Immunomodulatory molecules and uses thereof

Abstract

The present application relates to immunomodulatory molecules comprising a first binding domain (e.g., an immunostimulatory cytokine, such as IL-2 or IL-12, or a variant thereof) that specifically recognizes a first target molecule (e.g., a receptor for an immunostimulatory cytokine) and a second binding domain (e.g., an agonist ligand, such as PD-L1 or PD-L2, or a variant thereof, or an agonist antigen-binding fragment, such as an anti-PD-1 agonist Fab, scFv, VHH, or full length antibody) that specifically recognizes a second target molecule (e.g., an inhibitory checkpoint molecule, such as PD-1), wherein the first binding domain, upon binding to the first target molecule, upregulates an immune response and the second binding domain, upon binding to the second target molecule, downregulates an immune response. Methods of making and using such immunomodulatory molecules are also provided.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 159,441, filed March 10, 2021, and International Patent Application No. PCT / US2021 / 073107, filed December 23, 2021, the contents of each of which are incorporated herein by reference in their entirety.

[0002] Submission of sequence listing as an ASCII text file The contents of the following ASCII text file submission are incorporated herein by reference in their entirety: Sequence Listing in Computer Readable Form (CRF) (Filename: 754392000740SEQLIST.TXT, Recorded: March 10, 2022, Size: 789,099 bytes).

[0003] The present invention relates to immunomodulatory molecules that both up- and down-regulate immune responses, methods for making and uses thereof. [Background technology]

[0004] Current immunotherapies often induce unwanted immune responses, such as overactivation of immune cells and cytokine storms.

[0005] Cytokines are key regulators of the innate and adaptive immune systems, enabling immune cells to communicate with each other. Cytokine therapy to stimulate the immune system in cancer patients has always been a major area of ​​interest in clinical cancer research. A key challenge with cytokine monotherapy is achieving effective antitumor responses without causing treatment-limiting toxicities. The low response rates and notorious toxicities of IL-2 and IL-12 therapy exemplify this dilemma. High doses of IL-2 have been shown to induce blood leak syndrome (VLS), tumor tolerance caused by activation-induced cell death (AICD), and immunosuppression caused by the activation of regulatory T cells (Tregs). These severe side effects often limit optimal IL-2 dosing and therefore limit the number of patients who successfully respond to this therapy. IL-12 has demonstrated modest antitumor responses in clinical trials, but is often accompanied by significant toxicity issues (Lasek et al., Cancer Immunol Immunother., 2014). IL-12 treatment has been found to be associated with systemic flu-like symptoms (e.g., fever, chills, fatigue, erythromelalgia, and headache) and toxic effects on the bone marrow and liver. Dose-finding studies have shown that patients can only tolerate IL-12 at doses below 1 μg / kg, far below the therapeutically effective dose. IL-12, whether used as monotherapy or in combination with other drugs, has failed to demonstrate strong and sustained therapeutic efficacy in clinical trials (Lasek et al., 2014).

[0006] Several approaches have been taken to overcome the problems associated with cytokine monotherapy. Recently, NKTR-214, a recombinant human IL-2 conjugated with polyethylene glycol (PEG) ("IL-2-PEG"), has shown promising results in animal models. IL-2-PEG offers two benefits. First, steric hindrance of PEG masks the region on IL-2 that interacts with the IL-2 receptor alpha (IL-2Rα) subunit, which is involved in the activation of immunosuppressive Tregs, thereby biasing activity toward tumor-killing CD8+ T cells (Charych et al., Clin Cancer Res., 2016). Second, PEG conjugation significantly improves plasma half-life and proteolytic stability, reducing immunogenicity and hepatic uptake (Chaffee et al., J Clin Invest., 1992; Pyatak et al., Res Commun Chem Pathol Pharmacol., 1980). Targeted delivery of cytokines (e.g., IL-12) to tumor sites by local injection or by using immunocytokines (cytokines fused to antibodies, antibody fragments, or ligand / receptor-Fc fusion proteins) has also been developed to overcome the side effects of cytokine therapy. Immunocytokines can target cytokines to cells or tissues of interest, such as tumor cells or immune effector cells (Klein et al., Oncoimmunology, 2017; King et al., J Clin Oncol., 2004).

[0007] The disclosures of all publications, patents, patent applications and published patent applications mentioned herein are hereby incorporated by reference in their entireties. Summary of the Invention [Means for solving the problem]

[0008] One aspect of the present application provides an immunomodulatory molecule comprising a first binding domain (e.g., an immunostimulatory cytokine such as IL-2 or IL-12 or a variant thereof) that specifically recognizes a first target molecule (e.g., a receptor for the immunostimulatory cytokine) and a second binding domain (e.g., an agonist ligand or variant thereof such as PD-L1 or PD-L2, or an agonist antigen-binding fragment such as an anti-PD-1 agonist Fab, scFv, VHH, or full-length antibody) that specifically recognizes a second target molecule (e.g., an inhibitory checkpoint molecule such as PD-1), wherein the first binding domain upregulates an immune response when bound to the first target molecule (e.g., IL-2 or IL-12 receptor), and the second binding domain downregulates an immune response when bound to the second target molecule (e.g., PD-1).

[0009] Another aspect of the present application provides a method of modulating an immune response in an individual, comprising administering to the individual an effective amount of any of the immunomodulatory molecules described herein.

[0010] Additionally provided are isolated nucleic acids encoding any one of the immunomodulatory molecules described herein, vectors (e.g., lentiviral vectors) comprising such nucleic acids, host cells (e.g., CHO cells) comprising such nucleic acids or vectors, and methods of making any one of the immunomodulatory molecules described herein.

[0011] Also provided are compositions (e.g., pharmaceutical compositions), kits, and articles of manufacture comprising any of the immunomodulatory molecules described herein. Methods of treating a disease or disorder in an individual (e.g., cancer, an infectious disease, an autoimmune disease, an allergy, transplant rejection, or graft-versus-host disease (GvHD)) using an effective amount of any of the immunomodulatory molecules or compositions (e.g., pharmaceutical compositions) described herein are also provided. [Brief explanation of the drawings]

[0012] [Figure 1-1]1A-1W show exemplary immunomodulatory molecule structures of the invention. FIG. 1A shows an exemplary immunomodulatory structure comprising a cytokine or variant thereof fused to the N-terminus of a subunit of the Fc fragment of a parent full-length antibody. FIG. 1B shows a dimeric (homodimeric or heterodimeric) cytokine or variant thereof (e.g., IFN-γ, IL-10, IL-12, or IL-23) expressed as a single chain and located in the hinge region of one heavy chain of the parent full-length antibody. FIGS. 1A-1B show exemplary immunomodulatory structures of the invention in which an immunostimulatory cytokine or variant thereof (e.g., IFN-γ, IL-2, IL-12, or IL-23) expressed as a single chain and located in the hinge region of one heavy chain of a dimeric parent ligand / receptor / Fab-hinge-Fc fusion protein is immunostimulatory. FIG. 1A shows that the Fab of the dimeric parent ligand / receptor / Fab-hinge-Fc fusion protein can be an agonist. Figure 1B shows that the Fab of the dimeric parent ligand / receptor / Fab-hinge-Fc fusion protein can be agonistic or nonagonistic. Figure 1C depicts an exemplary immunomodulatory structure of the invention that is immunostimulatory, in which an immunostimulatory cytokine or variant thereof (e.g., IFN-γ, IL-2, IL-12, or IL-23) is expressed as a single chain and located in the hinge region of one heavy chain of a parent full-length agonist antibody (e.g., an anti-PD-1 agonist). Figure 1D depicts an alternative exemplary immunomodulatory structure of the invention in which an immunostimulatory cytokine or variant thereof is located between the VH (e.g., within the Fab of an agonist antibody) and the Fc fragment subunit. [Figure 1-2]Figures 1A-1W show exemplary immunomodulatory molecule structures of the invention. Figure 1E shows an exemplary immunomodulatory structure comprising an immunostimulatory cytokine or variant thereof (e.g., IFN-γ, IL-2, IL-12, or IL-23) positioned in the hinge region of one polypeptide of a dimeric parent ligand / receptor-hinge-Fc fusion protein. Figure 1F shows an exemplary immunomodulatory structure comprising an immunostimulatory cytokine or variant thereof (e.g., IFN-γ, IL-2, IL-12, or IL-23) fused to the N-terminus of a subunit of the Fc fragment of a parent full-length agonist antibody (e.g., an anti-PD agonist). Figure 1G shows an exemplary immunomodulatory structure comprising a cytokine or variant thereof (e.g., IFN-γ, IL-2, IL-12, or IL-23) positioned in the hinge region of one polypeptide of a parent ligand / receptor-hinge-Fc fusion protein. FIG. 1H shows an exemplary immunomodulatory structure comprising an immunostimulatory cytokine or variant thereof (e.g., IFN-γ, IL-2, IL-12, or IL-23) positioned in the hinge region of one polypeptide of a dimeric parent ligand / receptor-hinge-Fc fusion protein. [Figure 1-3] Figures 1A-1W show exemplary immunomodulatory molecule structures of the invention. Figure 1I shows an exemplary immunostimulatory structure of the invention in which an immunostimulatory cytokine or variant thereof (e.g., IFN-γ, IL-2, IL-12, or IL-23) is located at the C-terminus of the Fc domain of a parent ligand / receptor-hinge-Fc fusion protein. Figure 1J shows an exemplary immunomodulatory structure comprising an immunostimulatory cytokine or variant thereof (e.g., IFN-γ, IL-2, IL-12, or IL-23) located at the C-terminus of the Fc domain of a parent full-length agonist antibody (e.g., an anti-PD-1 agonist). Figure 1K shows an exemplary immunomodulatory structure comprising an immunostimulatory cytokine or variant thereof (e.g., IFN-γ, IL-2, IL-12, or IL-23) located at the C-terminus of the Fc domain of a dimeric parent ligand / receptor / agonist Fab-hinge-Fc fusion protein. [Figure 1-4]Figures 1A-1W show exemplary immunomodulatory molecule structures of the present invention. Figure 1L shows two cytokines or variants thereof, each located in the hinge region of one polypeptide of a parent ligand / receptor-hinge-Fc fusion protein, or a dimeric (homodimeric or heterodimeric) cytokine or variants thereof, where each subunit is located in the hinge region of one polypeptide of a parent ligand / receptor-hinge-Fc fusion protein. Figure 1M shows two cytokines or variants thereof, each located in the hinge region of one polypeptide of a dimeric parent ligand / receptor-hinge-Fc fusion protein, or a dimeric (homodimeric or heterodimeric) cytokine or variants thereof, where each subunit is located in the hinge region of one polypeptide of a parent ligand / receptor-hinge-Fc fusion protein. Figures 1N-1O show two cytokines or variants thereof, each located in the hinge region of one polypeptide of a parent full-length agonist antibody, or a dimeric (homodimeric or heterodimeric) cytokine or variant thereof, with each subunit located in the hinge region of one polypeptide of a parent full-length agonist antibody. Figure 1N shows that the Fabs are the same and both are derived from agonist antibodies. Figure 1O shows that the Fabs can be different, with one being the Fab of an agonist antibody and the other being a different Fab (which can be non-agonist or agonist). [Figure 1-5]Figures 1A-1W show exemplary immunomodulatory molecule structures of the present invention. Figure 1P shows two cytokines or variants thereof, each located at the C-terminus of the Fc domain of a parent ligand / receptor-hinge-Fc fusion protein, or a dimeric (homodimeric or heterodimeric) cytokine or variant thereof, with each subunit located at the C-terminus of one polypeptide of the parent ligand / receptor-hinge-Fc fusion protein and the other at the hinge region of one polypeptide of the parent ligand / receptor-hinge-Fc fusion protein. Figures 1Q-1R show two cytokines or variants thereof, each located at the C-terminus of one polypeptide of a parent full-length antibody, or a dimeric (homodimeric or heterodimeric) cytokine or variant thereof, with each subunit located at the C-terminus of one polypeptide of the Fc domain of an agonist antibody and the other at the C-terminus of the Fc domain of the other polypeptide. Figure 1Q shows that the Fabs can be the same, both derived from an agonist antibody. Figure 1R shows that the Fabs can be different, one being the Fab of an agonist antibody and the other being a different Fab (which can be non-agonist or agonist). Figure 1S shows an exemplary immunomodulatory construct comprising an immunostimulatory cytokine or variant thereof fused to the C-terminus of the light chain constant region (CL) of a parent full-length agonist antibody. [Figure 1-6]Figures 1A-1W show exemplary immunomodulatory molecule structures of the invention. Figure 1T shows an exemplary immunomodulatory structure comprising an immunostimulatory cytokine or variant thereof fused to the N-terminus of the heavy chain variable domain (VH) of a parent full-length antibody. Figure 1U shows an exemplary immunomodulatory structure comprising an immunostimulatory cytokine or variant thereof fused to the N-terminus of one polypeptide of a dimeric parent ligand / receptor-hinge-Fc fusion protein. Figure 1V shows an exemplary immunomodulatory structure comprising an immunostimulatory cytokine or variant thereof fused to the N-terminus of one polypeptide of a parent ligand / receptor / agonist Fab-hinge-Fc fusion protein. Figure 1W shows an exemplary immunomodulatory structure comprising an immunostimulatory cytokine or variant thereof fused to the N-terminus of the heavy chain variable domain (VH) of a parent ligand / receptor / agonist Fab-hinge-Fc fusion protein. [Figure 2-1] Figures 2A-2C show tumor volumes in mice with CT26 syngeneic tumors treated with IL-12(F60A) / PD-L2-Fc, hinge (IW-#30) immunomodulatory molecule, IL-12(F60A) / PD-L2-Fc, C-terminus of HC (IW-#34) immunomodulatory molecule, or PBS (negative control). The responses of individual mice in each group are shown in Figures 2B-2C. [Figure 2-2] Figures 2A-2C show tumor volumes in mice bearing CT26 syngeneic tumors treated with IL-12(F60A) / PD-L2-Fc, hinge (IW-#30) immunomodulatory molecule, IL-12(F60A) / PD-L2-Fc, C-terminus of HC (IW-#34) immunomodulatory molecule, or PBS (negative control). Black arrows indicate the day of injection. The responses of individual mice in each group are shown in Figures 2B-2C. [Figure 3] Figures 3A-3B show the growth of CT26 and EMT6 tumor volume over time in cured CT26 mice (previously cured in Figures 2A-2C). [Figure 4]Figure 4A shows tumor volume in mice bearing CT26 syngeneic tumors treated with IL-12(E59A / F60A) / PD-L2-Fc, hinge (IW-#29) immunomodulatory molecule, IL-12(F60A) / PD-L2-Fc, hinge (IW-#30) immunomodulatory molecule, or PBS (negative control). Black arrows indicate the day of injection. Figure 4B shows a series of photographs taken in one mouse over the course of treatment with IL-12(F60A) / PD-L2-Fc, hinge (IW-#30) immunomodulatory molecule. [Figure 5-1] Figures 5A-5D show tumor volumes in mice bearing EMT6 syngeneic tumors treated with IL-12(E59A / F60A) / PD-L2-Fc, hinge (IW-#29) immunomodulatory molecule, IL-12(F60A) / PD-L2-Fc, hinge (IW-#30) immunomodulatory molecule, IL-12(E59A / F60A) / anti-PD-1, hinge (IW-#48) immunomodulatory molecule, or PBS (negative control). Black arrows indicate the day of injection. Responses of individual mice in each group are shown in Figures 5B-5D. [Figure 5-2] Figures 5A-5D show tumor volumes in mice bearing EMT6 syngeneic tumors treated with IL-12(E59A / F60A) / PD-L2-Fc, hinge (IW-#29) immunomodulatory molecule, IL-12(F60A) / PD-L2-Fc, hinge (IW-#30) immunomodulatory molecule, IL-12(E59A / F60A) / anti-PD-1, hinge (IW-#48) immunomodulatory molecule, or PBS (negative control). Black arrows indicate the day of injection. Responses of individual mice in each group are shown in Figures 5B-5D. [Figure 6] Figures 6A-6C show the growth of CT26 and EMT6 tumor volume over time in cured EMT6 mice (previously cured in Figures 5A-5D). [Figure 7]Figures 7A-7D show tumor volumes in mice bearing 4T1 syngeneic tumors treated with increasing concentrations (0, 1, 3, 10, and 50 mg / kg) of IL-12(F60A) / PD-L2-Fc, hinge (IW-#30) immunomodulatory molecule, IL-12(F60A) / PD-L2-Fc, C-terminal HC (IW-#34) immunomodulatory molecule, IL-12(F60A) / anti-PD-1, hinge (IW-#46) immunomodulatory molecule, IL-12(E59A / F60A) / anti-PD-1, hinge (IW-#48) immunomodulatory molecule, or PBS (negative control). Black arrows indicate the day of injection. [Figure 8] Figures 8A-8C show tumor volumes in mice bearing B16-F10 syngeneic tumors treated with IL-12(F60A) / PD-L2-Fc, hinge (IW-#30), PD-L2-Fc / IL-12(F60A) (IW-#34; C-terminal fusion) immunomodulatory molecules, or PBS (negative control). Black arrows indicate the day of injection. Figure 8A shows the mean tumor volume for all mouse groups, with the mean tumor size (±STD) at the time of administration of the first treatment indicated in parentheses. Figures 8B-8C show tumor volumes for individual mice treated with the indicated IL-12 immunomodulatory molecules. [Figure 9] Figures 9A-9C show tumor volumes in mice bearing LL2 syngeneic tumors treated with IL-12(F60A) / PD-L2-Fc, hinge (IW-#30) immunomodulatory molecule, IL-12(F60A) / PD-L2-Fc, C-terminus of HC (IW-#34) immunomodulatory molecule, or PBS (negative control). Black arrows indicate the day of injection. The responses of individual mice in each group are shown in Figures 9B-9C. [Figure 10]Figure 10 shows two approaches to activating the immune system against disease (e.g., cancer). The left panel shows a target-independent activation mechanism ("transactivation"), in which an immunomodulatory molecule can bind to a target antigen on an immune cell (e.g., a T cell) and a target antigen on a target cell (e.g., a tumor cell), thereby bringing the immune cell into close proximity with the target cell for therapeutic effect. However, this approach can be associated with systemic toxicity because binding domains that target immune cells (e.g., wild-type immunostimulatory cytokines such as IL-12 or IL-2) can stimulate an immune response even in the absence of target cells. The right panel shows a target cell antigen (e.g., a tumor antigen)-independent activation mechanism ("cisactivation"), in which an immunomodulatory molecule can both up- and down-regulate the immune response, more closely mimicking the natural regulation and balance of the immune system. In further aspects, the immunomodulatory molecules in both the left and right panels can further have a "restricted activation mechanism," in which the first binding domain is modified to reduce activity (binding and / or biological activity) upon binding to an immune cell (e.g., an immunostimulatory cytokine such as IL-12 or IL-2) that upregulates the immune response, and / or is in a "masked" configuration (e.g., located in the hinge region) until binding of the second binding domain to a second target antigen (e.g., a tumor antigen or an immune cell surface molecule) occurs. Exemplary immunomodulatory molecules of the present invention can function via restricted activation, cis-activation, trans-activation, or all mechanisms. [Figure 11-1]11A-11L show exemplary multispecific immunomodulatory molecules of the invention. The immunomodulatory molecules may comprise various combinations of binding domain types: i) a first binding domain, labeled "1" in the figures, that upregulates an immune response upon binding to a first target molecule; ii) a second binding domain, labeled "2" in the figures, that downregulates an immune response upon binding to a second target molecule; and iii) an optional third binding domain, labeled "3" in the figures, that helps localize the immunomodulatory molecule to a target site (e.g., the tumor microenvironment) by targeting a third target molecule (e.g., a marker of exhausted T cells, a T cell surface marker, or a tumor antigen). An immunomodulatory molecule may comprise one or more of any of the first, second, and / or third binding domains. The multiple first binding domains may be the same or different from each other. The multiple second binding domains may be the same or different from each other. The multiple third binding domains may be the same or different from each other. The various binding domains within an immunomodulatory molecule can be constructed in a variety of configurations, including but not limited to those shown in Figures 11A-11L. As illustrated in Figures 11A-11L, an IL-12 moiety located in C' of one or both Fc subunits (e.g., a mutant IL-12 moiety with reduced IL-12 activity, either constructed as a single-chain fusion or as two separate subunits) can be a type of first binding domain that upregulates an immune response upon binding to IL-12R on an immune cell. Thus, in Figures 11G, 11I, and 11J, the IL-12 moiety functions as the "first binding domain." The first binding domain (e.g., an immunostimulatory cytokine moiety or a variant thereof) can be positioned in the hinge region between the Fc subunit and the second or third binding domain, such as in the exemplary configurations shown in Figures 11A-11F, 11H, 11K, and 11L.Such a "limited access" configuration of the first binding domain to its first target molecule can allow i) reduced, minimized or absent binding / activity of the first binding domain to its first target molecule in the absence of binding of the second binding domain to the second target molecule and / or binding of the third binding domain to the third target molecule (any domain in the N' of the first binding domain), and ii) rescued / restored binding / activity of the first binding domain in the presence of binding between the second binding domain to the second target molecule and / or binding between the third binding domain to the third target molecule (any domain in the N' of the first binding domain). The first binding domain (e.g., an immunostimulatory cytokine moiety or variant thereof) can also be located at C' of one or both Fc subunits of an Fc fusion protein, such as the IL-12 moieties exemplified in Figures 11A-11L (either constructed as a single-chain fusion and fused to one Fc subunit, or constructed as two separate subunits each fused to one Fc subunit of the Fc domain). Such a configuration does not or hardly limits the binding / activity of the first binding domain. [Figure 11-2]11A-11L show exemplary multispecific immunomodulatory molecules of the invention. The immunomodulatory molecules may comprise various combinations of binding domain types: i) a first binding domain, labeled "1" in the figures, that upregulates an immune response upon binding to a first target molecule; ii) a second binding domain, labeled "2" in the figures, that downregulates an immune response upon binding to a second target molecule; and iii) an optional third binding domain, labeled "3" in the figures, that helps localize the immunomodulatory molecule to a target site (e.g., the tumor microenvironment) by targeting a third target molecule (e.g., a marker of exhausted T cells, a T cell surface marker, or a tumor antigen). An immunomodulatory molecule may comprise one or more of any of the first, second, and / or third binding domains. The multiple first binding domains may be the same or different from each other. The multiple second binding domains may be the same or different from each other. The multiple third binding domains may be the same or different from each other. The various binding domains within an immunomodulatory molecule can be constructed in a variety of configurations, including but not limited to those shown in Figures 11A-11L. As illustrated in Figures 11A-11L, an IL-12 moiety located in C' of one or both Fc subunits (e.g., a mutant IL-12 moiety with reduced IL-12 activity, either constructed as a single-chain fusion or as two separate subunits) can be a type of first binding domain that upregulates an immune response upon binding to IL-12R on an immune cell. Thus, in Figures 11G, 11I, and 11J, the IL-12 moiety functions as the "first binding domain." The first binding domain (e.g., an immunostimulatory cytokine moiety or a variant thereof) can be positioned in the hinge region between the Fc subunit and the second or third binding domain, such as in the exemplary configurations shown in Figures 11A-11F, 11H, 11K, and 11L.Such a "limited access" configuration of the first binding domain to its first target molecule can allow i) reduced, minimized or absent binding / activity of the first binding domain to its first target molecule in the absence of binding of the second binding domain to the second target molecule and / or binding of the third binding domain to the third target molecule (any domain in the N' of the first binding domain), and ii) rescued / restored binding / activity of the first binding domain in the presence of binding between the second binding domain to the second target molecule and / or binding between the third binding domain to the third target molecule (any domain in the N' of the first binding domain). The first binding domain (e.g., an immunostimulatory cytokine moiety or variant thereof) can also be located at C' of one or both Fc subunits of an Fc fusion protein, such as the IL-12 moieties exemplified in Figures 11A-11L (either constructed as a single-chain fusion and fused to one Fc subunit, or constructed as two separate subunits each fused to one Fc subunit of the Fc domain). Such a configuration does not or hardly limits the binding / activity of the first binding domain. [Figure 11-3]11A-11L show exemplary multispecific immunomodulatory molecules of the invention. The immunomodulatory molecules may comprise various combinations of binding domain types: i) a first binding domain, labeled "1" in the figures, that upregulates an immune response upon binding to a first target molecule; ii) a second binding domain, labeled "2" in the figures, that downregulates an immune response upon binding to a second target molecule; and iii) an optional third binding domain, labeled "3" in the figures, that helps localize the immunomodulatory molecule to a target site (e.g., the tumor microenvironment) by targeting a third target molecule (e.g., a marker of exhausted T cells, a T cell surface marker, or a tumor antigen). An immunomodulatory molecule may comprise one or more of any of the first, second, and / or third binding domains. The multiple first binding domains may be the same or different from each other. The multiple second binding domains may be the same or different from each other. The multiple third binding domains may be the same or different from each other. The various binding domains within an immunomodulatory molecule can be constructed in a variety of configurations, including but not limited to those shown in Figures 11A-11L. As illustrated in Figures 11A-11L, an IL-12 moiety located in C' of one or both Fc subunits (e.g., a mutant IL-12 moiety with reduced IL-12 activity, either constructed as a single-chain fusion or as two separate subunits) can be a type of first binding domain that upregulates an immune response upon binding to IL-12R on an immune cell. Thus, in Figures 11G, 11I, and 11J, the IL-12 moiety functions as the "first binding domain." The first binding domain (e.g., an immunostimulatory cytokine moiety or a variant thereof) can be positioned in the hinge region between the Fc subunit and the second or third binding domain, such as in the exemplary configurations shown in Figures 11A-11F, 11H, 11K, and 11L.Such a "limited access" configuration of the first binding domain to its first target molecule can allow i) reduced, minimized or absent binding / activity of the first binding domain to its first target molecule in the absence of binding of the second binding domain to the second target molecule and / or binding of the third binding domain to the third target molecule (any domain in the N' of the first binding domain), and ii) rescued / restored binding / activity of the first binding domain in the presence of binding between the second binding domain to the second target molecule and / or binding between the third binding domain to the third target molecule (any domain in the N' of the first binding domain). The first binding domain (e.g., an immunostimulatory cytokine moiety or variant thereof) can also be located at C' of one or both Fc subunits of an Fc fusion protein, such as the IL-12 moieties exemplified in Figures 11A-11L (either constructed as a single-chain fusion and fused to one Fc subunit, or constructed as two separate subunits each fused to one Fc subunit of the Fc domain). Such a configuration does not or hardly limits the binding / activity of the first binding domain. [Figure 12]Figures 12A-12D show exemplary immunomodulatory molecules with a first binding domain (e.g., an immunostimulatory cytokine, such as IL-12 or a variant thereof, constructed as a single-chain fusion, for example) located in the hinge region of one polypeptide chain of a parent (ligand / receptor / antigen-binding domain)-hinge-Fc fusion protein, which may be homodimeric or heterodimeric. Figure 12A shows an exemplary immunomodulatory molecule in which a PD-L1 or PD-L2 extracellular domain (wild-type or mutant) is fused via the hinge to the N-terminus of the Fc domain, and an immunostimulatory cytokine moiety (e.g., IL-12 or a variant constructed as a single-chain fusion) is located in the hinge region of one of the (PD-L1 or PD-L2)-hinge-Fc polypeptide chains. These may be referred to as IL-12 / PD-L1-Fc or IL-12 / PD-L2-Fc. Figure 12B shows an exemplary immunomodulatory molecule in which a PD-L1 or PD-L2 extracellular domain (wild-type or mutant) is fused via a first hinge to the N-terminus of a first Fc subunit, a CD155 extracellular domain (wild-type or mutant) is fused via a second hinge to the N-terminus of a second Fc subunit, and an immunostimulatory cytokine moiety (e.g., IL-12 or a variant constructed as a single-chain fusion) is located in the hinge region of one of the paired polypeptide chains (e.g., PD-L1 / PD-L2-hinge-Fc chain), which may be referred to as IL-12 / PD-L1-Fc / CD155-Fc or IL-12 / PD-L2-Fc / CD155-Fc. Figure 12C shows an exemplary immunomodulatory molecule in which a PD-L1 or PD-L2 extracellular domain (wild-type or mutant) is fused to the N-terminus of a first Fc subunit via a first hinge, an antibody moiety (e.g., an sdAb or scFv) that specifically recognizes a target molecule (which may be an agonist, antagonist, or neutral Ab that may or may not modulate an immune response) is fused to the N-terminus of a second Fc subunit via a second hinge, and an immunostimulatory cytokine moiety (e.g., IL-12 or a variant constructed as a single-chain fusion) is located in the hinge region of one of the paired polypeptide chains (e.g., PD-L1 / PD-L2-hinge-Fc chain). These may be referred to as sdAb / IL-12 / PD-L1-Fc or sdAb / IL-12 / PD-L2-Fc.Figure 12D shows an exemplary immunomodulatory molecule in which a PD-L1 or PD-L2 extracellular domain (wild-type or mutant) is fused to the N-terminus of a first Fc subunit via a first hinge, a Fab that specifically recognizes a target molecule (which may be an agonist, antagonist, or neutral Ab that may or may not modulate an immune response) is fused through CH1 to the N-terminus of a second Fc subunit via a second hinge, and an immunostimulatory cytokine portion (e.g., IL-12 or a variant constructed as a single-chain fusion) is located in the hinge region of one of the paired polypeptide chains (e.g., PD-L1 / PD-L2-hinge-Fc chain), which may be referred to as Fab / IL-12 / PD-L1-Fc or Fab / IL-12 / PD-L2-Fc. [Figure 13-1]Figures 13A-13D show exemplary immunomodulatory molecules with a first binding domain (e.g., an immunostimulatory cytokine, e.g., IL-12 or a variant thereof, e.g., constructed as a single-chain fusion) positioned C-terminal to the Fc domain (one or both Fc subunits) of a parent (ligand / receptor / antigen-binding domain)-hinge-Fc fusion protein, which can be homodimeric or heterodimeric. Figure 13A shows an exemplary immunomodulatory molecule in which a PD-L1 or PD-L2 extracellular domain (wild-type or mutant) is fused to the N-terminus of the Fc domain via an optional hinge, and an immunostimulatory cytokine moiety (e.g., IL-12 or a variant constructed as a single-chain fusion) is positioned C-terminal to one or both Fc subunits. These molecules may be referred to as PD-L1-Fc / IL-12 or PD-L2-Fc / IL-12. Figure 13B shows an exemplary immunomodulatory molecule in which a PD-L1 or PD-L2 extracellular domain (wild-type or mutant) is fused to the N-terminus of a first Fc subunit via a first optional hinge, a CD155 extracellular domain (wild-type or mutant) is fused to the N-terminus of a second Fc subunit via a second optional hinge, and an immunostimulatory cytokine moiety (e.g., IL-12 or a variant constructed as a single-chain fusion) is located at the C-terminus of the Fc domain of one or both subunits (e.g., the C' end of PD-L1 / PD-L2-hinge-Fc chain), which may be referred to as PD-L1-Fc / CD155-Fc / IL-12 or PD-L2-Fc / CD155-Fc / IL-12. [Figure 13-2]Figures 13A-13D show exemplary immunomodulatory molecules with a first binding domain (e.g., an immunostimulatory cytokine, such as IL-12 or a variant thereof, e.g., constructed as a single-chain fusion) positioned C-terminal to the Fc domain (one or both Fc subunits) of a parent (ligand / receptor / antigen binding domain)-hinge-Fc fusion protein, which can be homodimeric or heterodimeric. Figure 13C shows an exemplary immunomodulatory molecule in which a PD-L1 or PD-L2 extracellular domain (wild-type or mutant) is fused to the N-terminus of a first Fc subunit via a first optional hinge, an antibody moiety (e.g., an sdAb or scFv) that specifically recognizes a target molecule (which may be an agonist, antagonist, or neutral Ab that may or may not modulate an immune response) is fused to the N-terminus of a second Fc subunit via a second optional hinge, and an immunostimulatory cytokine moiety (e.g., IL-12 or a variant constructed as a single-chain fusion) is located at the C-terminus of the Fc domain of one or both subunits (e.g., at the C' end of PD-L1 / PD-L2-hinge-Fc chain). These may be referred to as sdAb / PD-L1-Fc / IL-12 or sdAb / PD-L2-Fc / IL-12. Figure 13D shows an exemplary multi-target immunomodulatory molecule in which a PD-L1 or PD-L2 extracellular domain (wild-type or mutant) is fused to the N-terminus of a first Fc subunit via a first optional hinge, a Fab that specifically recognizes a target molecule (which may be an agonist, antagonist, or neutral Ab that may or may not modulate an immune response) is fused via its CH1 to the N-terminus of a second Fc subunit via a second optional hinge, and an immunostimulatory cytokine moiety (e.g., IL-12 or a variant constructed as a single-chain fusion) is located at the C-terminus of the Fc domain of one or both subunits (e.g., the C' end of PD-L1 / PD-L2-hinge-Fc chain). This molecule may be referred to as Fab / PD-L1-Fc / IL-12 or Fab / PD-L2-Fc / IL-12. [Figure 14-1]Figures 14A-14D show exemplary immunomodulatory molecules with two first binding domains (e.g., immunostimulatory cytokines such as IL-12, IL-2 or variants thereof, e.g., constructed as single-chain fusions) each positioned in the hinge region of one polypeptide chain of a parent (ligand / receptor / antigen binding domain)-hinge-Fc fusion protein, which can be homodimeric or heterodimeric. Figure 14A shows an exemplary immunomodulatory molecule in which a PD-L1 or PD-L2 extracellular domain (wild-type or mutant) is fused via a hinge to the N-terminus of an Fc domain, a first immunostimulatory cytokine moiety (e.g., IL-12 or a mutant constructed as a single-chain fusion) is positioned in the hinge region of one of the (PD-L1 or PD-L2)-hinge-Fc polypeptide chains, and a second immunostimulatory cytokine moiety (e.g., IL-2 or a variant thereof) is positioned in the hinge region of the other (PD-L1 or PD-L2)-hinge-Fc polypeptide chain. This may be referred to as IL-12 / IL-2 / PD-L1-Fc or IL-12 / IL-2 / PD-L2-Fc. Figure 14B shows an exemplary immunomodulatory molecule in which a PD-L1 or PD-L2 extracellular domain (wild-type or mutant) is fused via a first hinge to the N-terminus of a first Fc subunit, a CD155 extracellular domain (wild-type or mutant) is fused via a second hinge to the N-terminus of a second Fc subunit, a first immunostimulatory cytokine moiety (e.g., IL-12 or a variant constructed as a single-chain fusion) is positioned in the hinge region of one of the paired polypeptide chains (e.g., PD-L1 / PD-L2-hinge-Fc chain), and a second immunostimulatory cytokine moiety (e.g., IL-2 or a variant thereof) is positioned in the hinge region of the other paired polypeptide chain (e.g., CD155-hinge-Fc chain). It may be referred to as IL-12 / IL-2 / PD-L1-Fc / CD155-Fc or IL-12 / IL-2 / PD-L2-Fc / CD155-Fc. [Figure 14-2]Figures 14A-14D show exemplary immunomodulatory molecules with two first binding domains (e.g., immunostimulatory cytokines such as IL-12, IL-2 or variants thereof, e.g., constructed as single-chain fusions) each positioned in the hinge region of one polypeptide chain of a parent (ligand / receptor / antigen binding domain)-hinge-Fc fusion protein, which can be homodimeric or heterodimeric. Figure 14C shows an exemplary immunomodulatory molecule in which a PD-L1 or PD-L2 extracellular domain (wild-type or mutant) is fused to the N-terminus of a first Fc subunit via a first hinge, an antibody moiety (e.g., an sdAb or scFv) that specifically recognizes a target molecule (which may be an agonist, antagonist, or neutral Ab that may or may not modulate an immune response) is fused to the N-terminus of a second Fc subunit via a second hinge, a first immunostimulatory cytokine moiety (e.g., IL-12 or a variant constructed as a single-chain fusion) is positioned in the hinge region of one of the paired polypeptide chains (e.g., PD-L1 / PD-L2-hinge-Fc chain), and a second immunostimulatory cytokine moiety (e.g., IL-2 or a variant thereof) is positioned in the hinge region of the other of the paired polypeptide chains (e.g., CD155-hinge-Fc chain). It can be referred to as sdAb / IL-12 / IL-2 / PD-L1-Fc or sdAb / IL-12 / IL-2 / PD-L2-Fc. Figure 14D shows an exemplary immunomodulatory molecule in which a PD-L1 or PD-L2 extracellular domain (wild-type or mutant) is fused to the N-terminus of a first Fc subunit via a first hinge; a Fab that specifically recognizes a target molecule (which may be an agonist, antagonist, or neutral Ab that may or may not modulate an immune response) is fused through its CH1 to the N-terminus of a second Fc subunit via a second hinge; a first immunostimulatory cytokine moiety (e.g., IL-12 or a variant constructed as a single-chain fusion) is positioned in the hinge region of one of the paired polypeptide chains (e.g., PD-L1 / PD-L2-hinge-Fc chain); and a second immunostimulatory cytokine moiety (e.g., IL-2 or a variant thereof) is positioned in the hinge region of the other of the paired polypeptide chains (e.g., CD155-hinge-Fc chain).It may be referred to as Fab / IL-12 / IL-2 / PD-L1-Fc or Fab / IL-12 / IL-2 / PD-L2-Fc. [Figure 15-1] Figures 15A-15D show exemplary immunomodulatory molecules having two first binding domains (e.g., immunostimulatory cytokines such as IL-12, IL-2, or variants thereof, e.g., constructed as single-chain fusions), one located in the hinge region of one polypeptide chain of the parent (ligand / receptor / antigen-binding domain)-hinge-Fc fusion protein and the other located at the C-terminus of one or both Fc subunits of the parent (ligand / receptor / antigen-binding domain)-hinge-Fc fusion protein. Figure 15A shows an exemplary immunomodulatory molecule in which a PD-L1 or PD-L2 extracellular domain (wild-type or mutant) is fused via a hinge to the N-terminus of an Fc domain, a first immunostimulatory cytokine moiety (e.g., IL-2 or a variant) is positioned in the hinge region of one of the (PD-L1 or PD-L2)-hinge-Fc polypeptide chains, and a second immunostimulatory cytokine moiety (e.g., IL-12 or a variant constructed as a single-chain fusion) is positioned in the C' region of the Fc subunit of the other (PD-L1 or PD-L2)-hinge-Fc polypeptide chain. This molecule may be referred to as IL-2 / PD-L1-Fc / IL-12 or IL-2 / PD-L2-Fc / IL-12. Figure 15B shows an exemplary immunomodulatory molecule in which a PD-L1 or PD-L2 extracellular domain (wild-type or mutant) is fused via a first hinge to the N-terminus of a first Fc subunit, a CD155 extracellular domain (wild-type or mutant) is fused via a second hinge to the N-terminus of a second Fc subunit, a first immunostimulatory cytokine moiety (e.g., IL-12 or a variant constructed as a single-chain fusion) is positioned at the C' of the Fc subunit of one of the paired polypeptide chains (e.g., PD-L1 / PD-L2-hinge-Fc chain), and a second immunostimulatory cytokine moiety (e.g., IL-2 or a variant thereof) is positioned in the hinge region of the other paired polypeptide chain (e.g., CD155-hinge-Fc chain). It may be referred to as IL-2 / PD-L1-Fc / CD155-Fc / IL-12 or IL-2 / PD-L2-Fc / CD155-Fc / IL-12. [Figure 15-2]Figures 15A-15D show exemplary immunomodulatory molecules having two first binding domains (e.g., immunostimulatory cytokines such as IL-12, IL-2, or variants thereof, e.g., constructed as single-chain fusions), one located in the hinge region of one polypeptide chain of the parent (ligand / receptor / antigen-binding domain)-hinge-Fc fusion protein and the other located at the C-terminus of one or both Fc subunits of the parent (ligand / receptor / antigen-binding domain)-hinge-Fc fusion protein. Figure 15C shows an exemplary immunomodulatory molecule in which a PD-L1 or PD-L2 extracellular domain (wild-type or mutant) is fused to the N-terminus of a first Fc subunit via a first hinge, an antibody moiety (e.g., an sdAb or scFv) that specifically recognizes a target molecule (which may be an agonist, antagonist, or neutral Ab that may or may not modulate an immune response) is fused to the N-terminus of a second Fc subunit via a second hinge, a first immunostimulatory cytokine moiety (e.g., IL-12 or a variant constructed as a single-chain fusion) is positioned at the C' of one Fc subunit of the paired polypeptide chains (e.g., PD-L1 / PD-L2-hinge-Fc chain), and a second immunostimulatory cytokine moiety (e.g., IL-2 or a variant thereof) is positioned in the hinge region of the other chain of the paired polypeptide chains (e.g., CD155-hinge-Fc chain). It can be referred to as sdAb / IL-2 / PD-L1-Fc / IL-12 or sdAb / IL-2 / PD-L2-Fc / IL-12.Figure 15D shows an exemplary immunomodulatory molecule in which PD-L1 or PD-L2 (wild-type or mutant) is fused to the N-terminus of a first Fc subunit via a first hinge, a Fab that specifically recognizes a target molecule (which may be an agonist, antagonist, or neutral Ab that may or may not modulate an immune response) is fused through its CH1 to the N-terminus of a second Fc subunit via a second hinge, a first immunostimulatory cytokine moiety (e.g., IL-12 or a variant constructed as a single-chain fusion) is positioned at the C' of one Fc subunit of the paired polypeptide chains (e.g., PD-L1 / PD-L2-hinge-Fc chain), and a second immunostimulatory cytokine moiety (e.g., IL-2 or a variant thereof) is positioned in the hinge region of the other chain of the paired polypeptide chains (e.g., CD155-hinge-Fc chain). It may be referred to as Fab / IL-2 / PD-L1-Fc / IL-12 or Fab / IL-2 / PD-L2-Fc / IL-12. [Figure 16] Figure 16 shows 4T1 mouse breast cancer tumors extracted from the mammary fat pads of mice treated with IL-12(E59A / F60A) / PD-L2-Fc(IW-#29), IL-12(F60A) / PD-L2-Fc(IW-#30), a combination of anti-PD-1 and anti-CTLA-4 antibodies, or PBS (negative control). [Figure 17] Figure 17 shows 4T1 mouse breast cancer cells metastasizing to the lungs in mice injected with 4T1 cells into the mammary fat pad and treated with IL-12(E59A / F60A) / PD-L2-Fc(IW-#29), IL-12(IW-F60A) / PD-L2-Fc(IW-#30), a combination of anti-PD-1 and anti-CTLA-4 antibodies, or PBS (negative control). [Figure 18] Figure 18 shows tumor volumes in mice with 4T1 syngeneic tumors treated with IL-12 (E59A / F60A) / anti-PD-1 (IW-#48), IL-12 (E59A / F60A) / PD-L2-Fc (IW-#29), IL-12 (E59A / F60A) / IL-2 (R38D / K43E / E61R) / anti-PD-1 (IW-#54) immunomodulatory molecules, or PBS (negative control). Black arrows indicate the day of injection. [Figure 19] Figure 1 shows tumor volume in mice with EMT6 syngeneic tumors treated with IL-12 (E59A / F60A) / anti-PD-1 (IW-#48), IL-12 (E59A / F60A) / PD-L2-Fc (IW-#29), IL-2 (R38D / K43E / E61R) / PD-L2-Fc (IW-#11) immunomodulatory molecules, or PBS (negative control). Black arrows indicate the day of injection. DETAILED DESCRIPTION OF THE INVENTION

