L-5N). While the antibody in Figures 5L-5N is depicted as an Fv, particularly an scFv, the scFv may be substituted with a Fab, as shown in Figures 39A-39B. [Figure 5T] 5A-5C are diagrams depicting exemplary mechanisms of action of targeted IL12-Fc fusion proteins disclosed herein, eg, in FIGS. 5B-5S and 5V-5X. [Figure 5U] 5A-5C are diagrams depicting exemplary mechanisms of action of targeted IL12-Fc fusion proteins disclosed herein, eg, in FIGS. 5B-5S and 5V-5X. [Figure 5V]5A-5X , which are schematic diagrams showing the structural orientation of additional embodiments of IL12-Fc fusion proteins that may be incorporated into the IL12 receptor agonists 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 may be incorporated into the IL12 fusion proteins of FIGS. 5A-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 FIGS. 5B-5S and 5V-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 of 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 (shown in Figures 5H-5K, 5O, 5R, 5S, and 5V-5X) or an antibody (shown in Figures 5L-5N). While the antibody in Figures 5L-5N is depicted as an Fv, particularly an scFv, the scFv may be substituted with a Fab, as shown in Figures 39A-39B. [Figure 5W]5A-5X , which are schematic diagrams showing the structural orientation of additional embodiments of IL12-Fc fusion proteins that may be incorporated into the IL12 receptor agonists 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 may be incorporated into the IL12 fusion proteins of FIGS. 5A-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 FIGS. 5B-5S and 5V-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 of 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 (shown in Figures 5H-5K, 5O, 5R, 5S, and 5V-5X) or an antibody (shown in Figures 5L-5N). While the antibody in Figures 5L-5N is depicted as an Fv, particularly an scFv, the scFv may be substituted with a Fab, as shown in Figures 39A-39B. [Figure 5X]5A-5X , which are schematic diagrams showing the structural orientation of additional embodiments of IL12-Fc fusion proteins that may be incorporated into the IL12 receptor agonists 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 may be incorporated into the IL12 fusion proteins of FIGS. 5A-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 FIGS. 5B-5S and 5V-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 of 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 (shown in Figures 5H-5K, 5O, 5R, 5S, and 5V-5X) or an antibody (shown in Figures 5L-5N). While the antibody in Figures 5L-5N is depicted as an Fv, particularly an scFv, the scFv may be substituted with a Fab, as shown in Figures 39A-39B. [Figure 6] Figure 6 shows an alignment of mouse and human IL12 p35, with arrows indicating examples of representative mutein positions. Figure 6 discloses SEQ ID NOs: 126 and 6, respectively, in order of appearance. [Figure 7] Figure 7 shows an alignment of mouse and human IL12 p40, with arrows indicating examples of representative mutein positions. Figure 7 discloses SEQ ID NOs: 127 and 5, respectively, in order of appearance. [Figure 8] Figure 8 shows a sequence alignment of human IL12 p35 and other representative IL6 family cytokines. Arrows indicate the positions of representative amino acid substitutions. Figure 8 discloses SEQ ID NOS: 6, 128 to 132 in order of appearance. [Figure 9]The three-dimensional structures of IL12 (p35 and p40) are shown, highlighting residues potentially involved in the interaction of p35 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] Figure 10A is a photograph of an SDS-PAGE gel showing the size of IL12-Fc fusion proteins: Lane 1) Monovalent: IL12(p35xp40)-Fc; Lane 2) Monovalent: Fc-IL12(p35xp40); Lane 3) Monovalent: IL12*(p35*xp40*)-Fc. Figure 10B: 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 10B] Figure 10A is a photograph of an SDS-PAGE gel showing the size of IL12-Fc fusion proteins: Lane 1) Monovalent: IL12(p35xp40)-Fc; Lane 2) Monovalent: Fc-IL12(p35xp40); Lane 3) Monovalent: IL12*(p35*xp40*)-Fc. Figure 10B: 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]Figure 1 shows a trace from size-exclusion ultra-performance liquid chromatography (SEC) coupled with multi-angle light scattering (MALS) (SEC-MALS) demonstrating the size and configuration of monovalent IL12(p35 x 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 molecular weight of 122.5 kDa upon glycosylation. The fusion protein exhibited a 125.6 kDa monomeric protein with a peak area of ​​approximately 75% and two high molecular weight (HMV) species (peak 2, molecular weight approximately 258 kDa, peak area 18.1%) and (peak 1, peak area 6.0%). [Figure 12] Figure 1 shows a trace from SEC-MALS demonstrating the size and arrangement of Fc-monovalent:Fc-IL12 (p35 x p40). The predicted molecular weight of the fusion protein is 110.5 kDa. The fusion protein has six predicted glycosylation sites, resulting in a predicted molecular weight of 122.5 kDa upon 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] SEC-MALS trace showing the size and configuration 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 a predicted molecular weight of 122.5 kDa upon glycosylation. The fusion protein exhibited a peak area of ​​approximately 72% as a 128.6 kDa monomeric protein, with predicted dimeric and tetrameric oligomers detected (peaks 2 and 3). [Figure 14]

[0033] Figure 1 shows a trace from SEC-MALS demonstrating the size and configuration of 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 molecular weight of 190.0 kDa upon glycosylation. The fusion protein represents a largely aggregated protein, with high molecular weight species accounting for approximately 45% of the total peak area and an apparent molar mass of 1.7 MDa. [Figure 15] Figure 1 shows a trace from SEC-MALS demonstrating the size and configuration of 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 a predicted molecular weight of 195.2 kDa upon glycosylation. The fusion protein represented a 195.2 kDa monomeric protein with a total peak area of ​​approximately 70%. A predicted dimer was also detected (peak 2). [Figure 16] Figure 1 shows a trace from SEC-MALS demonstrating 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 molecular weight of 195.2 kDa upon glycosylation. The fusion protein represented a largely aggregated protein, with the predominant species at approximately 450 kDa with a peak area of ​​47.7%. [Figure 17] Figure 1 shows a trace from SEC-MALS demonstrating the size and arrangement of 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 a predicted molecular weight of 190.0 kDa upon glycosylation. The fusion protein exhibited a total peak area of ​​approximately 82% and represented a 198.9 kDa monomeric protein. A predicted dimer was also detected (peak 2). [Figure 18]SEC-MALS trace showing the size and configuration of 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 a predicted molecular weight of 195.2 kDa upon glycosylation. The fusion protein consisted primarily of monomeric species (total peak area approximately 60%) with an apparent molar mass of 201.0 kDa. [Figure 19] Curves depicting the biological activity of the indicated control or IL12-Fc fusion proteins on CTLL2 / STAT3-Luc cells are shown. [Figure 20A] Curves depicting the biological activity of the indicated control or IL12-Fc fusion proteins or muteins on CTLL2 / STAT3-Luc cells are shown. [Figure 20B] Curves depicting the biological activity of the indicated control or IL12-Fc fusion proteins or muteins on CTLL2 / STAT3-Luc cells are shown. [Figure 21] FIG. 1 is a schematic diagram depicting the experimental protocol in which C57BL / 6 mice were implanted with MC38 cancer cells and subsequently administered a test fusion protein. [Figure 22] 1 is a graph showing the effect of the indicated control or fusion proteins on tumor volume in a Mc38 tumor model. [Figure 23A] 1 is a graph showing the effect of the indicated control or fusion proteins on individual tumor growth in the MC38 tumor model. [Figure 23B] 1 is a graph showing the effect of the indicated control or fusion proteins on individual tumor growth in the MC38 tumor model. [Figure 23C] 1 is a graph showing the effect of the indicated control or fusion proteins on individual tumor growth in the MC38 tumor model. [Figure 23D] 1 is a graph showing the effect of the indicated control or fusion proteins on individual tumor growth in the MC38 tumor model. [Figure 23E]1 is a graph showing the effect of the indicated control or fusion proteins on individual tumor growth in the MC38 tumor model. [Figure 23F] 1 is a graph showing the effect of the indicated control or fusion proteins on individual tumor growth in the MC38 tumor model. [Figure 24] 1 is a graph showing the effect of the indicated control or fusion proteins on body weight change in mice in the MC38 tumor model. [Figure 25A] 1 shows the effect of the indicated control or fusion protein or mutein fusion protein on tumor volume and body weight change in the MC38 tumor model. [Figure 25B] 1 shows the effect of the indicated control or fusion protein or mutein fusion protein on tumor volume and body weight change in the MC38 tumor model. [Figure 26A] Traces from binding assays are shown, demonstrating binding of the indicated IL12-Fc fusion proteins to primary murine T cells. [Figure 26B] Traces from binding assays are shown, demonstrating binding of the indicated IL12-Fc fusion proteins to primary murine T cells. [Figure 27A] 1 shows traces from a pSTAT4-based bioassay demonstrating the effect of the indicated IL12-Fc fusion proteins on pSTAT4 activity in primary mouse T cells. [Figure 27B] 1 shows traces from a pSTAT4-based bioassay demonstrating the effect of the indicated IL12-Fc fusion proteins on pSTAT4 activity in primary mouse T cells. [Figure 28] 1 shows traces from a STAT3-based bioassay demonstrating the effect of the indicated IL12-Fc fusion proteins on STAT3 activity in NK92 cells (NK92 / STAT3-Luc cl.7F7). [Figure 29]1 shows traces from a STAT3-based bioassay demonstrating the effect of the indicated IL12-Fc fusion proteins on STAT3 activity in NK92 cells (NK92 / STAT3-Luc cl.7F7). [Figure 30] 1 shows traces from a pSTAT4-based bioassay demonstrating the effect of the indicated IL12-Fc fusion proteins on pSTAT4 activity in primary mouse T cells. [Figure 31] 1 shows traces from a STAT3-based bioassay demonstrating the effect of the indicated IL12-Fc fusion proteins on STAT3 activity in NK92 cells (NK92 / STAT3-Luc cl.7F7). [Figure 32] 1 shows traces from a STAT3-based bioassay demonstrating 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 shows traces from a STAT3-based bioassay demonstrating the effect of the indicated IL12-Fc fusion proteins on STAT3 activity in human NK92 cells (NK92 / STAT3-Luc cl.7F7). [Figure 34] 1 shows traces from a STAT3-based bioassay demonstrating the effect of the indicated IL12-Fc fusion proteins on STAT3 activity in murine HT-2 cells. [Figure 35A] We show that receptor-masked Fc-IL12 reduces toxicity and maintains levels of antitumor activity in vivo. [Figure 35B] We show that receptor-masked Fc-IL12 reduces toxicity and maintains levels of antitumor activity in vivo. [Figure 35C] We show that receptor-masked Fc-IL12 reduces toxicity and maintains levels of antitumor activity in vivo. [Figure 36A]We show that PD1-targeted receptor-masked IL12 has targeted antitumor efficacy without concomitant weight loss and reduction in systemic IFNγ. [Figure 36B] We show that PD1-targeted receptor-masked IL12 has targeted antitumor efficacy without concomitant weight loss and reduction in systemic IFNγ. [Figure 36C] We show that PD1-targeted receptor-masked IL12 has targeted antitumor efficacy without concomitant weight loss and reduction in systemic IFNγ. [Figure 36D] We show that PD1-targeted receptor-masked IL12 has targeted antitumor efficacy without concomitant weight loss and reduction in systemic IFNγ. [Figure 36E] We show that PD1-targeted receptor-masked IL12 has targeted antitumor efficacy without concomitant weight loss and reduction in systemic IFNγ. [Figure 37A] We show that PD1-targeted receptor-masked IL12 has superior antitumor efficacy to PD-1 blockade or the combination of non-targeted receptor-masked IL12 and PD-1 blockade. [Figure 37B] We show that PD1-targeted receptor-masked IL12 has superior antitumor efficacy to PD-1 blockade or the combination of non-targeted receptor-masked IL12 and PD-1 blockade. [Figure 38A] We show that PD1-targeted receptor-masked IL12 has targeted antitumor efficacy without weight loss and minimal systemic IFNγ. [Figure 38B-1] We show that PD1-targeted receptor-masked IL12 has targeted antitumor efficacy without weight loss and minimal systemic IFNγ. [Figure 38B-2] We show that PD1-targeted receptor-masked IL12 has targeted antitumor efficacy without weight loss and minimal systemic IFNγ. [Figure 38C-1]We show that PD1-targeted receptor-masked IL12 has targeted antitumor efficacy without weight loss and minimal systemic IFNγ. [Figure 38C-2] We show that PD1-targeted receptor-masked IL12 has targeted antitumor efficacy without weight loss and minimal systemic IFNγ. [Figure 38C-3] We show that PD1-targeted receptor-masked IL12 has targeted antitumor efficacy without weight loss and minimal systemic IFNγ. [Figure 38C-4] We show that PD1-targeted receptor-masked IL12 has targeted antitumor efficacy without weight loss and minimal systemic IFNγ. [Figure 38C-5] We show that PD1-targeted receptor-masked IL12 has targeted antitumor efficacy without weight loss and minimal systemic IFNγ. [Figure 39A] 39A and 39B are diagrams depicting exemplary antibody-masked IL12 / Fc fusion constructs. The masking antibody is depicted as a Fab, although as shown in Figures 5L-5N, the Fab may be replaced with an Fv. The constructs depicted in Figures 39A and 39B may further comprise a targeting moiety. Figures 39C and 39D depict embodiments of the constructs of Figures 39A and 39B, respectively, with a targeting moiety at their N-terminus. In Figures 39C and 39D, the targeting moiety is depicted as a Fab, although other formats can be used. Thus, embodiments described herein (including Section 6.2, Groups A and B, numbered embodiments, and claims) encompassing the formats of Figures 39A and 39B may further comprise a targeting moiety, e.g., a targeting moiety described in Section 6.5.2, along with the Fab formats illustrated in Figures 39A and 39D. [Figure 39B]39A and 39B are diagrams depicting exemplary antibody-masked IL12 / Fc fusion constructs. The masking antibody is depicted as a Fab, although as shown in Figures 5L-5N, the Fab may be replaced with an Fv. The constructs depicted in Figures 39A and 39B may further comprise a targeting moiety. Figures 39C and 39D depict embodiments of the constructs of Figures 39A and 39B, respectively, with a targeting moiety at their N-terminus. In Figures 39C and 39D, the targeting moiety is depicted as a Fab, although other formats can be used. Thus, embodiments described herein (including Section 6.2, Groups A and B, numbered embodiments, and claims) encompassing the formats of Figures 39A and 39B may further comprise a targeting moiety, e.g., a targeting moiety described in Section 6.5.2, along with the Fab formats illustrated in Figures 39A and 39D. [Figure 39C] 39A and 39B are diagrams depicting exemplary antibody-masked IL12 / Fc fusion constructs. The masking antibody is depicted as a Fab, although as shown in Figures 5L-5N, the Fab may be replaced with an Fv. The constructs depicted in Figures 39A and 39B may further comprise a targeting moiety. Figures 39C and 39D depict embodiments of the constructs of Figures 39A and 39B, respectively, with a targeting moiety at their N-terminus. In Figures 39C and 39D, the targeting moiety is depicted as a Fab, although other formats can be used. Thus, embodiments described herein (including Section 6.2, Groups A and B, numbered embodiments, and claims) encompassing the formats of Figures 39A and 39B may further comprise a targeting moiety, e.g., a targeting moiety described in Section 6.5.2, along with the Fab formats illustrated in Figures 39A and 39D. [Figure 39D]39A and 39B are diagrams depicting exemplary antibody-masked IL12 / Fc fusion constructs. The masking antibody is depicted as a Fab, although as shown in Figures 5L-5N, the Fab may be replaced with an Fv. The constructs depicted in Figures 39A and 39B may further comprise a targeting moiety. Figures 39C and 39D depict embodiments of the constructs of Figures 39A and 39B, respectively, with a targeting moiety at their N-terminus. In Figures 39C and 39D, the targeting moiety is depicted as a Fab, although other formats can be used. Thus, embodiments described herein (including Section 6.2, Groups A and B, numbered embodiments, and claims) encompassing the formats of Figures 39A and 39B may further comprise a targeting moiety, e.g., a targeting moiety described in Section 6.5.2, along with the Fab formats illustrated in Figures 39A and 39D. [Figure 40A-1] Shown are traces from a STAT3-based bioassay in NK92 cells for PD1-targeted mIL12 with an R1 mask or an scFv mask. [Figure 40A-2] Shown are traces from a STAT3-based bioassay in NK92 cells for PD1-targeted mIL12 with an R1 mask or an scFv mask. [Figure 40B] Shown are traces from a STAT3-based bioassay in NK92 cells for PD1-targeted mIL12 with an R1 mask or an scFv mask. [Figure 41A] Figure 1 shows that IL12 masked with a PD1-targeting antibody inhibits tumor growth without weight loss and minimal systemic IFNγ. [Figure 41B] Figure 1 shows that IL12 masked with a PD1-targeting antibody inhibits tumor growth without weight loss and minimal systemic IFNγ. [Figure 41C] Figure 1 shows that IL12 masked with a PD1-targeting antibody inhibits tumor growth without weight loss and minimal systemic IFNγ. [Figure 41D]Figure 1 shows that IL12 masked with a PD1-targeting antibody inhibits tumor growth without weight loss and minimal systemic IFNγ. [Figure 42] Combining a receptor mask for one IL12 subunit with a p40 mutein shows further attenuation of activity compared to the receptor mask alone. [Figure 43] Combining a receptor mask for one IL12 subunit with a p35 mutein shows further attenuation of activity compared to the receptor mask alone. [Figure 44] We show that a receptor-masked "three-chain" format protein construct attenuates the biological activity of IL12. [Figure 45] 1 shows an exemplary format of receptor-masked IL12 with target-enhanced bioactivity. [Figure 46A] The protocol for in vivo administration of a receptor-masked "three-chain" format protein construct is shown (Figure 46A), and the resulting activity on tumor growth (Figure 46B), weight loss (Figure 46C), and IFNγ production (Figure 46D) is shown. [Figure 46B] The protocol for in vivo administration of a receptor-masked "three-chain" format protein construct is shown (Figure 46A), and the resulting activity on tumor growth (Figure 46B), weight loss (Figure 46C), and IFNγ production (Figure 46D) is shown. [Figure 46C] The protocol for in vivo administration of a receptor-masked "three-chain" format protein construct is shown (Figure 46A), and the resulting activity on tumor growth (Figure 46B), weight loss (Figure 46C), and IFNγ production (Figure 46D) is shown. [Figure 46D] The protocol for in vivo administration of a receptor-masked "three-chain" format protein construct is shown (Figure 46A), and the resulting activity on tumor growth (Figure 46B), weight loss (Figure 46C), and IFNγ production (Figure 46D) is shown. DETAILED DESCRIPTION OF THE INVENTION