[0013] Current immunotherapies often induce unwanted immune responses, such as immune cell overactivation and cytokine storms. For example, cytokine therapy (e.g., for cancer treatment) has shown limited success because severe toxicity limits dose setting far below the therapeutically effective dose. Immunocytokines are constructs in which a cytokine is fused to an antibody, antigen-binding fragment, ligand-Fc fusion protein, or receptor-Fc fusion protein (collectively referred to as "ligand / receptor-Fc fusion protein" or "ligand / receptor-hinge-Fc fusion protein" hereinafter). Recognition of the target antigen by an antibody or antigen-binding fragment (e.g., antibody fragment, ligand, or receptor) within an immunomodulatory molecule allows the cytokine to be delivered to target cells (e.g., tumor cells or immune effector cells) or tissues, thereby reducing nonspecific (off-target) cytokine activity and / or associated toxicity (e.g., toxicity to healthy cells or tissues) and concentrating the cytokine therapeutic effect at the target site (e.g., disease site). Activation of immunomodulatory molecules can occur via transactivation, which requires specific binding of an antibody or antigen-binding fragment to a target antigen on tumor cells; or via cisactivation, which requires specific binding of an antibody or antigen-binding fragment to a target antigen on immune cells (see Figure 10). Many immunocytokines developed today have cytokine moieties fused to the N- or C-terminus of the heavy or light chain of a full-length antibody (e.g., Hu14.8-IL2, NHS-IL2LT, NHS-IL12, BC1-IL12; see, e.g., Figures 1C-1E) or to the N- or C-terminus of an antigen-binding fragment (e.g., diabodies, scFvs, e.g., L19-IL2 or F16-IL2), so cytokine-receptor binding / activation can still occur in the absence of antibody-antigen recognition, which can lead to off-target toxicity. Recently developed immune checkpoint inhibitors (e.g., anti-PD-1, anti-CTLA-4 Abs) have shown some great clinical success in cancer patients, but also focus on upregulating immune responses, which may exacerbate systemic toxicity when further used in conjunction with pro-inflammatory cytokines.

[0014] The present invention provides immunomodulatory molecules that have opposing effects in modulating immune responses and have demonstrated significantly better toxicity profiles and therapeutic efficacy. The immunomodulatory molecules comprise a first binding domain (e.g., an immunostimulatory cytokine or a variant thereof, e.g., IL-12, IL-2, IFN-γ) that specifically recognizes a first target molecule (e.g., a receptor for the immunostimulatory cytokine or a variant thereof) and a second binding domain (e.g., a ligand such as PD-L1, PD-L2, CD155 extracellular domain or a variant thereof) that specifically recognizes a second target molecule (e.g., PD-1 or TIGIT on an immune effector cell), wherein upon binding to the first target molecule, the first binding domain upregulates the immune response, and upon binding to the second target molecule, the second binding domain downregulates the immune response. For example, if the IL-12 cytokine (pro-inflammatory) is located in the hinge region of a PD-L2 extracellular domain-hinge-Fc fusion protein, the resulting IL-12 / PD-L2-Fc immunomodulatory molecule not only specifically targets IL-12 activity (e.g., IL-12 receptor binding activity and / or IL-12 pro-inflammatory activity) on PD-1+ target cells, but also stimulates PD-1 inhibitory immune checkpoint signaling via PD-L2-PD-1 binding, thus creating an immunosuppressive signal that "balances" or "counters" the immunostimulatory activity of IL-12. Any agonist antibody or ligand (e.g., PD-L2, PD-L1, CD80, or CD86) that can activate or stimulate immunosuppressive signaling pathways (e.g., by binding to an inhibitory immune checkpoint molecule such as PD-1 or CTLA-4), or any antagonist antibody, ligand, or receptor that can reduce or block immunostimulatory signaling pathways (e.g., by binding to a stimulatory immune checkpoint molecule such as CD27 or CD28, or an immunostimulatory receptor such as IL-2R), can be used in combination with an immunostimulatory cytokine or variant thereof (e.g., IL-2, IL-12, IFN-γ, or IL-23) to construct an immunomodulatory molecule having any of the immunomodulatory molecule configurations described herein.Any antagonistic antibody, ligand, or receptor capable of reducing or blocking an immunosuppressive signaling pathway (e.g., by binding to an inhibitory immune checkpoint molecule such as PD-1 or CTLA-4), or any agonistic antibody or ligand (e.g., CD70, CD80, CD86, or IL-2) capable of activating or stimulating an immunostimulatory signaling pathway (e.g., by binding to a stimulatory immune checkpoint molecule such as CD27 or CD28, or an immunostimulatory receptor such as IL-2R), can be used in combination with an immunosuppressive cytokine or variant thereof (e.g., IL-10, IL-27, IL-35, TGF-β) to construct an immunomodulatory molecule having any of the immunomodulatory molecule configurations described herein. The immunomodulatory molecules described herein can include one or more first binding domains and / or one or more second binding domains to achieve multiple immune response modulation. The multiple first binding domains can be the same or different. The multiple second binding domains can be the same or different. See, for example, Figures 1A-1W and 11A-15D.

[0015] The first binding domain can comprise a molecule such as an immunostimulatory cytokine, a ligand, or an agonist antibody (e.g., a ligand or agonist Ab that stimulates a stimulatory checkpoint molecule such as OX40) that targets immune cells such as T cells, NK cells, DC cells, macrophages, and B cells. In some embodiments, the present invention provides first binding domains that have reduced activity (e.g., reduced binding or restimulatory activity for its target) compared to the unmodified parent first binding domain. See, for example, the cytokine variants described herein that exhibit dramatically reduced activity compared to the wild-type cytokine. The reduced binding affinity of the first binding domain can skew the mechanism of action toward target-dependent activation (cis-activation) and away from target-independent activation (trans-activation).

[0016] The second binding domain may comprise a molecule such as an immunosuppressive cytokine, ligand, or agonist antibody (e.g., a ligand (PD-L1, PD-L2, CD155, etc.) or agonist Ab that stimulates an inhibitory checkpoint molecule such as PD-1 or TIGIT) to downregulate an immune response. In some embodiments, the present invention provides anti-PD-1 antibodies (antagonist Abs) with reduced binding affinity for PD-1, thus reducing the immune response that may be induced by a wild-type anti-PD-1 antibody (antagonist Ab such as nivolumab) (see Example 22). In some embodiments, the present invention also provides ligands with increased binding affinity for inhibitory checkpoint molecules such as PD-1, which can further downregulate an immune response compared to the wild-type ligand. See, e.g., the mutant PD-L1 and PD-L2 molecules generated in Example 23. Immunomodulatory molecules containing mutant PD-L1 or PD-L2 extracellular domains as the second binding domain reduced adverse events compared with those with wild-type PD-L1 or PD-L2 extracellular domains. -8 MK d low binding affinity of PD-L2(mut) or PD-L1(mut) to PD-1 compared with wild-type anti-PD-1 antibodies (>10 -9 MK d Mutant anti-PD-1 antibodies (antagonist Abs; 10 -8 MK d The low binding affinity of PD-1 allows the immunomodulatory molecule to target not only PD-1-positive cells but also cancer cells that express much higher levels of PD-1, such as exhausted T cells and the tumor microenvironment, which attempt to bypass anti-tumor activity.

[0017] For example, the IL-12(E59A / F60A) / PD-L2(S58V)-Fc immunomodulatory molecule described herein provides both a positive signal (IL-12 / IL-12R signaling) and a negative signal (PD-1 / PD-L2 signaling). The immunomodulatory molecules with opposing effects described herein mimic the natural T cell activation process, modulate the T cell activation process, and allow overactivation of the immune system to be overcome.

[0018] The immunomodulatory molecules comprising the first and second binding domains described herein can further comprise a third binding domain that specifically recognizes a third target molecule. The third binding domain can help localize the immunomodulatory molecule to a target site (e.g., the tumor microenvironment) by binding to the third target molecule (e.g., a marker for exhausted T cells, a T cell surface marker, or a tumor antigen). Upon binding to the third target molecule, the third binding domain can i) upregulate the above-mentioned immune response or another immune response, ii) downregulate the above-mentioned immune response or another immune response, or iii) not regulate any immune response by itself. For example, the third binding domain can function solely as a tumor antigen targeting domain to deliver the immunomodulatory molecule to the tumor site, or as an immune effector cell targeting domain to deliver the immunomodulatory molecule to or enhance its binding to immune effector cells. The tumor microenvironment contains relatively high levels of exhausted T cells that express several markers, such as TIGIT, TIM3, LAG3, and PD-1. Because the expression patterns and levels of exhaustion markers in the tumor microenvironment (TME) vary greatly, a third binding domain can be used to target additional exhaustion markers to broadly target the TME. Alternatively, the third binding domain can be used to target specific cancers to specific tumor antigens, including, but not limited to, Her2, CEACAM, Her3, EGFR, Trop2, CLDN18.2, prostate-specific antigen, MUC1, EpCAM, GPC3, mesothelin (MSLN), nectin-4, folate receptor alpha, tissue factor, and the like. The third binding domain can also target T cell markers, including, but not limited to, CD4, CD8, CD3, CD2, CD5, CD7, CD40L, CD25, CD137, CD69, CTLA4, CD127, ICOS, and the like. The third binding domain may also target a dendritic cell marker, including, but not limited to, CD1c, CD11c, CD141, CD123, BDCA-2, BDCA-4, CLEC9A, XCR1, CD80, CD86, PD-L1, PD-L2, etc.The third binding domain may also target monocyte / macrophage markers, including but not limited to CSF1R, CD80, CD86, CD11, CD14, CD68, CD163, CD16, CD32, CD64, etc. The third binding domain may also target neutrophil cell markers, including but not limited to CD11, CD16, CD32, etc. The immunomodulatory molecules described herein can comprise one or more third binding domains to achieve multiple immune response modulation or to enhance targeting. The multiple third binding domains can be the same or different.

[0019] Furthermore, the present invention also provides immunomodulatory molecules with specific unique configurations that address problems faced by current cytokine / immunocytokine therapies. In particular, some immunomodulatory molecules of the present invention comprise a first binding domain (e.g., a cytokine or a variant thereof) bound to a second binding domain (e.g., a ligand, receptor, VHH, scFv, or Fab) at the hinge region between the Fc domain subunit or a portion thereof (e.g., a CH2-CH3 fragment, or CH2 only, or CH3 only), e.g., the hinge region between an scFv and an Fc domain subunit (e.g., an antigen-binding polypeptide comprising a VH-VL-cytokine-Fc subunit or a VL-VH-cytokine-Fc subunit), the Fab and Fc domain of a full-length antibody, or a portion thereof (e.g., a CH2-CH3 fragment, or CH2 only, or CH3 only). Placing a steric hindrance between the first binding domain (e.g., a ligand, receptor, VHH, scFv, Fab) and the Fc domain or portion thereof in the hinge region between the first binding domain (e.g., an antigen-binding polypeptide comprising a VH-CH1-cytokine-Fc subunit) or the hinge region between the ligand (or receptor) and the Fc domain subunit (e.g., an antigen-binding polypeptide comprising a ligand-cytokine-Fc subunit or a receptor-cytokine-Fc subunit) reduces the nonspecific activity (i.e., antibody- or antigen-binding fragment-independent binding) and increases the specific activity (i.e., antibody- or antigen-binding fragment-dependent binding) of the first binding domain (e.g., an immunostimulatory cytokine). Without being bound by theory, steric hindrance between the second binding domain (e.g., a ligand, receptor, VHH, scFv, Fab) and the Fc domain or portion thereof is thought to reduce the accessibility of the first binding domain (e.g., an immunomodulatory cytokine or variant thereof) to its target molecule (e.g., a receptor for an immunomodulatory cytokine) in the absence of binding of the second binding domain to the second target molecule, or to "mask" the first binding domain from binding to its first target molecule. On the other hand, binding of the second binding domain to a second target molecule activates the first binding domain.Surprisingly, unlike other immunocytokine designs that "expose" a cytokine moiety at its N- or C-terminus, the unique immunomodulatory molecule configuration of the present invention requires binding of a second binding domain (e.g., a ligand, receptor, VHH, scFv, or Fab) to a second target molecule (e.g., a receptor) before binding of a first binding domain (e.g., an immunomodulatory cytokine moiety) to its first target molecule can occur, thus ensuring that upregulation of an immune response (e.g., cytokine signaling activation) is entirely dependent (on-target) on second binding domain binding. This enhanced targeting specificity design, optionally further combined with the reduced activity of the first binding domain (e.g., a cytokine variant described herein), allows the desired immune response (e.g., cytokine signaling activation) to be safely delivered to a target site (e.g., a tumor cell or immune cell) to achieve a therapeutic effect. Such a unique targeting specificity design adds an additional layer of regulation to the current "balancing" or "countering" immune response design, further fine-tuning the biological activity and toxicity of the immunomodulatory molecules described herein.

[0020] Accordingly, one aspect of the present application provides an immunomodulatory molecule comprising a first binding domain (e.g., a ligand, VHH, scFv, or VH, e.g., an immunostimulatory cytokine such as IL-2 or IL-12) that specifically recognizes a first target molecule (e.g., a cell surface antigen or receptor, e.g., a receptor for an immunostimulatory cytokine), and a second binding domain (e.g., a ligand, VHH, scFv, or VH, e.g., an agonist ligand such as PD-L1 or PD-L2, or an agonist antigen-binding fragment such as an anti-PD-1 agonist Fab, scFv, VH, VHH, or full-length antibody) that specifically recognizes a second target molecule (e.g., a cell surface antigen or receptor, e.g., an inhibitory checkpoint molecule such as PD-1), wherein the first binding domain upregulates an immune response when bound to the first target molecule, and the second binding domain downregulates an immune response when bound to the second target molecule.

[0021] Also provided are isolated nucleic acids encoding such immunomodulatory molecules, vectors containing such nucleic acids, host cells containing such nucleic acids or vectors, methods for producing such immunomodulatory molecules, pharmaceutical compositions and articles containing such immunomodulatory molecules, methods for modulating immune responses with such immunomodulatory molecules or pharmaceutical compositions thereof, and methods for treating diseases (e.g., cancer, viral infections, autoimmune diseases) with such immunomodulatory molecules or pharmaceutical compositions thereof.

[0022] I. Definition The practice of the present invention will employ, unless specifically indicated to the contrary, conventional methods of virology, immunology, microbiology, molecular biology and recombinant DNA techniques within the skill of the art, many of which are described below by way of example, and such techniques are fully explained in the literature. For example, Current Protocols in Molecular Biology or Current Protocols in Immunology,John Wiley & Sons,New York,NY(2009);Ausubel et al.,Short Protocols in Molecular Biology,3rd ed.,John Wiley & Sons,1995;Sambrook and Russell,Molecular Cloning:A Laboratory Manual(3rd Edition,2001);Maniatis et al.,Molecular Cloning:A Laboratory Manual(1982);DNA Cloning: A Practical Approach,vol.I&II(D.Glover,ed.);Oligonucleotide Synthesis(N.Gait,ed.,1984);Nucleic Acid Hybridization(B.Hames & S.Higgins,eds.,1985);Transcription and Translation(B.Hames & S.Higgins,eds.,1984);Animal Cell Culture(R.Freshney,ed.,1986);Perbal,A See Practical Guide to Molecular Cloning (1984) and other similar references.

[0023] The term "immunocytokine," as used herein, refers to an antigen-binding protein (e.g., an antibody, or antigen-binding fragment (e.g., a ligand, receptor, or antibody fragment)) format that is fused to a cytokine molecule. The antigen-binding protein (e.g., an antibody, or antigen-binding fragment (e.g., a ligand, receptor, or antibody fragment)) format can be any of those described herein, and the cytokine can be fused to the antigen-binding protein format directly or by a linker or by chemical conjugation.

[0024] The term "cytokine storm," also known as "cytokine cascade" or "hypercytokinemia," is a potentially fatal immune response that typically consists of a positive feedback loop between cytokines and immune cells, and is accompanied by highly elevated levels of various cytokines (e.g., INF-γ, IL-10, IL-6, CCL2, etc.).

[0025] As used herein, when a binding domain (e.g., an antibody, antigen-binding fragment, or ligand) is referred to as an "antagonist" of a target molecule (e.g., a receptor or immune checkpoint molecule), it means that, upon target antigen binding, the binding domain (e.g., an antibody, antigen-binding fragment, or ligand) blocks, inhibits, or reduces (e.g., by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) the biological activity of the target molecule (e.g., blocks receptor signaling). For example, an anti-PD-1 antagonist antibody is an antibody that reduces or blocks PD-1 signaling, and an antagonist ligand of the IL-12 receptor reduces or blocks IL-12 receptor signaling. When a binding domain (e.g., an antibody, antigen-binding fragment, or ligand) is referred to as an "agonist" of a target molecule (e.g., a receptor or immune checkpoint molecule), it means that, upon binding to the target molecule, the binding domain (e.g., an antibody, antigen-binding fragment, or ligand) stimulates, activates, or enhances (e.g., enhances by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more) the biological activity of the target molecule (e.g., activates receptor signaling). For example, wild-type PD-L2 ligand (e.g., the extracellular domain) is an agonist that activates PD-1 signaling. For example, an anti-PD-1 agonist antibody is an antibody that induces or enhances PD-1 signaling.

[0026] As used herein, "treatment" or "treating" refers to an approach for obtaining beneficial or desired results, including clinical results. For purposes of the present invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms caused by a disease, reducing the extent of the disease, stabilizing the disease (e.g., preventing or slowing the progression of the disease), preventing or slowing the spread of the disease (e.g., metastasis), preventing or slowing the recurrence of the disease, slowing or slowing the progression of the disease, ameliorating the disease state, providing remission (partial or complete) of the disease, reducing the dose of one or more other medications required to treat the disease, slowing the progression of the disease, improving quality of life, and / or prolonging survival. "Treatment" also encompasses the reduction of the pathological consequences of a disease. The methods of the present invention contemplate any one or more of these aspects of treatment. For example, an individual is successfully "treated" if one or more symptoms associated with a viral infection are alleviated or eliminated, including, but not limited to, reducing the proliferation of (or destroying) the infectious virus, reducing symptoms caused by the disease (e.g., cytokine storm), improving the quality of life of an individual affected by the disease, reducing the dose of other medications required to treat the disease, and / or prolonging the survival of the individual.

[0027] The term "prevent," and similar phrases such as "prevented," "preventing," and the like, refer to an approach for preventing, inhibiting, or reducing the likelihood of a disease or condition, such as cancer, recurring. It also refers to delaying the recurrence of a disease or condition, or delaying the recurrence of symptoms of a disease or condition. As used herein, "prevention" and similar phrases also refer to reducing the intensity, effect, symptoms, and / or burden of a disease or condition before the disease or condition recurs.

[0028] As used herein, "delaying" the onset of a disease means to postpone, prevent, slow, retard, stabilize, and / or postpone the onset of the disease. This delay can be of varying lengths of time depending on the disease under treatment and / or the individual's medical history. A method that "delays" the onset of a disease is one that reduces the probability of disease onset in a given time frame and / or reduces the extent of disease in a given time frame when compared to the absence of the method. Such comparisons are typically based on clinical trials using a statistically significant number of individuals. The onset of cancer may be detectable using standard methods, including, but not limited to, computerized axial tomography (CAT scan), magnetic resonance imaging (MRI), abdominal ultrasound, coagulation tests, arteriography, or biopsy. Onset may also refer to disease (e.g., cancer) progression, which may be initially undetectable and includes emergence, recurrence, and development.

[0029] As used herein, the term "effective amount" refers to an amount of an agent or combination of agents sufficient to treat, such as ameliorating, alleviating, mitigating, and / or delaying one or more of the symptoms of, the specified disorder, condition, or disease. With respect to cancer, an effective amount includes an amount sufficient to cause tumor shrinkage and / or a reduction in the rate of tumor growth (such as tumor growth inhibition), or to prevent or delay other undesirable cell proliferation. In some embodiments, an effective amount is an amount sufficient to delay onset. In some embodiments, an effective amount is an amount sufficient to prevent or delay recurrence. An effective amount can be administered in one or more administrations. An effective amount of a drug or composition may (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) inhibit, delay, slow to some extent, and preferably stop cancer cell invasion into peripheral organs; (iv) inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay the appearance and / or recurrence of tumors; (vii) alleviate to some extent one or more symptoms associated with cancer; (viii) stimulate or activate immune cells (e.g., immune effector cells) for an immune response, such as by producing one or more cytokines, or for immune cell proliferation and / or differentiation; and / or (ix) prevent, reduce, or eliminate an inflammatory or autoimmune response, such as by inhibiting pro-inflammatory cytokine secretion.In the case of a viral infection, an effective amount of an agent may inhibit (i.e., reduce to some extent, preferably neutralize) viral activity; control and / or attenuate and / or inhibit inflammation or cytokine storm induced by said viral pathogen; prevent, arrest and / or ameliorate at least one symptom of said viral infection or damage to said subject or said subject's organs or tissues resulting from or associated with said viral infection from worsening; control, reduce and / or inhibit cell necrosis in infected and / or non-infected tissues and / or organs; control, ameliorate and / or prevent infiltration of inflammatory cells (e.g., NK cells, cytotoxic T cells, neutrophils) in infected or non-infected tissues and / or organs; and / or stimulate or activate immune cells (e.g., immune effector cells) for an immune response, such as by producing one or more cytokines, or for immune cell proliferation and / or differentiation.

[0030] As used herein, "individual" or "subject" refers to a mammal, including, but not limited to, a human, cow, horse, cat, dog, rodent, or primate. In some embodiments, the individual is a human.

[0031] The term "antibody" is used in its broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies, and antigen-binding fragments thereof so long as they exhibit the desired antigen-binding activity. The term "antibody" includes traditional four-chain antibodies, single-domain antibodies, and antigen-binding fragments thereof.

[0032] The basic four-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. IgM antibodies consist of five of these basic heterotetrameric units, together with an additional polypeptide called the J chain, and contain ten antigen-binding sites, while IgA antibodies contain two to five of these basic four-chain units, which can combine with the J chain to form multivalent aggregates. In the case of IgG, the four-chain unit is generally about 150,000 daltons. Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain contains an N-terminal variable domain (V H ), followed by three constant domains (C for each of the α and γ chains) H ), and four C for μ and ε isotypes H Each L chain has an N-terminal variable domain (V L ), followed by a constant domain at the other end. L is V H Align with C L is the first constant domain of the heavy chain (C H 1). Particular amino acid residues are believed to form an interface between the light-chain variable domain and the heavy-chain variable domain. H and V L pair together to form a single antigen-binding site. For the structure and properties of different classes of antibodies, see, e.g., Basic and Clinical Immunology, 8th Edition, Daniel P. Sties, Abba I. Terr and Tristram G. Parsolw (eds), Appleton & Lange, Norwalk, Conn., 1994, page 71 and Chapter 6. L chains from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequence of their constant domains. Immunoglobulins are characterized by the constant domains of their heavy chains (C H) can be assigned to different classes or isotypes depending on the amino acid sequence. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, each with a heavy chain designated α, δ, ε, γ, and μ. The γ and α classes are H They are further divided into subclasses based on relatively minor differences in sequence and function; for example, humans express the following subclasses: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1, and IgA2.

[0033] An "isolated" antibody (or construct) is one that has been identified, separated, and / or recovered from components of its production environment (e.g., natural or recombinant). Preferably, an isolated polypeptide is free from association with any other components from its production environment. Contaminant components of its production environment, such as those arising from recombinantly transfected cells, are materials that would typically interfere with research, diagnostic, or therapeutic uses for the antibody, and can include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the polypeptide will be purified (1) to greater than 95% by weight of the antibody, and in some embodiments, greater than 99% by weight, as determined, for example, by the Lowry assay; (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator; or (3) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue, or preferably silver staining. Isolated antibody (or construct) includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated polypeptide, antibody, or construct will be prepared by at least one purification step.

[0034] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domains of the heavy or light chain of the antibody. The variable domains of the heavy and light chains are respectively referred to as "V H " and "V LThese domains are generally the most variable parts of the antibody (relative to other antibodies of the same class) and contain the antigen-binding sites. Heavy chain-only antibodies from camelid species have a single heavy chain variable region, which is called a "V" domain. H H". Therefore, V H H is a special kind of V H is.

[0035] The term "variable" refers to the fact that certain sections of the variable domains differ extensively in sequence among antibodies. V domains mediate antigen binding and define the specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed across the entire width of the variable domains. Instead, variability is concentrated in three sections, called complementarity-determining regions (CDRs) or hypervariable regions (HVRs), in both the heavy and light chain variable domains. The more highly conserved portions of the variable domains are called framework regions (FRs). Native heavy and light chain variable domains each contain four FR regions that largely adopt a β-sheet configuration, linked by three CDRs that form loops that connect, and in some cases form part of, the β-sheet structure. The CDRs of each chain are held together in close proximity by the FR regions, and the CDRs from the other chain contribute to forming the antigen-binding site of antibodies (see Kabat et al., Sequences of Immunological Interest, Fifth Edition, National Institutes of Health, Bethesda, Md. (1991)). The constant domains are not directly involved in binding an antibody to an antigen but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity.

[0036] The term "monoclonal antibody," as used herein, refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation), which may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous antibody population, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies used in the present invention can be produced by, for example, the hybridoma method (Kohler and Milstein, Nature, 256:495-97 (1975); Hongo et al., Hybridoma, 14(3):253-260 (1995); Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2002) nded. 1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, NY, 1981)), recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567), phage display technology (see, e.g., Clackson et al., Nature, 352:624-628 (1991); Marks et al., J. Mol. Biol. 222:581-597 (1992); Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al., J. Mol. Biol. 340(5):1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34):12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2):119-132 (2004), and techniques for producing human or human-like antibodies in animals that have some or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences (e.g., WO 1998 / 24893; WO 1996 / 34096; WO 1996 / 33735; WO 1991 / 10741; Jakobovits et al., Proc. Natl. Acad. Sci. USA 90:2551 (1993); Jakobovits et al., Nature 362:255-258 (1993); Bruggemann et al., Year in Immunol. 7:33 (1993); US Patent No. 5,545,807; US Patent No. 5,545,806; US Patent No. 5,569,825; US Patent No. 5,625,126; US Patent No. 5,633,425; al.,Bio / Technology 10:779-783(1992);Lonberg et al.,Nature 368:856-859(1994);Morrison,Nature 368:812-813(1994);Fishwild et al., Nature Biotechnol. 14:845-851 (1996); Neuberger, Nature Biotechnol. 14:826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13:65-93 (1995)).

[0037] The terms "full-length antibody," "intact antibody," or "whole antibody" are used interchangeably to refer to an antibody in its substantially intact form, as opposed to an antibody fragment. Specifically, full-length four-chain antibodies include those having heavy and light chains, including an Fc region. Full-length heavy-chain-only antibodies include those having a heavy chain variable domain (V H The antibody comprises a heavy chain-only antibody (e.g., a heavy chain-specific antibody, such as a nucleotide sequence, ...

[0038] An "antibody fragment," "antigen-binding domain," or "antigen-binding fragment" comprises a portion of an intact antibody, preferably the antigen-binding and / or variable region of the intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (see U.S. Pat. No. 5,641,870, Example 2; Zapata et al., Protein Eng. 8(10):1057-1062 (1995)); single-chain antibody (scFv) molecules; single-domain antibodies (V HThese include antibodies with specificity (e.g., H), and multispecific antibodies formed from antibody fragments. Papain digestion of antibodies yields two identical antigen-binding fragments called "Fab" fragments and a residual "Fc" fragment, a designation reflecting their ability to be readily crystallized. The Fab fragment contains the entire L chain along with the variable domain of the H chain (V H ), and the first constant domain of one heavy chain (C H 1). Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site. Pepsin treatment of an antibody yields a single large F(ab')2 fragment, which roughly corresponds to two disulfide-linked Fab fragments with different antigen-binding activities, still capable of cross-linking antigen. The Fab' fragment is C H They differ from Fab fragments by having several additional residues at the carboxy terminus of one domain, including one or more cysteines from the antibody hinge region. Fab'-SH is the designation used herein for Fab' in which one or more cysteine ​​residues in the constant domains bear a free thiol group. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known. It should be understood that, for the purposes of the present invention, the expression "antigen-binding domain" or "antigen-binding fragment" also includes a ligand capable of specifically recognizing a target receptor, or a receptor capable of specifically recognizing a target ligand.

[0039] The term "constant domain" refers to a portion of an immunoglobulin molecule that has a more conserved amino acid sequence than the other portion of the immunoglobulin, the variable domain, which contains the antigen-binding site. The constant domain is the C H 1. C H 2 and C H 3 domains (collectively, C H ) and light chain CHL (or C L ) domain.

[0040] The "heavy chain" of an antibody (immunoglobulin) can be divided into three functional regions: the Fd region, the hinge region, and the Fc region (a crystallizable fragment). The Fd region is VH and CH1 domains, which combine with the light chain to form the Fab-antigen-binding fragment. The Fc fragment is responsible for immunoglobulin effector functions, including, for example, complement fixation and binding to cognate Fc receptors on effector cells. The hinge region, found in IgG, IgA, and IgD immunoglobulin classes, acts as a flexible spacer that allows the Fab portion to move freely in space relative to the Fc region. In contrast to the constant region, the hinge domain is structurally diverse, varying in both sequence and length between immunoglobulin classes and subclasses. For heavy-chain-only antibodies, the "heavy chain" refers to the heavy chain variable domain (V H H), a hinge region, and an Fc region. For the purposes of the present invention, the term "heavy chain" should also be understood to include a heavy chain comprising a VH domain, a hinge region, and an Fc domain or a portion thereof (e.g., a VL-VH-hinge-Fc domain subunit, or a VH-VL-hinge-Fc domain subunit), and a heavy chain comprising a first binding domain (e.g., a cytokine moiety) located in the hinge region (e.g., the heavy chain of a full-length four-chain antibody, a VH-hinge-Fc-containing antibody, or a heavy chain-only antibody) (see, e.g., Figures 1C, 1D, 1N, 10).

[0041] The "light chains" of antibodies (immunoglobulins) from any mammalian species can be assigned to one of two clearly distinct types, called kappa ("κ") and lambda ("λ"), based on the amino acid sequences of their constant domains.

[0042] An "Fv" is the minimum antibody fragment containing a complete antigen-recognition and binding site. This fragment consists of a dimer of one heavy-chain and one light-chain variable domain in tight, noncovalent association. The folding of these two domains generates six hypervariable loops (three loops from each heavy and light chain) that contribute to antigen binding by amino acid residues and confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three antigen-specific CDRs) has the ability to recognize and bind antigen, although with lower affinity than the entire binding site.

[0043] "Single-chain Fv", also abbreviated as "sFv" or "scFv", is a V linked to a single polypeptide chain. H and V L Preferably, the scFv polypeptide is an antibody fragment comprising a V H Domains and V L Because scFvs further contain a polypeptide linker between the domains, they are able to form the desired structure for antigen binding. For a review of scFvs, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0044] The term "diabody" refers to a V domain that is fragmented such that interchain, but not intrachain, pairing of V domains occurs, resulting in a bivalent fragment, i.e., a fragment with two antigen-binding sites. H Domains and V L This refers to small antibody fragments prepared by constructing sFv fragments (see previous paragraph) with a short linker (approximately 5-10 residues) between the domains. Bispecific diabodies are small antibody fragments prepared by constructing sFv fragments (see previous paragraph) with a short linker (approximately 5-10 residues) between the domains. H and V L Diabodies are heterodimers of two "crossover" sFv fragments whose domains are present on different polypeptide chains. Diabodies are described in further detail in, for example, EP 404,097; WO 93 / 11161; Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993).

[0045] The monoclonal antibodies of this specification particularly include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and / or light chain is identical to or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of one or more chains is identical to or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). The term "humanized antibody" is used as a subset of "chimeric antibody."

[0046] "Humanized" forms of non-human (e.g., llama or camelid) antibodies are chimeric antibodies which contain minimal sequence derived from non-human immunoglobulin. In some embodiments, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a CDR (defined below) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, rabbit, camel, llama, alpaca, or non-human primate having the desired specificity, affinity, and / or capacity. In some instances, framework ("FR") residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or the donor antibody. These modifications may be made to further refine antibody performance, such as binding affinity. Generally, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin sequence and all or substantially all of the FR regions are those of a human immunoglobulin sequence, although the FR regions may include one or more individual substitutions of FR residues which improve antibody performance, such as binding affinity, isomerization, immunogenicity, etc. The number of these amino acid substitutions in the FR typically does not exceed six in the H chain and three in the L chain. The humanized antibody also optionally will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see, e.g., Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).See also, e.g., Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994); and U.S. Pat. Nos. 6,982,321 and 7,087,409.

[0047] A "human antibody" is an antibody having an amino acid sequence corresponding to that of an antibody produced by a human and / or an antibody generated using any of the techniques for generating human antibodies as disclosed herein. This definition of a human antibody specifically excludes humanized antibodies containing non-human antigen-binding residues. Human antibodies can be generated using a variety of techniques known in the art, including phage display libraries. Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991). Human monoclonal antibodies can also be prepared using the methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol., 147(1):86-95 (1991). See also van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:368-74 (2001). Human antibodies can be prepared by administering antigen to transgenic animals, e.g., immunized xenomouse, that have been modified to produce such antibodies in response to antigen challenge, but in which the endogenous gene locus has been disabled (see, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 regarding XENOMOUSE™ technology). See also, e.g., Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006), regarding human antibodies generated by human B cell hybridoma technology.

[0048] The terms "hypervariable region," "HVR," or "HV," as used herein, refer to regions in an antibody variable domain that are hypervariable in sequence and / or form structurally defined loops. Generally, single-domain antibodies comprise three HVRs (or CDRs): HVR1 (or CDR1), HVR2 (or CDR2), and HVR3 (or CDR3). HVR3 (or CDR3) exhibits the highest degree of diversity among the three HVRs and is believed to play a unique role in conferring precise specificity to antibodies. See, e.g., Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al., Nature Struct. Biol. 3:733-736 (1996).

[0049] The term "complementarity determining region" or "CDR" is used to refer to a hypervariable region as defined by the Kabat system. See Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991).

[0050] Several HVR designations are in use and are encompassed herein. Kabat complementarity-determining regions (CDRs) are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Chothia, instead, refers to the location of structural loops (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). AbM HVRs represent a compromise between Kabat HVRs and Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software. "Contact" HVRs are based on analysis of available complex crystal structures. Residues from each of these HVRs are listed in Table A below.

[0051] [Table 1]

[0052] HVRs may include "extended HVRs" as follows: L 24-36 or 24-34 (L1), 46-56 or 50-56 (L2) and 89-97 or 89-96 (L3) and V H 26-35 (H1), 50-65 or 49-65 (H2) and 93-102, 94-102, or 95-102 (H3) in the variable domain. The variable domain residues are numbered according to Kabat et al., supra, for each of these definitions.

[0053] The phrases "variable domain residue numbering as in Kabat" or "amino acid position numbering as in Kabat," and variations thereof, refer to the numbering system used for the heavy or light chain variable domains of the antibody sequences in Kabat et al., supra. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, the FRs or HVRs of the variable domain. For example, a heavy chain variable domain may contain a single amino acid insertion after residue 52 of H2 (residue 52a according to Kabat) and inserted residues after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c, etc. according to Kabat). The Kabat numbering of residues may be determined for a given antibody by aligning regions of homology in the antibody sequence with sequences according to the "standard" Kabat numbering system.

[0054] Unless otherwise indicated herein, the numbering of residues in an immunoglobulin heavy chain is that of the EU index as in Kabat et al., supra. "EU index as in Kabat" refers to the numbering of residues in a human IgG1 EU antibody.

[0055] "Framework" or "FR" residues are those variable domain residues other than the HVR residues as herein defined.

[0056] "Human consensus framework" or "acceptor human framework" refers to the human immunoglobulin V L or V H The framework corresponds to the most frequently occurring amino acid residue in a series of alternative framework sequences. L or V H The set of sequence choices is from a subgroup of variable domain sequences. Generally, the subgroup of sequences is as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5 thSubgroups as in Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). For example, V L For VH, the subgroup can be subgroup kappa I, kappa II, kappa III, or kappa IV as in Kabat et al., supra. Additionally, for VH, the subgroup can be subgroup I, subgroup II, or subgroup III as in Kabat et al. Alternatively, a human consensus framework can be derived from the above at specific residues, such as when human framework residues are selected based on their homology with the donor framework by aligning the donor framework sequence with a panel of different human framework sequences. An acceptor human framework "derived" from a human immunoglobulin framework or human consensus framework can comprise the same amino acid sequence, or it can have pre-existing amino acid sequence changes. In some embodiments, the number of pre-existing amino acid changes is 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, or 2 or fewer.

[0057] An "affinity matured" antibody is one that has one or more modifications in one or more of its CDRs that result in an improved affinity of the antibody for an antigen compared to a parent antibody that does not possess such one or more modifications. In some embodiments, the affinity matured antibody has nanomolar or even picomolar affinity for the target antigen. Affinity matured antibodies are produced by procedures known in the art. For example, Marks et al., Bio / Technology 10:779-783 (1992) describe affinity matured antibodies. H and V LAffinity maturation by domain shuffling has been described. Random mutagenesis of CDR and / or framework residues has been described, for example, by Barbas et al. Proc Nat. Acad. Sci. USA 91:3809-3813 (1994); Schier et al. Gene 169:147-155 (1995); Yelton et al. J. Immunol. 155:1994-2004 (1995); Jackson et al., J. Immunol. 154(7):3310-9 (1995); and Hawkins et al., J. Mol. Biol. 226:889-896 (1992).

[0058] The term "epitope" refers to a protein determinant capable of specific binding to an antibody or antigen-binding fragment (e.g., ligand, receptor, VHH, scFv, Fab, etc.). Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that the binding to the former, but not the latter, is lost in the presence of denaturing solvents.

[0059] As used herein, the terms "specifically bind," "specifically recognize," or "specific for" refer to a measurable and reproducible interaction, such as binding between a target molecule and a binding domain (or between a cytokine and a cytokine receptor), that is determinative of the presence of a target (or cytokine) in the presence of a heterogeneous population of molecules, including biomolecules. For example, an antigen-binding protein (such as a Fab) that specifically binds to a target molecule (which may be an epitope) is an antigen-binding protein that binds this target with higher affinity, avidity, more readily, and / or with a longer duration than it binds other target molecules. A cytokine that specifically binds to a cytokine receptor is a cytokine that binds this cytokine receptor with higher affinity, avidity, more readily, and / or with a longer duration than it binds other cytokine receptors. In some embodiments, the extent to which a binding domain (or cytokine) binds to an unrelated target molecule (or an unrelated cytokine receptor) is less than about 10% of the binding of the binding domain (or cytokine) to the target molecule (or cytokine receptor as measured), as measured, for example, by radioimmunoassay (RIA). In some embodiments, an antigen binding protein that specifically binds a target (or cytokine that specifically binds a cytokine receptor) has a binding affinity of ≦10 -5 M, ≤10 -6 M, ≤10 -7 M, ≤10 -8 M, ≤10 -9 M, ≤10 -10 M, ≤10 -11 M, or ≦10 -12 Dissociation constant of M (K D). In some embodiments, the antigen binding protein or binding domain (or cytokine receptor) specifically binds an epitope on a protein (or cytokine) that is conserved among proteins from different species. In some embodiments, specific binding can, but does not necessarily, include exclusive binding. The binding specificity of an antigen binding protein (or cytokine and cytokine receptor) can be experimentally determined by any protein binding method known in the art. Such methods include, but are not limited to, Western blot, ELISA test, RIA test, ECL test, IRMA test, EIA test, BIACORE™ test, and peptide scan.

[0060] The term "specificity" refers to the selective recognition of a binding domain for a specific epitope on a target molecule. Natural antibodies, for example, are monospecific. The term "multispecificity" as used herein means that an antigen-binding protein has multiepitope specificity (i.e., specific binding to two, three, or more different epitopes on a single biomolecule, or specific binding to epitopes on two, three, or more different biomolecules). "Bispecificity" as used herein means that an antigen-binding protein has two different antigen-binding specificities. Unless otherwise specified, the antigens bound by a bispecific antibody may be listed in any order. For example, the terms "anti-CD3 / HER2," "anti-HER2 / CD3," "CD3xHER2," and "HER2xCD3" may be used interchangeably to refer to a bispecific antibody that specifically binds to both CD3 and HER2. The term "monospecificity" as used herein refers to an antigen-binding protein having one or more binding sites, each of which binds to the same epitope on the same antigen.

[0061] The term "valent," as used herein, refers to the presence of a specified number of binding sites in an antigen-binding protein. For example, a natural antibody, or a full-length antibody, has two binding sites and is bivalent. Thus, the terms "trivalent," "tetravalent," "pentavalent," and "hexavalent" refer to the presence of two, three, four, five, and six binding sites, respectively, in an antigen-binding protein.

[0062] "Antibody effector function" refers to a biological activity attributable to the Fc region of an antibody (a native sequence Fc region or an amino acid sequence variant Fc region) and varies depending on the antibody isotype. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation. A "reduced or minimized" antibody effector function means that it is reduced by at least 50% (alternatively, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) from the wild-type or unmodified antibody. Determination of antibody effector function is readily determinable and measurable by one of skill in the art. In preferred embodiments, the antibody effector functions of complement fixation, complement-dependent cytotoxicity, and antibody-dependent cellular cytotoxicity are affected. In some embodiments, effector function is abolished by a mutation in the constant region that abolishes glycosylation, e.g., an "effectorless mutation." In some embodiments, the effectorless mutation is HThe N297A or DANA mutation (D265A + N297A) in the two regions is shown in Shields et al., J. Biol. Chem. 276(9):6591-6604 (2001). Alternatively, additional mutations that result in reduced or eliminated effector function include K322A and L234A / L235A (LALA). Alternatively, effector function can be reduced or eliminated through production techniques, such as expression in non-glycosylated host cells (e.g., E. coli) or host cells that result in an altered glycosylation pattern that is ineffective or less effective in promoting effector function (e.g., Shinkawa et al., J. Biol. Chem. 278(5):3466-3473 (2003)).

[0063] "Antibody-dependent cell-mediated cytotoxicity" or ADCC refers to a form of cytotoxicity in which secreted Ig binds to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages), enabling specific binding of these cytotoxic effector cells to antigen-bearing target cells and subsequent killing of the target cells by cytotoxins. Antibodies "arm" the cytotoxic cells and are required for target cell killing by this mechanism. The primary cells for mediating ADCC, NK cells, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. Fc expression on hematopoietic cells is summarized in Table 2 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991). To assess the ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in U.S. Patent No. 5,500,362 or U.S. Patent No. 5,821,337, may be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, or additionally, the ADCC activity of a molecule of interest can be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al., PNAS USA 95:652-656 (1998).

[0064] "Complement-dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to an antibody (of the appropriate subclass) that binds to its cognate antigen. To assess complement activation, a CDC assay, e.g., as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), may be performed. Antibody variants with altered Fc region amino acid sequences and increased or decreased C1q binding ability are described in U.S. Pat. No. 6,194,551 B1 and WO 99 / 51642, the contents of which are specifically incorporated herein by reference. See also Idusogie et al., J. Immunol. 164:4178-4184 (2000).

[0065] The terms "Fc region," "fragment crystallizable region," "Fc fragment," or "Fc domain" are used herein to define the C-terminal region of an immunoglobulin heavy chain, including native-sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy chain Fc region is usually defined to stretch from the amino acid residue at position Cys226, or from Pro230, to its carboxyl-terminus. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) may be removed, for example, during production or purification of the antibody or Fc fusion protein, or by recombinantly engineering a nucleic acid encoding the heavy chain of the antibody or Fc fusion protein. Accordingly, a composition of intact antibodies can include antibody populations in which all K447 residues have been removed, antibody populations in which none of the K447 residues have been removed, and antibody populations having a mixture of antibodies with and without the K447 residue. Native sequence Fc regions suitable for use in the immunomodulatory molecules described herein include human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4.

[0066] The term IgG "isotype" or "subclass," as used herein, refers to any of the subclasses of immunoglobulins defined by the chemical and antigenic properties of their constant regions. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, γ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the different immunoglobulin classes are well known and are outlined, for example, in Abbas et al. Cellular and Mol. Immunology, 4th ed. (WB Saunders, Co., 2000).

[0067] "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody or Fc fusion protein. A preferred FcR is a native-sequence human FcR. Furthermore, a preferred FcR is one that binds IgG antibodies (gamma receptors) and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses (including allelic variants and alternatively spliced ​​forms of these receptors). FcγRII receptors include FcγRIIA (an "activating receptor") and FcγRIIB (an "inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain. (See M. Daeron, Annu. Rev. Immunol. 15:203-234 (1997). FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs, including those to be identified in the future, are encompassed by the term "FcR" herein.

[0068] The term "Fc receptor" or "FcR" also includes the neonatal receptor, FcRn, which is involved in the transfer of maternal IgG to the fetus. Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994). Methods for measuring binding to FcRn are known (see, e.g., Ghetie and Ward, Immunol. Today 18:(12):592-8 (1997); Ghetie et al., Nature Biotechnology 15(7):637-40 (1997); Hinton et al., J. Biol. Chem. 279(8):6213-6 (2004); WO 2004 / 92219 (Hinton et al.)). In vivo binding to FcRn and serum half-life of human FcRn high affinity binding polypeptides can be assayed, for example, in transgenic mice or transfected human cell lines expressing human FcRn, or in primates to which polypeptides having variant Fc regions are administered. WO 2004 / 42072 (Presta) describes antibody variants with improved or reduced binding to FcR. See, e.g., Shields See also, et al., J. Biol. Chem. 9(2):6591-6604 (2001).

[0069] "Binding affinity" generally refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an antibody, antigen-binding fragment (ligand, receptor, VHH, scFv, etc.), or cytokine) and its binding partner (e.g., an antigen (cell surface molecule, receptor, ligand, etc.), or cytokine receptor). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair. Binding affinity is measured by the K d , K. off , K. on , or K a The term "K" can be used to refer to off " as used herein refers to the unit s -1The term "K" is intended to refer to the off-rate constant for dissociation of an antibody (or antigen-binding fragment) from an antibody (or antigen-binding fragment) / antigen complex (e.g., a ligand-receptor complex), or the off-rate constant for dissociation of a cytokine from a cytokine / cytokine receptor complex, as determined from a kinetic selection setup, expressed as on ", as used herein, refers to the unit M -1 s -1 The equilibrium dissociation constant "K" is intended to refer to the on-rate constant for the association of an antibody (or antigen-binding fragment) with an antigen to form an antibody (or antigen-binding fragment) / antigen complex, or the on-rate constant for the association of a cytokine with a cytokine receptor to form a cytokine / cytokine receptor complex, which is expressed as D " or "K d " as used herein refers to the dissociation constant of a particular antibody (or antigen-binding fragment)-antigen interaction (or cytokine-cytokine receptor interaction), and represents the concentration of antigen (or cytokine) required to occupy half of all antibody binding domains (or antigen-binding fragments) present at equilibrium in a solution of antibody (or antigen-binding fragment) molecules (or cytokine receptors), and is expressed in units of M. off / K on is equal to K d Measurement of K assumes that all of the binding agent is in solution. When the antibody (or antigen-binding fragment) is anchored to the cell wall, for example in a yeast expression system, the corresponding equilibrium rate constant is expressed as EC50, which is K d gives a good approximation of the affinity constant K a is the dissociation constant K d is the reciprocal of the unit M -1 It is expressed as the dissociation constant (K D or K d) is used as an indicator of the affinity of an antibody (or antigen-binding fragment) for an antigen (or a cytokine for a cytokine receptor). For example, simple analysis is possible by the Scatchard method using antibodies (or antigen-binding fragments) labeled with various markers, or by using a commercially available measurement kit, BIACORE™ X (manufactured by Amersham Biosciences), or a similar kit, following the user manual and experimental procedures provided with the kit. The K that can be derived using these methods D The value is expressed in units of M (Mol). An antibody or antigen-binding fragment thereof (or cytokine) that specifically binds to a target (or cytokine receptor) has a binding activity of, for example, ≦10 -5 M, ≤10 -6 M, ≤10 -7 M, ≤10 -8 M, ≤10 -9 M, ≤10 -10 M, ≤10 -11 M, or ≦10 -12 Dissociation constant of M (K d ).

[0070] 50% inhibitory concentration (IC 50 IC ) is a measure of the effectiveness of a substance (such as an antibody or antigen-binding fragment) in inhibiting a specific biological or biochemical function. It indicates how much of a particular drug or other substance (such as an antibody or antigen-binding fragment, an inhibitor) is needed to inhibit a given biological process by half. This value is typically expressed as a molar concentration. 50 The EC2000 standard for agonist drugs or other substances (such as antibodies, antigen-binding fragments, or cytokines) is 50 " is equivalent to EC 50 Also refers to the plasma concentration required to achieve 50% of the maximal effect in vivo. As used herein, "IC 50 " is used to refer to the effective concentration of an antibody or antigen-binding fragment required to neutralize 50% of the antigen's biological activity in vitro. 50 or EC 50can be measured by bioassays such as inhibition of ligand binding by FACS analysis (competitive binding assays), cell-based cytokine release assays, or amplified luminescence proximity homogeneous assays (AlphaLISA).

[0071] As used herein, a "covalent bond" refers to a stable bond between two atoms that share one or more electrons. Examples of covalent bonds include, but are not limited to, peptide bonds and disulfide bonds. As used herein, a "peptide bond" refers to a covalent bond formed between a carboxyl group of an amino acid and an amine group of an adjacent amino acid. A "disulfide bond" refers to a covalent bond formed between two sulfur atoms, such as the combination of two Fc fragments (or cytokine subunits) through one or more disulfide bonds. One or more disulfide bonds may be formed between two fragments by linking thiol groups present in the two fragments. In some embodiments, one or more disulfide bonds can be formed between one or more cysteines of two Fc fragments. A disulfide bond can be formed by oxidation of two thiol groups. In some embodiments, the covalent linkage is a direct covalent linkage. In some embodiments, the covalent linkage is a direct peptide bond or disulfide bond.

[0072] "Percent (%) amino acid sequence identity" and "homology," with respect to peptide, polypeptide, or antibody sequences, are defined as the percentage of amino acid residues in a candidate sequence that are identical with amino acid residues in a particular peptide or polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment for the purposes of determining percent amino acid sequence identity can be achieved in a variety of ways that are within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN™ (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including which algorithms are needed to achieve maximal alignment over the full length of the sequences under comparison.

[0073] As used herein, the "C-terminus" of a polypeptide refers to the last amino acid residue of the polypeptide that donates its amine group to form a peptide bond with the carboxyl group of an adjacent amino acid residue. The "N-terminus" of a polypeptide, as used herein, refers to the first amino acid of the polypeptide that donates its carboxyl group to form a peptide bond with the amine group of an adjacent amino acid residue.

[0074] An "isolated" nucleic acid molecule encoding a construct, antibody, or antigen-binding fragment thereof described herein is a nucleic acid molecule that has been identified and separated from at least one contaminant nucleic acid molecule with which it is normally associated in the environment in which it is produced. Preferably, an isolated nucleic acid is free from association with all components associated with the production environment. An isolated nucleic acid molecule encoding a construct, polypeptide, or antibody described herein is in a form other than the form or setting in which it is found in nature. Thus, an isolated nucleic acid molecule is distinguished from nucleic acids encoding the constructs, polypeptides, and antibodies described herein that are naturally present in a cell. Isolated nucleic acid includes a nucleic acid molecule that is contained in a cell that ordinarily contains the nucleic acid molecule, but that is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0075] The term "control sequence" refers to a DNA sequence necessary for the expression of an operably linked coding sequence in a particular host organism. Control sequences suitable for prokaryotes include, for example, a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

[0076] A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to promote translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading frame. However, enhancers need not be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

[0077] The term "vector," as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors as autonomously replicating nucleic acid structures as well as vectors that integrate into the genome of a host cell into which they are introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0078] The terms "transfected" or "transformed" or "transduced," as used herein, refer to the process for transferring or introducing exogenous nucleic acid into a host cell. A "transfected" or "transformed" or "transduced" cell is one that has been transfected, transformed, or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0079] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," including the primary transformed cell and its progeny without regard to the number of passages. Progeny may not be completely identical to the parent cell in nucleic acid content and may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are encompassed herein.

[0080] The term "pharmaceutical formulation" of "pharmaceutical composition" refers to a preparation in a form that allows the biological activity of the active ingredient to be effective and that does not contain additional ingredients that are unacceptably toxic to the subject to whom the formulation is to be administered. Such formulations are sterile. A "sterile" formulation is aseptic or free of all living microorganisms and their spores.

[0081] It is understood that embodiments of the invention described herein include "consisting of" and / or "consisting essentially of" embodiments.

[0082] As used herein, reference to "about" a value or parameter includes (and represents) a variation on the value or parameter itself. For example, the reference to "about X" includes the reference to "X."

[0083] As used herein, the phrase "not" a value or parameter generally means and describes "other than" the value or parameter. For example, "this method is not used to treat cancer type X" means that the method is used to treat cancer types other than X.

[0084] As used herein, the term "about X to Y" has the same meaning as "about X to about Y."

[0085] As used in this specification and the appended claims, the singular forms "a," "or," and "the" include plural referents unless the context clearly dictates otherwise.

[0086] II. Immunomodulatory molecules In one aspect, the present invention provides an immunomodulatory molecule comprising a first binding domain (e.g., an immunostimulatory cytokine, such as IL-2 or IL-12, or a variant thereof) that specifically recognizes a first target molecule (e.g., a receptor for the immunostimulatory cytokine) and a second binding domain (e.g., an agonist ligand or variant thereof, such as PD-L1 or PD-L2, or an agonist antigen-binding fragment, such as an anti-PD-1 agonist Fab, scFv, VHH, or full-length antibody) that specifically recognizes a second target molecule (e.g., an inhibitory checkpoint molecule, such as PD-1), wherein the first binding domain upregulates an immune response when bound to the first target molecule and the second binding domain downregulates an immune response when bound to the second target molecule. In some embodiments, the immunomodulatory molecule further comprises a third binding domain (e.g., an antigen-binding fragment) that specifically recognizes a third target molecule, such as a cell surface antigen on an immune effector cell (e.g., CD3, PD-1, CTLA-4) or a cancer cell (e.g., a tumor antigen). In some embodiments, the third binding domain upregulates or downregulates an immune response upon binding to the third target molecule. In some embodiments, the third binding domain does not regulate an immune response upon binding to the third target molecule.