[0012] 6.1.Definition About, Approximately: The terms "about," "approximately," and the like are used throughout this specification preceding numerical values ​​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 number, should also be understood to 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 stated, 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 (A or B, where the selection of A is mutually exclusive of B) and the simultaneous selection of features (A or B, where both A and B are selected). In some places in this text, the term "and / or" is used interchangeably and should not be interpreted to suggest that "or" is used to refer to mutually exclusive alternatives.

[0014] Antigen Binding Domain or ABD: As used herein, the term "antigen binding domain" or "ABD" refers to a 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 one another, e.g., non-covalently via molecular interactions, or covalently via 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 a 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 via the bloodstream and lymphatic system to other parts of the body. Examples of various cancers are described herein and include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colon cancer, kidney cancer, liver cancer, brain tumors, 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: As used herein, the term "complementarity-determining region" or "CDR" 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 for identifying CDR sequences within antibodies are also available.

[0019] EC50: The term "EC50" refers to the half-maximal effective concentration of a molecule (e.g., an IL12 receptor agonist) that induces a response halfway between baseline and maximum after a particular 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 a targeting moiety of the present disclosure refers to a pair of polypeptide chains, the first of which comprises the antibody variable heavy (VH) domain N-terminal to a first constant domain (referred to herein as C1), and the second of which comprises the antibody variable light (VL) domain N-terminal to 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 of the heavy chain (CH1), and the VL is N-terminal to the light chain constant domain (CL). The Fabs of the present disclosure may be oriented according to their native orientation or may include domain substitutions or swaps that facilitate correct VH and VL pairing. For example, replacing the CH1 and CL domain pair in a Fab with a CH3 domain pair can facilitate correct modified Fab chain pairing in a heterodimeric molecule. It is also possible to reverse CH1 and CL, so that CH1 is attached to VL and CL to VH; this configuration is generally 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 the same 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 allow 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 to the progeny or potential progeny of such a cell. Because certain modifications may occur in successive generations, either due to mutation or environmental influences, such progeny may not actually be identical to the parent cell, but are still 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 and refer to a molecule that comprises or consists of an IL12 mutein and has IL12 activity. The IL12 activity may 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 "internal IL12 moiety linker" refers to a linker connecting two IL12 moieties, for example 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, for example, (i) a multimerization moiety (e.g., a dimerization domain such as an Fc domain) domain and / or (ii) a targeting moiety and / or (iii) a stabilizing moiety and / or (iv) an IL12βR moiety.

[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" optionally refers to the core components of a variant IL12 molecule, i.e., the p35 and p40 moieties, and optionally also to a multimerizing moiety, e.g., an Fc domain and any / or associated linker moieties; it will be understood that unless the context indicates otherwise, the term "IL12 mutein" also extends to an IL12 molecule comprising 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.

[0028] Thus, an IL12 mutein can comprise 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 may comprise an IL12Rβ2 portion, and the p40 portion may comprise an IL12Rβ1 portion. The p35 portion and the IL12Rβ2 portion may be on the same or different polypeptide chains. The p40 portion and the IL12Rβ1 portion may be on the same or different polypeptide chains. The IL12Rβ1 portion and the IL12Rβ2 portion generally function as masking moieties and, therefore, when present, are typically configured to interact with the p40 portion and the p35 portion, 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 subparagraphs therein, 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 contained on the same polypeptide chain or on different polypeptide chains. Exemplary configurations of 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. Exemplary masking moieties of the present disclosure are disclosed in, among other places, Figures 4B-4E, 4G-4W, 5H-5O, 5R-5S, 5V-5X, and 39A-39D, and in the numbered embodiments disclosed in Section 6.4 and in Section 7 below, which refer to these Figures and / or their constituent exemplary monomers.

[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 in the numbered embodiments disclosed, inter alia, in Figures 4O-4R, 5L-5N, and 39A-39D, Sections 6.2 and 6.4, and in Section 7 below, which refers to these figures and / or their constituent exemplary monomers. Exemplary receptor-based masking moieties, and IL12 receptor agonists comprising them, are disclosed in the numbered embodiments 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 Section 7 below, which refers to these figures and / or their constituent exemplary monomers.

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

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

[0035] 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).

[0036] 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 has one or more amino acid substitutions, as defined in Section 6.3.2, below.

[0037] In various embodiments, the p35 portion of an IL12 mutein of the present disclosure retains (a) none, either one, or both interchain disulfide bonds, and / or (b) any combination of cysteines that form interchain bonds with p40.

[0038] 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)).

[0039] The p40 portion preferably comprises the D2 and D3 domains of mammalian, e.g., human or murine, 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.

[0040] The p40 portion can also comprise the D1 domain of mammalian, e.g., human or murine, 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 D1 domain, optionally with one or more amino acid substitutions as defined in Section 6.3.1 below.

[0041] In various embodiments, the p40 portion of an IL12 mutein of the present disclosure retains any combination of (a) none, any one, any two, or all three interchain disulfide bonds, and / or (b) a cysteine ​​that forms an interchain bond with p35, and / or (c) the conserved WSXWS motif (SEQ ID NO: 3).

[0042] IL12Rβ1 Moiety: An IL12Rβ1 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 IL12 p40-binding portion of a mammalian, e.g., human or murine, IL12 receptor subunit beta-1 (IL12Rβ1). The IL12 p40-binding portion of IL12Rβ1 comprises or consists of the extracellular domain of the receptor subunit. 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).

[0043] The IL12Rβ1 portion preferably comprises 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 extracellular domain).

[0044] IL12Rβ moiety: As used herein, the term IL12Rβ moiety refers to an IL12Rβ1 moiety or an IL12Rβ2 moiety. IL12Rβ2 Moiety: An IL12Rβ2 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 IL12 p35-binding portion of a mammalian, e.g., human or murine, IL12 receptor subunit beta-2 (IL12Rβ2). The IL12 p35-binding portion of IL12Rβ2 comprises or consists of the extracellular domain of the receptor subunit. The sequence of human IL12Rβ 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).

[0045] The IL12Rβ2 portion preferably comprises 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 extracellular domain).

[0046] 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), mutants, and various derivatives thereof (including fusion proteins), which retain the ability to present 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 chain that can accommodate peptides of approximately 8-10 amino acids. Despite the fact that both MHC classes 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 α 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 are typically 13-17 amino acids long, although shorter or longer lengths are not uncommon. As a result, peptides may shift within the MHC class II peptide-binding groove, and which 9-mer is directly located within the groove may change at any given time. Conventional identification of specific MHC variants is used herein. This term encompasses "human leukocyte antigen" or "HLA."

[0047] 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 moiety or an IL12Rβ2 moiety). In other embodiments, the masking moiety is an anti-IL12 (e.g., an anti-p35 or anti-p40) antibody fragment.

[0048] 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 the second polypeptide chain; (c) comprises a p40 portion and is capable of associating with a p35 portion on the 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 the second polypeptide chain; or (e) is any combination of (a), (b), (c), and (d) above. Thus, a monomer can associate with other monomers via p35 / p40 portion pairing and / or multimerization portion (e.g., Fc domain) pairing. In some embodiments, one or more associations between monomers are stabilized via interchain disulfide bridges, such as those at the p35 / p40 interface, or via 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 may 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 may contain more than two polypeptide chains, for example, three or four polypeptide chains.

[0049] Monomeric p40 or monomeric p40 polypeptide chain: The terms "monomeric p40," "monomeric p40 polypeptide chain," and the like refer to a polypeptide chain that does not include a dimerization moiety, e.g., does not include an Fc domain, and includes an IL12-p40 moiety. The monomeric p40 polypeptide chain can optionally include a p40 masking moiety (e.g., a p40-binding moiety of IL12Rβ1 or an anti-p40 antibody-based masking moiety). Such a polypeptide chain may be referred to herein as a "masked monomeric p40."

[0050] 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 that has only a single IL12 heterodimer (i.e., one p40 x p35 heterodimer) and / or targeting moiety, respectively.

[0051] Operably linked: As used herein, the term "operably linked" refers to a functional relationship between two or more regions of a polypeptide chain, which are linked to provide a functional polypeptide or two or more nucleic acid sequences, e.g., to provide an in-frame fusion of two polypeptide components or to link a regulatory sequence to a coding sequence.

[0052] Peptide-MHC complex, pMHC complex, intragroove peptide: "Peptide-MHC complex," "pMHC complex," and "intragroove peptide" refer to (i) an MHC domain (e.g., a human MHC molecule or a 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 a portion thereof), wherein the MHC domain, antigenic peptide, and optional β2 microglobulin domain are complexed in a manner that enables 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.

[0053] 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.

[0054] Specific (or selective) binding: As used herein, the term "specifically (or selectively) binding" means that a targeting moiety, e.g., an antibody, or antigen-binding domain ("ABD") thereof, forms a complex with a target molecule that is relatively stable under physiological conditions. Specific binding is defined as a binding affinity of approximately 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 component targeting moiety, to a target molecule are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance (e.g., Biacore assay), fluorescence-activated cell sorting (FACS) binding assay, etc. However, 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 have cross-reactivity to target molecules from one or more other species.

[0055] 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. Unless otherwise specified, the terms "patient" and "subject" are used interchangeably herein.

[0056] 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.

[0057] 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 a site where an IL12 agonist of the present disclosure is localized, e.g., on a tumor cell or on a lymphocyte in the tumor microenvironment. A targeting moiety may also have functional activity in addition to localizing an IL12 receptor agonist to a specific site. For example, a targeting moiety that is an anti-PD1 antibody or an antigen-binding portion thereof may also exhibit anti-tumor activity or enhance the anti-tumor activity of an IL12 mutein by inhibiting PD1 signaling.

[0058] 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 specific 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 inhibiting the progression of a proliferative disorder physically (e.g., by stabilization of a discernible symptom), physiologically (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.

[0059] 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 pre-malignant and malignant cancers and tumors.

[0060] 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 entirely or as fragments (e.g., MHC / peptide), and that is useful for preferential targeting of drugs to cancer cells. In some embodiments, a TAA is a marker expressed by both normal and cancer cells, such as a lineage marker, e.g., CD19 on B cells. In some embodiments, a TAA is a cell surface molecule that is overexpressed in cancer cells compared to normal cells, e.g., a cell surface molecule that is overexpressed 1-fold, 2-fold, 3-fold, or more compared to normal cells. In some embodiments, a 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, a TAA is expressed entirely or as fragments (e.g., MHC / peptide) only on the cell surface of cancer cells and is not synthesized or expressed on the surface of normal cells. Thus, the term "TAA" encompasses antigens specific to cancer cells, sometimes referred to in the art as tumor-specific antigens ("TSAs").

[0061] Universal light chain: As used herein in the context of a targeting moiety, the term "universal light chain" refers to a light chain polypeptide that can pair with a heavy chain region of a targeting moiety and also with other heavy chain regions. A universal light chain is also known as a "common light chain."

[0062] 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.

[0063] 6.2. IL-12 receptor agonists The present disclosure provides IL12 receptor agonists comprising or consisting of an IL12 mutein. The IL12 mutein comprises a p35 portion and a p40 portion, which differ from wild-type IL12 by (a) primary amino acid sequence (e.g., amino acid insertion, deletion, or substitution compared to p35 and / or p40, or any combination of the foregoing), and / or (b) association with an additional domain not naturally associated with IL12, such as (i) a multimerization moiety (e.g., a dimerization domain such as an Fc domain) domain and / or (ii) a targeting moiety and / or (iii) a stabilizing moiety and / or (iv) an IL12β receptor (IL12βR1 and / or IL12βR2) sequence.

[0064] The IL12 receptor agonists of the present disclosure and / or IL12 muteins therein may have amino acid modifications that result in reduced binding affinity to the IL12 receptor complex (e.g., a receptor complex comprising IL12Rβ1 and IL12Rβ2) compared to wild-type IL12. Overall, the IL12 receptor agonists of the present disclosure and / or IL12 muteins therein may have normal or reduced 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 reduced 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.

[0065] 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 thereafter. For example, in some embodiments, an IL12 mutein can reduce binding to the human IL12 receptor complex 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, e.g., substitution W37A, that reduces binding to IL12Rβ1.

[0066] Certain aspects of the present disclosure relate to IL12 receptor agonists comprising: (a) a first polypeptide chain comprising, from N- to C-terminus, a first targeting moiety or targeting moiety component, a first Fc domain, and a p35 moiety; (b) a second polypeptide chain comprising, from N- to C-terminus, 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 the p40 moiety in the form of a 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 p35 moiety may have attenuating substitutions, e.g., as described in Section 6.3. In some embodiments, the p40 moiety has an amino acid substitution, e.g., substitution W37A, at a position corresponding to amino acid W37 of full-length human p40 or full-length mouse p40.

[0067] A further aspect of the present disclosure relates to an IL12 receptor agonist comprising an IL12 mutein, the IL12 receptor agonist having at least 500-fold attenuation compared to wild-type IL12, the IL12 receptor agonist comprising: (a) a first polypeptide chain and a second polypeptide chain dimerized via 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.

[0068] 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 via a first Fc domain and a second Fc domain, wherein: (i) the p35 portion comprises an attenuating amino acid substitution, optionally the attenuating amino acid substitution being: (A) amino acid Y189 of full-length human p35 or amino acid Y185 of full-length mouse p35 (the substitution is optionally A, V, R, or E); (B) amino acid 1193 of full-length human p35 or amino acid M189 of full-length mouse p35 (wherein the substitution is optionally A, V, or E); (C) amino acid R211 of full-length human p35 or amino acid R207 of full-length mouse p35 (wherein the substitution is optionally A or K); or (D) any combination of (A)-(C); and / or (ii) the p40 portion comprises an attenuating amino acid substitution, optionally the attenuating amino acid substitution being (A) amino acid K28 of full-length human p40 or amino acid K28 of full-length mouse p40 ( (B) amino acid W37 of full length human p40 or amino acid W37 of full length mouse p40 (the substitution is optionally A); (C) amino acid D115 of full length human p40 or amino acid E115 of full length mouse p40 (the substitution is optionally A); (D) amino acid K118 of full length human p40 or amino acid K118 of full length mouse p40 (the substitution is optionally A); (E) amino acid K126 of full length human p40 or amino acid K126 of full length mouse p40 (the substitution is optionally A); (F) an IL12 mutein comprising a p35 portion and a p40 portion, the mutein comprising amino acid Y268 of full-length human p40 or amino acid Y265 of full-length mouse p40 (optionally with a V or F substitution), (G) amino acid Y314 of full-length human p40 or amino acid Y318 of full-length mouse p40 (optionally with a F substitution), or (H) any combination of (A) to (G); and (c) an IL12Rβ portion or an IL12 antibody fragment configured to mask the p35 portion or the p40 portion.

[0069] 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).

[0070] In some embodiments, the IL12 receptor agonists and IL12 muteins of the present disclosure may comprise an IL12 receptor sequence, e.g., an IL12Rβ1 sequence and / or an IL12Rβ2 sequence, which is believed to attenuate the side effects of IL12 receptor agonist treatment, as described in Section 6.4 and thereafter.

[0071] 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 portion and / or a p35 portion, and in some embodiments, also includes a multimerization portion.

[0072] The IL12 receptor agonist or IL12 mutein may further comprise one or more targeting moieties and / or 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 may be a dual-purpose domain, conferring the stabilizing properties of the stabilizing moiety, as described in Section 6.7.

[0073] Exemplary targeting moieties are described in Section 6.4 and include antigen-binding domains (e.g., scFv or Fab) that bind to tumor-associated antigens, bind to tumor microenvironment antigens, or bind to tumor lymphocytes, and peptide-MHC complexes that recognize tumor lymphocytes.

[0074] 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.

[0075] 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.

[0076] 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 may 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 an Fc domain.

[0077] It has been discovered that incorporating the p40 and p35 moieties at the C-terminus of an Fc domain configured such that the p40 moiety is N-terminal to the p35 moiety improves expression yields and reduces aggregation, resulting 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 and Fc domains), 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 a p35 moiety in one of the two monomers.

[0078] In some embodiments, an IL12 mutein or IL12 agonist can comprise one or more linker sequences connecting various components of one or more polypeptide chains thereof, such as: (1) the p35 portion and p40 portion of IL12 when present on the same polypeptide chain; (2) the p35 portion and a multimerization domain (e.g., an Fc domain); (3) the p40 portion and a multimerization domain (e.g., an Fc domain); (4) the p35 portion and a targeting portion or a component thereof (e.g., the heavy chain of an scFv or Fab); (5) the p40 portion and a targeting portion or a component thereof (e.g., the heavy chain of an scFv or Fab); (6) a multimerization domain (e.g., an Fc domain) and a targeting portion or a component thereof (e.g., the heavy chain of an scFv or Fab); (7) the p35 portion, the p40 portion, the multimerization domain, or the targeting portion or a component thereof, and an IL12βR portion, e.g., an IL12βR1 portion or an IL12βR2 portion; or (8) any combination of the above. Exemplary linkers are described in Section 6.8.

[0079] 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 polypeptide chains comprising 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 "monomers" or "IL12 monomers," respectively. Below are some illustrative examples of IL12 monomers of the present disclosure, listed from N- to C-terminal. The individual elements of each monomer are described in detail herein, e.g., in the following sections and numbered embodiments.

[0080] (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)).

[0081] (2) Exemplary Monomer 2: IL12 p40 moiety-optional linker-multimerization moiety (see, for example, the right monomer in Figure 2B and the right monomer in Figure 2C). (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)).

[0082] (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)).

[0083] (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)).

[0084] (6) Exemplary Monomer 6: IL12 p35 moiety-optional linker-IL12 p40 moiety-optional linker-multimerization moiety (see, e.g., both monomers in Figure 2J).

[0085] (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).

[0086] (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)).

[0087] (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.

[0088] (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.

[0089] (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.

[0090] (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).

[0091] (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.

[0092] (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.

[0093] (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.

[0094] (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.

[0095] (17) Exemplary Monomer 17: IL12 p35 moiety-optional linker-multimerization moiety-optional linker-targeting moiety (see, for example, the left monomer in Figure 5G). (18) Exemplary Monomer 18: IL12 p40 moiety-optional linker-multimerization moiety-optional linker-targeting moiety (see, for example, the right monomer in Figure 5G).

[0096] (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)).

[0097] (20) Exemplary monomer 20: targeting moiety-optional linker-multimerization moiety-optional linker-IL12 p40 moiety (see, e.g., the right monomer in Figure 5B). (21) Exemplary monomer 21: targeting moiety-optional linker-IL12 p35 moiety-optional linker-multimerization moiety.

[0098] (22) Exemplary Monomer 22: targeting moiety-optional linker-IL12 p40 moiety-optional linker-multimerization moiety. (23) Exemplary Monomer 23: multimerization moiety-optional linker-IL12 p35 moiety-optional linker-targeting moiety.

[0099] (24) Exemplary Monomer 24: multimerization moiety-optional linker-IL12 p40 moiety-optional linker-targeting moiety. (25) Exemplary Monomer 25: IL12 p40 moiety-optional linker-IL12 p35 moiety-optional linker-multimerization moiety-optional linker-targeting moiety (see, e.g., both monomers in Figure 5F).