[0087] In some embodiments, the first binding domain and / or the second binding domain and / or the third binding domain is a VHH. In some embodiments, the first binding domain and / or the second binding domain and / or the third binding domain is an scFv. In some embodiments, the first binding domain and / or the second binding domain and / or the third binding domain is a Fab. In some embodiments, the first binding domain and / or the second binding domain and / or the third binding domain is a single-chain ligand (e.g., a PD-L2 extracellular domain or a cytokine) or receptor. For example, the first domain can be a dimeric cytokine moiety formed by a first cytokine subunit recombinantly linked to a second cytokine subunit via an optional linker. In some embodiments, the first binding domain and / or the second binding domain and / or the third binding domain is a ligand or receptor formed by two polypeptide chains. For example, the first domain can be a dimeric cytokine moiety formed by a first cytokine subunit in one polypeptide chain and a second cytokine subunit in another polypeptide chain. In some embodiments, the first binding domain or portion thereof is fused to the N-terminus of the second binding domain or portion thereof. In some embodiments, the first binding domain or portion thereof is fused to the C-terminus of the second binding domain or portion thereof. In some embodiments, the first binding domain or portion thereof is fused to the N-terminus of the third binding domain or portion thereof. In some embodiments, the first binding domain or portion thereof is fused to the C-terminus of the third binding domain or portion thereof. In some embodiments, the third binding domain or portion thereof is fused to the N-terminus of the second binding domain or portion thereof. In some embodiments, the third binding domain or portion thereof is fused to the C-terminus of the second binding domain or portion thereof. The immunomodulatory molecule can have any of the configurations / components illustrated in Figures 1A-1W and 11A-15D, and described in any of the Examples and Sequence Listings herein.

[0088] In some embodiments, the first binding domain is a VHH. In some embodiments, the first binding domain is an scFv. In some embodiments, the first binding domain is a single-chain ligand (e.g., PD-L2 or a cytokine) or receptor. In some embodiments, the second binding domain is a Fab. In some embodiments, the first binding domain is fused to the N-terminus of the VH of a Fab. In some embodiments, the first binding domain is fused to the N-terminus of the VL of a Fab. In some embodiments, the first binding domain is fused to the C-terminus of the CH of a Fab. In some embodiments, the first binding domain is fused to the C-terminus of the CL of a Fab. In some embodiments, the first binding domain is a Fab.

[0089] In some embodiments, the second binding domain is a VHH. In some embodiments, the second binding domain is an scFv. In some embodiments, the second binding domain is a single-chain ligand (e.g., PD-L2 or a cytokine) or receptor. In some embodiments, the first binding domain is a Fab. In some embodiments, the second binding domain is fused to the N-terminus of the VH of a Fab. In some embodiments, the second binding domain is fused to the N-terminus of the VL of a Fab. In some embodiments, the second binding domain is fused to the C-terminus of the CH of a Fab. In some embodiments, the second binding domain is fused to the C-terminus of the CL of a Fab. In some embodiments, the second binding domain is a Fab.

[0090] In some embodiments, the third binding domain is a VHH. In some embodiments, the third binding domain is an scFv. In some embodiments, the third binding domain is a Fab. In some embodiments, the third binding domain is a ligand or receptor (e.g., the extracellular domain of a ligand or receptor).

[0091] In some embodiments, the first binding domain is located in the hinge region of the immunomodulatory molecule, e.g., the hinge region between the second binding domain and an Fc domain subunit or portion thereof. In some embodiments, the first binding domain is not located in the hinge region of the immunomodulatory molecule, e.g., is located in the C' region of one or both Fc subunits of a parent Fc fusion protein or Fc-containing parent antibody.

[0092] In some embodiments, the immunomodulatory molecule comprises: i) an antigen-binding protein comprising an antigen-binding polypeptide; and ii) a first binding domain (e.g., an immunostimulatory cytokine, such as IL-2 or IL-12, or a variant thereof), which antigen-binding polypeptide comprises, from N-terminus to C-terminus: a second binding domain or portion thereof (e.g., an agonist ligand, such as PD-L1 or PD-L2, or a variant thereof, or an agonist antigen-binding fragment, such as an anti-PD-1 agonist Fab, scFv, or VHH), a hinge region, and an Fc domain subunit or portion thereof, wherein the first binding domain is located in the hinge region. Thus, in some embodiments, there is provided an immunomodulatory molecule comprising: i) an antigen-binding protein comprising an antigen-binding polypeptide; and ii) a first binding domain (e.g., an immunostimulatory cytokine, such as IL-2 or IL-12, or a variant thereof) that specifically recognizes a first target molecule (e.g., a receptor for the immunostimulatory cytokine), wherein the antigen-binding polypeptide comprises, from N-terminus to C-terminus: a second binding domain or portion thereof (e.g., an agonist ligand or variant thereof, such as PD-L1 or PD-L2, or an agonist antigen-binding fragment, such as an anti-PD-1 agonist Fab, scFv, or VHH) that specifically recognizes a second target molecule (e.g., an inhibitory checkpoint molecule, such as PD-1), a hinge region, and an Fc domain subunit or portion thereof, wherein the first binding domain is located in the hinge region, and wherein the first binding domain bound to the first target molecule upregulates an immune response, and the second binding domain bound to the second target molecule downregulates an immune response. In some embodiments, in the presence of binding of the second binding domain to the second target molecule, the activity of the first binding domain is increased by at least about 20% (e.g., at least about any of 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or more) compared to the activity in the absence of binding of the second binding domain to the second target molecule.In some embodiments, in the absence of binding of the second binding domain to a second target molecule, the activity of the first binding domain located in the hinge region is about 70% or less (e.g., about any of 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or 0%) of the activity of the corresponding first binding domain in the free state. In some embodiments, the antigen-binding protein comprises two antigen-binding polypeptides, each comprising a hinge region, and only one of the antigen-binding polypeptides comprises a first binding domain located in the hinge region. In some embodiments, the antigen-binding protein comprises two antigen-binding polypeptides, each comprising a hinge region, and each antigen-binding polypeptide comprises a first binding domain located in the hinge region. In some embodiments, the immunomodulatory molecule comprises two or more first binding domains, wherein the two or more first binding domains are arranged in tandem in the hinge region of the antigen-binding polypeptide. In some embodiments, the first binding domain is an immunostimulatory cytokine or variant thereof. In some embodiments, the immunostimulatory cytokine is selected from the group consisting of IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-12, IL-15, IL-17, IL-18, IL-21, IL-22, IL-23, IL-27, IFN-α, IFN-β, IFN-γ, TNF-α, erythropoietin, thrombopoietin, G-CSF, M-CSF, SCF, and GM-CSF. In some embodiments, the first binding domain is an immunostimulatory cytokine variant, and the activity of the immunostimulatory cytokine variant in the free state is about 80% or less (e.g., about 70%, 60%, 50%, 40%, 30%, 20%, 10%, or 5% or less) of the activity of the corresponding wild-type immunostimulatory cytokine in the free state. In some embodiments, the immunostimulatory cytokine or variant thereof is a monomeric immunostimulatory cytokine or variant thereof.In some embodiments, the immunostimulatory cytokine or variant thereof is a dimeric immunostimulatory cytokine or variant thereof. In some embodiments, both subunits of the dimeric immunostimulatory cytokine or variant thereof are arranged in tandem in the hinge region of an antigen-binding polypeptide. In some embodiments, the antigen-binding protein comprises two antigen-binding polypeptides, each comprising a hinge region, wherein one subunit of the dimeric immunostimulatory cytokine or variant thereof is located in the hinge region of one antigen-binding polypeptide, and the other subunit of the dimeric immunostimulatory cytokine or variant thereof is located in the hinge region of the other antigen-binding polypeptide. In some embodiments, the immunostimulatory cytokine or variant thereof is IL-2 or a variant thereof. In some embodiments, the IL-2 variant comprises one or more mutations at positions selected from the group consisting of F24, K35, R38, F42, K43, E61, and P65 relative to wild-type IL-2. In some embodiments, the IL-2 variant comprises one or more mutations selected from the group consisting of F24A, R38D, K43E, E61R, and P65L compared to wild-type IL-2. In some embodiments, the IL-2 variant comprises R38D / K43E / E61R mutations compared to wild-type IL-2. In some embodiments, the immunostimulatory cytokine or variant thereof is IL-12 or a variant thereof. In some embodiments, the IL-12 variant comprises one or more mutations within the p40 subunit at positions selected from the group consisting of E45, Q56, V57, K58, E59, F60, G61, D62, A63, G64, Q65, and C177 compared to the wild-type p40 subunit. In some embodiments, the IL-12 variant comprises one or more mutations within the p40 subunit relative to the wild-type p40 subunit selected from the group consisting of Q56A, V57A, K58A, E59A, F60A, G61A, D62A, A63S, G64A, and Q65A, hi some embodiments, the IL-12 variant comprises E59A / F60A mutations within the p40 subunit relative to the wild-type p40 subunit.In some embodiments, the IL-12 variant comprises an F60A mutation within the p40 subunit compared to the wild-type p40 subunit. In some embodiments, the p40 subunit and the p35 subunit of IL-12 or a variant thereof are connected by a linker. In some embodiments, the two or more first binding domains are the same. In some embodiments, the two or more first binding domains are different. In some embodiments, the second binding domain is an agonist ligand of an inhibitory checkpoint molecule or a variant thereof. In some embodiments, the inhibitory checkpoint molecule is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, LAG-3, TIM-3, HHLA2, CD47, CXCR4, CD160, CD73, BLTA, B7-H4, TIGIT, Siglec7, Siglec9, and VISTA. In some embodiments, the second binding domain is PD-L1 or a variant thereof. In some embodiments, the PD-L1 variant has increased binding affinity to PD-1 compared to wild-type PD-L1. In some embodiments, the PD-L1 variant comprises one or more mutations at positions selected from the group consisting of I54, Y56, E58, R113, M115, S117, and G119 compared to wild-type PD-L1. In some embodiments, the PD-L1 variant comprises one or more mutations selected from the group consisting of I54Q, Y56F, E58M, R113T, M115L, S117A, and G119K compared to wild-type PD-L1. In some embodiments, the PD-L1 variant comprises I54Q / Y56F / E58M / R113T / M115L / S117A / G119K mutations compared to wild-type PD-L1. In some embodiments, the second binding domain is PD-L2 or a variant thereof. In some embodiments, the PD-L2 variant has increased binding affinity for PD-1 compared to wild-type PD-L2. In some embodiments, the second binding domain is an agonist antibody or antigen-binding fragment thereof of an inhibitory checkpoint molecule.In some embodiments, the inhibitory checkpoint molecule is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, LAG-3, TIM-3, HHLA2, CD47, CXCR4, CD160, CD73, BLTA, B7-H4, TIGIT, Siglec7, Siglec9, and VISTA. In some embodiments, the agonist antibody or antigen-binding fragment thereof specifically recognizes PD-1 (an "anti-PD-1 agonist antibody or antigen-binding fragment thereof"). In some embodiments, the agonist antibody or antigen-binding fragment thereof is a Fab. In some embodiments, the agonist antibody or antigen-binding fragment thereof is an scFv. In some embodiments, the antigen-binding protein comprises two or more second binding domains. In some embodiments, two or more second binding domains or portions thereof are arranged in tandem at the N-terminus of the antigen-binding polypeptide. In some embodiments, the antigen-binding protein comprises two antigen-binding polypeptides, each comprising a hinge region, and only one of the antigen-binding polypeptides comprises two or more second binding domains or portions thereof arranged in tandem at the N-terminus of the antigen-binding polypeptide. In some embodiments, the antigen-binding protein comprises two antigen-binding polypeptides, each comprising a hinge region, and each antigen-binding polypeptide comprises one or more second binding domains or portions thereof at the N-terminus of the respective antigen-binding polypeptide. In some embodiments, the antigen-binding protein comprises two antigen-binding polypeptides, each comprising a hinge region, and a first antigen-binding polypeptide comprises one or more second binding domains or portions thereof at the N-terminus of the first antigen-binding polypeptide, and a second antigen-binding polypeptide comprises a third binding domain or portion thereof at the N-terminus of the second antigen-binding polypeptide, wherein the third binding domain specifically recognizes a third target molecule. In some embodiments, the third binding domain and the second binding domain are the same. In some embodiments, the third binding domain and the second binding domain are different. In some embodiments, the third target molecule and the second target molecule are the same. In some embodiments, the third target molecule and the second target molecule are different.

[0093] In some embodiments, the antibody comprises: i) a first antigen-binding polypeptide comprising, from N-terminus to C-terminus: a first secondary binding domain (e.g., PD-L2 or PD-L1 or a variant thereof), a second secondary binding domain (e.g., PD-L2 or PD-L1 or a variant thereof), a first binding domain located in a first hinge region (e.g., the p35 subunit and p40 subunit of IL-12 or a variant thereof (e.g., p40 E59A / F60A or F60A) connected in tandem), and a first subunit of an Fc domain or a portion thereof; and ii) a second antigen-binding polypeptide comprising, from N-terminus to C-terminus: a VH, an optional CH1, a second hinge region, and a second subunit of an Fc domain or a portion thereof. and iii) a third antigen-binding polypeptide comprising, from N-terminus to C-terminus: a VL and an optional CL, wherein the VH and VL, and optionally the CH1 and CL, form a third binding domain that specifically recognizes a third target molecule, the first binding domain specifically recognizes the first target molecule, the second binding domain specifically recognizes a second target molecule (e.g., PD-1), and the first binding domain, upon binding to the first target molecule (e.g., IL-12 receptor), upregulates an immune response, and the first and / or second binding domains, upon binding to the second target molecule (e.g., PD-L2 or PD-L1 or a variant thereof), downregulates an immune response. See, e.g., Figure 1B. In some embodiments, the third binding domain is an agonistic antigen-binding fragment that specifically recognizes PD-1. See, e.g., Figure 1A. In some embodiments, the first and second secondary binding domains are the same. In some embodiments, the first and second secondary binding domains are different. In some embodiments, the first and second secondary binding domains specifically recognize the same epitope. In some embodiments, the first and second secondary binding domains specifically recognize different epitopes.

[0094] In some embodiments, the polypeptide comprises: i) a first antigen-binding polypeptide comprising, from N-terminus to C-terminus: a first VH, an optional first CH1, a first binding domain arranged in a first hinge region (e.g., the p35 subunit and p40 subunit of IL-12 or a variant thereof connected in tandem), and a first subunit of an Fc domain or a portion thereof; ii) a second antigen-binding polypeptide comprising, from N-terminus to C-terminus: a second VH, an optional second CH1, a second hinge region, and a second subunit of an Fc domain or a portion thereof; iii) a third antigen-binding polypeptide comprising, from N-terminus to C-terminus: a first VL and an optional first CL; and iv) a second VL and an optional second and a fourth antigen-binding polypeptide comprising a CL, wherein the first VH and first VL and optional first CH1 and first CL form a second binding domain that specifically recognizes a second target molecule (e.g., an agonist antigen-binding fragment that specifically recognizes PD-1), the first binding domain specifically recognizes the first target molecule (e.g., an IL-12 receptor), and the second VH and second VL and optional second CH1 and second CL form a third binding domain that specifically recognizes a third target molecule, wherein the first binding domain, upon binding to the first target molecule (e.g., an IL-12 receptor), upregulates an immune response, and the second binding domain, upon binding to the second target molecule (e.g., PD-1), downregulates an immune response. See, e.g., Figure ID. In some embodiments, the third binding domain is an agonist antigen-binding fragment that specifically recognizes PD-1.Thus, in some embodiments, a polypeptide may comprise: i) a first antigen-binding polypeptide comprising, from N-terminus to C-terminus: a first VH, an optional first CH1, a p35 subunit and a p40 subunit of IL-12 or a variant thereof arranged in tandem in a first hinge region, and a first subunit of an Fc domain or a portion thereof; ii) a second antigen-binding polypeptide comprising, from N-terminus to C-terminus: a second VH, an optional second CH1, a second hinge region, and a second subunit of an Fc domain or a portion thereof; iii) a third antigen-binding polypeptide comprising, from N-terminus to C-terminus: a first VL and an optional first CL; and iv) a third antigen-binding polypeptide comprising, from N-terminus to C-terminus: a first VL and an optional first CL. and a fourth antigen-binding polypeptide comprising, towards the end: a second VL and an optional second CL, wherein the first VH and first VL and the optional first CH1 and first CL form a second binding domain that is an agonist antigen-binding fragment that specifically recognizes PD-1; the second VH and second VL and the optional second CH1 and second CL form a third binding domain that is an agonist antigen-binding fragment that specifically recognizes PD-1; and wherein the IL-12 or variant upon binding to the IL-12 receptor upregulates an immune response, and the second binding domain and / or the third binding domain downregulates an immune response upon binding to PD-1. See, e.g., FIG. 1C . In some embodiments, the third binding domain and the second binding domain are the same. In some embodiments, the third binding domain and the second binding domain are different. In some embodiments, the third binding domain and the second binding domain specifically recognize the same epitope. In some embodiments, the third binding domain and the second binding domain specifically recognize different epitopes.

[0095] In some embodiments, the antigen-binding polypeptide comprises: i) a first antigen-binding polypeptide comprising, from N-terminus to C-terminus: a first secondary binding domain (e.g., PD-L2 or PD-L1 or a variant thereof), a first hinge region-located first binding domain (e.g., the p35 and p40 subunits of IL-12 or a variant thereof, connected in tandem), and a first subunit or portion thereof of an Fc domain; and ii) a second secondary binding domain (e.g., , PD-L2 or PD-L1 or a variant thereof), a second hinge region, and a second antigen-binding polypeptide comprising a second subunit of an Fc domain or a portion thereof, wherein the first binding domain specifically recognizes a first target molecule (e.g., IL-12 receptor), the first binding domain bound to the first target molecule (e.g., IL-12 receptor) upregulates an immune response, and the second binding domain bound to the second target molecule (e.g., PD-1) downregulates an immune response. See, e.g., FIG. 1G. In some embodiments, the first and second secondary binding domains are the same. In some embodiments, the first and second secondary binding domains are different. In some embodiments, the first and second secondary binding domains specifically recognize the same epitope. In some embodiments, the first and second secondary binding domains specifically recognize different epitopes.

[0096] In some embodiments, the polypeptide comprises: i) a first antigen-binding polypeptide comprising, from N-terminus to C-terminus: a first secondary binding domain (e.g., PD-L2 or PD-L1 or a variant thereof), a second secondary binding domain (e.g., PD-L2 or PD-L1 or a variant thereof), a first hinge region-disposed first binding domain (e.g., the p35 and p40 subunits of IL-12 or a variant thereof, connected in tandem), and a first subunit of an Fc domain or a portion thereof; and ii) a third secondary binding domain (e.g., PD-L2 or PD-L1 or a variant thereof), a fourth secondary binding domain. and a second antigen-binding polypeptide comprising a first target molecule (e.g., PD-L2 or PD-L1 or a variant thereof), a second hinge region, and a second subunit of an Fc domain or portion thereof, wherein the first binding domain specifically recognizes a first target molecule (e.g., IL-12 receptor), and the first, second, third, and / or fourth second binding domains specifically recognize a second target molecule (e.g., PD-1), wherein the first binding domain upregulates an immune response upon binding to the first target molecule (e.g., IL-12 receptor), and the first, second, third, and / or fourth second binding domains downregulate an immune response upon binding to the second target molecule (e.g., PD-1). See, e.g., Figure 1H. In some embodiments, the first, second, third, and / or fourth second binding domains are the same. In some embodiments, the first, second, third, and / or fourth second binding domains are different. In some embodiments, the first, second, third, and / or fourth second binding domains specifically recognize the same epitope. In some embodiments, the first, second, third, and / or fourth second binding domains specifically recognize different epitopes.

[0097] In some embodiments, the antibody comprises: i) a first antigen-binding polypeptide comprising, from N-terminus to C-terminus: a first secondary binding domain (e.g., PD-L2 or PD-L1 or a variant thereof), a portion of the first binding domain located in the first hinge region (e.g., the p35 subunit of IL-12 or a variant thereof), and a first subunit of an Fc domain or a portion thereof; and ii) a second secondary binding domain (e.g., PD-L2 or PD-L1 or a variant thereof), another portion of the first binding domain located in the second hinge region (e.g., the p35 subunit of IL-12 or a variant thereof). and a second antigen-binding polypeptide comprising a second subunit of an Fc domain or a portion thereof, wherein the first binding domain specifically recognizes a first target molecule (e.g., an IL-12 receptor), the first and second secondary binding domains specifically recognize a second target molecule (e.g., PD-1), the first binding domain upregulates an immune response upon binding to the first target molecule (e.g., an IL-12 receptor), and the first and / or second secondary binding domain downregulates an immune response upon binding to the second target molecule (e.g., PD-1). See, e.g., FIG. 1L. In some embodiments, the first and second secondary binding domains are the same. In some embodiments, the first and second secondary binding domains are different. In some embodiments, the first and second secondary binding domains specifically recognize the same epitope. In some embodiments, the first and second second binding domains specifically recognize different epitopes.

[0098] In some embodiments, the polypeptide comprises: i) a first antigen-binding polypeptide comprising, from N-terminus to C-terminus: a portion of a first binding domain located in the first hinge region (e.g., the p35 or p40 subunit of IL-12 or a variant thereof), and a first subunit of an Fc domain or a portion thereof; and ii) a first antigen-binding polypeptide comprising, from N-terminus to C-terminus: a first second binding domain (e.g., PD-L2 or PD-L1 or a variant thereof), a second second binding domain (e.g., PD-L2 or PD-L1 or a variant thereof), a first binding domain located in the second hinge region (e.g., IL-12 or a variant thereof). and a second antigen-binding polypeptide comprising a second subunit of an Fc domain or a portion thereof, wherein the first binding domain specifically recognizes a first target molecule (e.g., the p40 subunit or the p35 subunit of the Fc domain of an IL-12 receptor), the first and second secondary binding domains specifically recognize a second target molecule (e.g., PD-1), the first binding domain upregulates an immune response upon binding to the first target molecule (e.g., the IL-12 receptor), and the first and / or second secondary binding domain downregulates an immune response upon binding to the second target molecule (e.g., PD-1). See, e.g., FIG. 1M. In some embodiments, the first and second secondary binding domains are the same. In some embodiments, the first and second secondary binding domains are different. In some embodiments, the first and second secondary binding domains specifically recognize the same epitope. In some embodiments, the first and second second binding domains specifically recognize different epitopes.

[0099] In some embodiments, the polypeptide comprises: i) a first antigen-binding polypeptide comprising, from N-terminus to C-terminus: a first VH, an optional first CH1, a portion of the first binding domain located in the first hinge region (e.g., the p35 subunit or p40 subunit of IL-12 or a variant thereof), and a first subunit of the Fc domain or a portion thereof; ii) a second antigen-binding polypeptide comprising, from N-terminus to C-terminus: a second VH, an optional second CH1, another portion of the first binding domain located in the second hinge region (e.g., the p40 subunit or p35 subunit of IL-12 or a variant thereof), and a second subunit of the Fc domain or a portion thereof; iii) a third antigen-binding polypeptide comprising, from N-terminus to C-terminus: a first VL and an optional first CL; and iv) a N and a fourth antigen-binding polypeptide comprising, from the C-terminus to the C-terminus: a second VL and an optional second CL, wherein the first VH and first VL and the optional first CH1 and first CL form a second binding domain that specifically recognizes a second target molecule (e.g., an agonist antigen-binding fragment that specifically recognizes PD-1), the second VH and second VL and the optional second CH1 and second CL form a third binding domain that specifically recognizes a third target molecule, the first binding domain specifically recognizes the first target molecule (e.g., IL-12 receptor), and the first binding domain up-regulates an immune response when bound to the first target molecule (e.g., IL-12 receptor), and the second binding domain down-regulates an immune response when bound to the second target molecule (e.g., PD-1). See, e.g., Figure 1O. In some embodiments, the third binding domain is an agonistic antigen-binding fragment that specifically recognizes PD-1. See, e.g., Figure 1N. In some embodiments, the third binding domain and the second binding domain are the same. In some embodiments, the third binding domain and the second binding domain are different. In some embodiments, the third binding domain and the second binding domain specifically recognize the same epitope. In some embodiments, the third binding domain and the second binding domain specifically recognize different epitopes.

[0100] In some embodiments, the immunomodulatory molecule comprises an antigen-binding protein comprising an antigen-binding polypeptide, the antigen-binding polypeptide comprising, from N' to C': a first binding domain or portion thereof, a second binding domain or portion thereof, an optional hinge region, and an Fc domain subunit or portion thereof. Thus, in some embodiments, there is provided an immunomodulatory molecule comprising an antigen-binding protein comprising an antigen-binding polypeptide comprising, from N' to C': a first binding domain or portion thereof (e.g., an immunostimulatory cytokine or variant thereof, such as IL-2 or IL-12), a second binding domain or portion thereof (e.g., an agonist ligand or variant thereof, such as PD-L1 or PD-L2, or an agonist antigen-binding fragment such as an anti-PD-1 agonist Fab, scFv or VHH), an optional hinge region, and an Fc domain subunit or portion thereof, wherein the first binding domain specifically recognizes a first target molecule (e.g., IL-12 receptor) and the second binding domain specifically recognizes a second target molecule (e.g., PD-1), wherein the first binding domain upregulates an immune response when bound to the first target molecule (e.g., IL-12 receptor) and the second binding domain downregulates an immune response when bound to the second target molecule (e.g., PD-1). In some embodiments, the second binding domain is an agonistic Fab or agonistic scFv that specifically recognizes an inhibitory checkpoint molecule. In some embodiments, the second binding domain is an agonistic ligand of an inhibitory checkpoint molecule or a variant thereof. In some embodiments, the second binding domain is PD-L1 or PD-L2 or a variant thereof. In some embodiments, the first binding domain is an immunostimulatory cytokine or a variant thereof. In some embodiments, the immunostimulatory cytokine or a variant thereof is IL-2 or IL-12 or a variant thereof.In some embodiments, the antigen-binding protein comprises two antigen-binding polypeptides, each comprising a hinge region, wherein the first antigen-binding polypeptide comprises, from N' to C': a first binding domain or portion thereof, a second binding domain or portion thereof, a first hinge region, and a first subunit of an Fc domain or portion thereof; and the second antigen-binding polypeptide comprises, from N' to C': a third binding domain or portion thereof, a second hinge region, and a second subunit of an Fc domain or portion thereof, wherein the third binding domain specifically recognizes a third target molecule. In some embodiments, the third binding domain and the second binding domain are the same. In some embodiments, the third binding domain and the second binding domain are different. In some embodiments, the third target molecule and the second target molecule are the same. In some embodiments, the third target molecule and the second target molecule are different.

[0101] In some embodiments, the antibody comprises: i) a first antigen-binding polypeptide comprising, from N-terminus to C-terminus: a first binding domain that specifically recognizes a first target molecule (e.g., the p35 and p40 subunits of IL-12 or a variant thereof fused in tandem), a first VH, an optional first CH1, a first hinge region, and a first subunit or portion thereof of an Fc domain; ii) a second antigen-binding polypeptide comprising, from N-terminus to C-terminus: a second VH, an optional second CH1, a second hinge region, and a second subunit or portion thereof of an Fc domain; iii) a third antigen-binding polypeptide comprising, from N-terminus to C-terminus: a first VL and an optional first CL; and iv) a N and a fourth antigen-binding polypeptide comprising, from the C-terminus to the C-terminus: a second VL and an optional second CL, wherein the first VH and first VL and the optional first CH1 and first CL form a second binding domain that specifically recognizes a second target molecule (e.g., an agonist antigen-binding fragment that specifically recognizes PD-1), and the second VH and second VL and the optional second CH1 and second CL form a third binding domain that specifically recognizes a third target molecule, wherein the first binding domain upregulates an immune response when bound to the first target molecule (e.g., IL-12 receptor), and the second binding domain downregulates an immune response when bound to the second target molecule (e.g., PD-1). In some embodiments, the third binding domain is an agonist antigen-binding fragment that specifically recognizes PD-1. See, e.g., Figure 1T. In some embodiments, the third binding domain and the second binding domain are the same. In some embodiments, the third binding domain and the second binding domain are different. In some embodiments, the third target molecule and the second target molecule are the same. In some embodiments, the third target molecule and the second target molecule are different.

[0102] In some embodiments, the polypeptide comprises: i) a first antigen-binding polypeptide comprising, from N-terminus to C-terminus: a first binding domain that specifically recognizes a first target molecule (e.g., the p35 and p40 subunits of IL-12 or a variant thereof fused in tandem), a first secondary binding domain (e.g., PD-L2 or PD-L1 or a variant thereof), a second secondary binding domain (e.g., PD-L2 or PD-L1 or a variant thereof), a first hinge region, and a first subunit or portion thereof of an Fc domain; and ii) from N-terminus to C-terminus: a third secondary binding domain (e.g., PD-L2 or PD-L1 or a variant thereof) that specifically recognizes a first target molecule (e.g., the p35 and p40 subunits of IL-12 or a variant thereof fused in tandem). and a second antigen-binding polypeptide comprising a first second binding domain (e.g., PD-L1 or a variant thereof), a fourth second binding domain (e.g., PD-L2 or PD-L1 or a variant thereof), a second hinge region, and a second subunit of an Fc domain or a portion thereof, wherein the first, second, third, and / or fourth second binding domains specifically recognize a second target molecule (e.g., PD-1), the first binding domain up-regulates an immune response upon binding to the first target molecule (e.g., IL-12 receptor), and the first, second, third, and / or fourth second binding domains down-regulate an immune response upon binding to the second target molecule (e.g., PD-1). See, e.g., Figure 1U. In some embodiments, the first, second, third, and / or fourth second binding domains are the same. In some embodiments, the first, second, third, and / or fourth second binding domains are different. In some embodiments, the first, second, third, and / or fourth second binding domains specifically recognize the same epitope, hi some embodiments, the first, second, third, and / or fourth second binding domains specifically recognize different epitopes.

[0103] In some embodiments, the antibody comprises: i) a first antigen-binding polypeptide comprising, from N-terminus to C-terminus: a first binding domain that specifically recognizes a first target molecule (e.g., the p35 and p40 subunits of IL-12 or a variant thereof fused in tandem), a first secondary binding domain (e.g., PD-L2 or PD-L1 or a variant thereof), a second secondary binding domain (e.g., PD-L2 or PD-L1 or a variant thereof), a first hinge region, and a first subunit or portion of an Fc domain; and ii) from N-terminus to C-terminus: a VH, an optional CH1, a second hinge region, and a second subunit or portion of an Fc domain. and iii) a third antigen-binding polypeptide comprising, from N-terminus to C-terminus: a VL and an optional CL, wherein the VH and VL and the optional CH1 and CL form a third binding domain that specifically recognizes a third target molecule, and the first and / or second secondary binding domains specifically recognize the second target molecule (e.g., PD-1), wherein the first binding domain upregulates an immune response upon binding to the first target molecule (e.g., IL-12 receptor), and the first and / or second secondary binding domain downregulates an immune response upon binding to the second target molecule (e.g., PD-1). In some embodiments, the third binding domain is an agonist antigen-binding fragment that specifically recognizes PD-1. See, e.g., Figure 1V. In some embodiments, the first and second secondary binding domains are the same. In some embodiments, the first and second secondary binding domains are different. In some embodiments, the first and second secondary binding domains specifically recognize the same epitope. In some embodiments, the first and second secondary binding domains specifically recognize different epitopes.

[0104] In some embodiments, the polypeptide comprises: i) a first antigen-binding polypeptide comprising, from N-terminus to C-terminus: a first binding domain that specifically recognizes a first target molecule (e.g., the p35 and p40 subunits of IL-12 or a variant thereof fused in tandem), a VH, an optional CH1, a first hinge region, and a first subunit of an Fc domain or a portion thereof; and ii) from N-terminus to C-terminus: a first third binding domain (e.g., PD-L2 or PD-L1 or a variant thereof), a second third binding domain (e.g., PD-L2 or PD-L1 or a variant thereof), a second hinge region, and a second subunit of an Fc domain or a portion thereof. and iii) a third antigen-binding polypeptide comprising, from N-terminus to C-terminus: a VL and an optional CL, wherein the VH and VL and the optional CH1 and CL form a second binding domain that specifically recognizes a second target molecule (e.g., an agonist antigen-binding fragment that specifically recognizes PD-1), the first and / or second tertiary binding domains specifically recognize a third target molecule (e.g., PD-1), and the first binding domain upregulates an immune response when bound to the first target molecule (e.g., IL-12 receptor), and the second binding domain downregulates an immune response when bound to the second target molecule (e.g., PD-1). See, e.g., Figure 1W. In some embodiments, the first and second tertiary binding domains are the same. In some embodiments, the first and second tertiary binding domains are different. In some embodiments, the first and second tertiary binding domains specifically recognize the same epitope. In some embodiments, the first and second third binding domains specifically recognize different epitopes.

[0105] In some embodiments, the polypeptide comprises: i) a first antigen-binding polypeptide comprising, from N-terminus to C-terminus: a first VH, an optional first CH1, a first hinge region, and a first subunit of an Fc domain or a portion thereof; ii) a second antigen-binding polypeptide comprising, from N-terminus to C-terminus: a second VH, an optional second CH1, a second hinge region, and a second subunit of an Fc domain or a portion thereof; iii) a third antigen-binding polypeptide comprising, from N-terminus to C-terminus: a first binding domain that specifically recognizes a first target molecule (e.g., the p35 and p40 subunits of IL-12 or a variant thereof fused in tandem), a first VL, and an optional first CL; and iv) a N and a fourth antigen-binding polypeptide comprising, from the C-terminus to the C-terminus: a second VL and an optional second CL, wherein the first VH and first VL and the optional first CH1 and first CL form a second binding domain that specifically recognizes a second target molecule (e.g., an agonist antigen-binding fragment that specifically recognizes PD-1), and the second VH and second VL and the optional second CH1 and second CL form a third binding domain that specifically recognizes a third target molecule, wherein the first binding domain upregulates an immune response when bound to the first target molecule (e.g., IL-12 receptor), and the second binding domain downregulates an immune response when bound to the second target molecule (e.g., PD-1). In some embodiments, the third binding domain is an agonist antigen-binding fragment that specifically recognizes PD-1. In some embodiments, the third binding domain and the second binding domain are the same. In some embodiments, the third binding domain and the second binding domain are different. In some embodiments, the third target molecule and the second target molecule are the same. In some embodiments, the third target molecule and the second target molecule are different.

[0106] In some embodiments, the polypeptide comprises: i) a first antigen-binding polypeptide comprising, from N-terminus to C-terminus: a VH, an optional CH1, a first hinge region, and a first subunit of an Fc domain or a portion thereof; ii) a second antigen-binding polypeptide comprising, from N-terminus to C-terminus: a first tertiary binding domain (e.g., PD-L2 or PD-L1 or a variant thereof), a second tertiary binding domain (e.g., PD-L2 or PD-L1 or a variant thereof), a second hinge region, and a second subunit of an Fc domain or a portion thereof; and iii) a first binding domain that specifically recognizes a first target molecule (e.g., a tandemly fused I and a third antigen-binding polypeptide comprising a VH and VL and optional CH1 and CL, wherein the VH and VL and optional CH1 and CL form a second binding domain that specifically recognizes a second target molecule (e.g., an agonist antigen-binding fragment that specifically recognizes PD-1), the first and / or second third binding domains specifically recognize the third target molecule (e.g., PD-1), and the first binding domain upregulates an immune response when bound to the first target molecule (e.g., IL-12 receptor), and the second binding domain downregulates an immune response when bound to the second target molecule (e.g., PD-1). In some embodiments, the first and second third binding domains are the same. In some embodiments, the first and second third binding domains are different. In some embodiments, the first and second third binding domains specifically recognize the same epitope. In some embodiments, the first and second third binding domains specifically recognize different epitopes.

[0107] In some embodiments, the immunomodulatory molecule comprises an antigen binding protein comprising a first antigen-binding polypeptide and a second antigen-binding polypeptide, wherein the first antigen-binding polypeptide comprises, from N-terminus to C-terminus: a second antigen-binding domain or portion thereof, a first hinge domain, and a first subunit of an Fc domain or portion thereof, and the second antigen-binding polypeptide comprises, from N-terminus to C-terminus: the first antigen-binding domain or portion thereof, a second hinge domain, and a second subunit of an Fc domain or portion thereof. Thus, in some embodiments, an immune modulatory molecule is provided comprising an antigen binding protein comprising a first antigen-binding polypeptide and a second antigen-binding polypeptide, wherein the first antigen-binding polypeptide comprises, from N-terminus to C-terminus: a second antigen-binding domain or portion thereof, a first hinge domain, and a first subunit of an Fc domain or portion thereof; and the second antigen-binding polypeptide comprises, from N-terminus to C-terminus: a first antigen-binding domain or portion thereof, a second hinge domain, and a second subunit of an Fc domain or portion thereof, wherein the first binding domain specifically recognizes a first target molecule and the second binding domain specifically recognizes a second target molecule, wherein the first binding domain upregulates an immune response when bound to the first target molecule (e.g., IL-12 receptor) and the second binding domain downregulates an immune response when bound to the second target molecule (e.g., PD-1). In some embodiments, the second binding domain is an agonistic Fab or agonistic scFv that specifically recognizes an inhibitory checkpoint molecule. In some embodiments, the second binding domain is an agonistic ligand of an inhibitory checkpoint molecule or a variant thereof. In some embodiments, the second binding domain is PD-L1 or PD-L2 or a variant thereof. In some embodiments, the first binding domain is an immunostimulatory cytokine or a variant thereof. In some embodiments, the immunostimulatory cytokine or a variant thereof is IL-2 or IL-12 or a variant thereof.

[0108] In some embodiments, the antibody comprises: i) a first antigen-binding polypeptide comprising, from N-terminus to C-terminus: a VH, an optional CH1, a first hinge region, and a first subunit of an Fc domain or a portion thereof; ii) a second antigen-binding polypeptide comprising, from N-terminus to C-terminus: a first binding domain that specifically recognizes a first target molecule (e.g., the p35 and p40 subunits of IL-12 or a variant thereof fused in tandem), a second hinge region, and a second subunit of an Fc domain or a portion thereof; ii) a third antigen-binding polypeptide comprising, from N-terminus to C-terminus: a VL, and an optional CL, wherein the VH and VL, and optionally the CH1 and CL, form a second binding domain that specifically recognizes a second target molecule (e.g., an agonist antigen-binding fragment that specifically recognizes PD-1), and wherein the first binding domain bound to the first target molecule (e.g., IL-12 receptor) upregulates an immune response, and the second binding domain bound to the second target molecule (e.g., PD-1) downregulates an immune response. See, e.g., Figure 1F.

[0109] In some embodiments, the antibody comprises: i) a first antigen-binding polypeptide comprising, from N-terminus to C-terminus: a first secondary binding domain (e.g., PD-L2 or PD-L1 or a variant thereof), a second secondary binding domain (e.g., PD-L2 or PD-L1 or a variant thereof), a first hinge region, and a first subunit or portion of an Fc domain; and ii) from N-terminus to C-terminus: a first binding domain that specifically recognizes a first target molecule (e.g., p3 of IL-12 or a variant thereof fused in tandem). and a second antigen-binding polypeptide comprising a second subunit or portion thereof of a first subunit (a p5 subunit and a p40 subunit), a second hinge region, and an Fc domain, wherein the first and / or second secondary binding domains specifically recognize a second target molecule (e.g., PD-1), the first binding domain upregulates an immune response when bound to the first target molecule (e.g., an IL-12 receptor), and the first and / or second secondary binding domain downregulates an immune response when bound to the second target molecule (e.g., PD-1). See, e.g., FIG. 1E. In some embodiments, the first and second secondary binding domains are the same. In some embodiments, the first and second secondary binding domains are different. In some embodiments, the first and second secondary binding domains specifically recognize the same epitope. In some embodiments, the first and second secondary binding domains specifically recognize different epitopes.

[0110] In some embodiments, the immunomodulatory molecule comprises an antigen binding protein comprising an antigen binding polypeptide comprising, from N-terminus to C-terminus: a second binding domain or portion thereof, an optional hinge region, an Fc domain subunit or portion thereof, and a first binding domain or portion thereof. Thus, in some embodiments, an immunomodulatory molecule is provided comprising an antigen binding protein comprising an antigen binding polypeptide comprising, from N-terminus to C-terminus: a second binding domain or portion thereof, an optional hinge region, an Fc domain subunit or portion thereof, and a first binding domain or portion thereof, wherein the first binding domain specifically recognizes a first target molecule and the second binding domain specifically recognizes a second target molecule (e.g., PD-1), wherein the first binding domain upregulates an immune response when bound to the first target molecule (e.g., IL-12 receptor) and the second binding domain downregulates an immune response when bound to the second target molecule (e.g., PD-1). In some embodiments, the second binding domain is an agonistic Fab or agonistic scFv that specifically recognizes an inhibitory checkpoint molecule. In some embodiments, the second binding domain is an agonistic ligand of the inhibitory checkpoint molecule or a variant thereof. In some embodiments, the second binding domain is PD-L1 or PD-L2 or a variant thereof. In some embodiments, the first binding domain is an immunostimulatory cytokine or a variant thereof. In some embodiments, the immunostimulatory cytokine or variant thereof is IL-2 or IL-12 or a variant thereof. In some embodiments, the immunostimulatory cytokine or variant thereof is a monomeric immunostimulatory cytokine or variant thereof. In some embodiments, the immunostimulatory cytokine or variant thereof is a dimeric immunostimulatory cytokine or variant thereof. In some embodiments, both subunits of the dimeric immunostimulatory cytokine or variant thereof are arranged in tandem at the C-terminus of the antigen-binding polypeptide.In some embodiments, the antigen-binding protein comprises two antigen-binding polypeptides, each comprising a hinge region and an Fc domain subunit or portion thereof, wherein one subunit of the dimeric immunostimulatory cytokine or variant thereof is fused to the C-terminus of the Fc domain subunit or portion thereof of one of the antigen-binding polypeptides, and the other subunit of the dimeric immunostimulatory cytokine or variant thereof is fused to the C-terminus of the Fc domain subunit or portion thereof of the other antigen-binding polypeptide. In some embodiments, the antigen-binding polypeptide that does not comprise the second binding domain or portion thereof comprises, from N-terminus to C-terminus: a third binding domain or portion thereof that specifically recognizes a third target molecule, a hinge region, an Fc domain subunit or portion thereof, and a dimeric immunostimulatory cytokine subunit or variant thereof. In some embodiments, the antigen-binding protein comprises a first antigen-binding polypeptide and a second antigen-binding polypeptide, wherein the first antigen-binding polypeptide comprises, from N-terminus to C-terminus: a second binding domain or portion thereof, a first hinge region, a first subunit of an Fc domain or portion thereof, and the first binding domain or portion thereof; and the second antigen-binding polypeptide comprises, from N' to C': a third binding domain or portion thereof that specifically recognizes a third target molecule, a second hinge region, and a second subunit of an Fc domain or portion thereof. In some embodiments, the third binding domain and the second binding domain are the same. In some embodiments, the third binding domain and the second binding domain are different. In some embodiments, the third target molecule and the second target molecule are the same. In some embodiments, the third target molecule and the second target molecule are different.

[0111] In some embodiments, the antibody comprises: i) a first antigen-binding polypeptide comprising, from N-terminus to C-terminus: a first secondary binding domain (e.g., PD-L2 or PD-L1 or a variant thereof), a first hinge region, a first subunit or portion thereof of an Fc domain, and a first binding domain that specifically recognizes a first target molecule (e.g., the p35 subunit and p40 subunit of IL-12 or a variant thereof fused in tandem); and ii) from N-terminus to C-terminus: a second secondary binding domain (e.g., PD-L2 or PD-L1 or a variant thereof). and a second antigen-binding polypeptide comprising a first and / or second second binding domain (e.g., PD-L2 or PD-L1 or a variant thereof), a second hinge region, and a second subunit of an Fc domain or a portion thereof, wherein the first and / or second second binding domain specifically recognize a second target molecule (e.g., PD-1), the first binding domain bound to the first target molecule (e.g., IL-12 receptor) upregulates an immune response, and the first and / or second second binding domain bound to the second target molecule (e.g., PD-1) downregulates an immune response. See, e.g., Figure 1I.

[0112] In some embodiments, the polypeptide comprises: i) a first antigen-binding polypeptide comprising, from N-terminus to C-terminus: a first VH, an optional first CH1, a first hinge region, a first subunit of an Fc domain or a portion thereof, and a first binding domain that specifically recognizes a first target molecule (e.g., the p35 subunit and p40 subunit of IL-12 or a variant thereof fused in tandem); ii) a second antigen-binding polypeptide comprising, from N-terminus to C-terminus: a second VH, an optional second CH1, a second hinge region, and a second subunit of an Fc domain or a portion thereof; iii) a third antigen-binding polypeptide comprising, from N-terminus to C-terminus: a first VL and an optional first CL; and iv) an N-terminus and a fourth antigen-binding polypeptide comprising, from C-terminus to C-terminus: a second VL and an optional second CL, wherein the first VH and first VL and the optional first CH1 and first CL form a second binding domain that specifically recognizes a second target molecule (e.g., an agonist antigen-binding fragment that specifically recognizes PD-1), and the second VH and second VL and the optional second CH1 and second CL form a third binding domain that specifically recognizes a third target molecule, wherein the first binding domain upregulates an immune response when bound to the first target molecule (e.g., IL-12 receptor), and the second binding domain downregulates an immune response when bound to the second target molecule (e.g., PD-1). In some embodiments, the third binding domain is an agonist antigen-binding fragment that specifically recognizes PD-1. See, e.g., Figure 1J. In some embodiments, the third binding domain and the second binding domain are the same. In some embodiments, the third binding domain and the second binding domain are different. In some embodiments, the third target molecule and the second target molecule are the same. In some embodiments, the third target molecule and the second target molecule are different.

[0113] In some embodiments, the antibody comprises: i) a first antigen-binding polypeptide comprising, from N-terminus to C-terminus: a VH, an optional CH1, a first hinge region, a first subunit or portion thereof of an Fc domain, and a first binding domain that specifically recognizes a first target molecule (e.g., the p35 subunit and p40 subunit of IL-12 or a variant thereof fused in tandem); and ii) from N-terminus to C-terminus: a first third binding domain (e.g., PD-L2 or PD-L1 or a variant thereof), a second third binding domain (e.g., PD-L2 or PD-L1 or a variant thereof), a second hinge region, and an Fc domain. and iii) a third antigen-binding polypeptide comprising, from N-terminus to C-terminus: a VL, and optionally a CL, wherein the VH and VL, and optionally the CH1 and CL, form a second binding domain that specifically recognizes a second target molecule (e.g., an agonist antigen-binding fragment that specifically recognizes PD-1), wherein the first binding domain upregulates an immune response when bound to a first target molecule (e.g., an IL-12 receptor), and the second binding domain downregulates an immune response when bound to a second target molecule (e.g., PD-1). See, e.g., Figure 1K. In some embodiments, the first and second third binding domains are the same. In some embodiments, the first and second third binding domains are different. In some embodiments, the first and second third binding domains specifically recognize the same epitope. In some embodiments, the first and second third binding domains specifically recognize different epitopes.

[0114] In some embodiments, the antibody comprises: i) a first antigen-binding polypeptide comprising, from N-terminus to C-terminus: a first second binding domain (e.g., PD-L2 or PD-L1 or a variant thereof), a first hinge region, a first subunit of an Fc domain or a portion thereof, and a portion of a first binding domain (e.g., the p35 subunit or p40 subunit of IL-12 or a variant thereof); and ii) a second second binding domain (e.g., PD-L2 or PD-L1 or a variant thereof), a second hinge region, and a second subunit of an Fc domain or a portion thereof, and and a second antigen-binding polypeptide comprising another portion of the first binding domain (e.g., the p40 subunit or p35 subunit of IL-12 or a variant thereof), wherein the first binding domain specifically recognizes a first target molecule (e.g., an IL-12 receptor), the first and / or second secondary binding domains specifically recognize a second target molecule, the first binding domain upregulates an immune response upon binding to the first target molecule (e.g., an IL-12 receptor), and the first and / or second secondary binding domain downregulates an immune response upon binding to the second target molecule (e.g., PD-1). See, e.g., FIG. 1P. In some embodiments, the first and second secondary binding domains are the same. In some embodiments, the first and second secondary binding domains are different. In some embodiments, the first and second secondary binding domains specifically recognize the same epitope. In some embodiments, the first and second second binding domains specifically recognize different epitopes.

[0115] In some embodiments, the polypeptide comprises: i) a first antigen-binding polypeptide comprising, from N-terminus to C-terminus: a first VH, an optional first CH1, a first hinge region, a first subunit of an Fc domain or a portion thereof, and a portion of a first binding domain (e.g., the p35 or p40 subunit of IL-12 or a variant thereof); ii) a second antigen-binding polypeptide comprising, from N-terminus to C-terminus: a second VH, an optional second CH1, a second hinge region, a second subunit of an Fc domain or a portion thereof, and another portion of the first binding domain (e.g., the p40 or p35 subunit of IL-12 or a variant thereof); iii) a third antigen-binding polypeptide comprising, from N-terminus to C-terminus: a first VL, and an optional first CL; and iv) a third antigen-binding polypeptide comprising, from N-terminus to C-terminus: a first VL, and an optional first CL; and a fourth antigen-binding polypeptide comprising a second VL and an optional second CL, wherein the first VH and first VL and the optional first CH1 and first CL form a second binding domain that specifically recognizes a second target molecule (e.g., an agonist antigen-binding fragment that specifically recognizes PD-1), the second VH and second VL and the optional second CH1 and second CL form a third binding domain that specifically recognizes a third target molecule, the first binding domain specifically recognizes the first target molecule (e.g., IL-12 receptor), and the first binding domain upregulates an immune response when bound to the first target molecule (e.g., IL-12 receptor), and the first and / or second second binding domain downregulates an immune response when bound to the second target molecule (e.g., PD-1). See, e.g., Figure 1R. In some embodiments, the third binding domain is an agonistic antigen-binding fragment that specifically recognizes PD-1. See, e.g., Figure 1Q. In some embodiments, the third binding domain and the second binding domain are the same. In some embodiments, the third binding domain and the second binding domain are different. In some embodiments, the third target molecule and the second target molecule are the same. In some embodiments, the third target molecule and the second target molecule are different.

[0116] In some embodiments, the immunomodulatory molecule comprises an antigen binding protein comprising a first antigen binding polypeptide and a second antigen binding polypeptide, wherein the first antigen binding polypeptide comprises, from N-terminus to C-terminus: VH, CH1, optional hinge region, Fc domain subunit or portion thereof, and the second antigen binding polypeptide comprises, from N-terminus to C-terminus: VL, CL and the first binding domain or portion thereof, wherein the VH and VL and optionally CH1 and CL form the second binding domain. Thus, in some embodiments, there is provided an immunomodulatory molecule comprising: i) a first antigen-binding polypeptide and a second antigen-binding polypeptide, wherein the first antigen-binding polypeptide comprises, from N-terminus to C-terminus: VH, CH1, optional hinge region, Fc domain subunit or portion thereof; and the second antigen-binding polypeptide comprises, from N-terminus to C-terminus: VL, CL and a first binding domain or portion thereof, wherein the VH and VL, and optionally the CH1 and CL, form a second binding domain that specifically recognizes a second target molecule, the first binding domain specifically recognizing the first target molecule (e.g., IL-12 receptor), and wherein the first binding domain upregulates an immune response when bound to the first target molecule (e.g., IL-12 receptor), and the second binding domain downregulates an immune response when bound to the second target molecule (e.g., PD-1). In some embodiments, the first antigen-binding polypeptide comprises, from N-terminus to C-terminus: VH, CH1, a first hinge region, a first subunit of an Fc domain or a portion thereof, and the antigen-binding protein further comprises a third antigen-binding polypeptide comprising, from N-terminus to C-terminus: a third binding domain or a portion thereof that specifically recognizes a third target molecule, a second hinge region, and a second subunit of an Fc domain or a portion thereof. In some embodiments, the third binding domain and the second binding domain are the same. In some embodiments, the third binding domain and the second binding domain are different. In some embodiments, the third target molecule and the second target molecule are the same. In some embodiments, the third target molecule and the second target molecule are different.In some embodiments, the immunomodulatory molecule comprises an antigen-binding protein comprising four antigen-binding polypeptides, wherein the first antigen-binding polypeptide comprises, from N-terminus to C-terminus: a first VH, a first CH1, a first hinge region, a first subunit of an Fc domain or a portion thereof; the second antigen-binding polypeptide comprises, from N-terminus to C-terminus: a first VL, a first CL, and a first binding domain or a portion thereof; the third antigen-binding polypeptide comprises, from N-terminus to C-terminus: a second VH, a second CH1, a second hinge region, and a second subunit of an Fc domain or a portion thereof; and the fourth antigen-binding polypeptide comprises, from N-terminus to C-terminus: a second VL and a second CL, wherein the first VH and first VL and the first CH1 and first CL form a second binding domain, and the second VH and second VL and the second CH1 and second CL form a third binding domain that specifically recognizes a third target molecule. In some embodiments, the first binding domain is an immunostimulatory cytokine or variant thereof. In some embodiments, the immunostimulatory cytokine or variant thereof is IL-2 or IL-12 or variants thereof. In some embodiments, the immunostimulatory cytokine or variant thereof is a monomeric immunostimulatory cytokine or variant thereof. In some embodiments, the immunostimulatory cytokine or variant thereof is a dimeric immunostimulatory cytokine or variant thereof. In some embodiments, the dimeric immunostimulatory cytokine or variant thereof is arranged in tandem at the C-terminus of the second antigen-binding polypeptide and / or the fourth antigen-binding polypeptide. In some embodiments, one subunit of the dimeric immunostimulatory cytokine or variant thereof is fused to the C-terminus of the first CL of the second antigen-binding polypeptide, and the other subunit of the dimeric immunostimulatory cytokine or variant thereof is fused to the second CL of the fourth antigen-binding polypeptide.

[0117] In some embodiments, the polypeptide comprises: i) a first antigen-binding polypeptide comprising, from N-terminus to C-terminus: a first VH, a first CH1, a first hinge region, and a first subunit or portion thereof of an Fc domain; ii) a second antigen-binding polypeptide comprising, from N-terminus to C-terminus: a first VL, a first CL, and a first first binding domain (e.g., the p35 and p40 subunits of IL-12 or a variant thereof fused in tandem); iii) a third antigen-binding polypeptide comprising, from N-terminus to C-terminus: a second VH, a second CH1, a second hinge region, and a second subunit or portion thereof of an Fc domain; and iv) a second VL, a second CL, and a second first binding domain (e.g., the p35 and p40 subunits of IL-12 or a variant thereof fused in tandem). and a fourth antigen-binding polypeptide comprising a p5 subunit and a p40 subunit, wherein the first VH and first VL and the first CH1 and first CL form a second binding domain that specifically recognizes a second target molecule (e.g., an agonist antigen-binding fragment that specifically recognizes PD-1), and the second VH and second VL and the second CH1 and second CL form a third binding domain that specifically recognizes a third target molecule, wherein the first and / or second first binding domains specifically recognize the first target molecule (e.g., IL-12 receptor), and the first and / or second first binding domains up-regulate an immune response when bound to the first target molecule (e.g., IL-12 receptor), and the second binding domain down-regulates an immune response when bound to the second target molecule (e.g., PD-1). In some embodiments, the third binding domain is an agonistic antigen-binding fragment that specifically recognizes PD-1. See, e.g., Figure 1S. In some embodiments, the third binding domain and the second binding domain are the same. In some embodiments, the third binding domain and the second binding domain are different. In some embodiments, the third target molecule and the second target molecule are the same. In some embodiments, the third target molecule and the second target molecule are different. In some embodiments, the first and second first binding domains are the same.In some embodiments, the first and second first binding domains are different. In some embodiments, the first and second first binding domains specifically recognize the same epitope. In some embodiments, the first and second first binding domains specifically recognize different epitopes.

[0118] In some embodiments, an immunomodulatory molecule is provided that comprises: i) a first antigen-binding polypeptide comprising, from N-terminus to C-terminus: a second binding domain, a first first binding domain, a first hinge region, a first subunit of an Fc domain or a portion thereof, and a second first binding domain (e.g., a p35 subunit and a p40 subunit of IL-12 or a variant thereof connected in tandem); and ii) a second antigen-binding polypeptide comprising, from N-terminus to C-terminus: a third binding domain, optionally a third first binding domain, a second hinge region, and a second subunit of an Fc domain or a portion thereof, wherein the first first binding domain specifically recognizes a first target molecule and the second first binding domain specifically recognizes a first target molecule. The first binding domain specifically recognizes a second target molecule (e.g., IL-12 receptor), the second binding domain specifically recognizes a third target molecule, the third binding domain specifically recognizes a fourth target molecule, and optionally, the optional third first binding domain recognizes a fifth target molecule; the first first binding domain, upon binding to the first target molecule, upregulates an immune response; the second first binding domain, upon binding to the second target molecule, upregulates an immune response; the second binding domain, upon binding to the third target molecule, downregulates an immune response; the third binding domain, upon binding to the fourth target molecule, localizes an immunomodulatory molecule to the tumor microenvironment; and optionally, the third first binding domain, upon binding to the fifth target molecule, upregulates an immune response. See, e.g., Figures 11A-11B. In some embodiments, the first, second, and / or third first binding domains are different. In some embodiments, the first, second, and / or third first binding domains specifically recognize the same epitope, hi some embodiments, the first, second, and / or third first binding domains specifically recognize different epitopes.

[0119] In some embodiments, an immunomodulatory molecule is provided that comprises: i) a first antigen-binding polypeptide comprising, from N-terminus to C-terminus: a second binding domain, a first first binding domain, a first hinge region, a first subunit of an Fc domain or a portion thereof, and a second first binding domain subunit (e.g., a p35 subunit or a p40 subunit of IL-12 or a variant thereof); and (ii) a second antigen-binding polypeptide comprising, from N-terminus to C-terminus: a third binding domain, optionally a third first binding domain, a second hinge region, a second subunit of an Fc domain or a portion thereof, and a second first binding domain subunit (e.g., a p35 subunit or a p40 subunit of IL-12 or a variant thereof), wherein the first first binding domain The main first binding domain specifically recognizes a first target molecule, the second first binding domain specifically recognizes a second target molecule (e.g., IL-12 receptor), the second binding domain specifically recognizes a third target molecule, the third binding domain specifically recognizes a fourth target molecule, and optionally the third first binding domain recognizes a fifth target molecule; the first first binding domain upregulates an immune response when bound to the first target molecule, the second first binding domain upregulates an immune response when bound to the second target molecule, the second binding domain downregulates an immune response when bound to the third target molecule, the third binding domain bound to the fourth target molecule localizes an immunomodulatory molecule to the tumor microenvironment, and optionally the third first binding domain bound to the fifth target molecule upregulates an immune response. See, e.g., Figures 11C-11F. In some embodiments, the first, second, and / or third first binding domains are different. In some embodiments, the first, second, and / or third first binding domains specifically recognize the same epitope. In some embodiments, the first, second, and / or third first binding domains specifically recognize different epitopes.

[0120] In some embodiments, the polypeptide comprises: i) a first antigen-binding polypeptide comprising, from N-terminus to C-terminus: a second binding domain, a first hinge region, a first subunit or portion thereof of an Fc domain, and a first first binding domain subunit (e.g., a p35 or p40 subunit of IL-12 or a variant thereof); and ii) a second antigen-binding polypeptide comprising, from N-terminus to C-terminus: a third binding domain, optionally a second first binding domain, a second hinge region, a second subunit or portion thereof of an Fc domain, and a first first binding domain subunit (e.g., a p35 or p40 subunit of IL-12 or a variant thereof); wherein a first first binding domain specifically recognizes a first target molecule (e.g., an IL-12 receptor), a second binding domain specifically recognizes a second target molecule, a third binding domain specifically recognizes a third target molecule, and optionally an optional second first binding domain recognizes a fourth target molecule, wherein the first first binding domain upon binding to the first target molecule upregulates an immune response, the second binding domain upon binding to the second target molecule downregulates an immune response, the third binding domain upon binding to the third target molecule localizes the immunomodulatory molecule to the tumor microenvironment, and optionally the second first binding domain upon binding to the fourth target molecule upregulates an immune response. See, e.g., Figures 11I-11L. In some embodiments, the first and / or second first binding domains are different. In some embodiments, the first and / or second first binding domains specifically recognize the same epitope. In some embodiments, the first and / or second first binding domains specifically recognize different epitopes.

[0121] In some embodiments, the antibody comprises: i) a first antigen-binding polypeptide comprising, from N-terminus to C-terminus: a first second binding domain (e.g., PD-L2 or PD-L1 or a variant thereof) located in a first hinge region, and a first subunit or portion of an Fc domain; and ii) a second second binding domain (e.g., PD-L2 or PD-L1 or a variant thereof), a first binding domain (e.g., the p35 and p40 subunits of IL-12 or a variant thereof connected in tandem), a second hinge region, or a portion thereof. and a second antigen-binding polypeptide comprising a first Fc domain or a second subunit or portion thereof, wherein the first binding domain specifically recognizes a first target molecule (e.g., an IL-12 receptor), the first and / or second secondary binding domains specifically recognize a second target molecule (e.g., PD-1), the first binding domain upregulates an immune response upon binding to the first target molecule (e.g., an IL-12 receptor), and the first and / or second secondary binding domain downregulates an immune response upon binding to the second target molecule (e.g., PD-1). See, e.g., FIG. 12A. In some embodiments, the first and / or second secondary binding domains are the same. In some embodiments, the first and / or second secondary binding domains are different. In some embodiments, the first and / or second secondary binding domains specifically recognize the same epitope. In some embodiments, the first and / or second second binding domains specifically recognize different epitopes.

[0122] In some embodiments, the antibody comprises: i) a first antigen-binding polypeptide comprising, from N-terminus to C-terminus: a first second binding domain (e.g., CD155 or a variant thereof) located at a first hinge region, and a first subunit of an Fc domain or a portion thereof; and ii) a second second binding domain (e.g., PD-L2 or PD-L1 or a variant thereof), a first binding domain (e.g., the p35 and p40 subunits of IL-12 or a variant thereof connected in tandem), a second hinge region, and a second subunit of an Fc domain or a portion thereof. and a second antigen-binding polypeptide comprising a portion thereof, wherein the first binding domain specifically recognizes a first target molecule (e.g., IL-12 receptor), the first secondary binding domain specifically recognizes a second target molecule (e.g., TIGIT), and the second secondary binding domain specifically recognizes a third target molecule (e.g., PD-1), wherein the first binding domain bound to the first target molecule (e.g., IL-12 receptor) upregulates an immune response, and the first and / or second secondary binding domains bound to the second target molecule (e.g., TIGIT and / or PD-1) downregulate an immune response. See, e.g., Figure 12B.

[0123] In some embodiments, an immunomodulatory molecule is provided that comprises: i) a first antigen-binding polypeptide comprising, from N-terminus to C-terminus: a third binding domain (e.g., an sdAb), a first hinge region, and a first subunit of an Fc domain or a portion thereof; and ii) a second antigen-binding polypeptide comprising, from N-terminus to C-terminus: a second binding domain (e.g., PD-L2 or PD-L1 or a variant thereof), a first binding domain (e.g., the p35 and p40 subunits of IL-12 or a variant thereof connected in tandem), a second hinge region, and a second subunit of an Fc domain or a portion thereof, wherein the first binding domain bound to a first target molecule (e.g., an IL-12 receptor) upregulates an immune response, and the second binding domain upregulates an immune response. The first binding domain specifically recognizes a second target molecule (e.g., PD-1), the third binding domain specifically recognizes a third target molecule (e.g., TIGIT, TIM3, LAG3, CTLA4, CD16A, HER2, Nectin-4, Trop2, or CLDN18.2, or a variant thereof), the first binding domain bound to the first target molecule (e.g., IL-12 receptor) upregulates an immune response, the second binding domain bound to the second target molecule (e.g., PD-1) downregulates an immune response, and the third binding domain bound to the third target molecule (e.g., TIGIT, TIM3, LAG3, CTLA4, CD16A, HER2, Nectin-4, Trop2, or CLDN18.2, or a variant thereof) localizes the immunomodulatory molecule to the tumor microenvironment. See, e.g., Figure 12C.

[0124] In some embodiments, an immunomodulatory molecule is provided that comprises: i) a first antigen-binding polypeptide comprising, from N-terminus to C-terminus: a third binding domain (e.g., a Fab comprising a VH and optional CH1), a first hinge region, and a first subunit of an Fc domain or a portion thereof; and ii) a second antigen-binding polypeptide comprising, from N-terminus to C-terminus: a second binding domain (e.g., PD-L2 or PD-L1 or a variant thereof), a first binding domain (e.g., the p35 and p40 subunits of IL-12 or a variant thereof connected in tandem), a second hinge region, and a second subunit of an Fc domain or a portion thereof, wherein the first binding domain bound to a first target molecule (e.g., an IL-12 receptor) enhances an immune response. The first binding domain specifically recognizes a second target molecule (e.g., PD-1), the third binding domain specifically recognizes a third target molecule (e.g., TIGIT, TIM3, LAG3, CTLA4, CD16A, HER2, Nectin-4, Trop2, or CLDN18.2, or a variant thereof), the first binding domain bound to the first target molecule (e.g., IL-12 receptor) upregulates an immune response, the second binding domain bound to the second target molecule (e.g., PD-1) downregulates an immune response, and the third binding domain bound to the third target molecule (e.g., TIGIT, TIM3, LAG3, CTLA4, CD16A, HER2, Nectin-4, Trop2, or CLDN18.2) localizes the immunomodulatory molecule to the tumor microenvironment. See, e.g., Figure 12D.