[0100] (26) Exemplary Monomer 26: IL12 p35 moiety-optional linker-IL12 p40 moiety-optional linker-multimerization moiety-optional linker-targeting moiety. (27) Exemplary Monomer 27: targeting moiety-optional linker-multimerization moiety-optional linker-IL12 p35 moiety-optional linker-IL12 p40 moiety.

[0101] (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)).

[0102] (29) Exemplary Monomer 29: targeting moiety-optional linker-IL12 p40 moiety-optional linker-IL12 p35 moiety-optional linker-multimerization moiety. (30) Exemplary monomer 30: targeting moiety-optional linker-IL12 p35 moiety-optional linker-IL12 p40 moiety-optional linker-multimerization moiety.

[0103] (31) Exemplary monomer 31: multimerization moiety-optional linker-IL12 p35 moiety-optional linker-IL12 p40 moiety-optional linker-targeting moiety. (32) Exemplary Monomer 32: multimerization moiety-optional linker-IL12 p40 moiety-optional linker-IL12 p35 moiety-optional linker-targeting moiety.

[0104] (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)).

[0105] (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.

[0106] (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.

[0107] (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.

[0108] (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.

[0109] (39) Exemplary Monomer 39: 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-optional linker-targeting moiety. In some embodiments, the IL12Rβ moiety is an IL12Rβ1 moiety. In other embodiments, the IL12Rβ moiety is an IL12Rβ2 moiety.

[0110] (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.

[0111] (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.

[0112] (42) Exemplary Monomer 42: multimerization 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-targeting moiety. In some embodiments, the IL12Rβ moiety is an IL12Rβ1 moiety. In other embodiments, the IL12Rβ moiety is an IL12Rβ2 moiety.

[0113] (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.

[0114] (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.

[0115] (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.

[0116] (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.

[0117] (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.

[0118] (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.

[0119] (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.

[0120] (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.

[0121] (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.

[0122] (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.

[0123] (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.

[0124] (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)).

[0125] (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)).

[0126] (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.

[0127] (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)).

[0128] (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)).

[0129] (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)).

[0130] (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.

[0131] (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.

[0132] (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)).

[0133] 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).

[0134] 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 an IL12 receptor agonist comprising exemplary monomer 3 and exemplary monomer 4 (see, eg, Figures 2D and 2E).

[0135] 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.

[0136] 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 an IL12 receptor agonist 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.

[0137] 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).

[0138] In some embodiments, the present disclosure provides IL12 receptor agonists comprising exemplary monomer 5 and exemplary monomer 33 (see, eg, Figures 5E, 5P, and 5Q). In some embodiments, the present disclosure provides an IL12 receptor agonist comprising two monomers according to exemplary monomer 6 (see, eg, Figure 2J).

[0139] In some embodiments, the present disclosure provides an IL12 receptor agonist comprising two monomers according to exemplary monomer 7 (see, eg, Figure 2L). In some embodiments, the present disclosure provides an IL12 receptor agonist comprising exemplary monomer 7 and exemplary monomer 60 (see, eg, FIG. 2G).

[0140] 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). In some embodiments, the present disclosure provides IL12 receptor agonists comprising exemplary monomer 8 and exemplary monomer 60 (see, eg, Figures 2F and 4F).

[0141] In some embodiments, the present disclosure provides IL12 receptor agonists comprising exemplary monomer 8 and exemplary monomer 51 (see, eg, Figures 4J, 4K, and 4S). 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).

[0142] 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 may both be IL12Rβ1 moieties, both be IL12Rβ2 moieties, or a combination of an IL12Rβ1 moiety and an IL12Rβ2 moiety.

[0143] In some embodiments, the present disclosure provides IL12 receptor agonists comprising exemplary monomer 9 and exemplary monomer 60 (see, eg, Figures 4I and 4T). 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 the monomer according to exemplary monomer 11 may both be IL12Rβ1 moieties, both be IL12Rβ2 moieties, or a combination of an IL12Rβ1 moiety and an IL12Rβ2 moiety.

[0144] In some embodiments, the present disclosure provides IL12 receptor agonists comprising exemplary monomer 11 and exemplary monomer 60 (see, eg, Figures 4G and 4H). In some embodiments, the present disclosure provides an IL12 receptor agonist comprising exemplary monomer 15 and exemplary monomer 33 (see, e.g., FIG. 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.

[0145] 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).

[0146] In some embodiments, the present disclosure provides an IL12 receptor agonist comprising 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.

[0147] 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.

[0148] 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).

[0149] In some embodiments, the present disclosure provides an IL12 receptor agonist 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.

[0150] In some embodiments, the present disclosure provides IL12 receptor agonists comprising exemplary monomer 28 and exemplary monomer 59 (see, eg, Figures 5M, 5N, 39C, and 39D).

[0151] In some embodiments, the present disclosure provides IL12 receptor agonists comprising 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.

[0152] In some embodiments, the present disclosure provides an IL12 receptor agonist 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 an IL12Rβ1 moiety and an IL12Rβ2 moiety. 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.

[0153] In some embodiments, the present disclosure provides IL12 receptor agonists comprising exemplary monomer 55 and exemplary monomer 60 (see, eg, Figures 4O and 4P). In some embodiments, the present disclosure provides an IL12 receptor agonist comprising exemplary monomer 57 and exemplary monomer 63 (see, eg, FIG. 5S).

[0154] Further embodiments of exemplary monomer pairings are described in the numbered embodiments disclosed in Section 7. In the aforementioned IL12 receptor agonist embodiments comprising two exemplary monomers having IL12Rβ moieties, the IL12Rβ moieties may both be IL12Rβ1 moieties, both be IL12Rβ2 moieties, or a combination of an IL12Rβ1 moiety and an IL12Rβ2 moiety.

[0155] 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 polypeptide chain having a heavy chain variable region and the other polypeptide chain having a light chain variable region. The targeting moiety itself can comprise a heavy chain variable domain and a light chain variable domain on separate polypeptide chains. For example, with respect to 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.

[0156] 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 greater than 1, 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.

[0157] 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.

[0158] 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 may be rapidly cleared by phagocytosis, leading to reduced antibody efficacy. Large complexes can also increase the immunogenicity of therapeutic antibodies. See, for example, International Publication No. WO2020047067(A1). 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, the IL12 receptor agonists of the present disclosure exhibit at least 50%, at least 60%, at least 70%, at least 80%, at least 95%, or at least 99% less aggregation during recombinant production in mammalian cell lines than IL12 receptor agonists with alternative structures. The oligomerization state of the IL12 receptor agonist can be determined, for example, by size-exclusion ultra-performance liquid chromatography.

[0159] 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 result in 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, such as differential scanning fluorimetry (DSF).

[0160] 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 with 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.

[0161] 6.3.1.IL12 p40 part Each IL12 p40 moiety of an IL12 receptor agonist of the present disclosure comprises a wild-type or variant IL12 p40 moiety. In some embodiments, an IL12 receptor agonist of the present disclosure comprises a single IL12 p40 moiety (e.g., an IL12 p40 moiety on the first monomer or on the second monomer in embodiments in which the IL12 receptor agonist is monovalent for IL12). In some embodiments, an IL12 receptor agonist of the present disclosure comprises two IL12 p40 moieties (e.g., a first IL12 p40 moiety on the first monomer and a second IL12 p40 moiety on the second monomer in embodiments in which the IL12 agonist is bivalent for IL12). In such embodiments, the two IL12 p40 moieties may be the same or different.

[0162] 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).

[0163] In some embodiments, the p40 portion includes the p40 D2 and D3 domains, but excludes the p40 D1 domain. In other embodiments, the p40 portion includes the p40 D1, D2, and D3 domains.

[0164] Full-length human IL12 p40 has the following amino acid sequence (signal sequence = underlined; D1 domain = italics; D2 domain = bold; D3 domain = bold and underlined).

[0165] [ka]

[0166] 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 may have up to 1,000-fold attenuated binding to human IL12Rβ1 compared to wild-type human IL12 p40. In some embodiments, the IL12 moiety may 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.

[0167] Other features 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 specified. 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 position listed.

[0168] [Table 1]

[0169] 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.

[0170] Exemplary amino acid substitutions in mature human E32 include E32Q and E32A. Exemplary amino acid substitutions in mature human E33 include E33Q and E33A.

[0171] Exemplary amino acid substitutions in mature human D34 include D34N, D34K, and D34A. An exemplary amino acid substitution in mature human Q42 is Q42E.

[0172] Exemplary amino acid substitutions in mature human S43 include S43E and S34K. An exemplary amino acid substitution in mature human E45 is E45Q.

[0173] An exemplary amino acid substitution in mature human Q56 is Q56E. Exemplary amino acid substitutions in mature human E59 include E59K, E59Q, and E59A.

[0174] 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.

[0175] 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.

[0176] An exemplary amino acid substitution in mature human K96 is E93A. Exemplary amino acid substitutions in mature human K99 include K99E, K99Y, and K99A.

[0177] An exemplary amino acid substitution in mature human E100 is E100Q. Exemplary amino acid substitutions in mature human N103 include N103D and N103Q.

[0178] An exemplary amino acid substitution in mature human K104 is K104A. Exemplary amino acid substitutions in mature human N113 include N113D and N113Q.

[0179] 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.

[0180] 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.

[0181] 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.

[0182] Exemplary amino acid substitutions in mature human Y246 include Y246V and Y246F. An exemplary amino acid substitution in mature human C252 is C252S.

[0183] 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.

[0184] 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.

[0185] 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.

[0186] In some embodiments, the p40 moiety is fused directly or indirectly to the IL12 p40 binding domain of IL12Rβ1 (i.e., an IL12Rβ1 moiety, e.g., one described in Section 6.4.1), optionally via a linker (e.g., one described in Section 6.8). If present, the IL12 p40 binding domain of IL12Rβ1 can be N-terminal or C-terminal to the IL12 p40 moiety. When the p40 moiety is fused "directly" to the IL12 p40 binding domain of IL12Rβ1, the p40 moiety and the IL12 p40 binding domain of IL12Rβ1 are positioned adjacent to each other on the same monomer, separated only by a linker, if present. 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., an IL12 p35 moiety) on the same monomer or are located on separate monomers.

[0187] 6.3.2.IL12 p35 part Each IL12 p35 moiety of an IL12 receptor agonist of the present disclosure comprises a wild-type or variant IL12 p35 moiety. In some embodiments, an IL12 receptor agonist of the present disclosure comprises a single IL12 p35 moiety (e.g., an IL12 p35 moiety on the first monomer or on the second monomer in embodiments in which the IL12 receptor agonist is monovalent for IL12). In some embodiments, an IL12 receptor agonist of the present disclosure comprises two IL12 p35 moieties. In such embodiments, the two IL12 p35 moieties may be the same or different.

[0188] 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).

[0189] Full-length human IL12 p35 has the following amino acid sequence (signal sequence = underlined):

[0190] [ka]

[0191] 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 may have up to 1,000-fold reduced binding to human IL12Rβ1 compared to wild-type human IL12 p35. In some embodiments, the IL12 moiety may 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 reduced binding to human IL12Rβ2 compared to wild-type human IL12 p35.

[0192] 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.

[0193] [Table 2]

[0194] 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.

[0195] An exemplary amino acid substitution in mature human E45 is E45Q. Exemplary amino acid substitutions in mature human D55 include D55Q and D55K.

[0196] 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.

[0197] An exemplary amino acid substitution in mature human E79 is E79Q. Exemplary amino acid substitutions in mature human N85 include N85D and N85Q.

[0198] 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.

[0199] 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.

[0200] 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.

[0201] 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.

[0202] 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.

[0203] In some embodiments, the p35 moiety is fused directly or indirectly to the IL12 p35 binding domain of IL12Rβ2 (i.e., an IL12Rβ2 moiety, e.g., one described in Section 6.4.2), optionally via a linker (e.g., one described in Section 6.8). If present, the IL12 p35 binding domain of IL12Rβ2 can be N-terminal 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 adjacent to each other 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.

[0204] 6.4.IL12 Masking Part The present disclosure provides IL12 receptor agonists having one or more IL12 masking moieties capable of binding to an IL12 p40 moiety and / or a p35 moiety. In some embodiments, the IL12 masking moieties described herein bind to an IL12 p40 moiety and / or a p35 moiety, thereby attenuating IL12 activity against target cells. In some embodiments, the IL12 masking moiety is an IL12Rβ1 moiety capable of binding to an IL12 p40 moiety. In other embodiments, the IL12 masking moiety is an IL12Rβ2 moiety capable of binding to an 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.