[0125] In some embodiments, the polypeptide comprises: i) a first antigen-binding polypeptide comprising, from N-terminus to C-terminus: a first second binding domain (e.g., PD-L2 or PD-L1 or a variant thereof) located in a first hinge region, and a first subunit or portion thereof of an Fc domain, and a first binding domain (e.g., the p35 subunit and p40 subunit of IL-12 or a variant thereof connected in tandem); and ii) a second second binding domain (e.g., PD-L2 or PD-L1 or a variant thereof), a second and a second antigen-binding polypeptide comprising a hinge region and a second subunit of an Fc domain or a portion thereof, wherein the first binding domain specifically recognizes a first target molecule (e.g., an IL-12 receptor), the first and second secondary binding domains specifically recognize a second target molecule (e.g., PD-1), and the first binding domain, upon binding to the first target molecule (e.g., an IL-12 receptor), upregulates an immune response, and the first and / or second secondary binding domains, upon binding to the second target molecule (e.g., PD-1), downregulates an immune response. See, e.g., Figure 13A. In some embodiments, the first and / or second secondary binding domains are the same. In some embodiments, the first and / or second secondary binding domains are different. In some embodiments, the first and / or second secondary binding domains specifically recognize the same epitope. In some embodiments, the first and / or second second binding domains specifically recognize different epitopes.

[0126] In some embodiments, the antigen-binding polypeptide comprises: i) a first antigen-binding polypeptide comprising, from N-terminus to C-terminus: a first second binding domain (e.g., CD155 or a variant thereof) located in a first hinge region, and a first subunit or portion thereof of an Fc domain; and ii) a second antigen-binding polypeptide comprising, from N-terminus to C-terminus: a second second binding domain (e.g., PD-L2 or PD-L1 or a variant thereof), a second hinge region, and a second subunit or portion thereof of an Fc domain, a first binding domain (e.g., the p35 subunit and p40 subunit of IL-12 or a variant thereof connected in tandem). and a second antigen-binding polypeptide comprising a first binding domain specifically recognizing a first target molecule (e.g., IL-12 receptor), a first second binding domain specifically recognizing a second target molecule (e.g., TIGIT), and a second second binding domain specifically recognizing a third target molecule (e.g., PD-1), wherein the first binding domain bound to the first target molecule (e.g., IL-12 receptor) upregulates an immune response, and the first and / or second second binding domains bound to the second target molecule (e.g., TIGIT and / or PD-1) downregulate an immune response. See, e.g., Figure 13B.

[0127] In some embodiments, an immunomodulatory molecule is provided that comprises: i) a first antigen-binding polypeptide comprising, from N-terminus to C-terminus: a second binding domain located in a first hinge region (e.g., PD-L2 or PD-L1 or a variant thereof), and a first subunit or portion thereof of an Fc domain, and a first binding domain (e.g., the p35 subunit and p40 subunit of IL-12 or a variant thereof connected in tandem); and ii) a second antigen-binding polypeptide comprising, from N-terminus to C-terminus: a third binding domain (e.g., an sdAb), a second hinge region, and a second subunit or portion thereof of an Fc domain, wherein the first binding domain bound to a first target molecule (e.g., an IL-12 receptor) upregulates an immune response. the first binding domain specifically recognizes a second target molecule (e.g., PD-1), the third binding domain specifically recognizes a third target molecule (e.g., TIGIT, TIM3, LAG3, CTLA4, CD16A, HER2, Nectin-4, Trop2, or CLDN18.2, or a variant thereof); the first binding domain bound to the first target molecule (e.g., IL-12 receptor) upregulates an immune response; the second binding domain bound to the second target molecule (e.g., PD-1) downregulates an immune response; and the third binding domain bound to the third target molecule (e.g., TIGIT, TIM3, LAG3, CTLA4, CD16A, HER2, Nectin-4, Trop2, or CLDN18.2) localizes the immunomodulatory molecule to the tumor microenvironment. See, e.g., Figure 13C.

[0128] In some embodiments, an immunomodulatory molecule is provided that comprises: i) a first antigen-binding polypeptide comprising, from N-terminus to C-terminus: a second binding domain located in a first hinge region (e.g., PD-L2 or PD-L1 or a variant thereof), and a first subunit or portion thereof of an Fc domain, and a first binding domain (e.g., the p35 subunit and p40 subunit of IL-12 or a variant thereof connected in tandem); and ii) a second antigen-binding polypeptide comprising, from N-terminus to C-terminus: a third binding domain (e.g., a Fab comprising a VH and optional CH1), a second hinge region, and a second subunit or portion thereof of an Fc domain, wherein the first binding domain bound to a first target molecule (e.g., an IL-12 receptor) regulates the immune response. the first binding domain specifically recognizes a second target molecule (e.g., PD-1), and the third binding domain specifically recognizes a third target molecule (e.g., TIGIT, TIM3, LAG3, CTLA4, CD16A, HER2, Nectin-4, Trop2, or CLDN18.2, or a variant thereof); the first binding domain bound to the first target molecule (e.g., IL-12 receptor) upregulates an immune response; the second binding domain bound to the second target molecule (e.g., PD-1) downregulates an immune response; and the third binding domain bound to the third target molecule (e.g., TIGIT, TIM3, LAG3, CTLA4, CD16A, HER2, Nectin-4, Trop2, or CLDN18.2) localizes an immunomodulatory molecule to the tumor microenvironment. See, e.g., Figure 13D.

[0129] In some embodiments, the antibody comprises: i) a first antigen-binding polypeptide comprising, from N-terminus to C-terminus: a first second binding domain (e.g., PD-L2 or PD-L1 or a variant thereof), a first first binding domain (e.g., IL-2 or a variant thereof), a first hinge region, a first subunit of an Fc domain or a portion thereof; and ii) a second antigen-binding polypeptide comprising, from N-terminus to C-terminus: a second second binding domain (e.g., PD-L2 or PD-L1 or a variant thereof), a second first binding domain (e.g., the p35 and p40 subunits of IL-12 or a variant thereof connected in tandem), a second hinge region, and a second subunit of an Fc domain or a portion thereof. and an antigen-binding polypeptide, wherein a first first binding domain specifically recognizes a first target molecule (e.g., IL-2 receptor), a second first binding domain specifically recognizes a second target molecule (e.g., IL-12 receptor), and the first and second secondary binding domains specifically recognize a third target molecule (e.g., PD-1), wherein the first first binding domain upregulates an immune response upon binding to the first or target molecule (e.g., IL-2 receptor), the second first binding domain upregulates an immune response upon binding to the second target molecule (e.g., IL-12 receptor), and the first and / or second secondary binding domain downregulates an immune response upon binding to the third target molecule (e.g., PD-1). See, e.g., Figure 14A. In some embodiments, the first and / or second first binding domains are the same. In some embodiments, the first and / or second first binding domains are different. In some embodiments, the first and / or second first binding domains specifically recognize the same epitope. In some embodiments, the first and / or second first binding domains specifically recognize different epitopes. In some embodiments, the first and / or second second binding domains are the same. In some embodiments, the first and / or second second binding domains are different. In some embodiments, the first and / or second second binding domains specifically recognize the same epitope.In some embodiments, the first and / or second second binding domains specifically recognize different epitopes.

[0130] In some embodiments, an immunomodulatory molecule is provided that comprises: i) a first antigen-binding polypeptide comprising, from N-terminus to C-terminus: a first second binding domain (e.g., CD155 or a variant thereof), a first first binding domain (e.g., IL-2 or a variant thereof), a first hinge region, a first subunit of an Fc domain or a portion thereof; and ii) a second antigen-binding polypeptide comprising, from N-terminus to C-terminus: a second second binding domain (e.g., PD-L2 or PD-L1 or a variant thereof), a second first binding domain (e.g., the p35 subunit and p40 subunit of IL-12 or a variant thereof connected in tandem), a second hinge region, and a second subunit of an Fc domain or a portion thereof, wherein the first first binding domain binds to a first target. The first first binding domain specifically recognizes a first target molecule (e.g., IL-2 receptor), the second first binding domain specifically recognizes a second target molecule (e.g., IL-12 receptor), the first second binding domain specifically recognizes a third target molecule (e.g., TIGIT), and the second second binding domain specifically recognizes a fourth target molecule (e.g., PD-1); the first first binding domain upregulates an immune response when bound to the first or target molecule (e.g., IL-2 receptor), the second first binding domain upregulates an immune response when bound to the second target molecule (e.g., IL-12 receptor), the first second binding domain downregulates an immune response when bound to the third target molecule (e.g., TIGIT), and the second second binding domain downregulates an immune response when bound to the fourth target molecule (e.g., PD-1). See, e.g., Figure 14B.

[0131] In some embodiments, an immunomodulatory molecule is provided that comprises: i) a first antigen-binding polypeptide comprising, from N-terminus to C-terminus: a third binding domain (e.g., an sdAb), a first first binding domain (e.g., IL-2 or a variant thereof), a first hinge region, a first subunit of an Fc domain or a portion thereof; and ii) a second antigen-binding polypeptide comprising, from N-terminus to C-terminus: a second binding domain (e.g., PD-L2 or PD-L1 or a variant thereof), a second first binding domain (e.g., the p35 and p40 subunits of IL-12 or a variant thereof connected in tandem), a second hinge region, and a second subunit of an Fc domain or a portion thereof, wherein the first first binding domain specifically recognizes a first target molecule (e.g., an IL-2 receptor) and the second first binding domain specifically recognizes a second target molecule (e.g., an IL-12 receptor). The first binding domain specifically recognizes a first target molecule (e.g., PD-1), the second binding domain specifically recognizes a third target molecule (e.g., PD-1), and the third binding domain specifically recognizes a fourth target molecule (e.g., TIGIT, TIM3, LAG3, CTLA4, CD16A, HER2, Nectin-4, Trop2, or CLDN18.2), and when bound to the first or target molecule (e.g., IL-2 receptor), the first binding domain upregulates an immune response, and the second binding domain specifically recognizes a fourth target molecule (e.g., TIGIT, TIM3, LAG3, CTLA4, CD16A, HER2, Nectin-4, Trop2, or CLDN18.2). The second first binding domain upregulates an immune response when bound to a third target molecule (e.g., IL-12 receptor), the second binding domain downregulates an immune response when bound to a third target molecule (PD-1), and the third binding domain localizes the immunomodulatory molecule to the tumor microenvironment when bound to a fourth target molecule (e.g., TIGIT, TIM3, LAG3, CTLA4, CD16A, HER2, Nectin-4, Trop2, or CLDN18.2). See, e.g., Figure 14C.

[0132] In some embodiments, an immunomodulatory molecule is provided that comprises: i) a first antigen-binding polypeptide comprising, from N-terminus to C-terminus: a third binding domain (e.g., a Fab comprising a VH and optional CH1), a first first binding domain (e.g., IL-2 or a variant thereof), a first hinge region, a first subunit of an Fc domain or a portion thereof; and ii) a second antigen-binding polypeptide comprising, from N-terminus to C-terminus: a second binding domain (e.g., PD-L2 or PD-L1 or a variant thereof), a second first binding domain (e.g., the p35 and p40 subunits of IL-12 or a variant thereof connected in tandem), a second hinge region, and a second subunit of an Fc domain or a portion thereof, wherein the first first binding domain specifically recognizes a first target molecule (e.g., an IL-2 receptor) and the second first binding domain specifically recognizes a second target molecule (e.g., an IL-2 receptor). the first binding domain specifically recognizes a third target molecule (e.g., PD-1), the second binding domain specifically recognizes a fourth target molecule (e.g., TIGIT, TIM3, LAG3, CTLA4, CD16A, HER2, Nectin-4, Trop2, or CLDN18.2), and when bound to the first or target molecule (e.g., IL-2 receptor), the first binding domain upregulates an immune response, and the second binding domain specifically recognizes a third target molecule (e.g., PD-1), and the third binding domain specifically recognizes a fourth target molecule (e.g., TIGIT, TIM3, LAG3, CTLA4, CD16A, HER2, Nectin-4, Trop2, or CLDN18.2). The second first binding domain upregulates an immune response when bound to a target molecule (e.g., IL-12 receptor), the second binding domain downregulates an immune response when bound to a third target molecule (PD-1), and the third binding domain localizes the immunomodulatory molecule to the tumor microenvironment when bound to a fourth target molecule (e.g., TIGIT, TIM3, LAG3, CTLA4, CD16A, HER2, Nectin-4, Trop2, or CLDN18.2). See, e.g., Figure 14D.

[0133] In some embodiments, the antigen-binding polypeptide comprises: i) a first antigen-binding polypeptide comprising, from N-terminus to C-terminus: a first second binding domain (e.g., PD-L2 or PD-L1 or a variant thereof) located in a first hinge region, and a first subunit or portion thereof of an Fc domain, and a first first binding domain (e.g., the p35 subunit and p40 subunit of IL-12 or a variant thereof connected in tandem); and ii) a second second binding domain (e.g., PD-L2 or PD-L1 or a variant thereof), a second first binding domain (e.g., IL-2 or a variant thereof), a second hinge region, and a second subunit or portion thereof of an Fc domain. and a second antigen-binding polypeptide comprising a first first binding domain and a second antigen-binding polypeptide comprising a first first binding domain and a second antigen-binding polypeptide comprising a first first binding domain and a second antigen-binding polypeptide comprising a first first binding domain and a second antigen-binding polypeptide comprising a second first binding domain and a second antigen-binding polypeptide comprising a first first binding domain and a second antigen-binding polypeptide comprising a second first binding domain and a second antigen-binding polypeptide comprising a second antigen-binding domain and a second antigen-binding polypeptide comprising a first first binding domain and a second antigen-binding polypeptide comprising a second antigen-binding ... third antigen-binding domain, wherein the first first binding domain specifically recognizes a first target molecule (e.g., IL-12 receptor), the second first binding domain specifically recognizes a second target molecule (e.g., IL-2 receptor), the first first binding domain upregulates an immune response when bound to the first target molecule (e.g., IL-12 receptor), the second first binding domain upregulates an immune response when bound to the second target molecule (e.g., IL-2 receptor), and the first and / or second secondary binding domain downregulates an immune response when bound to the third target molecule (e.g., PD-1). See, e.g., Figure 15A. In some embodiments, the first and / or second first binding domains are the same. In some embodiments, the first and / or second first binding domains are different. In some embodiments, the first and / or second first binding domains specifically recognize the same epitope. In some embodiments, the first and / or second first binding domains specifically recognize different epitopes. In some embodiments, the first and / or second second binding domains are the same. In some embodiments, the first and / or second second binding domains are different. In some embodiments, the first and / or second second binding domains specifically recognize the same epitope.In some embodiments, the first and / or second second binding domains specifically recognize different epitopes.

[0134] In some embodiments, an immunomodulatory molecule is provided that comprises: i) a first antigen-binding polypeptide comprising, from N-terminus to C-terminus: a first second binding domain (e.g., PD-L2 or PD-L1 or a variant thereof) located in a first hinge region, and a first subunit or portion thereof of an Fc domain, and a first first binding domain (e.g., the p35 subunit and p40 subunit of IL-12 or a variant thereof connected in tandem); and ii) a second antigen-binding polypeptide comprising, from N-terminus to C-terminus: a second second binding domain (e.g., CD155 or a variant thereof), a second first binding domain (e.g., IL-2 or a variant thereof), a second hinge region, and a second subunit or portion thereof of an Fc domain, wherein the first first binding domain The first first binding domain specifically recognizes a first target molecule (e.g., IL-12 receptor), the second first binding domain specifically recognizes a second target molecule (e.g., IL-2 receptor), the first second binding domain specifically recognizes a third target molecule (e.g., PD-1), and the second second binding domain recognizes a fourth target molecule (e.g., TIGIT), wherein the first first binding domain upregulates an immune response when bound to the first target molecule (e.g., IL-12 receptor), the second first binding domain upregulates an immune response when bound to the second target molecule (e.g., IL-2 receptor), the first second binding domain downregulates an immune response when bound to the third target molecule (e.g., PD-1), and the second second binding domain downregulates an immune response when bound to the third target molecule (e.g., TIGIT). See, e.g., Figure 15B.

[0135] In some embodiments, an immunomodulatory molecule is provided that comprises: i) a first antigen-binding polypeptide comprising, from N-terminus to C-terminus: a first second binding domain (e.g., PD-L2 or PD-L1 or a variant thereof) located in a first hinge region, and a first subunit or portion thereof of an Fc domain, and a first first binding domain (e.g., the p35 subunit and p40 subunit of IL-12 or a variant thereof connected in tandem); and ii) a second antigen-binding polypeptide comprising, from N-terminus to C-terminus: a third binding domain (e.g., an sdAb), a second first binding domain (e.g., IL-2 or a variant thereof), a second hinge region, and a second subunit or portion thereof of an Fc domain, wherein the first first binding domain specifically recognizes a first target molecule (e.g., an IL-12 receptor) and the second first binding domain specifically recognizes a second target molecule (e.g., an IL-12 receptor). the first binding domain specifically recognizes a third target molecule (e.g., PD-1), the third binding domain specifically recognizes a fourth target molecule (e.g., TIGIT, TIM3, LAG3, CTLA4, CD16A, HER2, Nectin-4, Trop2, or CLDN18.2), and when bound to the first or target molecule (e.g., IL-12 receptor), the first binding domain upregulates an immune response, and the second binding domain specifically recognizes a third target molecule (e.g., PD-1), and the third binding domain specifically recognizes a fourth target molecule (e.g., TIGIT, TIM3, LAG3, CTLA4, CD16A, HER2, Nectin-4, Trop2, or CLDN18.2). The second first binding domain upregulates an immune response when bound to a target molecule (e.g., IL-2 receptor), the second binding domain downregulates an immune response when bound to a third target molecule (PD-1), and the third binding domain localizes the immunomodulatory molecule to the tumor microenvironment when bound to a fourth target molecule (e.g., TIGIT, TIM3, LAG3, CTLA4, CD16A, HER2, Nectin-4, Trop2, or CLDN18.2). See, e.g., Figure 15C.

[0136] In some embodiments, an immunomodulatory molecule is provided that comprises: i) a first antigen-binding polypeptide comprising, from N-terminus to C-terminus: a first second binding domain (e.g., PD-L2 or PD-L1 or a variant thereof) located in a first hinge region, and a first subunit or portion thereof of an Fc domain, and a first first binding domain (e.g., the p35 subunit and p40 subunit of IL-12 or a variant thereof connected in tandem); and ii) a second antigen-binding polypeptide comprising, from N-terminus to C-terminus: a third binding domain (e.g., a Fab comprising a VH and optional CH1), a second first binding domain (e.g., IL-2 or a variant thereof), a second hinge region, and a second subunit or portion thereof of an Fc domain, wherein the first first binding domain specifically recognizes a first target molecule (e.g., an IL-12 receptor) and the second first binding domain specifically recognizes a second target molecule (e.g., an IL-12 receptor). the first binding domain specifically recognizes a target molecule (e.g., IL-2 receptor), the second binding domain specifically recognizes a third target molecule (e.g., PD-1), the third binding domain specifically recognizes a fourth target molecule (e.g., TIGIT, TIM3, LAG3, CTLA4, CD16A, HER2, Nectin-4, Trop2, or CLDN18.2), and the first binding domain upregulates an immune response when bound to the first or target molecule (e.g., IL-12 receptor); The second first binding domain upregulates an immune response when bound to a second target molecule (e.g., IL-2 receptor), the second binding domain downregulates an immune response when bound to a third target molecule (PD-1), and the third binding domain localizes the immunomodulatory molecule to the tumor microenvironment when bound to a fourth target molecule (e.g., TIGIT, TIM3, LAG3, CTLA4, CD16A, HER2, Nectin-4, Trop2, or CLDN18.2). See, e.g., Figure 15D.

[0137] In some embodiments, there is provided an immunomodulatory molecule described herein in any of Figures 1A-1W and 11A-15D, the Examples, and the Sequence Listing.

[0138] A binding domain that specifically recognizes a target molecule The immunomodulatory molecules described herein comprise a first binding domain that specifically recognizes a first target molecule and a second binding domain that specifically recognizes a second target molecule, wherein upon binding to the first target molecule, the first binding domain upregulates an immune response, and upon binding to the second target molecule, the second binding domain downregulates an immune response.

[0139] In some embodiments, the first binding domain, upon binding to the first target molecule, upregulates the immune response through an activity (an "upregulated activity") selected from one or more of: upregulating the release of immunostimulatory cytokines, downregulating the release of immunosuppressive cytokines, upregulating immune cell proliferation, upregulating immune cell differentiation, upregulating immune cell activation, upregulating cytotoxicity against tumor cells, and upregulating clearance of infectious agents.

[0140] In some embodiments, the second binding domain, upon binding to the second target molecule, downregulates the immune response through an activity (a "downregulated activity") selected from one or more of: downregulating the release of immunostimulatory cytokines, upregulating the release of immunosuppressive cytokines, downregulating immune cell proliferation, downregulating immune cell differentiation, downregulating immune cell activation, downregulating cytotoxicity against tumor cells, and downregulating clearance of infectious agents.

[0141] In some embodiments, the first binding domain, upon binding to a first target molecule, and the second binding domain, upon binding to a second target molecule, modulate an immune response (e.g., modulate by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more) with an activity independently selected from one or more of: upregulation of cytokine release, immune cell proliferation, immune cell differentiation, immune cell activation, cytotoxicity against tumor cells, and clearance of infectious agents. For example, in some embodiments, the first binding domain, upon binding to the first target molecule, upregulates the immune response (e.g., by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more) through an activity (an "upregulated activity") selected from one or more of: upregulating the release of immunostimulatory cytokines, downregulating the release of immunosuppressive cytokines, upregulating immune cell proliferation, upregulating immune cell differentiation, upregulating immune cell activation, upregulating cytotoxicity against tumor cells, and upregulating clearance of infectious agents. In some embodiments, the second binding domain, upon binding to the second target molecule, downregulates the immune response (e.g., downregulates (or upregulates in the case of release of immunosuppressive cytokines) by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) through an activity (a "downregulated activity") selected from one or more of: downregulating release of immunostimulatory cytokines, upregulating release of immunosuppressive cytokines, downregulating immune cell proliferation, downregulating immune cell differentiation, downregulating immune cell activation, downregulating cytotoxicity against tumor cells, and downregulating clearance of infectious agents.In some embodiments, the immunostimulatory cytokine is selected from the group consisting of IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-12, IL-15, IL-17, IL-18, IL-21, IL-22, IL-23, IL-27, IFN-α, IFN-β, IFN-γ, TNF-α, erythropoietin, thrombopoietin, G-CSF, M-CSF, SCF, and GM-CSF. In some embodiments, the immunosuppressive cytokine is selected from the group consisting of IL-1Ra, IL-4, IL-5, IL-6, IL-10, IL-11, IL-13, IL-27, IL-33, IL-35, IL-37, IL-39, IFN-α, LIF, and TGF-β.

[0142] In some embodiments, the first target molecule and / or the second target molecule is a stimulatory checkpoint molecule. In some embodiments, the stimulatory checkpoint molecule is selected from the group consisting of CD27, CD28, CD40, CD122, CD137, OX40, GITR, and ICOS. In some embodiments, the first binding domain is an agonist antibody or antigen-binding fragment thereof. In some embodiments, the agonist ligand is selected from the group consisting of CD27L (TNFSF7, CD70), CD40L (CD154), CD80, CD86, CD137L, OX40L (CD252), GITRL, and ICOSLG (CD275). In some embodiments, the first binding domain is a variant of an agonist ligand, wherein the variant of the agonist ligand has increased activity (e.g., binding affinity and / or biological activity) for the first target molecule compared to the agonist ligand (e.g., at least about a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more increase). In some embodiments, the first binding domain is a variant of an agonist ligand, wherein the variant of the agonist ligand has decreased activity (e.g., binding affinity and / or biological activity) for the first target molecule compared to the agonist ligand (e.g., at least about a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% decrease). In some embodiments, the second binding domain is an antagonist antibody or antigen-binding fragment thereof (e.g., VH, VHH, scFv, Fab, full-length Ab). In some embodiments, the second binding domain is an antagonist ligand or a variant thereof. In some embodiments, the second binding domain is a variant of the antagonist ligand, which has increased activity (e.g., binding affinity and / or biological activity) for the second target molecule compared to the antagonist ligand (e.g., at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more increase).In some embodiments, the second binding domain is a variant of the antagonist ligand, and the variant of the antagonist ligand has reduced activity (e.g., binding affinity and / or biological activity) for the second target molecule compared to the antagonist ligand (e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduced).

[0143] In some embodiments, the first target molecule and / or the second target molecule is a receptor for an immunostimulatory cytokine. In some embodiments, the immunostimulatory cytokine is selected from the group consisting of IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-12, IL-15, IL-17, IL-18, IL-21, IL-22, IL-23, IL-27, IFN-α, IFN-β, IFN-γ, TNF-α, erythropoietin, thrombopoietin, G-CSF, M-CSF, SCF, and GM-CSF. In some embodiments, the first binding domain is an immunostimulatory cytokine or a variant thereof. In some embodiments, the first binding domain is a variant of an immunostimulatory cytokine that has increased (e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more increased) or decreased (e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% decreased) activity (e.g., binding affinity and / or biological activity) for the first target molecule compared to the immunostimulatory cytokine. In some embodiments, the first binding domain is IL-2 or a variant thereof. In some embodiments, the first binding domain is an IL-2 variant that has reduced activity (e.g., binding affinity and / or biological activity) for the IL-2 receptor compared to wild-type IL-2 (e.g., at least about a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% decrease). In some embodiments, the first binding domain is IL-12 or a variant thereof. In some embodiments, the first binding domain is an IL-12 variant that has reduced activity (e.g., binding affinity and / or biological activity) for the IL-12 receptor compared to wild-type IL-12 (e.g., at least about a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% decrease).In some embodiments, the first binding domain is an agonist antibody or antigen-binding fragment thereof (e.g., VH, VHH, scFv, Fab, full-length Ab, e.g., an agonist of IL-12 receptor signaling). In some embodiments, the second binding domain is an antagonist antibody or antigen-binding fragment thereof (e.g., VH, VHH, scFv, Fab, full-length Ab). In some embodiments, the second binding domain is an antagonist ligand or variant thereof (e.g., that blocks or reduces IL-12 receptor signaling). In some embodiments, the second binding domain is a variant of an antagonist ligand, wherein the variant of the antagonist ligand has increased (e.g., at least about a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more increase) or decreased (e.g., at least about a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% decrease) activity (e.g., binding affinity and / or biological activity) for the second target molecule compared to the antagonist ligand.

[0144] The receptor for IL-2, the interleukin-2 receptor (IL-2R), is a heterotrimeric protein expressed on the surface of certain immune cells, such as lymphocytes. IL-2R consists of an α chain (IL-2Rα, CD25, Tac antigen), a β chain (IL-2Rβ, CD122), and a γ chain (IL-2Rγ, γ cIL-2 has three forms resulting from different combinations of IL-2Rα, IL-2Rβ, IL-2Rγ, and IL-2Rβ (common γ chain, or CD132). IL-2Rα binds IL-2 with low affinity, primarily on memory T cells and NK cells, while the IL-2Rβ and IL-2Rγ complex binds IL-2 with intermediate affinity. The α, β, and γ chain complex binds IL-2 with high affinity on activated T cells and regulatory T cells (Tregs). CD25 (IL-2Rα) plays a critical role in the development and maintenance of Tregs and may play a role in Treg expression of CD62L, which is required for Tregs to enter lymph nodes (Malek and Bayer, 2004). CD25 is a marker for activated T cells and Tregs. Experimental data suggest the immunosuppressive potential of antagonistic anti-CD25 antibodies, which significantly delayed rejection of cardiac allografts in mice (Kirkman et al., 1985) and renal allografts in non-human primates (Reed et al., 1989). Exemplary antagonistic anti-CD25 antibodies include, but are not limited to, basiliximab (e.g., Simulect®) and daclizumab (e.g., Zinbryta®).

[0145] In some embodiments, the first target molecule and / or the second target molecule is an activating immune cell surface receptor. In some embodiments, the activating immune cell surface receptor is selected from the group consisting of CD2, CD3, CD4, CD8, CD16, CD56, CD96, CD161, CD226, NKG2C, NKG2D, NKG2E, NKG2F, NKG2H, NKp30, NKp44, NKp46, CD11c, CD11b, CD13, CD45RO, CD33, CD123, CD62L, CD45RA, CD36, CD163, and CD206. In some embodiments, the first binding domain is an agonist antibody or an antigen-binding fragment thereof (e.g., VH, VHH, scFv, Fab, full-length Ab). In some embodiments, the first binding domain is an agonist ligand or a variant thereof. In some embodiments, the first binding domain is a variant of an agonist ligand, and the variant of the agonist ligand has increased (e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more increased) or decreased (e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% decreased) activity (e.g., binding affinity and / or biological activity) for the first target molecule compared to the agonist ligand. In some embodiments, the second binding domain is an antagonist antibody or an antigen-binding fragment thereof (e.g., VH, VHH, scFv, Fab, full-length Ab). In some embodiments, the second binding domain is an antagonist ligand or a variant thereof. In some embodiments, the second binding domain is a variant of an antagonist ligand, wherein the variant of the antagonist ligand has increased (e.g., at least about a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more increase) or decreased (e.g., at least about a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% decrease) activity (e.g., binding affinity and / or biological activity) for the second target molecule compared to the antagonist ligand.

[0146] In some embodiments, the first target molecule and / or the second target molecule is an inhibitory checkpoint molecule. In some embodiments, the inhibitory checkpoint molecule is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, LAG-3, TIM-3, HHLA2, CD47, CXCR4, CD160, CD73, BLTA, B7-H4, TIGIT, Siglec7, Siglec9, and VISTA. In some embodiments, the first binding domain is an antagonistic ligand or a variant thereof (e.g., that blocks or reduces PD-1 signaling). In some embodiments, the first binding domain is an antagonistic ligand of PD-1 or a variant thereof. In some embodiments, the first binding domain is a variant of an antagonist ligand, which has increased (e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more increased) or decreased (e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% decreased) activity (e.g., binding affinity and / or biological activity) for the first target molecule compared to the antagonist ligand. In some embodiments, the first binding domain is an antagonist antibody or antigen-binding fragment thereof (e.g., VH, VHH, scFv, Fab, full-length Ab). In some embodiments, the first binding domain is an antagonist anti-PD-1 antibody or antigen-binding fragment thereof. In some embodiments, the second binding domain is an agonist antibody or antigen-binding fragment thereof (e.g., VH, VHH, scFv, Fab, full-length Ab). In some embodiments, the agonist antibody or antigen-binding fragment thereof specifically recognizes PD-1, TIGIT, LAG-3, TIM-3, or CTLA-4. In some embodiments, the second binding domain is an agonist ligand or a variant thereof. In some embodiments, the second target molecule is PD-1 and the second binding domain is PD-L1, PD-L2, or a variant thereof.In some embodiments, the second target molecule is TIGIT, and the second binding domain is CD112 (PVRL2, Nectin-2), CD155 (PVR), or a variant thereof. In some embodiments, the second target molecule is LAG-3, and the second binding domain is MHC II, LSECtin, or a variant thereof. In some embodiments, the second target molecule is TIM-3, and the second binding domain is galectin-9, Caecam-1, HMGB-1, phosphatidylserine, or a variant thereof. In some embodiments, the second target molecule is CTLA-4, and the second binding domain is CD80, CD86, or a variant thereof. In some embodiments, the second binding domain is a variant of an agonist ligand, and the variant of the agonist ligand has increased (e.g., at least about a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more increase) or decreased (e.g., at least about a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% decrease) activity (e.g., binding affinity and / or biological activity) for the second target molecule compared to the agonist ligand. In some embodiments, the second binding domain is a variant of PD-L1 (or PD-L2) that has increased (e.g., at least about a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more increase) or decreased (e.g., at least about a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% decrease) activity (e.g., binding affinity and / or biological activity) towards PD-1 relative to wild-type PD-L1 (or PD-L2). In some embodiments, the second binding domain comprises the extracellular domain of an agonist ligand or a variant thereof.

[0147] PD-1 (programmed cell death protein 1) is part of the B7 / CD28 family of costimulatory molecules that regulate T cell activation and tolerance; therefore, antagonistic anti-PD-1 antibodies or PD-1 ligand-Fc fusion proteins may be useful for breaking tolerance. PD-1 is defined as the receptor for B7-4. B7-4 can inhibit immune cell activation by binding to inhibitory receptors on immune cells. Engagement of the PD-1 / PD-L1 pathway results in inhibition of T cell effector function, cytokine secretion, and proliferation (Turnis et al., OncoImmunology 1(7):1172-1174, 2012). High levels of PD-1 are associated with T cell exhaustion or chronic stimulation. Furthermore, increased PD-1 expression correlates with decreased survival in cancer patients. Drugs that downregulate PD-1, B7-4, and the interaction of inhibitory signals between B7-4 and PD-1 on immune cells can result in enhanced immune responses. Exemplary antagonist anti-PD-1 antibodies include, but are not limited to, pembrolizumab (e.g., Keytruda®), cemiplimab (Libtayo®), and nivolumab (e.g., Opdivo®).