[0205] 6.4.1.IL12Rβ1 part The IL12 receptor agonist of the present disclosure optionally comprises one or more IL12Rβ1 moieties, each of which is capable of binding to an IL12 p40 moiety of the present disclosure. The IL12Rβ1 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 IL12 p40-binding portion of mammalian, e.g., human or murine, IL12 receptor subunit beta-1 (IL12Rβ1). The IL12 p40-binding portion of IL12Rβ1 comprises or consists of the extracellular domain of the receptor subunit. 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 24-545 constituting the extracellular domain.

[0206] In some embodiments, the IL12Rβ1 portion comprises 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 extracellular domain).

[0207] In certain embodiments, the IL12Rβ1 portion can comprise an amino acid sequence 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% sequence identity to amino acids 24 to 545 of full-length human IL12Rβ1 (i.e., Uniprot identifier P42701). or consisting of, optionally the binding moiety has an amino acid sequence of (a) at least 160 amino acids, at least 161 amino acids, at least 162 amino acids, at least 164 amino acids or at least 165 amino acids, and / or (b) up to 251, up to 240, up to 230, up to 220, up to 210, up to 200, up to 190, up to 180 or up to 170 amino acids from amino acids 24 to 545 of full-length human IL12Rβ1. In certain embodiments, the portion of human IL12Rβ1 is constrained by any one of (a) and (b) of the preceding sentence, e.g., at least 160 and at most 180 amino acids from human IL12Rβ1, at least 162 and at most 200 amino acids from human IL12Rβ1, at least 160 and at most 220 amino acids from human IL12Rβ1, at least 164 and at most 190 amino acids from human IL12Rβ1, etc.

[0208] 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 amino acids 24-545 of full-length human IL12Rβ1, with or without up to 5 amino acids, up to 10 amino acids, up to 15 amino acids, up to 20 amino acids, up to 30 amino acids, or up to 40 additional amino acids C-terminal to amino acid residue 545 of IL12Rβ1.

[0209] The IL12Rβ1 portion-containing IL12 receptor agonist of the present disclosure can have the IL12Rβ1 extracellular domain at the N-terminus or C-terminus of the IL12 p40 portion when located on the same monomer. In some embodiments, the IL12Rβ1 portion-containing IL12 receptor agonist of the present disclosure preferably has the IL12Rβ1 extracellular domain at the N-terminus of the IL12 p40 portion.

[0210] 6.4.2.IL12Rβ2 part The IL12 receptor agonist of the present disclosure optionally includes one or more IL12Rβ2 moieties. Each of the one or more IL12Rβ2 moieties can bind to an IL12 p35 moiety of the present disclosure. The IL12Rβ2 moiety 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 IL12 p35-binding portion of mammalian, e.g., human or murine, IL12 receptor subunit beta-2 (IL12Rβ2). The IL12 p35-binding portion of IL12Rβ2 comprises or consists of the extracellular domain of the receptor subunit. The sequence of human IL12Rβ has Uniprot identifier Q99665 (uniprot.org / uniprot / Q99665), with amino acids 24 to 622 constituting the extracellular domain.

[0211] In some embodiments, the IL12Rβ2 portion comprises 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 extracellular domain).

[0212] In certain embodiments, the IL12Rβ2 portion can comprise amino acids 24-637 or an amino acid sequence 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% sequence identity to full-length human IL12Rβ2 (i.e., Uniprot identifier Q99665). or consisting of, optionally the binding moiety has an amino acid sequence of (a) at least 160 amino acids, at least 161 amino acids, at least 162 amino acids, at least 164 amino acids or at least 165 amino acids from amino acids 24 to 637 of full-length IL12Rβ2, and / or (b) up to 251, up to 240, up to 230, up to 220, up to 210, up to 200, up to 190, up to 180 or up to 170 amino acids. In certain embodiments, the portion of human IL12Rβ2 is constrained by any one of (a) and (b) of the preceding sentence, e.g., at least 160 and at most 180 amino acids from human IL12Rβ2, at least 162 and at most 200 amino acids from human IL12Rβ2, at least 160 and at most 220 amino acids from human IL12Rβ2, at least 164 and at most 190 amino acids from human IL12Rβ2, etc.

[0213] 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 amino acids 24-637 of full-length IL12Rβ2, with or without up to 5 amino acids, up to 10 amino acids, up to 15 amino acids, up to 20 amino acids, up to 30 amino acids, or up to 40 additional amino acids C-terminal to amino acid residue 637 of IL12Rβ2.

[0214] The IL12Rβ2 portion-containing IL12 receptor agonist of the present disclosure can have the IL12Rβ2 extracellular domain at the N-terminus or C-terminus of the IL12 p35 portion when located on the same monomer. In some embodiments, the IL12Rβ2 portion-containing IL12 receptor agonist of the present disclosure preferably has the IL12Rβ2 extracellular domain at the N-terminus of the IL12 p35 portion.

[0215] 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 moiety and the p35 moiety (see, e.g., the left monomer in Figures 4O and 4P). In other embodiments, an IL12 monomer lacking both the p40 moiety and the p35 moiety 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., the two left monomers of FIG. 4O, the two left monomers of FIG. 4P, or one left monomer of FIG. 4O and one left monomer of FIG. 4P). In such embodiments, the two IL12 antibody fragments may be the same or different. In some embodiments, a first IL12 antibody fragment may target the p40 portion, and a second IL12 antibody fragment may target the p35 portion. In other embodiments, both IL12 antibody fragments may target the p40 portion, or both IL12 antibody fragments may target the p35 portion.

[0216] In some embodiments, the IL12 antibody fragment comprises the antibody-binding domain of any known anti-IL12 antibody. Examples of known anti-IL12 antibodies include, but are 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, to p40, or to both p35 and p40).

[0217] 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. Then, a first antibody and a second antibody are added. One of the two antibodies is labeled. If the labeled and unlabeled antibodies 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 overlap, the unlabeled antibody will compete, reducing the amount of labeled antibody bound to the antigen. When excess unlabeled antibody is present, the labeled antibody will bind very little, if at all. In some embodiments, a competing antibody is one 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%. Detailed 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., the same antibody is used 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 are plotted and the concentrations required to achieve the desired degree of binding inhibition are compared. In some embodiments, binding competition to target molecules can be determined using real-time label-free biolayer interference assays, for example, on an Octet HTX biosensor platform (Pall ForteBio Corp.).

[0218] For example, the IL12 antibody fragment can be formatted according to any of the formats described for targeting moieties in Section 6.5.2. 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.

[0219] 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, for example, as represented by the exemplary monomers described in Section 6.2. As described in Section 6.2, an IL12 antibody fragment 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 described in detail in Section 6.2.

[0220] 6.5. Targeting part The incorporation of a targeting moiety in 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 resulting in fewer side effects than those achieved with wild-type IL12.

[0221] Suitable targeting moiety formats are described in Section 6.5.2. The targeting moiety is preferably an antigen-binding moiety, e.g., an antibody or 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.

[0222] Antibodies and antigen-binding moieties generally bind to a specific antigenic determinant and can direct the IL12 receptor agonist to a target site, such as a specific type of tumor cell 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.

[0223] 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.

[0224] 6.5.1.Target molecules The target molecules recognized by the targeting moieties of the IL12 receptor agonists 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 bodies in serum, in the extracellular matrix (ECM), or immune cells present at the target site, e.g., tumor-reactive lymphocytes. When immune cells are exogenously administered (e.g., chimeric antigen receptor ("CAR")-expressing T cells), the targeting moiety may recognize the chimeric antigen receptor (CAR) or another molecule found on the surface of the CAR T cell. In various embodiments, the CAR comprises CDRs or VH and VL sequences (e.g., in scFv format) that specifically recognize a TAA or pMHC complex.

[0225] 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, colon-related 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, MAGE-A7), -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 (TC R-related heteromultimer), 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, IGF1 receptor, 5T4, ROR1, Nkp30, NKG2D, tumor stromal antigen, CA166-9, extra domain A (EDA) and extra domain B (EDB) of fibronectin, and the A1 domain of tenascin-C (TnC A1).

[0226] 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 an antigen binding domain derived from one of the following heavy chain variable regions (VH) and light chain variable regions (VL):

[0227] [ka]

[0228] [ka]

[0229] [ka]

[0230] [ka]

[0231] [ka]

[0232] [ka]

[0233] [ka]

[0234] 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).

[0235] Non-limiting examples of ECM antigens include syndecans, heparanase, integrins, osteopontin, link, cadherins, laminins, laminin-type EGFs, lectins, fibronectin, fibronectin extra domain B (ED-B), notch, tenascin, collagens, and matrixins.

[0236] 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.

[0237] 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.

[0238] 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 an antigen-binding domain derived from the heavy chain variable region (VH) and light chain variable region (VL) of the following:

[0239] [ka]

[0240] [ka]

[0241] Additional CD20 and PD1 targeting moieties are listed in Table 3 below. In some embodiments, the targeting moiety targets an exemplary target molecule set forth in Table 3 below, which also lists exemplary antibodies or antibody sequences on which the targeting moiety may be based.

[0242] [Table 3-1]

[0243] [Table 3-2]

[0244] [Table 3-3]

[0245] [Table 3-4]

[0246] [Table 3-5]

[0247] [Table 3-6]

[0248] [Table 3-7]

[0249] [Table 3-8]

[0250] [Table 3-9]

[0251] [Table 3-10]

[0252] In some embodiments, the targeting moiety competes with an antibody listed above in Table 3 for binding to a target molecule. In further embodiments, the targeting moiety comprises a CDR having the CDR sequence of an antibody listed above in Table 3. In some embodiments, the targeting moiety comprises all six CDR sequences of an antibody listed above, including an antibody listed 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 embodiments, the targeting moiety comprises a VH comprising the amino acid sequence of the VH of an antibody listed above, e.g., listed in Table 3. In some embodiments, the targeting moiety further comprises a VL comprising the amino acid sequence of the VL of an antibody listed above, e.g., listed in Table 3. In other embodiments, the targeting moiety further comprises a universal light chain VL sequence.

[0253] In some embodiments, the checkpoint inhibitor targeting moiety is a non-blocking or low-blocking ligand-receptor binding antibody. 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 WO 2015 / 112800 (A1), SEQ ID NOs: 16 / 17 in U.S. Pat. No. 11,034,765 (B2), 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. Pat. No. 10,294,299 (B2). Examples of non-blocking or low-blocking anti-LAG 3 antibodies include antibodies having the VH / VL amino acid sequences of SEQ ID NOs: 23 / 24, 3 / 4 and 11 / 12 of US Patent Application Publication No. 2022 / 0056126(A1).

[0254] Additional target molecules that can be targeted by IL12 receptor agonists are disclosed in Table 6 below and, for example, in Hafeez et al., 2020, Molecules 25:4764, doi:10.3390 / molecules25204764, specifically Table 1. Table 1 of Hafeez et al. is incorporated herein by reference in its entirety.

[0255] 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 IgG class immunoglobulin molecule, 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.

[0256] 6.5.2.1.scFv Single-chain Fv or "scFv" antibody fragments comprise the VH and VL domains of an antibody in 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 that enables the scFv to form the desired structure for target binding. Examples of suitable linkers for connecting the VH and VL chains of an scFv are the linkers identified in Section 6.5.3.

[0257] Unless otherwise specified, as used herein, an scFv can have the VL and VH variable regions 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.

[0258] The scFv may 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, e.g., one of the linkers described in Section 6.5.3 (typically a repeat of a sequence containing the amino acids glycine and serine, such as the amino acid sequence (Gly4-Ser)3 (SEQ ID NO: 16)), thereby enabling the VH and VL sequences to be expressed as a contiguous single-chain protein in which the VL and VH regions are linked 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).

[0259] 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.

[0260] The Fab domain may comprise constant domain and variable region sequences from any suitable species, and may therefore be murine, chimeric, human or humanized. The Fab domain typically comprises a CH1 domain linked to a VH domain, which is paired with a CL domain linked 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.

[0261] 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 strategies shown in Table 4 below can be used.

[0262] [Table 4]

[0263] Thus, in certain embodiments, correct association between the two polypeptides of a Fab is facilitated by swapping the VL and VH domains of the Fab, or swapping the CH1 and CL domains, as described, for example, in WO 2009 / 080251.

[0264] Correct Fab pairing can also be promoted by introducing one or more amino acid modifications in the CH1 domain and one or more amino acid modifications in the CL domain of the Fab, and / or one or more amino acid modifications in the VH domain and one or more amino acid modifications in the VL domain 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.

[0265] In one embodiment, the one or more amino acid modifications are restricted to conserved framework residues in the variable (VH, VL) and constant (CH1, CL) domains, as indicated by the Kabat numbering of residues. Almagro, 2008, Frontiers In Bioscience 13:1619-1633, provides framework residue definitions based on the Kabat, Chothia, and IMGT numbering schemes.

[0266] In one embodiment, the modifications introduced into 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 compatibility between the two interacting surfaces.

[0267] In one embodiment, the one or more introduced modifications introduce new hydrogen bonds across the interface of the Fab component. In one embodiment, the one or more introduced modifications introduce new salt bridges across the interface of the Fab component. Exemplary substitutions are described in WO 2014 / 150973 and WO 2014 / 082179, the contents of which are incorporated herein by reference.

[0268] In some embodiments, the Fab domain comprises a 192E substitution in the CH1 domain and 114A and 137K substitutions in the CL domain, which introduce a salt bridge between the CH1 and CL domains (see, e.g., Golay et al., 2016, J Immunol 196:3199-211).

[0269] 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 the hydrophobic and polar regions of contact between the CH1 and CL domains (see, e.g., Golay et al., 2016, J Immunol 196:3199-211).

[0270] In some embodiments, the Fab domain can contain 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 one embodiment, 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.

[0271] The Fab domain can also be modified to replace the native CH1:CL disulfide bond with an engineered disulfide bond to increase the pairing efficiency of the Fab component. 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).

[0272] 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 from a T cell receptor, replacing the CL domain with a b domain from a T cell receptor, and introducing a 38D modification in the VL domain and a 39K modification in the VH domain, thereby pairing these domain replacements with additional charge-charge interactions between the VL and VH domains.

[0273] Instead of, or in addition to, using a Fab heterodimerization strategy to promote precise VH-VL pairing, a VL of a common light chain (also called 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 incorrect 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 includes human heavy chain variable gene segments rearranged in vivo in mouse B cells. The mouse B cells have been engineered to express a limited human light chain repertoire or a single human light chain cognate to the human heavy chain, and to generate, in response to exposure to an antigen of interest, an antibody repertoire containing one possible human VL or multiple human VH cognate to one of two possible human VLs, where the 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) versions. See, e.g., U.S. Patent No. 10,412,940.

[0274] 6.5.3.MHC-peptide fusions The targeting moiety of the IL12 receptor agonist of the present disclosure may be a peptide-MHC complex ("pMHC complex"), such as a peptide complexed with an MHC class I domain, or a peptide complexed with an MHC class II domain, either of which optionally has a β2 microglobulin domain.

[0275] The peptide in the pMHC complex can have an amino acid sequence that allows the peptide to associate with, e.g., be presented by, an MHC class I molecule. In certain embodiments, the sequence can include 6 to 20 contiguous amino acids. In certain embodiments, the peptide sequence can be the sequence of a protein fragment, e.g., a protein derived from a portion of 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.

[0276] 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 WO 2021 / 127487(A2), 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 described in Section 6.7.1 of WO 2021 / 127487(A2), which section is specifically incorporated herein by reference.

[0277] The peptides in the pMHC complexes of the present disclosure are typically at least a portion, e.g., an antigenic determinant, of a protein of an infectious agent (e.g., a bacterium, a virus, or a parasite), an allergen, or a tumor-associated protein. Preferably, the pMHC complex includes an antigenic determinant of a cancer cell. 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), and 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, 371 D), and WT1(126-134). Additional antigenic determinants of cancer cells are described in Section 6.4.3 and Table 3 of WO 2021 / 127487(A2) (this Section and Table are specifically incorporated herein by reference).

[0278] 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).

[0279] 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.

[0280] 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.

[0281] One embodiment of the present disclosure relates to a dimer comprising two Fc-fusion polypeptides created by fusing one or more IL12 moieties (e.g., a p35 moiety and a p40 moiety) to an Fc region of an antibody (e.g., by fusing both the p35 moiety and the p40 moiety to an Fc domain, which upon expression allows the formation of an IL12 monomer capable of homodimerization, or by fusing the p35 moiety to a first Fc domain and the p40 moiety to a second Fc domain, which upon expression allows the formation of two different IL12 monomers capable of heterodimerization). Dimers can be created, for example, by inserting gene fusions encoding the fusion proteins into an appropriate expression vector, expressing the gene fusions in a host cell transformed with the recombinant expression vector, allowing the expressed fusion proteins to assemble like antibody molecules, and allowing interchain bonds to form between the Fc moieties to form a dimer.

[0282] 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.

[0283] The two Fc domains within an Fc region can be the same 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 can advantageously be different to allow heterodimerization, as described in Section 6.6.1.2, below.

[0284] 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 heavy chain Fc domain of IgE and IgM consists of three heavy chain constant domains (CH2, CH3, and CH4), which dimerize to form the Fc region.

[0285] In the IL12 receptor agonists of the present disclosure, the Fc region and / or Fc domains therein can comprise heavy chain constant domains of one or more different classes of antibodies, for example, one, two, or three different classes.

[0286] In one embodiment, the Fc region comprises CH2 and CH3 domains derived from IgG1. In one embodiment, the Fc region comprises CH2 and CH3 domains derived from IgG2.

[0287] In one embodiment, the Fc region comprises CH2 and CH3 domains derived from IgG3. In one embodiment, the Fc region comprises CH2 and CH3 domains derived from IgG4.

[0288] 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.

[0289] It will be understood that heavy chain constant domains for use in producing the Fc region of the IL12 receptor agonist 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 constant domain of a variant can be longer or shorter than the wild-type constant domain. Preferably, the constant domain of a variant is at least 60% identical or similar to the wild-type constant domain. In another example, the constant domain of a variant is at least 70% identical or similar. In another example, the constant domain of a variant is at least 80% identical or similar. In another example, the constant domain of a variant is at least 90% identical or similar. In another example, the constant domain of a variant is at least 95% identical or similar.

[0290] IgM and IgA naturally occur in humans as covalently linked multimers of a common H2L2 antibody unit. IgM exists as a pentamer when it incorporates a J chain or as a hexamer when it lacks a J chain. IgA exists in 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 in 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 comprise a tail.

[0291] The Fc domain incorporated into the IL12 receptor agonists of the present disclosure can include 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 structure, or altered glycosylation pattern. Exemplary Fc modifications that alter effector function are described in Section 6.6.1.1.

[0292] The Fc domain can also be engineered to contain modifications that improve the manufacturability of asymmetric IL12 receptor agonists, for example, by allowing heterodimerization, which is the preferential pairing of non-identical Fc domains over 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 that contain Fc domains that differ in sequence. Examples of heterodimerization strategies are illustrated in Section 6.6.1.2.

[0293] 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 can be combined with other modifications to alter the properties of the IL12 receptor agonist.

[0294] 6.6.1.1. Effector Function-Altered Fc Domains In some embodiments, the Fc domain comprises one or more amino acid substitutions that reduce binding to an Fc receptor and / or effector function.

[0295] 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.

[0296] 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 specific 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 specific embodiment, the amino acid substitution is P329A or P329G, particularly P329G (numbering according to the EU index of Kabat). 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 EU index of Kabat). In a more specific 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 EU index of Kabat). In a more specific embodiment, the Fc domain comprises the amino acid mutations L234A, L235A and P329G (“P329G LALA,” “PGLALA,” or “LALAPG”).

[0297] Typically, the same one or more amino acid substitutions are present in each of the two Fc domains of the Fc region. Thus, in a specific embodiment, each Fc domain of the Fc region comprises amino acid substitutions L234A, L235A, and P329G (Kabat EU index numbering), i.e., in each of the first and second Fc domains of 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).

[0298] 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.

[0299] 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.

[0300] [Table 5-1]

[0301] [Table 5-2]

[0302] [Table 5-3]

[0303] In certain embodiments, the IgG4 with reduced effector function comprises the bolded portion of the amino acid sequence of SEQ ID NO: 31 of WO 2014 / 121087 (SEQ ID NO: 20), and is sometimes referred to herein as IgG4 or hIgG4.

[0304] The heterodimeric Fc region may incorporate a combination of the above-mentioned variant IgG4 Fc sequences, for example an Fc region comprising an Fc domain comprising the amino acid sequence of SEQ ID NO: 30 of WO 2014 / 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 WO 2014 / 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 WO 2014 / 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 WO 2014 / 121087 (SEQ ID NO: 22) (or a bolded portion thereof).

[0305] Fc Heterodimerization Variants Certain IL12 receptor agonists, unlike native immunoglobulins, involve dimerization between two Fc domains operably linked to non-identical N-terminal regions (e.g., one Fc domain is connected to a Fab and the other Fc domain is connected to an IL12 moiety). Incorrect heterodimerization of two Fc domains to form an Fc region can be an obstacle to increasing the yield of the desired heterodimerized molecule, presenting 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, and are disclosed, for example, in EP 1870459(A1); U.S. Pat. Nos. 5,582,996; 5,731,168; 5,910,573; 5,932,448; 6,833,441; 7,183,076; U.S. Patent Application Publication No. 2006204493(A1); and WO 2009 / 089004(A1).

[0306] The present disclosure provides IL12 receptor agonists comprising an Fc heterodimer, i.e., an Fc region 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, as described in the previous section, preferably of the IgG (IgG1, IgG2, IgG3, and IgG4) class.

[0307] Heterodimerization of two different heavy chains at the CH3 domains yields the desired IL12 receptor agonist, whereas homodimerization of identical heavy chains 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 CH3 domains with modifications that favor heterodimeric association relative to unmodified Fc domains.

[0308] In a specific embodiment, the modification that promotes Fc heterodimer formation is a so-called "knob-into-hole" or "knob-in-hole" modification, comprising a "knob" modification in one of the Fc domains and a "hole" modification in the other Fc domain. Knob-into-hole technology is described, for example, in U.S. Pat. Nos. 5,731,168; 7,695,936; Ridgway et al., 1996, Prot Eng 9:617-621; and Carter, 2001, Immunol Meth 248:7-15. In general, the method involves introducing a protrusion ("knob") into the interface of a first polypeptide and a corresponding cavity ("hole") into the interface of a second polypeptide, such that the protrusion can be positioned within the cavity to promote heterodimer formation and prevent homodimer formation. The protrusion is constructed by replacing a small amino acid side chain from the interface of the first polypeptide with a larger side chain (e.g., tyrosine or tryptophan). Compensatory cavities of identical or similar size to the protrusions are created at the interface of the second polypeptide by replacing large amino acid side chains with smaller ones (eg, alanine or threonine).

[0309] 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 generating 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 generating a cavity in the CH3 domain of the second subunit that can be positioned within the protrusion in the CH3 domain of the first subunit. 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 protrusions and cavities can be generated by modifying a nucleic acid encoding the polypeptide, for example, by site-directed mutagenesis or by peptide synthesis. An exemplary substitution is Y470T.

[0310] In a specific such embodiment, in the first Fc domain, the threonine residue at position 366 is replaced with a tryptophan residue (T366W), and in the Fc domain, the tyrosine residue at position 407 is replaced with a valine residue (Y407V), and optionally, the threonine residue at position 366 is replaced with a serine residue (T366S), and the leucine residue at position 368 is replaced with an alanine residue (L368A) (numbering according to the Kabat EU index). In a further embodiment, the first Fc domain further comprises a replacement of the serine residue at position 354 with a cysteine ​​residue (S354C) or the glutamic acid residue at position 356 with a cysteine ​​residue (E356C) (particularly, the serine residue at position 354 is replaced with a cysteine ​​residue), and the second Fc domain further comprises a replacement of the tyrosine residue at position 349 with a cysteine ​​residue (Y349C) (numbering according to the Kabat EU index). In a specific embodiment, the first Fc domain comprises the amino acid substitutions S354C and T366W, and the second Fc domain comprises the amino acid substitutions Y349C, T366S, L368A, and Y407V (numbering according to the Kabat EU index).

[0311] 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 domains of the Fc region.

[0312] 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 binding to Protein A, thereby enabling a purification method that yields a heterodimeric protein. See, e.g., U.S. Pat. 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, such as a H95R modification (according to IMGT exon numbering; H435R according to EU numbering). The second CH3 may further comprise a Y96F modification (according to IMGT; Y436F according to EU). This class of modifications is referred to herein as a "star" mutation.

[0313] In some embodiments, the Fc may contain one or more mutations to promote heterodimerization (eg, knob and hole mutations) and a star mutation to facilitate purification.

[0314] 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 adding 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 may refer to half-life in humans or other mammals (e.g., mice or non-human primates).