[0148] In some embodiments, the second binding domain comprises an anti-PD-1 antibody fragment derived from nivolumab (antagonist). In some embodiments, the anti-PD-1 antibody fragment comprises a VH-CDR1, VH-CDR2, and VH-CDR3 comprising the sequence of SEQ ID NO: 48, and a VL-CDR1, VL-CDR2, and VL-CDR3 comprising the sequence of SEQ ID NO: 49. In some embodiments, the VH-CDR3 further comprises any one of the following mutations relative to SEQ ID NO: 48: D100N, D100G, D100R, N99G, N99A, or N99M. In some embodiments, anti-PD-1 antibody fragments comprising such VH-CDR3 mutations have reduced binding affinity to PD-1 compared to nivolumab (e.g., at least about a 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 20-, 50-, 100-, 1000-fold, or more).

[0149] In some embodiments, the second binding domain is an agonist antibody or antigen-binding fragment thereof that specifically recognizes PD-1 (an "anti-PD-1 agonist antibody or antigen-binding fragment thereof").

[0150] PD-L1 (programmed cell death ligand 1) is also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1). PD-L1 acts as a ligand for PD-1 and plays a key role in suppressing the immune system during certain events, such as pregnancy, tissue allografts, autoimmune diseases, and other disease states, such as hepatitis and cancer. Formation of the PD-1 receptor / PD-L1 ligand complex sends an inhibitory signal, leading to the suppression of CD8 expression in lymph nodes. + T cell proliferation is reduced. Exemplary antagonist anti-PD-L1 antibodies include, but are not limited to, atezolizumab (e.g., Tecentriq®), avelumab (e.g., Bavencio®), and durvalumab (e.g., IMFINZI™).

[0151] In some embodiments, the second binding domain is PD-L1 or a variant thereof. In some embodiments, the wt PD-L1 extracellular domain comprises the sequence of SEQ ID NO: 121. In some embodiments, the second binding domain is a PD-L1 variant, which has increased activity (e.g., binding affinity and / or biological activity) for PD-1 relative to wild-type PD-L1 (e.g., at least about a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more increase). In some embodiments, the PD-L1 variant comprises one or more mutations at positions selected from the group consisting of I54, Y56, E58, R113, M115, S117, and G119 relative to wild-type PD-L1 (SEQ ID NO: 120). In some embodiments, the PD-L1 variant comprises one or more mutations selected from the group consisting of I54Q, Y56F, E58M, R113T, M115L, S117A, and G119K compared to wild-type PD-L1 (SEQ ID NO: 120). In some embodiments, the PD-L1 variant comprises I54Q / Y56F / E58M / R113T / M115L / S117A / G119K mutations compared to wild-type PD-L1 (SEQ ID NO: 120). In some embodiments, the mutant PD-L1 extracellular domain comprises the sequence of any one of SEQ ID NOs: 122-129.

[0152] PD-L2 (programmed cell death 1 ligand 2, B7-DC, CD273) is another immune checkpoint receptor ligand for PD-1. PD-L2 plays a role in the negative regulation of adaptive immune responses. Engagement of PD-L2 with PD-1 dramatically inhibits T cell receptor (TCR)-mediated proliferation and cytokine production by T cells. At low antigen concentrations, PD-L2-PD-1 interaction inhibits strong B7-CD28 signaling. In contrast, at high antigen concentrations, PD-L2-PD-1 interaction reduces cytokine production but does not inhibit T cell proliferation.

[0153] In some embodiments, the second binding domain is PD-L2 or a variant thereof. In some embodiments, the second binding domain is a PD-L2 variant that has increased activity (e.g., binding affinity and / or biological activity) for PD-1 relative to wild-type PD-L2 (e.g., at least about a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more increase).

[0154] In some embodiments, the PD-L2 extracellular domain comprises the sequence of SEQ ID NO: 106. In some embodiments, the PD-L2 extracellular domain, or a portion thereof, is derived from wild-type (e.g., wild-type human) PD-L2. In some embodiments, the PD-L2 extracellular domain, or a portion thereof, comprises one or more mutations (e.g., deletions, insertions, or substitutions). In some embodiments, the PD-L2 variant comprises one or more mutations at positions selected from the group consisting of T56, S58, and Q60 (e.g., T56V, S58V, Q60L / T56V, S58V / Q60L) compared to wild-type PD-L2 (SEQ ID NO: 105). In some embodiments, the mutant PD-L2 extracellular domain, or a portion thereof, has increased binding affinity to PD-1 (e.g., about 2-, 3-, 4-, 5-, 10-, 50-, 100-, or more than 100-fold, etc.) compared to the wild-type PD-L2 extracellular domain, or a portion thereof. In some embodiments, the mutant PD-L2 extracellular domain comprises the sequence of any one of SEQ ID NOs: 107 to 110. In some embodiments, the mutant PD-L2 extracellular domain, or portion thereof, has reduced binding affinity for PD-1 compared to the wild-type PD-L2 extracellular domain, or portion thereof (e.g., by about 2, 3, 4, 5, 10, 50, 100, or less than 100-fold, etc.).

[0155] Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4, or CD152) is a homolog of CD28 and is known as an inhibitory immune checkpoint molecule upregulated on activated T cells. CTLA-4 also binds to B7-1 and B7-2, but with higher affinity than CD28. The interaction between B7 and CTLA-4 attenuates T cell activation, an important tumor immune escape mechanism. Antagonist anti-CTLA-4 antibody therapy has shown promise in several cancers, including melanoma. Exemplary antagonist anti-CTLA-4 antibodies include, but are not limited to, ipilimumab (e.g., Yervoy®).

[0156] In some embodiments, the second binding domain is CD155 (e.g., the extracellular domain) or a variant thereof. In some embodiments, the extracellular domain of wild-type human CD155 comprises the sequence of SEQ ID NO: 137. CD155 can bind to TIGIT and down-regulate the immune response.

[0157] In some embodiments, the first target molecule and / or the second target molecule is a receptor for an immunosuppressive cytokine. In some embodiments, the immunosuppressive cytokine is selected from the group consisting of IL-1Ra, IL-4, IL-5, IL-6, IL-10, IL-11, IL-13, IL-27, IL-33, IL-35, IFN-α, LIF, and TGF-β. In some embodiments, the second binding domain is an immunosuppressive cytokine or a variant thereof. In some embodiments, the second binding domain is a variant of an immunosuppressive cytokine, and the variant of the immunosuppressive cytokine has increased (e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more increased) or decreased (e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% decreased) activity (e.g., binding affinity and / or biological activity) for the second target molecule compared to the immunosuppressive cytokine. In some embodiments, the second binding domain is IL-10 or a variant thereof. In some embodiments, the second binding domain is TGF-β or a variant thereof. In some embodiments, the second binding domain is an agonist antibody or antigen-binding fragment thereof (e.g., VH, VHH, scFv, Fab, full-length Ab). In some embodiments, the first binding domain is an antagonist antibody or antigen-binding fragment thereof (e.g., VH, VHH, scFv, Fab, full-length Ab). In some embodiments, the first binding domain is an agonist ligand or a variant thereof.In some embodiments, the first binding domain is a variant of an antagonist ligand, and the variant of the antagonist ligand has increased (e.g., at least about a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more increase) or decreased (e.g., at least about a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% decrease) activity (e.g., binding affinity and / or biological activity) for the first target molecule compared to the antagonist ligand.

[0158] In some embodiments, the first target molecule and / or the second target molecule is an inhibitory immune cell surface receptor. In some embodiments, the inhibitory immune cell surface receptor is selected from the group consisting of CD5, NKG2A, NKG2B, KLRG1, FCRL4, Siglec2, CD72, CD244, GP49B, Lair-1, PirB, PECAM-1, CD200R, ILT2, and KIR2DL. In some embodiments, the second binding domain is an agonist antibody or antigen-binding fragment thereof (e.g., VH, VHH, scFv, Fab, full-length Ab). In some embodiments, the second binding domain is an agonist triligand or a variant thereof. In some embodiments, the second binding domain is a variant of an agonist ligand, which has increased (e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more increased) or decreased (e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% decreased) activity (e.g., binding affinity and / or biological activity) against the second target molecule compared to the agonist ligand. In some embodiments, the first binding domain is an antagonist antibody or antigen-binding fragment thereof (e.g., VH, VHH, scFv, Fab, full-length Ab, e.g., blocking or reducing NKG2B signaling). In some embodiments, the first binding domain is an agonist ligand or a variant thereof. In some embodiments, the first binding domain is a variant of an antagonist ligand, and the variant of the antagonist ligand has increased (e.g., at least about a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more increase) or decreased (e.g., at least about a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% decrease) activity (e.g., binding affinity and / or biological activity) for the first target molecule compared to the antagonist ligand.

[0159] In some embodiments, the first binding domain is IL-12 or a variant thereof, and the second binding domain is an agonist antibody or antigen-binding fragment thereof (e.g., VH, VHH, scFv, Fab, full-length Ab) that specifically recognizes PD-1. Hereinafter, such an immunomodulatory molecule is also referred to as an "IL-12 / anti-PD-1 agonist Ab." In some embodiments, the first binding domain is IL-12 or a variant thereof, and the second binding domain is PD-L1 (or its extracellular domain) or a variant thereof. Hereinafter, such an immunomodulatory molecule is also referred to as an "IL-12 / PD-L1 immunomodulatory molecule" or an "IL-12 / PD-L1 immunocytokine." In some embodiments, the second binding domain is a variant of PD-L1, and the PD-L1 variant has increased (e.g., at least about a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more increase) or decreased (e.g., at least about a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% decrease) activity (e.g., binding affinity and / or biological activity) for PD-1 compared to wild-type PD-L1. In some embodiments, the first binding domain is IL-12 or a variant thereof, and the second binding domain is PD-L2 (or the extracellular domain thereof) or a variant thereof. Such immunomodulatory molecules are also referred to hereinafter as "IL-12 / PD-L2 immunomodulatory molecules" or "IL-12 / PD-L2 immunocytokines." In some embodiments, the second binding domain is a variant of PD-L2, which has increased (e.g., at least about a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more increase) or decreased (e.g., at least about a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% decrease) activity (e.g., binding affinity and / or biological activity) for PD-1 compared to wild-type PD-L2.In some embodiments, the first binding domain is an IL-12 variant, wherein the IL-12 variant has increased (e.g., at least about any of a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more increase) or decreased (e.g., at least about any of a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% decrease) activity (e.g., binding affinity and / or biological activity) for the IL-12 receptor compared to wild-type IL-12.

[0160] In some embodiments, the first binding domain is IL-2 or a variant thereof, and the second binding domain is an agonist antibody or antigen-binding fragment thereof (e.g., VH, VHH, scFv, Fab, full-length Ab) that specifically recognizes PD-1. Hereinafter, such immunomodulatory molecules are also referred to as "IL-2 / anti-PD-1 agonist Abs." In some embodiments, the first binding domain is IL-2 or a variant thereof, and the second binding domain is PD-L1 (or its extracellular domain) or a variant thereof. Hereinafter, such immunomodulatory molecules are also referred to as "IL-2 / PD-L1 immunomodulatory molecules" or "IL-2 / PD-L1 immunocytokines." In some embodiments, the second binding domain is a variant of PD-L1, and the PD-L1 variant has increased (e.g., at least about a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more increase) or decreased (e.g., at least about a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% decrease) activity (e.g., binding affinity and / or biological activity) for PD-1 compared to wild-type PD-L1. In some embodiments, the first binding domain is IL-2 or a variant thereof, and the second binding domain is PD-L2 (or the extracellular domain thereof) or a variant thereof. Such immunomodulatory molecules are also referred to hereinafter as "IL-2 / PD-L2 immunomodulatory molecules" or "IL-2 / PD-L2 immunocytokines." In some embodiments, the second binding domain is a variant of PD-L2, which has increased (e.g., at least about a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more increase) or decreased (e.g., at least about a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% decrease) activity (e.g., binding affinity and / or biological activity) for PD-1 compared to wild-type PD-L2.In some embodiments, the first binding domain is an IL-2 variant, wherein the IL-2 variant has increased (e.g., at least about any of a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more increase) or decreased (e.g., at least about any of a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% decrease) activity (e.g., binding affinity and / or biological activity) for the IL-2 receptor compared to wild-type IL-2.

[0161] In some embodiments, the immunomodulatory molecule further comprises a third binding domain that specifically recognizes a third target molecule. In some embodiments, the third binding domain and the second binding domain are the same. In some embodiments, the third binding domain and the second binding domain are different. In some embodiments, the third target molecule and the second target molecule are the same. In some embodiments, the third target molecule and the second target molecule are different.

[0162] In some embodiments, the target molecule is a cell surface molecule (e.g., the extracellular domain of a receptor / ligand). In some embodiments, the target molecule acts as a cell surface marker on target cells (e.g., immune cells) associated with a particular disease state. The target molecule specifically recognized by the binding domain may be directly or indirectly involved in the disease.

[0163] The binding domains described herein can be in any format known in the art or can be derived from any suitable antibody or molecule. In some embodiments, the first binding domain is located in the hinge region between the second binding domain and an Fc domain subunit or portion thereof of an immunomodulatory molecule, and the second binding domain is in a format that ensures that binding of the first binding domain (e.g., an immunostimulatory cytokine moiety) to its first target molecule (e.g., a cytokine receptor) is reduced in the absence of second target molecule binding of the second binding domain, e.g., without second target molecule binding, reducing the activity of the first binding domain located in the hinge region (e.g., binding affinity for a cytokine receptor and / or biological activity) to about 70% or less of the activity of the corresponding first binding domain (e.g., a cytokine or variant thereof) in the free state. For example, the binding domain can be an antigen-binding fragment, such as an scFv, VH, VL, scFv-scFv, Fv, Fab, Fab', (Fab')2, minibody, diabody, domain antibody variant (dAb), camelid antibody (VHH) or VHH. NAR In some embodiments, the antigen-binding fragment is selected from single domain antibodies (sdAbs) such as IgG, fibronectin 3 domain variants, ankyrin repeat variants, and other target molecule-specific binding domains derived from other protein scaffolds. In some embodiments, the antigen-binding fragment is an scFv. In some embodiments, the antigen-binding fragment is a Fab. In some embodiments, the antigen-binding fragment is formed by a VH from a first polypeptide chain and a VL from a second polypeptide chain. In some embodiments, the antigen-binding fragment is human. In some embodiments, the antigen-binding fragment is humanized. In some embodiments, the antigen-binding fragment is chimeric. In some embodiments, the antigen-binding fragment is derived from a monoclonal antibody, such as a mouse, rat, monkey, or rabbit antibody.

[0164] In some embodiments, an immunomodulatory molecule comprises two or more first binding domains (e.g., immunostimulatory cytokine moieties). In some embodiments, an immunomodulatory molecule comprises two or more second binding domains (e.g., PD-L1 or PD-L2 extracellular domains, or anti-PD-1 agonist Fab, scFv, sdAb, etc.). In some embodiments, an immunomodulatory molecule further comprises one or more third binding domains. In some embodiments, two or more first binding domains (e.g., antigen-binding fragments or cytokine moieties) are linked in tandem via an optional linker. In some embodiments, two or more first binding domains are on different antigen-binding polypeptides. In some embodiments, two or more second binding domains (e.g., antigen-binding fragments or cytokine moieties) are linked in tandem via an optional linker. In some embodiments, two or more second binding domains are on different antigen-binding polypeptides. In some embodiments, two or more third binding domains (e.g., antigen-binding fragments or cytokine moieties) are linked in tandem via an optional linker. In some embodiments, two or more third binding domains are on different antigen-binding polypeptides. In some embodiments, two or more first binding domains are the same. In some embodiments, two or more first binding domains are different. In some embodiments, the target molecule epitopes specifically recognized by two or more first binding domains are the same. In some embodiments, the target molecule epitopes specifically recognized by two or more first binding domains are different. In some embodiments, two or more second binding domains are the same. In some embodiments, two or more second binding domains are different. In some embodiments, the target molecule epitopes specifically recognized by two or more second binding domains are the same. In some embodiments, the target molecule epitopes specifically recognized by two or more second binding domains are different. In some embodiments, two or more third binding domains are the same.In some embodiments, two or more third binding domains are different. In some embodiments, the target molecule epitopes specifically recognized by two or more third binding domains are the same. In some embodiments, the target molecule epitopes specifically recognized by two or more third binding domains are different. For example, in some embodiments, the immunomodulatory molecule comprises, from N' to C': Fab1 - optional linker 1 - Fab2 - optional linker 2 - (optional hinge or portion thereof - first binding domain (e.g., immunostimulatory cytokine moiety) - optional hinge or portion thereof) - Fc subunit. For example, CH1 or CL of Fab1 is linked to VH or VL of Fab2 via optional linker 1. In some embodiments, an immunomodulatory molecule comprises, from N' to C': scFv1 (or sdAb1)-optional linker 1-scFv2 (or sdAb2)-optional linker 2-(optional hinge or portion thereof-first binding domain (e.g., immunostimulatory cytokine moiety)-optional hinge or portion thereof)-Fc subunit. In some embodiments, an immunomodulatory molecule comprises, from N' to C': ligand 1 (e.g., PD-L2)-optional linker 1-ligand 2 (e.g., PD-L2)-optional linker 2-(optional hinge or portion thereof-first binding domain (e.g., immunostimulatory cytokine moiety)-optional hinge or portion thereof)-Fc subunit. The first binding domain (e.g., immunostimulatory cytokine moiety) in parentheses may not be present in the other paired immunomodulatory molecule chain. For example, an immunomodulatory molecule can comprise a first polypeptide chain comprising, from N' to C': scFv1 (or sdAb1)-optional linker 1-scFv2 (or sdAb2)-optional linker 2-first binding domain (e.g., an immunostimulatory cytokine moiety)-hinge or portion thereof-Fc subunit 1, and a second polypeptide chain comprising, from N' to C': scFv3 (or sdAb3)-optional linker 3-scFv4 (or sdAb4)-optional linker 4-hinge or portion thereof-Fc subunit 2.

[0165] The binding affinity of a binding domain (e.g., scFv, Fab, VHH, ligand, or receptor) and its target molecule can be experimentally determined by any suitable antibody / antigen binding assay or other protein binding assay (e.g., ligand-receptor binding) known in the art, such as Western blot, ELISA, MSD electrochemiluminescence, bead-based MIA, RIA, SPR, ECL, IRMA, EIA, Biacore assay, Octet analysis, peptide scan, FACS, etc. See also the "Binding Affinity" subsection below for exemplary methods. In some embodiments, the Kd of binding between an antibody or antigen-binding fragment and its target molecule is about ≦10 -5 M, ≤10 -6 M, ≤10 -7 M, ≤10 -8 M, ≤10 -9 M, ≤10 -10 M, ≤10 -11 M, or ≦10 -12 It is either M.

[0166] Amino acid sequence variants of antigen-binding proteins or binding domains (e.g., antigen-binding fragments) can be prepared by introducing appropriate modifications into the nucleic acid sequence encoding the antigen-binding protein or binding domain, or by peptide synthesis. Such modifications include, for example, deletions, insertions, and / or substitutions of residues within the amino acid sequence of the antigen-binding protein or binding domain. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., target molecule binding.

[0167] In some embodiments, an antigen binding protein (e.g., an antibody or ligand / receptor-hinge-Fc fusion protein) or binding domain (e.g., an scFv, Fab, VHH, ligand, or receptor) comprises one or more amino acid substitutions. Sites of interest for substitutional mutagenesis include the HVRs (or CDRs) and FRs of an antibody or antigen-binding fragment. Conservative substitutions are provided in Table B under the heading "Preferred Substitutions." More substantial changes are provided in Table B under the heading "Exemplary Substitutions," as further described below in connection with amino acid side chain classes. Amino acid substitutions can be introduced into a binding domain of interest and the products screened for the desired activity, e.g., retained / improved target molecule binding, reduced immunogenicity.

[0168] [Table 2]

[0169] Amino acids can be grouped according to common side chain properties: (1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that affect chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe. Non-conservative substitutions would involve exchanging a member from one of these classes for another.

[0170] One type of substitutional variant involves substituting one or more HVR residues of a parent antibody or antigen-binding fragment thereof. Generally, the resulting variant selected for further study will have altered (e.g., improved) specific biological properties (e.g., increased affinity, decreased immunogenicity) compared to the parent antibody or antigen-binding fragment thereof and / or will substantially retain specific biological properties of the parent antibody or antigen-binding fragment thereof. An exemplary substitutional variant is an affinity-matured antibody, which may be conveniently generated using, for example, phage-display-based affinity maturation techniques such as those described herein. Briefly, one or more HVR residues are mutated, and the variant antibodies are displayed on phage and screened for a specific biological activity (e.g., binding affinity).

[0171] In some embodiments, substitutions, insertions, or deletions may be made within one or more HVRs, so long as such modifications do not substantially reduce the ability of an antibody, or antigen-binding fragment thereof, to bind to an antigen. For example, HVRs may be made with conservative modifications (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity. Such modifications may be outside of HVR "hotspots" or CDRs.

[0172] Modifications (e.g., substitutions) may be made in HVRs, for example, to improve antibody affinity. Such modifications may be made in HVR "hotspots," i.e., residues encoded by codons that frequently undergo mutation during somatic maturation (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or in SDRs (a-CDRs), and the resulting variant VH or VL are tested for binding affinity. Affinity maturation by construction of a secondary library and subsequent selection therefrom is described, for example, in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then generated. This library is then screened to identify any antibody variants with the desired affinity. Another method for introducing diversity involves HVR-directed approaches, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding may be specifically identified using, for example, alanine scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are often targeted.

[0173] A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis," as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a residue or group of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) is identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the antibody's interaction with the antigen is affected. Further substitutions at these amino acid positions can demonstrate functional sensitivity to the initial substitutions. Alternatively or additionally, a crystal structure of the antigen-antibody complex can be used to identify contact points between the antibody and antigen. Such contact residues and neighboring residues can be targeted or eliminated as candidates for substitution. The mutants can be screened to determine whether they possess the desired properties.

[0174] In some embodiments, the first binding domain is an immunostimulatory cytokine moiety or variant thereof, e.g., any of the cytokine moieties described herein (e.g., any of SEQ ID NOs: 26-30, 41, 63-65, and 140). In some embodiments, the immunostimulatory cytokine moiety or variant thereof is selected from the group consisting of IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-12, IL-15, IL-17, IL-18, IL-21, IL-22, IL-23, IL-27, IFN-α, IFN-β, IFN-γ, TNF-α-G-CSF, M-CSF, SCF, and GM-CSF. In some embodiments, the first binding domain is an agonistic antibody to a T cell surface antigen, including, but not limited to, CD3ε, CD3δ, or CD3γ; or CD2, CD4, CD8, CD27, CD28, CD40, CD134, CD137, and CD278. In some embodiments, the first binding domain is an agonistic antibody to an NK cell surface antigen, including, but not limited to, CD16a, CD56 (NCAM), NKp46, NKp44, CD244, CD226, TIGIT, CD96, LAG3, TIM3, PD-1, KLRG1, CD161, CD94 / NKG2, KIR, NKG2D, and NKp30. In some embodiments, the first binding domain is an agonistic antibody to any of CD27, CD28, CD137, OX40, GITR, and HVEM. In some embodiments, the first binding domain is an agonist ligand, for example, CD80, CD86, or 4-1BB.

[0175] In some embodiments, the second binding domain is an agonist antibody to an inhibitory checkpoint molecule such as PD-1, TIGIT, or CTLT-4. In some embodiments, the second binding domain is a ligand of an inhibitory checkpoint molecule such as PD-L1, PD-L2, CD155, or a variant thereof. In some embodiments, the second binding domain comprises any of the sequences of SEQ ID NOs: 106-110, 121-128, and 137.

[0176] In some embodiments, the third binding domain is an antibody (agonist, antagonist, or neutral) against a T cell surface antigen, including, but not limited to, CD3ε, CD3δ, or CD3γ; or CD2, CD4, CD8, CD27, CD28, CD40, CD134, CD137, and CD278. In some embodiments, the third binding domain is an antibody (agonist, antagonist, or neutral) against a NK cell surface antigen, including, but not limited to, CD16a, CD56 (NCAM), NKp46, NKp44, CD244, CD226, TIGIT, CD96, LAG3, TIM3, PD-1, KLRG1, CD161, CD94 / NKG2, KIR, NKG2D, and NKp30. In some embodiments, the third binding domain is an antibody (agonist, antagonist, or neutral) against a T cell exhaustion marker, including but not limited to, PD-1, TIGIT, CTLA-4, LAG3, and TIM3. In some embodiments, the third binding domain is an antibody (agonist, antagonist, or neutral) against a tumor antigen, including but not limited to, Her2, Her3, CEA, Trop2, and CLDN18.2. In some embodiments, the third binding domain is a ligand for an immune cell surface antigen (e.g., PD-1 or TIGIT as the antigen), such as PD-L1, PD-L2, CD155, or a variant thereof. In some embodiments, the third binding domain comprises the sequence of any of SEQ ID NOs: 106-110, 121-128, and 137.

[0177] Cytokines or their variants Cytokines (also interchangeably referred to as "cytokine molecules" or "cytokine proteins") are secreted proteins that regulate the activity of cells of the immune system. Examples of cytokines include interleukins, interferons, chemokines, lymphokines, tumor necrosis factors, immune cell precursor colony-stimulating factors, and the like. In some embodiments, a cytokine is a wild-type cytokine. In some embodiments, a cytokine is a naturally occurring cytokine species variant. In some embodiments, a cytokine is a naturally occurring cytokine subtype. A "cytokine variant," as used herein, refers to any non-naturally occurring cytokine molecule, such as a cytokine-active fragment thereof (e.g., a cytokine fragment that retains at least about 10% of the biological activity or cytokine receptor binding activity of the full-length cytokine), mutant, or derivative thereof. A "cytokine or variant thereof" is also interchangeably referred to herein as a "cytokine portion," which may be a cytokine molecule, or a species variant, subtype, active fragment, mutant, or derivative thereof.

[0178] As used herein, a "heterodimeric cytokine" or "cytokine heterodimer" refers to a cytokine composed of two different protein subunits. Currently, the IL-12 family (including IL-12, IL-23, IL-27, and IL-35) is the only known naturally occurring heterodimeric cytokine family. However, artificial heterodimeric cytokines can be constructed. For example, IL-6 and a soluble IL-6R fragment can be combined to form a heterodimeric cytokine, such as CNTF and CNTF-Rα (Trinchieri (1994) Blood 84:4008). A "homodimeric cytokine" or "cytokine homodimer" herein refers to a cytokine composed of two identical protein subunits, such as IFN-γ or IL-10. A "monomeric cytokine" or "cytokine monomer" refers to a cytokine composed of a single cytokine molecule. In some embodiments, the cytokine or variant thereof is a monomeric cytokine or variant thereof. In some embodiments, the cytokine or variant thereof is a homodimeric cytokine or variant thereof. In some embodiments, the cytokine or variant thereof is a heterodimeric cytokine or variant thereof.

[0179] In some embodiments, the cytokine portion is a full-length cytokine molecule. In some embodiments, the cytokine portion is a functional fragment of a cytokine molecule capable of exhibiting a portion (e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%) or all of the biological activity and / or cytokine receptor binding activity of the full-length cytokine molecule. In some embodiments, the cytokine portion is a precursor cytokine molecule. In some embodiments, the cytokine portion is a mature cytokine molecule (e.g., lacking a signal peptide). In some embodiments, the cytokine portion is a wild-type cytokine. In some embodiments, the cytokine portion is a naturally occurring cytokine species variant. In some embodiments, the cytokine portion is a naturally occurring cytokine subtype. In some embodiments, the cytokine portion is a cytokine variant, such as a mutant cytokine capable of exhibiting a portion (e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%) or all of the biological activity and / or cytokine receptor binding activity of the wild-type cytokine. In some embodiments, the cytokine variant is a modified cytokine, such as a glycosylated cytokine. The cytokines or variants thereof described herein can be cytokines isolated from various sources, such as from human tissue types or from another source, or prepared by recombinant or synthetic methods. In some embodiments, the cytokine portion is a recombinant cytokine. In some embodiments, the cytokine portion described herein can be a cytokine derived from any organism, such as a mammal, including, but not limited to, livestock animals (e.g., cows, sheep, goats, cats, dogs, donkeys, and horses), primates (e.g., humans and non-human primates such as monkeys or chimpanzees), rabbits, and rodents (e.g., mice, rats, gerbils, and hamsters). In some embodiments, the cytokine portion is a human cytokine, such as a recombinant human cytokine.In some embodiments, the cytokine portion is a mouse cytokine, such as a recombinant mouse cytokine. In some embodiments, the cytokine portion is a mature human cytokine. In some embodiments, the cytokine portion includes a signal peptide at the N-terminus of the cytokine molecule, where the signal peptide may be from a different molecule or from the same cytokine molecule.

[0180] A cytokine variant may be a truncated version, a post-translationally modified version, a hybrid variant, a peptidomimetic, a biologically active fragment, a deletion variant, a substitution variant, or an addition variant that maintains at least some (e.g., at least about 10%) of the parent cytokine activity (cytokine receptor binding activity and / or biological activity). As used herein, "parent cytokine" or "parent cytokine" refers to the cytokine reference sequence from which the cytokine variant is engineered, modified, or derived.

[0181] When an immunomodulatory molecule of the subject invention is described as having two or more different cytokines (and optionally including additional protein moieties), it means that the immunomodulatory molecule has two or more different cytokine molecules (rather than two or more different cytokine subunits). For example, a homodimeric cytokine (e.g., IFN-α, IFN-β, IFN-γ, IL-5, IL-8, etc.) is referred to herein as a single cytokine molecule. For example, an immunomodulatory molecule containing two IL-5 monomers / subunits (either on the same polypeptide chain as a single-chain fusion or on different polypeptide chains) is considered to have only one cytokine molecule, i.e., IL-5. Similarly, a heterodimeric cytokine, such as IL-12, is a single cytokine, even though it has different subunits. For example, an immunomodulatory molecule containing a p35 subunit and a p40 subunit (either on the same polypeptide chain as a single-chain fusion or on different polypeptide chains) is considered to have only one cytokine molecule, i.e., IL-12. Furthermore, a dimeric form of a cytokine that is normally homodimeric, such as the MCP-1 / MCP-2 heterodimer, or a heterodimeric form of two alleles of a cytokine that is normally homodimeric (e.g., Zhang, J. Biol. Chem.

[1994] 269:15918-24), is a single cytokine. In some embodiments, a cytokine subunit on one polypeptide chain of an immunomodulatory molecule (e.g., p35 of IL-12) can dimerize with a paired cytokine subunit (e.g., p40) on the same or a different polypeptide chain within the same immunomodulatory molecule. In some embodiments, a cytokine subunit of an immunomodulatory molecule (e.g., p35 of IL-12) can dimerize with a paired cytokine subunit (e.g., p40) of a neighboring immunomodulatory molecule.

[0182] In some embodiments, the cytokine variant comprises a mutation or modification (e.g., a post-translational modification) that results in increased selectivity, measured as the ratio of activation of cells expressing a first type of receptor (e.g., a trimeric receptor or a high affinity receptor), relative to a second type of receptor (e.g., a dimeric receptor or a weaker affinity receptor) for the corresponding cytokine molecule. For example, in some embodiments, the cytokine variant is a mutant IL-2 (or post-translationally modified IL-2) that binds IL-2Rβγ with stronger affinity (e.g., at least about 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, or 10-fold stronger affinity) relative to IL-2Rαβγ, or activates cells expressing IL-2Rβγ to a greater extent than cells expressing IL-2Rαβγ (e.g., at least about 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, or 10-fold activation); or vice versa. In some embodiments, depending on the type of disease to be treated, the preferred mutation or modification increases the cytokine moiety of immune effector cell activation (e.g., CD8+ T cells for the treatment of cancer). For example, in some embodiments, the IL-2 variant has a mutation (or post-translational modification) that reduces activation of cells expressing the IL-2Rβγ receptor by the IL-2 variant compared to activation of cells expressing the IL-2Rαβγ receptor by the IL-2 variant.

[0183] In some embodiments, mutations or modifications in cytokine variants result in a differential effect (e.g., reduced binding or cell activation) compared to immunomodulatory molecules lacking such mutations or modifications in the cytokine portion. In one aspect, the differential effect is measured by the proliferative response of a cell or cell line to a growth-related cytokine (e.g., IL-2). This response of an immunomodulatory molecule is expressed as an EC50 value, which is obtained by plotting a dose-response curve and determining the protein concentration that results in a half-maximal response. In some embodiments, the ratio of EC50 values ​​obtained for cells expressing a first receptor type (e.g., IL-2Rβγ receptor) to cells expressing a second receptor type (e.g., IL-2Rαβγ receptor) for an immunomodulatory molecule of the invention (e.g., an IL-2 variant immunomodulatory molecule) to the ratio of EC50 values ​​for a reference immunomodulatory molecule (e.g., an IL-2 wild-type immunomodulatory molecule of the same configuration) provides a measure of the differential effect for that immunomodulatory molecule. In some embodiments, the EC50 value obtained for an immunomodulatory molecule of the invention (e.g., an IL-2 variant immunomodulatory molecule) is compared to the EC50 value for a reference immunomodulatory molecule (e.g., an IL-2 wild-type immunomodulatory molecule of the same configuration) to determine a measure of the differential effect for that immunomodulatory molecule.