[0315] Wild-type IL12 has a serum half-life of less than 10 minutes. The IL12 receptor agonists of the present disclosure preferably have a serum half-life of at least about 2 hours, at least about 4 hours, at least about 6 hours, or at least about 8 hours in humans and / or mice. In some embodiments, the IL12 receptor agonists of the present disclosure have 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.

[0316] Stabilizing moieties include polyoxyalkylene moieties (eg, polyethylene glycol), sugars (eg, sialic acid), and well-tolerated protein moieties (eg, Fc and fragments and variants thereof, transferrin, or serum albumin).

[0317] 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 products, polysialic acid conjugates, and fatty acid conjugates.

[0318] Thus, in some embodiments, the present disclosure provides an IL12 receptor agonist that includes a stabilizing moiety that is a polymeric sugar. Serum albumin may also contribute to the extension of half-life through modules capable of non-covalently interacting with albumin. Thus, the IL12 receptor agonist of the present disclosure can include an albumin-binding protein as a stabilizing moiety. The albumin-binding protein can be 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). Further examples of serum albumin-binding proteins include those described in U.S. Patent Application 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).

[0319] In other embodiments, the stabilizing moiety is human serum albumin. In other embodiments, the stabilizing moiety is transferrin. In some embodiments, the stabilizing moiety is an Fc domain, e.g., any of the Fc domains described in Section 6.6.1 and therein, incorporated herein by reference. The Fc domains described in Section 6.6.1 are generally capable of dimerization. However, for stabilization, the Fc domain may be a soluble monomeric Fc domain with a reduced ability to self-associate. See, e.g., 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 includes amino acid substitutions at positions corresponding to T366 and / or Y407 in CH3, as described in U.S. Patent Application 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 thereafter.

[0320] In yet other embodiments, the stabilizing moiety is a polyethylene glycol moiety or another polymer, as described below in Section 6.7.1. The stabilizing moiety may 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.

[0321] 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, propylene glycol homopolymer, prolypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyol (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. The polymer can be of any molecular weight and can be branched or unbranched.

[0322] Linker In certain embodiments, the present disclosure provides an IL12 receptor agonist, wherein 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 in an scFv).

[0323] The peptide linker can range from 2 to 60 or more amino acids in length, and in certain embodiments, the peptide linker ranges from 3 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 in length.

[0324] In particular 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.

[0325] In some of the aforementioned embodiments, the linker is between 5 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 aforementioned embodiments, the linker is between 6 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 aforementioned embodiments, the linker is between 7 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.

[0326] Charged linkers (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 (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 monomer or polymer of G4S (SEQ ID NO: 25), e.g., (GGGGS) n (SEQ ID NO: 26)

[0327] 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 (2 Gly), three consecutive glycines (3 Gly), four consecutive glycines (4 Gly) (SEQ ID NO: 27), five consecutive glycines (5 Gly) (SEQ ID NO: 28), six consecutive glycines (6 Gly) (SEQ ID NO: 29), seven consecutive glycines (7 Gly) (SEQ ID NO: 30), eight consecutive glycines (8 Gly) (SEQ ID NO: 31), or nine consecutive glycines (9 Gly) (SEQ ID NO: 32).

[0328] 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, e.g., 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 linkers described above, 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 an integer of at least 1 (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 therefore function as neutral tethers between components. Glycine polymers can be used; glycine has access to a much wider range of 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, incorporated herein by reference in its entirety). Exemplary linkers can include amino acid sequences including, but not limited to, 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), GGGGSGGGGSGGGGS (SEQ ID NO: 44), GGGASGGGGS (SEQ ID NO: 45), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 46), GCGGS (SEQ ID NO: 47), and the like. In some embodiments, the linker polypeptide comprises a cysteine ​​residue capable of forming a disulfide bond with a cysteine ​​residue present in another portion of the pMHC complex, hi certain embodiments, the linker comprises the amino acid sequence GCGGS (SEQ ID NO: 47).By substituting cysteine ​​for glycine in the G4S linker (SEQ ID NO: 25), a disulfide bond can be formed to form an MHC targeting moiety with the corresponding cysteine ​​substitution in, for example, HLA.A2 that stabilizes the MHC peptide within the MHC complex.

[0329] 6.8.2. Hinge arrangement In other embodiments, the IL12 receptor agonists of the present disclosure include 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. Hinge regions are typically found at the N-terminus of an Fc region. The term "hinge region," unless otherwise indicated by context, refers to a naturally occurring or non-naturally occurring hinge sequence, which can be a monomeric hinge domain in the context of a single or monomeric polypeptide chain and can comprise 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).

[0330] A native hinge region is the hinge region normally found between the Fab and Fc domains in naturally occurring antibodies. A modified hinge region is any hinge that differs in length and / or composition from the native hinge region. Such hinges may include hinge regions from other species, such as human, mouse, rat, rabbit, shark, pig, hamster, camel, llama, or goat hinge regions. Other modified hinge regions may 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, a modified hinge region may include a portion of a native hinge or a repeating unit in which each unit in the repeat is derived from a native hinge region. In a further alternative, the native hinge region can be modified by converting one or more cysteine ​​or other residues to neutral residues such as serine or alanine, or by converting appropriately placed residues to cysteine ​​residues. By such means, the number of cysteine ​​residues in the hinge region can be increased or decreased. Other modified hinge regions may be entirely synthetic and may be designed to have desired properties such as length, cysteine ​​composition, and flexibility.

[0331] Many modified hinge regions have been previously described, for example, in U.S. Pat. No. 5,677,425, WO 99 / 15549, WO 2005 / 003170, WO 2005 / 003169, WO 2005 / 003170, WO 98 / 25971, and WO 2005 / 003171, which are incorporated herein by reference.

[0332] In one embodiment, an IL12 receptor agonist of the present disclosure comprises an Fc region, where 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 unoccupied; G, G, unoccupied, and unoccupied; G, unoccupied, unoccupied, and unoccupied; or all unoccupied, positions numbered according to EU numbering.

[0333] 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).

[0334] In one embodiment, the IL12 receptor agonist of the present disclosure comprises an Fc region, wherein each Fc domain has an intact hinge region at its N-terminus, 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 increase flexibility in this region, allowing a portion of the molecule to form disulfide bonds within the same protein chain (intrachain disulfides) rather than cross-linking to other heavy chains in the IgG molecule to form interchain disulfides. (Angel et al., 1993, Mol Immunol 30(1):105-108). Changing the serine residues to proline to provide the same core sequence as IgG1 allows complete formation of interchain disulfides in the IgG4 hinge region, thereby reducing heterogeneity in the purified product. This modified isotype is called IgG4P.

[0335] 6.8.2.1. Chimeric Hinge Sequences The hinge region may 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 in combination with a "lower hinge" sequence derived from a human IgG1, human IgG2, or human IgG4 hinge region.

[0336] 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 WO 2014 / 121087, which is incorporated by reference in its entirety) or ESKYGPPCPPCPAPPVA (SEQ ID NO: 51) (previously disclosed as SEQ ID NO: 9 of WO 2014 / 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).

[0337] 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 International Publication No. WO 2016161010(A2), which is incorporated herein by reference in its entirety. In various embodiments, positions 233-236 of the modified hinge region are G, G, G, and unoccupied; G, G, unoccupied, and unoccupied; G, unoccupied, unoccupied, and unoccupied; or all unoccupied, with positions numbered according to EU numbering (as shown in Figure 1 of WO 2016161010(A2)). These segments can be represented as GGG-, GG--, G---, or ----, with "-" representing an unoccupied position.

[0338] Position 236 is unoccupied in standard human IgG2 but is occupied in other standard human IgG isotypes. Positions 233-235 are occupied by residues other than G in all four human isotypes (shown in Figure 1 of WO2016161010(A2)).

[0339] 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 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.

[0340] 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 WO 2016161010(A2)), CPPCPAPGG--GPSVF (SEQ ID NO: 53) (previously disclosed as SEQ ID NO: 2 in WO 2016161010(A2)), CPPCPAPG---GPSVF (SEQ ID NO: 54) (previously disclosed as SEQ ID NO: 3 in WO 2016161010(A2)), or CPPCPAP----GPSVF (SEQ ID NO: 55) (previously disclosed as SEQ ID NO: 4 in WO 2016161010(A2)).

[0341] The above-described modified hinge regions can be incorporated into heavy chain constant regions, which typically include CH2 and CH3 domains and may have additional hinge segments (e.g., upper hinges) flanking the designated regions. The additional constant region segments present are typically of the same isotype, preferably a human isotype, but may also be hybrids of different isotypes. The isotype of such additional human constant region segments is preferably human IgG4, but may 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 WO2016161010(A2).

[0342] In a specific embodiment, a modified hinge sequence may 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 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).

[0343] 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 containing 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.

[0344] 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 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 elements and separated by an internal ribosome entry site (IRES) sequence, allowing them to be translated into separate polypeptides.

[0345] 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).

[0346] The nucleic acids of the present disclosure can be DNA or RNA (e.g., mRNA). In another aspect, the disclosure provides host cells and vectors containing the nucleic acids of the disclosure. The nucleic acids may be present in a single vector or in separate vectors present in the same host cell or in separate host cells, as described in more detail herein below.

[0347] 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).

[0348] A number of 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.

[0349] Furthermore, 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 may 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 either be 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 synthesis of mRNA. These elements may include splice signals, as well as transcription promoters, enhancers, and termination signals.

[0350] Once the expression vector or DNA sequence containing the construct is prepared for expression, the expression vector can be transfected or introduced into a 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. Methods and conditions for culturing the resulting transfected cells and recovering the expressed polypeptide are known to those skilled in the art and can be modified or optimized based on the present description depending on the specific expression vector and mammalian host cell used.

[0351] 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.

[0352] 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 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.

[0353] The present disclosure also provides host cells comprising the vectors described herein. The cell may 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.

[0354] Pharmaceutical Compositions 6.10.1. Pharmaceutical Compositions Comprising IL12 Receptor Agonist Polypeptides The IL12 receptor agonists of the present disclosure may be in the form of a composition comprising an IL12 receptor agonist and one or more carriers, excipients, and / or diluents. The composition can be formulated for a particular use, such as veterinary use or pharmaceutical use in humans. The form of the composition used (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, for therapeutic use, on the mode of administration.

[0355] For therapeutic use, the composition may be supplied as part of a sterile pharmaceutical composition that includes a pharmaceutically acceptable carrier. This composition may be in any suitable form (depending on the desired method of administering it to a patient). Pharmaceutical compositions may be administered to a patient by a variety of routes, including oral, transdermal, subcutaneous, intranasal, intravenous, intramuscular, intratumoral, intrathecal, topically, or locally. The most suitable route of administration in any given case will depend on the particular antibody, the subject, and the nature and severity of the disease and the physical condition of the subject. Typically, pharmaceutical compositions are administered intravenously or subcutaneously.

[0356] 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 administration. The amount of IL12 receptor agonist contained in a unit dose varies depending on the disease being treated and other factors known in the art. Such unit dosage forms can be in the form of a lyophilized powder containing an amount of IL12 receptor agonist suitable for a single administration, or in liquid form. Dry powder unit dosage forms can be packaged in a kit together with a syringe, a suitable amount of diluent, and / or other components useful for administration. Liquid unit dosage forms can conveniently be supplied in the form of a syringe pre-filled with an amount of IL12 receptor agonist suitable for a single administration.

[0357] Pharmaceutical compositions may also be supplied in bulk form containing an amount of IL12 receptor agonist suitable for multiple administrations. Pharmaceutical compositions can be prepared for storage as lyophilized formulations or aqueous solutions by mixing an IL12 receptor agonist having the desired purity with any pharmaceutically acceptable carrier, excipient, or stabilizer (all of which are referred to herein as "carriers") typically used in the art, i.e., buffers, stabilizers, preservatives, isotonicifiers, 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 used.

[0358] Buffers help maintain pH in a range close to physiological conditions. They can be present in a wide range of concentrations, but are typically present at concentrations ranging from about 2 mM to about 50 mM. Suitable buffers 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.), succinic acid buffers (e.g., succinic acid-monosodium succinate mixtures, succinic acid-sodium hydroxide mixtures, succinic acid-disodium succinate mixtures, etc.), tartaric acid buffers (e.g., tartaric acid-sodium tartrate mixtures, tartaric acid-potassium tartrate mixtures, tartaric acid-sodium hydroxide mixtures, etc.), fumaric acid buffers (e.g., fumaric acid-monosodium fumarate mixtures, fumaric acid-monosodium fumarate mixtures, etc.), and the like. Examples of suitable buffers include: acid-disodium fumarate mixtures, monosodium fumarate-disodium fumarate mixtures, gluconic acid buffers (e.g., gluconic acid-sodium glyconate mixtures, gluconic acid-sodium hydroxide mixtures, gluconic acid-potassium glyconate mixtures, etc.), oxalic acid buffers (e.g., oxalic acid-sodium oxalate mixtures, oxalic acid-sodium hydroxide mixtures, oxalic acid-potassium oxalate mixtures, etc.), lactic acid buffers (e.g., lactic acid-sodium lactate mixtures, lactic acid-sodium hydroxide mixtures, lactic acid-potassium lactate mixtures, etc.), and acetate buffers (e.g., acetic acid-sodium acetate mixtures, acetic acid-sodium hydroxide mixtures, etc.). Phosphate buffers, histidine buffers, and trimethylamine salts (e.g., Tris) may also be used.

[0359] Preservatives may be added to retard microbial growth and can be added in amounts ranging from about 0.2% to 1% (w / v). Suitable preservatives for use in the present disclosure include phenol, benzyl alcohol, metacresol, methylparaben, propylparaben, octadecyldimethylbenzylammonium chloride, benzalkonium halides (e.g., chloride, bromide, and iodide), hexamethonium chloride, and alkylparabens, such as methyl or propylparaben, catechol, resorcinol, cyclohexanol, and 3-pentanol. Tonicity adjusting agents, sometimes referred to as "stabilizers," can be added to ensure the isotonicity of the liquid compositions of the present disclosure and include polyhydric sugar alcohols, such as trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol, and mannitol. Stabilizers refer to a broad category of excipients with a wide range of functions, from bulking agents to additives that help solubilize the therapeutic agent or prevent denaturation or adhesion to the container wall. Typical stabilizers include polyhydric sugar alcohols (listed above); amino acids such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, threonine, etc.; organic sugars or sugar alcohols such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myonisitol, galactitol, glycerol, etc. (including cyclitols such as inositol); polyethylene glycol; amino acid polymers; sulfur-containing reducing agents such as urea; The sugars may be glutathione, thioctic acid, sodium thioglycolate, thioglycerol, α-monothioglycerol, and sodium thiosulfate; low molecular weight polypeptides (e.g., peptides of 10 residues or less); proteins, such as human serum albumin, bovine serum albumin, gelatin, or immunoglobulins; hydr...

Claims

**Claim 1** An IL-12 receptor agonist, comprising: (a) a first polypeptide chain comprising, in the direction from the N-terminus to the C-terminus, a first targeting moiety or targeting moiety component, a first Fc domain, and a p35 moiety; (b) a second polypeptide chain comprising, in the direction from the N-terminus to the C-terminus, 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 a p40 moiety in the form of monomeric p40, optionally wherein the p40 moiety has a hypoactive 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 IL-12Rβ moiety or an IL-12 antibody fragment configured to mask the p35 moiety or the p40 moiety. **Claim 2** An IL-12 receptor agonist comprising an IL-12 mutein, wherein the IL-12 receptor agonist has at least 500-fold reduced activity compared to wild-type IL-12, and comprising: (a) a first polypeptide chain and a second polypeptide chain dimerized via 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; (d) an IL-12Rβ moiety or an IL-12 antibody fragment configured to mask the p35 moiety or the p40 moiety. **Claim 3** On a first polypeptide chain and a second polypeptide chain dimerized via a first Fc domain and a second Fc domain, there are: (a) an optional first targeting moiety and an optional second targeting moiety; (b) an IL-12 mutein comprising a p35 moiety and a p40 moiety, wherein: (i) the p35 portion comprises a weakened amino acid substitution, and optionally, the weakened amino acid substitution is (A) amino acid Y189 of full-length human p35 or amino acid Y185 of full-length mouse p35 (the substitution is optionally A, V, R or E); (B) amino acid I193 of full-length human p35 or amino acid M189 of full-length mouse p35 (the substitution is optionally A, V or E); (C) amino acid R211 of full-length human p35 or amino acid R207 of full-length mouse p35 (the substitution is optionally A or K); or (D) any combination of (A) to (C); and / or (ii) the p40 portion comprises a weakened amino acid substitution, and optionally, the weakened amino acid substitution is (A) amino acid K28 of full-length human p40 or amino acid K28 of full-length mouse p40 (the substitution is optionally A); (B) amino acid W37 of full-length human p40 or amino acid W37 of full-length mouse p40 (the substitution is optionally A); (C) amino acid D115 of full-length human p40 or amino acid E115 of full-length mouse p40 (the substitution is optionally A); (D) amino acid K118 of full-length human p40 or amino acid K118 of full-length mouse p40 (the substitution is optionally A); (E) amino acid K126 of full-length human p40 or amino acid K126 of full-length mouse p40 (the substitution is optionally A); (F) amino acid Y268 of full-length human p40 or amino acid Y265 of full-length mouse p40 (the substitution is optionally V or F); (G) amino acid Y314 of full-length human p40 or amino acid Y318 of full-length mouse p40 (the substitution is optionally F); or (H) any combination of (A) to (G), an IL-12 mutein; (c) an IL-12 receptor agonist comprising an IL-12Rβ portion or an IL-12 antibody fragment configured to mask the p35 portion or the p40 portion.

4. The IL-12 receptor agonist according to any one of Claims 1 to 3, comprising an IL-12Rβ1 portion configured to mask the p40 portion and an IL-12Rβ2 portion configured to mask the p35 portion.

5. The IL-12 receptor agonist according to Claim 4, which is monovalent with respect to the p35 portion and the p40 portion.

6. The IL12 receptor agonist according to any one of claims 1 to 3, 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.

7. The IL12 receptor agonist according to any one of claims 1 to 3, wherein the p40 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 p40, and comprises an amino acid sequence containing 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, and optionally, the substitution is optionally A.

8. The IL12 receptor agonist according to any one of claims 1 to 3, 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.

9. The IL12 receptor agonist according to any one of claims 1 to 3, comprising a first IL12 monomer having the structure of exemplary monomer 19 associated with monomeric p40 (optionally masked monomeric p40) and a second IL12 monomer having the structure of exemplary monomer 33.

10. The IL12 receptor agonist according to any one of claims 1 to 3, comprising a first IL12 monomer having the structure of exemplary monomer 19 associated with monomeric p40 (optionally masked monomeric p40) and a second IL12 monomer having the structure of exemplary monomer 57.

11. The IL12 receptor agonist according to any one of claims 1 to 3, comprising a first IL12 monomer having the structure of exemplary monomer 28 and a second IL12 monomer having the structure of exemplary monomer 57.

12. The IL12 receptor agonist according to any one of claims 1 to 3, comprising a first IL12 monomer having the structure of exemplary monomer 33 and a second IL12 monomer having the structure of exemplary monomer 35.

13. The IL12 receptor agonist according to any one of claims 1 to 3, comprising a first IL12 monomer having the structure of exemplary monomer 35 and a second IL12 monomer having the structure of exemplary monomer 57.

14. An IL-12 receptor agonist according to any one of claims 1 to 3, comprising a first IL-12 monomer having the structure of the exemplary monomer 57 and a second IL-12 monomer having the structure of the exemplary monomer 63.

15. An IL-12 receptor agonist according to any one of claims 1 to 3, comprising an IL-12Rβ1 portion configured to mask the p40 portion.

16. An IL-12 receptor agonist according to any one of claims 1 to 3, comprising an IL-12Rβ2 portion configured to mask the p35 portion.

17. An IL-12 receptor agonist according to any one of claims 1 to 3, comprising a first IL-12 monomer having the structure of the exemplary monomer 33 and a second IL-12 monomer having the structure of the exemplary monomer 58.

18. An IL-12 receptor agonist according to any one of claims 1 to 3, comprising a first IL-12 monomer having the structure of the exemplary monomer 28 and a second IL-12 monomer having the structure of the exemplary monomer 59.

19. An IL-12 receptor agonist according to any one of claims 1 to 3, comprising an antigen-binding fragment of an anti-IL-12 antibody configured to mask the p35 portion and / or the p40 portion.

20. The IL-12 receptor agonist according to claim 19, wherein the anti-IL-12 antibody binds to the p40 portion.

21. The IL-12 receptor agonist according to claim 19, wherein the anti-IL-12 antibody binds to the p35 portion.

22. An IL-12 receptor agonist according to any one of claims 1 to 3, wherein the p40 portion comprises a p40 D2 domain and a p40 D3 domain.

23. The IL-12 receptor agonist according to claim 22, wherein the p40 portion comprises a p40 D1 domain.

24. The first targeting moiety and / or the second targeting moiety is: (a) binds to a tumor-associated antigen; (b) binds to a tumor microenvironment antigen; (c) binds to a cell surface molecule of a tumor-reactive lymphocyte; (d) binds to a checkpoint inhibitor; (e) binds to a peptide-MHC complex; (f) is a peptide-MHC complex; (g) is associated with an autoimmune response or binds to an antigen targeted by an autoimmune response; (h) or is independently selected from (a) to (g) above, an IL-12 receptor agonist according to any one of claims 1 to 3.

25. The IL12 receptor agonist according to claim 24, wherein the first targeting moiety and / or the second targeting moiety is an antibody or an antigen-binding fragment of the antibody, and optionally, the targeting moiety is a Fab or scFv.

26. The IL12 receptor agonist according to claim 24, wherein the first targeting moiety and the second targeting moiety are the same.

27. The IL12 receptor agonist according to claim 24, wherein the first targeting moiety and / or the second targeting moiety binds to a tumor-associated antigen.

28. The IL12 receptor agonist according to claim 24, wherein the first targeting moiety and / or the second targeting moiety binds to a tumor microenvironment antigen.

29. The IL12 receptor agonist according to claim 24, wherein the first targeting moiety and / or the second targeting moiety binds to a cell surface molecule of a tumor lymphocyte.

30. The IL12 receptor agonist according to claim 29, wherein 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.

31. The IL12 receptor agonist according to claim 29, wherein the cell surface molecule is PD1 or LAG3.

32. The IL12 receptor agonist according to claim 31, wherein the first targeting moiety and / or the second targeting moiety is a blocking antibody.

33. The IL12 receptor agonist according to claim 24, wherein the first targeting moiety and / or the second targeting moiety binds to a checkpoint inhibitor.

34. The IL12 receptor agonist according to claim 33, wherein 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.

35. The IL12 receptor agonist according to claim 34, wherein the checkpoint inhibitor is PD1 or LAG3.

36. The IL12 receptor agonist according to claim 35, wherein the first targeting moiety and / or the second targeting moiety is a blocking antibody.

37. The IL12 receptor agonist according to claim 24, wherein the first targeting moiety and / or the second targeting moiety binds to an MHC-peptide complex.

38. The IL12 receptor agonist according to claim 24, wherein the first targeting moiety and / or the second targeting moiety is a peptide-MHC complex.

39. 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 and comprising 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, optionally wherein the substitution is optionally A, a p40 moiety.

40. One or more nucleic acids encoding the IL12 receptor agonist according to any one of claims 1 to 3.

41. A host cell engineered to express the IL12 receptor agonist according to any one of claims 1 to 3.

42. A method for producing the IL12 receptor agonist according to any one of claims 1 to 3, comprising culturing the host cell according to claim 41 and recovering the IL12 receptor agonist expressed thereby.

43. A pharmaceutical composition comprising the IL12 receptor agonist according to any one of claims 1 to 3 and an excipient.

44. An IL12 receptor agonist according to any one of claims 1 to 3 for use in a method of treating cancer, formulated to be administered to a subject in need of treatment for cancer.

45. A pharmaceutical composition according to claim 43 for use in a method of targeted treatment of cancer, formulated to be administered to a subject in need of targeted treatment of cancer.

46. An IL12 receptor agonist according to any one of claims 1 to 3 for use in a method of local delivery of IL12 protein, formulated to be administered to a subject in need of local delivery of IL12 protein.

47. An IL12 receptor agonist according to any one of claims 1 to 3 for use in a method of administering an IL12 therapeutic agent to a subject with reduced systemic exposure and / or reduced systemic toxicity, formulated to be administered to said subject.

48. An IL12 receptor agonist according to any one of claims 1 to 3 for use in a method of locally inducing an immune response in a target tissue, the IL12 receptor agonist being formulated to be administered to a subject in need thereof.

49. The IL12 receptor agonist according to claim 44, wherein the administration is systemic administration, and optionally intravenous administration.

50. The IL12 receptor agonist according to claim 44, wherein the administration is subcutaneous administration.

51. The IL12 receptor agonist according to claim 44, wherein the method further comprises administering an anti-PD1 antibody to the subject.

52. The IL12 receptor agonist according to claim 51, wherein the anti-PD1 antibody is MDX-1106 (nivolumab), MK-3475 (pembrolizumab), MEDI-0680 (AMP-514), PDR001, or BGB-108.