[0184] In some embodiments, a cytokine variant comprises a mutation at one or more amino acids of the parent cytokine molecule (e.g., a mature wild-type cytokine). In one embodiment, a cytokine variant comprises an amino acid substitution at one or more amino acid positions in the cytokine. In another embodiment, a cytokine variant comprises an amino acid deletion or insertion at one or more amino acid positions in the cytokine. In some embodiments, a cytokine variant comprises a modification of one or more amino acids in the cytokine.

[0185] In some embodiments, the cytokine or variant thereof is selected from the group consisting of IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, IL-22, IL-23, IL-27, IL-35, IFN-α, IFN-β, IFN-γ, TNF-α, TGF-β, VEGF, erythropoietin, thrombopoietin, G-CSF, M-CSF, SCF, and GM-CSF, or naturally occurring variants or subtypes thereof. In some embodiments, the cytokine or variant thereof is an anti-inflammatory or immunosuppressive cytokine or variant thereof, such as IL-1Ra, IL-4, IL-5, IL-6, IL-10, IL-11, IL-13, IL-27, IL-33, IL-35, IL-37, IL-39, IFN-α, LIF, or TGF-β. In some embodiments, the cytokine or variant thereof is a proinflammatory or immunostimulatory cytokine or variant thereof, such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-12, IL-15, IL-17, IL-18, IL-21, IL-22, IL-23, IL-27, IFN-α, IFN-β, IFN-γ, TNF-α, erythropoietin, thrombopoietin, G-CSF, M-CSF, SCF, or GM-CSF, or a variant or subtype thereof. In some embodiments, the cytokine or variant thereof is selected from the group consisting of IL-2, IL-10, IL-12, IL-23, IFN-α (e.g., IFN-α2 or IFN-α2b), IFN-β, and IFN-γ. In some embodiments, the immunostimulatory cytokine is IL-12 and the cytokine subunits are p35 and p40. In some embodiments, the immunostimulatory cytokine is IL-23 and the cytokine subunits are p40 and p19. In some embodiments, the cytokine is IL-27 and the cytokine subunits are Epstein-Barr virus-inducible gene 3 (EBI3) and IL-27p28. In some embodiments, the immunosuppressive cytokine is IL-35 and the cytokine subunits are IL-12α (p35) and IL-27β.In some embodiments, the cytokine variant is a single chain fusion of two or more subunits from different cytokines.

[0186] IL-2 In some embodiments, the immunostimulatory cytokine or variant thereof is IL-2 or a variant thereof. Interleukin-2 (IL-2), also known as T-cell growth factor (TCGF), is a 15.5 kDa monomeric protein that plays an important role in lymphocyte development, survival, and homeostasis. It is involved in the body's natural response to microbial infection and in distinguishing between "self" and "non-self." IL-2 is an interleukin, which belongs to a cytokine family that includes IL-4, IL-7, IL-9, IL-15, and IL-21. IL-2 mediates its effects by binding to the IL-2 receptor (IL-2R) expressed on lymphocytes. Activated CD4 + T cells and activated CD8 + T cells are the primary source of IL-2. IL-2's ability to expand lymphocyte populations and increase the effector functions of these cells makes it an attractive anticancer therapy. IL-2 has been proposed for the treatment of acute myeloid leukemia (AML), non-Hodgkin's lymphoma (NHL), cutaneous T-cell lymphoma (CTCL), breast cancer, and bladder cancer.

[0187] The IL-2 receptor (IL-2R) is a complex consisting of three chains: α (CD25, p55), β (CD122, p75), and γ (CD132, p65). The γ chain is shared by all IL-2 cytokine family members. The intermediate affinity dimer CD122 / CD132 IL-2R (IL-2Rβγ, Kd approximately 10 -9 M) or high-affinity trimeric CD25 / CD122 / CD132 IL-2R (IL-2Rαβγ, Kd approximately 10 -11Binding of IL-2 to either CD8 or CD25 (M) can lead to signal transduction, but binding to CD25 alone does not. The β chain is complexed with Janus kinase 1 (JAK1). The γ chain is complexed with JAK3. Binding of IL-2 to the IL-2R activates JAK1 and JAK3, which have the ability to add phosphate groups to molecules, thus initiating three intracellular signaling pathways: the MAP kinase pathway, the phosphoinositide 3-kinase (PI3K) pathway, and the JAK-STAT pathway. The dimeric IL-2Rβγ binds to memory CD8 and CD8+. + It is expressed on T cells, NK cells, and B cells, while high levels of the trimeric IL-2Rαβγ are expressed on regulatory T cells (Treg) and activated T cells.

[0188] Aldesleukin (Proleukin®), a recombinant human IL-2, was the first cancer immunotherapy and one of the earliest recombinant proteins approved by the FDA in 1992. Currently, aldesleukin is used by intravenous infusion to treat metastatic renal cell carcinoma (mRCC) and metastatic melanoma (mM). Because frequent intravenous infusions over multiple doses are required, aldesleukin is administered in a clinical setting. Aldesleukin has demonstrated complete cancer regression in approximately 10% of patients undergoing treatment for metastatic melanoma and renal cancer (Klapper et al., Cancer, 2008; Rosenberg, Sci Transl Med., 2012; Smith et al., Clin Cancer Res., 2008). Approximately 70% of patients who achieve a complete response are cured and maintain complete regression more than 25 years after initial treatment (Atkins et al., J Clin Oncol., 1999; Klapper et al., Cancer, 2008; Rosenberg, Sci Transl Med., 2012; Rosenberg et al., Ann Surg., 1998; Smith et al., Clin Cancer Res., 2008). However, high doses of IL-2 can induce vascular leak syndrome (VLS), tumor tolerance caused by activation-induced cell death (AICD), and immunosuppression caused by Treg activation. An additional concern with systemic IL-2 therapy relates to severe side effects when administered intravenously, including cardiovascular, pulmonary edema, hepatic, gastrointestinal (GI), neurological, and hematologic events (Proleukin (aldesleukin) Summary of Product Characteristics [SmPC]: http: / / www.medicines.org.uk / emc / medicine / 19322 / SPC). These severe side effects often limit optimal IL-2 dosing and therefore limit the number of patients who successfully respond to therapy. Concerns about IL-2 toxicity and its short half-life must be addressed before it can be widely used.

[0189] The native human IL-2 precursor polypeptide consists of 153 amino acid residues (amino acids 1-20 are a signal peptide), while the mature polypeptide consists of 133 amino acid residues (SEQ ID NO: 25). In some embodiments, the IL-2 moiety is human mature IL-2. In some embodiments, the IL-2 moiety is a polypeptide that is substantially homologous to the amino acid sequence of wild-type human IL-2 (SEQ ID NO: 25), e.g., has at least about 85% amino acid sequence identity (e.g., at least about any of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to wild-type human IL-2 (SEQ ID NO: 25). In some embodiments, the IL-2 moiety is unglycosylated. In some embodiments, the IL-2 moiety is glycosylated.

[0190] In some embodiments, the IL-2 moiety is (or consists essentially of) aldesleukin (e.g., Proleukin®; see, e.g., https: / / www.drugbank.ca / drugs / DB00041). Aldesleukin (desalanyl-1, serine-125 human interleukin-2) is an FDA-approved antineoplastic (anti-cancer) biological response modifier. It has a molecular weight of approximately 15.3 kDa and is also known as recombinant interleukin-2 human, interleukin-2 aldesleukin, 125-L-serine-2-133-interleukin-2 (human reduced), or interleukin-2(2-133),125-ser. Aldesleukin is recombinant IL-2 that differs from native IL-2 in the following ways: a) aldesleukin is produced from E. coli and is therefore not glycosylated; b) aldesleukin does not have an N-terminal alanine (A); c) aldesleukin has a cysteine ​​to serine substitution at position 125 (C125S); and d) aldesleukin is expected to differ from native IL-2 in its aggregation state. Thus, in some embodiments, the IL-2 variant contains a cysteine ​​to serine substitution at position 125 (C125S) from the mature form of human IL-2.

[0191] K. Sauve et al. (Proc Natl Acad Sci U S A. 1991;88(11):4636-4640) found that amino acid residues K35, R38, F42, and K43 of wild-type IL-2 are critical for IL-2 receptor binding (IL-2Rα, low affinity type), and that the R38A and F24A mutations retained substantial IL-2 biological activity. R. Vazquez-Lombardi et al. (Nat Commun. 2017;8:15373) found that the R38D, K43E, and E61R mutations of IL-2 minimized Treg expansion while significantly increasing CD25 expression compared to wild-type IL-2. - They found that the P65L mutation in IL-2 drove a potent expansion of the cytotoxic subset. The P65L mutation in IL-2 was found to exhibit reduced systemic toxicity and increased antitumor efficacy compared to wild-type IL-2 (Chen et al., Cell Death Dis. 2018;9(10):989).

[0192] In some embodiments, the IL-2 variant comprises one or more mutations at positions selected from the group consisting of L18, Q22, F24, K35, R38, F42, K43, E61, P65, Q126, and S130 compared to wild-type IL-2 (SEQ ID NO: 25). In some embodiments, the IL-2 variant comprises one or more mutations selected from the group consisting of L18R, Q22E, F24A, R38D, K43E, E61R, P65L, Q126T, and S130R compared to wild-type IL-2 (SEQ ID NO: 25). In some embodiments, the IL-2 variant comprises R38D / K43E / E61R mutations compared to wild-type IL-2 (SEQ ID NO: 25). In some embodiments, the IL-2 variant comprises the sequence of SEQ ID NO: 26. In some embodiments, the IL-2 variant comprises L18R / Q22E / R38D / K43E / E61R mutations compared to SEQ ID NO: 25. In some embodiments, the IL-2 variant comprises the sequence of SEQ ID NO: 27. In some embodiments, the IL-2 variant comprises R38D / K43E / E61R / Q126T mutations compared to SEQ ID NO: 25. In some embodiments, the IL-2 variant comprises the sequence of SEQ ID NO: 28. In some embodiments, the IL-2 variant comprises L18R / Q22E / R38D / K43E / E61R / Q126T mutations compared to SEQ ID NO: 25. In some embodiments, the IL-2 variant comprises the sequence of SEQ ID NO: 29. In some embodiments, the IL-2 variant comprises L18R / Q22E / R38D / K43E / E61R / Q126T / S130R mutations compared to SEQ ID NO: 25. In some embodiments, the IL-2 variant comprises the sequence of SEQ ID NO:30.

[0193] IFN-α In some embodiments, the immunostimulatory cytokine or variant thereof is IFN-α or a variant thereof, such as IFN-α2 or a variant thereof, or IFN-α2b or a variant thereof. Human type I interferons (IFNs) are a large group of IFNs that help regulate the activity of the immune system. They bind to a specific cell surface receptor complex known as the IFN-α receptor (IFNAR), which consists of the IFNAR1 and IFNAR2 chains. Mammalian type I IFNs include IFN-α, IFN-β, IFN-κ, IFN-δ, IFN-ε, IFN-τ, IFN-ω, and IFN-ζ (also known as limitin).

[0194] IFN-α protein is primarily produced by plasmacytoid dendritic cells (pDCs) and is primarily involved in innate immunity against viral infections. IFN-α protein is a 19-26 kDa monomeric protein and is widely used in the treatment of cancer and viral diseases such as hepatitis B and C. There are 13 genes involved in the synthesis of 13 IFN-α subtypes: IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNA10, IFNA13, IFNA14, IFNA16, IFNA17, and IFNA21.

[0195] Human IFN-α2a, IFN-α2b, and IFN-α2c represent allelic variants of the same gene. IFN-α2a and IFN-α2b have lysine and arginine, respectively, at position 23 of the mature protein. Human IFN-α2a and IFN-α2b are the only IFN-α subtypes that contain an O-glycosylation site (on Thr106). Interferon alpha-2a (IFN-α2a; marketed by Hoffmann-La Roche as Roferon-A®) and interferon alpha-2b (IFN-α2b, a recombinant form of IFN-α2; marketed by Schering-Plough as Intron-A®) have been approved for the treatment of hairy cell leukemia, melanoma, follicular lymphoma, renal cell carcinoma, AIDS-associated Kaposi's sarcoma, and chronic myeloid leukemia (M. Ferrantini et al., Biochimie. Jun-Jul 2007;89(6-7):884-893). Recent studies have highlighted novel immunomodulatory effects of IFN-α, including activity against T cells and dendritic cells, that may lead to the generation of durable antitumor responses. However, the use of IFN-α in clinical oncology is still generally based on exploiting the antiproliferative and antiangiogenic activities of these cytokines. To fully exploit the role of IFN-α as a regulator of immune responses and tumor immunity, novel approaches in the use of these cytokines may be required.

[0196] hIFN-α2b is a 166-amino acid glycoprotein with O-glycosylation at threonine 106. Each rhIFN-α2b consists of five α-helices (termed helices A to E) connected by loops AB, BC, CD, and DE. Residues important for receptor binding are the AB loop (Arg22, Leu26, Phe27, Leu30, Lys31, Arg33, and His34), helix B (Ser68), helix C (Thr79, Lys83, Tyr85, and Tyr89), helix D (Arg120, Lys121, Gln124, Lys131, and Glu132), and helix E (Arg144 and Glu146). The amino acid residues important for biological activity are Leu30, Lys31, Arg33, His34, Phe36, Arg120, Lys121, Gln124, Tyr122, Tyr129, Lys131, Glu132, Arg144, and Glu146 (Ratih Asmana Ningrum, Scientifica (Cairo). 2014; 2014: 970315).

[0197] In some embodiments, the IFN-α portion is IFN-α2. In some embodiments, the IFN-α portion is IFN-α2a. In some embodiments, the IFN-α portion is IFN-α2b. In some embodiments, the IFN-α portion is IFN-α2c. In some embodiments, the IFN-α portion is mature IFN-α. The native human IFN-α2b precursor polypeptide consists of 188 amino acid residues (amino acids 1-23 are a signal peptide), while the mature polypeptide consists of 165 amino acid residues (SEQ ID NO: 31). In some embodiments, the IFN-α portion is a polypeptide that is substantially homologous to wild-type IFN-α (SEQ ID NO: 31), e.g., having at least about 85% amino acid sequence identity (e.g., at least about any of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to wild-type IFN-α (SEQ ID NO: 31). In some embodiments, the IFN-α moiety is non-glycosylated. In some embodiments, the IFN-α moiety is glycosylated.

[0198] In some embodiments, the IFN-α variant (e.g., an IFN-α2b variant) comprises one or more mutations at positions selected from the group consisting of R22, L26, F27, L30, K31, D32, R33, H34, D35, F36, S68, T79, K83, Y85, Y89, R120, K121, Y122, Q124, Y129, K131, E132, R144, and E146 relative to an IFN-α (e.g., an IFN-α2b; SEQ ID NO: 31). In some embodiments, the IFN-α variant comprises the amino acid sequence of SEQ ID NO: 32. In some embodiments, the IFN-α variant (e.g., an IFN-α2b variant) comprises the sequence of any of SEQ ID NOs: 32-37.

[0199] IFN-β Two types of IFN-β are described: IFN-β1 and IFN-β3. In some embodiments, the immunostimulatory cytokine or variant thereof is IFN-β or a variant thereof, e.g., IFN-β1, IFN-β3, or a variant thereof. In some embodiments, the immunostimulatory cytokine or variant thereof is IFN-β1a or a variant thereof. In some embodiments, the IFN-β moiety is mature IFN-β. In some embodiments, the IFN-β moiety is wild-type (e.g., wild-type human) IFN-β. In some embodiments, the IFN-β moiety is mutant (e.g., mutant human) IFN-β. In some embodiments, the IFN-β moiety is unglycosylated. In some embodiments, the IFN-β moiety is glycosylated.

[0200] IFN-γ In some embodiments, the immunostimulatory cytokine or variant thereof is IFN-γ or a variant thereof. Interferon gamma (IFNγ) is a disulfide-bonded dimeric soluble cytokine and the only member of the type II class of interferons. IFN-γ is a homodimer of approximately 25 kDa with a tertiary fold structure constructed around an unusual pattern of interdigitated α-helices. It is produced primarily by T cells and NK cells in response to various inflammatory or immune stimuli. IFN-γ can act as both an activator and suppressor of the immune system. Studies have shown that cancer immunotherapy (checkpoint inhibitors) acts in part through increased IFN-γ expression, leading to the disappearance of cancer cells. Resistance to immunotherapy is due to defective IFN-γ signaling. However, IFN-γ can also contribute to tumorigenesis and angiogenesis, induce the expression of resistance molecules such as PD-L1, and evade cancer by inducing homeostatic programs. IFN-γ is not approved by the FDA for the treatment of cancer patients, except in cases of malignant osteoporosis, due to the opposing and competing effects it has on the immune system (L. Ni and J. Lu, Cancer Med. 2018;7(9):4509-4516).

[0201] The monomeric native human IFN-γ (hIFN-γ) prepropolypeptide consists of 166 amino acid residues (amino acids 1-23 are a signal peptide); the monomeric mature polypeptide consists of 138 amino acid residues (SEQ ID NO: 38), corresponding to amino acids 24-161 of the prepropolypeptide; amino acids 162-166 are the propeptide sequence of the prepropolypeptide. In some embodiments, the monomeric IFN-γ portion is a monomeric mature IFN-γ. In some embodiments, the monomeric IFN-γ portion is a polypeptide that is substantially homologous to wild-type IFN-γ (SEQ ID NO: 38), e.g., has at least about 85% amino acid sequence identity (e.g., at least about any of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to wild-type IFN-γ (SEQ ID NO: 38). In some embodiments, the IFN-γ portion (or subunit) is not glycosylated. In some embodiments, the IFN-γ portion (or subunit) is glycosylated. In some embodiments, the IFN-γ portion comprises two identical IFN-γ monomers / subunits. In some embodiments, the IFN-γ portion comprises two different IFN-γ monomers / subunits. For example, in some embodiments, the IFN-γ portion comprises one wild-type IFN-γ monomer and one IFN-γ mutant monomer. In some embodiments, the IFN-γ portion comprises two IFN-γ monomers (e.g., two IFN-γ mutant or wild-type monomers) linked together, such as via a peptide linker (e.g., any of SEQ ID NOs: 227-229, 245, and 246) or a chemical linker.

[0202] IFN-γ amino acid residues S20, A23, H111, and Q115 are important for receptor binding; amino acid residues V5, S20, A23, G26, and H111 are important for the biological activity of IFN-γ (M. Randal and AA Kossiakoff, Structure. 2001;9(2):155-63). Lander et al. (J Mol Biol. 2000;299(1):169-79) developed a biologically active single-chain mutant of hIFN-γ (IFN-γSC1) by linking two monomeric IFN-γ with a seven-amino acid residue linker and changing His111 in the first IFN-γ monomer to an aspartic acid residue. Due to this H111D mutation, IFN-γSC1 can only bind to one IFN-γRα, but can fully retain its biological activities in cell proliferation, MHC class I induction, and antiviral assays.

[0203] In some embodiments, the monomeric IFN-γ comprises the sequence of SEQ ID NO: 38. In some embodiments, the IFN-γ variant comprises one or more mutations within one or both IFN-γ subunits at positions selected from the group consisting of V5, S20, D21, V22, A23, D24, N25, G26, H111, and Q115 relative to the wild-type IFN-γ subunit (SEQ ID NO: 38). In some embodiments, the IFN-γ variant comprises one or more mutations within one or both IFN-γ subunits relative to the wild-type IFN-γ subunit (SEQ ID NO: 38) selected from the group consisting of S20A, D21A, D21K, V22A, A23S, A23E, A23Q, A23V, D24A, D24E, N25A, N25K, and H111D. In some embodiments, the IFN-γ variant comprises one or more mutations within one or both IFN-γ subunits selected from the group consisting of S20A / D21A, D21K, V22A / A23S, D24A / N25A, A23E / D24E / N25K, A23Q, and A23V relative to the wild-type IFN-γ subunit (SEQ ID NO: 38). In some embodiments, one or both subunits of the IFN-γ variant comprise the sequence of any of SEQ ID NOs: 39-45. In some embodiments, the IFN-γ variant comprises an A23V mutation within one or both IFN-γ subunits relative to the wild-type IFN-γ subunit (SEQ ID NO: 38). In some embodiments, one or both subunits of the IFN-γ variant comprise the sequence of SEQ ID NO: 41. In some embodiments, the two subunits of the IFN-γ or variant thereof are connected by a linker (e.g., any of SEQ ID NOs: 227-229, 245, and 246). In some embodiments, the IFN-γ variant comprises the sequence of SEQ ID NO: 47 or 252. In some embodiments, both IFN-γ subunits comprise the sequence of SEQ...

Claims

1. a first binding domain that specifically recognizes a first target molecule and a second binding domain that specifically recognizes a second target molecule; upon binding to said first target molecule, said first binding domain upregulates an immune response; upon binding to the second target molecule, the second binding domain downregulates the immune response, and the first binding domain is an IL-12 variant; the second binding domain is an agonist of an inhibitory checkpoint molecule; the inhibitory checkpoint molecule is PD-1; An immunomodulatory molecule, wherein the variant of IL-12 comprises one or more mutations in the p40 subunit at positions selected from the group consisting of E59 and F60 compared to the wild-type p40 subunit (SEQ ID NO: 62), and wherein the p40 subunit of said IL-12 variant has at least 95% amino acid sequence identity with the wild-type p40 subunit (SEQ ID NO: 62).

2. The immunomodulatory molecule of claim 1, wherein the IL-12 variant comprises one or more mutations in the p40 subunit selected from the group consisting of E59A, F60A, and F60D compared to the wild-type p40 subunit (SEQ ID NO: 62).

3. The immunomodulatory molecule of claim 2, wherein the IL-12 variant comprises an E59A mutation, an F60A mutation, or an E59A / F60A mutation in the p40 subunit compared to the wild-type p40 subunit (SEQ ID NO: 62).

4. The immunomodulatory molecule of claim 3, wherein the p40 subunit of the IL-12 variant comprises any of the sequences set forth in SEQ ID NOs: 63-65 and 140.

5. The immunomodulatory molecule of claim 1 , wherein the second binding domain is a ligand, antibody, or antigen-binding fragment of an inhibitory checkpoint molecule.

6. the second binding domain is PD-L1, PD-L2, or a variant thereof; The immunomodulatory molecule of claim 5 . (i) An immunomodulatory molecule comprising an antigen-binding protein comprising an antigen-binding polypeptide and the first binding domain, wherein the antigen-binding polypeptide comprises, from N-terminus to C-terminus: the second binding domain or a portion thereof, a hinge region, and an Fc domain subunit or a portion thereof, and the first binding domain is located in the hinge region; (ii) an immunomodulatory molecule comprising an antigen-binding protein comprising an antigen-binding polypeptide, the antigen-binding polypeptide comprising, from N'-terminus to C'-terminus: a first binding domain or portion thereof, a second binding domain or portion thereof, an optional hinge region, a subunit of an Fc domain or portion thereof; or (iii) an immunomodulatory molecule comprising an antigen-binding protein comprising an antigen-binding polypeptide, the antigen-binding polypeptide comprising, from N-terminus to C-terminus: a second binding domain or portion thereof, an optional hinge region, a subunit of an Fc domain or portion thereof, and a first binding domain or portion thereof; The immunomodulatory molecule of claim 1 .

8. (i) an immunomodulatory molecule comprising, from N-terminus to C-terminus: a first antigen-binding polypeptide comprising a first PD-L2 or PD-L1 or variant thereof, a second PD-L2 or PD-L1 or variant thereof, a p35 subunit and a p40 subunit of an IL-12 variant arranged in tandem in a first hinge region, and a first subunit of an Fc domain or a portion thereof; from N-terminus to C-terminus: a second antigen-binding polypeptide comprising a VH, an optional CH1, a second hinge region, and a second subunit of said Fc domain or a portion thereof; and from N-terminus to C-terminus: a third antigen-binding polypeptide comprising a VL and an optional CL, wherein said VH and said VL and optionally said CH1 and said CL form a third binding domain that specifically recognizes a third target molecule; (ii) From the N-terminus to the C-terminus: a first antigen-binding polypeptide comprising a first VH, an optional first CH1, a p35 subunit and a p40 subunit of an IL-12 variant arranged in tandem in a first hinge region, and a first subunit of an Fc domain or a portion thereof; From the N-terminus to the C-terminus: a second VH, an optional second CH1, a second hinge region, and a second subunit of the Fc domain or a portion thereof; From the N-terminus to the C-terminus: a first VL and an optional first C a third antigen-binding polypeptide comprising a first VH and a second VL; and a fourth antigen-binding polypeptide comprising, from N-terminus to C-terminus: a second VL and an optional second CL, wherein the first VH and the first VL and optionally the first CH1 and the first CL form the second binding domain which is an agonist antigen-binding fragment that specifically recognizes PD-1, and the second VH and the second VL and optionally the second CH1 and the second CL form a third binding domain that specifically recognizes a third target molecule; (iii) an immunomodulatory molecule comprising, from N-terminus to C-terminus: a first antigen-binding polypeptide comprising a first PD-L2 or PD-L1 or variant thereof, a p35 subunit and a p40 subunit of an IL-12 variant arranged in tandem in a first hinge region, and a first subunit of an Fc domain or a portion thereof; and, from N-terminus to C-terminus: a second antigen-binding polypeptide comprising a second PD-L2 or PD-L1 or variant thereof, a second hinge region, and a second subunit of an Fc domain or a portion thereof; (iv) an immunomodulatory molecule comprising, from N-terminus to C-terminus: a first antigen-binding polypeptide comprising a first PD-L2 or PD-L1 or variant thereof, a second PD-L2 or PD-L1 or variant thereof, the p35 subunit and the p40 subunit of an IL-12 variant arranged in tandem in a first hinge region, and a first subunit of an Fc domain or a portion thereof; and, from N-terminus to C-terminus: a second antigen-binding polypeptide comprising a third PD-L2 or PD-L1 or variant thereof, a fourth PD-L2 or PD-L1 or variant thereof, a second hinge region, and a second subunit of said Fc domain or a portion thereof; (v) an immunomodulatory molecule comprising, from N-terminus to C-terminus: a first antigen-binding polypeptide comprising a first PD-L2 or PD-L1 or variant thereof, a p35 subunit of an IL-12 variant located in a first hinge region, and a first subunit of an Fc domain or a portion thereof; and a second antigen-binding polypeptide comprising, from N-terminus to C-terminus: a second PD-L2 or PD-L1 or variant thereof, a p40 subunit of IL-12 or variant thereof located in a second hinge region, and a second subunit of the Fc domain or a portion thereof; (vi) an immunomodulatory molecule comprising, from N-terminus to C-terminus: a first antigen-binding polypeptide comprising a p35 or p40 subunit of an IL-12 variant located in a first hinge region and a first subunit of an Fc domain or a portion thereof; and a second antigen-binding polypeptide comprising, from N-terminus to C-terminus: a first PD-L2 or PD-L1 or variant thereof, a second PD-L2 or PD-L1 or variant thereof, a p40 or p35 subunit of an IL-12 variant located in a second hinge region, and a second subunit of said Fc domain or a portion thereof; or (vii) a first antigen-binding polypeptide comprising, from N-terminus to C-terminus: a first VH, an optional first CH1, a p35 or p40 subunit of an IL-12 variant located in a first hinge region, and a first subunit of an Fc domain or a portion thereof; a second antigen-binding polypeptide comprising, from N-terminus to C-terminus: a second VH, an optional second CH1, a p40 or p35 subunit of an IL-12 variant located in a second hinge region, and a second subunit of said Fc domain or a portion thereof; and, from N-terminus to C-terminus: a third antigen-binding polypeptide comprising a first VL and optionally a first CL; and a fourth antigen-binding polypeptide comprising a second VL and optionally a second CL, wherein the first VH and the first VL and optionally the first CH1 and the first CL form the second binding domain which is an agonist antigen-binding fragment that specifically recognizes PD-1, and the second VH and the second VL and optionally the second CH1 and the second CL form a third binding domain that specifically recognizes a third target molecule. The immunomodulatory molecule of claim 1 .

9. (i) a first antigen-binding polypeptide comprising, from N-terminus to C-terminus: a first PD-L2 or PD-L1 or variant thereof, a p35 subunit of an IL-12 variant located in a first hinge region, and a first subunit of an Fc domain or a portion thereof; a second antigen-binding polypeptide comprising, from N-terminus to C-terminus: a second PD-L2 or PD-L1 or variant thereof, a p40 subunit of an IL-12 variant located in a second hinge region, and a second subunit of an Fc domain or a portion thereof; or (ii) an immunomodulatory molecule comprising, from N-terminus to C-terminus: a first antigen-binding polypeptide comprising a first VH, an optional first CH1, the p35 and p40 subunits of an IL-12 variant located in a first hinge region, and a first subunit of an Fc domain or a portion thereof; from N-terminus to C-terminus: a second antigen-binding polypeptide comprising a second VH, an optional second CH1, the p35 and p40 subunits of an IL-12 variant located in a second hinge region, and a second subunit of an Fc domain or a portion thereof; from N-terminus to C-terminus: a third antigen-binding polypeptide comprising a first VL, and an optional first CL; and from N-terminus to C-terminus: a fourth antigen-binding polypeptide comprising a second VL, and an optional second CL; The immunomodulatory molecule of claim 1, wherein the first VH and first VL and the optional first CH1 and first CL form a second binding domain that specifically recognizes PD-1, and the second VH and second VL and the optional second CH1 and second CL form a third binding domain that specifically recognizes a third target molecule.

10. (i) a first antigen-binding polypeptide comprising, from N-terminus to C-terminus: a first PD-L2 or PD-L1 or variant thereof, an IL-12 variant, a first hinge region, and a first subunit of an Fc domain or a portion thereof; a second antigen-binding polypeptide comprising, from N-terminus to C-terminus: a second PD-L2 or PD-L1 or variant thereof, the p35 and p40 subunits of an IL-12 variant, a second hinge region, and a second subunit of an Fc domain or a portion thereof; or (ii) a first antigen-binding polypeptide comprising, from N-terminus to C-terminus: a third binding domain, an IL-12 variant, a first hinge region, a first subunit of an Fc domain or a portion thereof; and ii) a second antigen-binding polypeptide comprising, from N-terminus to C-terminus: PD-L2 or PD-L1 or a variant thereof, a p35 subunit and a p40 subunit of an IL-12 variant connected in tandem, a second hinge region, a second subunit of an Fc domain or a portion thereof, The immunomodulatory molecule of claim 1, wherein the third binding domain is TIGIT, TIM3, LAG3, CTLA4, CD16A, HER2, Nectin-4, Trop2, or CLDN18.

2.

11. (i) From N-terminus to C-terminus: a first antigen-binding polypeptide comprising a first VH, an optional first CH1, a first hinge region, and a first subunit of an Fc domain or a portion thereof; From N-terminus to C-terminus: a second antigen-binding polypeptide comprising a second VH, an optional second CH1, a second hinge region, and a second subunit of said Fc domain or a portion thereof; From N-terminus to C-terminus: a p35 subunit and a p40 subunit of an IL-12 variant fused in tandem, a first VL, and an optional first CL. and a fourth antigen-binding polypeptide comprising, from N-terminus to C-terminus: a second VL and an optional second CL, wherein the first VH and the first VL, and optionally the first CH1 and the first CL, form the second binding domain that is an agonist antigen-binding fragment that specifically recognizes PD-1, and the second VH and the second VL, and optionally the second CH1 and the second CL, form a third binding domain that specifically recognizes a third target molecule; or 2. The immunomodulatory molecule of claim 1, wherein the immunomodulatory molecule comprises: (ii) a first antigen-binding polypeptide comprising, from N-terminus to C-terminus: a VH, an optional CH1, a first hinge region, and a first subunit of an Fc domain or a portion thereof; a second antigen-binding polypeptide comprising, from N-terminus to C-terminus: a first PD-L2 or PD-L1 or variant thereof, a second PD-L2 or PD-L1 or variant thereof, a second hinge region, and a second subunit of the Fc domain or a portion thereof; and a third antigen-binding polypeptide comprising, from N-terminus to C-terminus: a p35 subunit and a p40 subunit of an IL-12 variant fused in tandem, a VL, and an optional CL, wherein the VH and the VL, and optionally the CH1 and the CL, form the second binding domain, which is an agonist antigen-binding fragment that specifically recognizes PD-1.

12. A pharmaceutical composition comprising an immunomodulatory molecule according to any one of claims 1 to 11, and optionally a pharmaceutically acceptable carrier.

13. An immunomodulatory molecule according to any one of claims 1 to 11 for treating a disease or disorder in an individual.

14. The immunomodulatory molecule of claim 13 , wherein the disease or disorder is cancer.

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