Heterodimeric FC cytokines and uses thereof

Abstract

The present disclosure provides IL12 and IL23 muteins as partial agonists comprising a modified human p40 molecule that associates with human p35 (hP35) and human P19 (hP19) to form modified hIL-12 and IL23 partial agonists, where the individual components of the IL12 and IL23 muteins are linked to an engineered Fc domain. TIFF2024539139000092.tif34128

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED PATENT APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 257,913, filed October 20, 2021. U.S. Provisional Patent Application No. 63 / 257,913 is incorporated by reference for all purposes. [Background technology]

[0002] 2. Background of the Invention Cytokine receptors are typically multimers of cell surface expressed proteins that stimulate signal transduction through the interaction of intracellular domains. Cytokines act as specific ligands for the extracellular domains of cytokine receptor subunits, facilitating the multimerization of such receptor subunits to bring the intracellular domains of such cytokine receptor subunits into close proximity so that intracellular signal transduction can occur. Certain cytokine receptor subunits are shared among different cytokines, and the nature of the cytokine determines which receptor subunits multimerize to form cytokine receptor complexes and the resulting intracellular signal transduction patterns. Thus, cytokines act to crosslink individual receptor subunits into receptor complexes that cause intracellular signal transduction.

[0003] Cytokine receptor subunit intracellular domains typically have JAK binding domains located in the box1 / box2 region of the cytokine receptor subunit intracellular domain, near the inner surface of the cell membrane. These JAK binding domains are associated with intracellular JAK kinases. Typically, the JAKs phosphorylate each other when the intracellular domains of the receptor subunits are brought into close proximity by binding of the cognate ligand for the receptor to the receptor subunit extracellular domain. Four Janus kinases have been identified in mammalian cells: JAK1, JAK2, JAK3, and TYK2. Ihle, et al. (1995) Nature 377(6550):591-4, 1995; O'Shea and Plenge (2012) Immunity 36(4):542-50. JAK phosphorylation induces a conformational change in the JAK that allows it to further phosphorylate other intracellular proteins initiating a cascade that activates multiple intracellular factors that transmit intracellular signals associated with that receptor. The resulting intracellular responses, such as gene transcription, are often collectively referred to as downstream signaling.

[0004] In many cases, the proteins phosphorylated by JAK are members of the signal transducer and activator of transcription (STAT) protein family. To date, seven members of the mammalian STAT family have been identified: STAT1, STAT2, STAT3, STAT4, STAT5a STAT5b, and STAT6. Delgoffe, et al., (2011) Curr Opin Immunol. 23(5):632-8; Levy and Darnell (2002) Nat Rev Mol Cell Biol. 3(9):651-62, and Murray, (2007) J Immunol. 178(5):2623-9. The selective interaction of activated JAK and STAT proteins is collectively referred to as the JAK / STAT pathway, which provides the wide variety of intracellular responses observed in response to cytokine binding.

[0005] Human IL-12 (hIL-12) is a heterodimeric cytokine composed of the human p35 subunit (also called hIL-12A; Uniprot Ref. 29459) and the human p40 subunit (also called hIL-12B; Uniprot Ref. 29460). The hIL-12 heterodimer is also called p70. hIL-12 is produced by dendritic cells, macrophages, and neutrophils. hIL-12 is typically identified as a T cell stimulatory factor capable of stimulating T cell proliferation and activation. hIL12 stimulates the production of IFNγ and TNFα and modulates the cytotoxic activity of NK cells and CD8+ cytotoxic T cells. hIL12 was first identified and mentioned as a cytotoxic lymphocyte maturation factor. Stern, et al (1990) Proc Natl Acad Sci USA 87:6808-6812 (Non-Patent Document 6) and Gately et al., U.S. Patent No. 6,683,046 issued January 27, 2004 (Patent Document 1). hIL-12 is also involved in immune cell differentiation, particularly differentiation of naive T cells to Th1 (CD4+) cells. hIL-12 has also been reported to provide anti-angiogenic activity. Since its discovery more than 30 years ago, hIL-12 has been proposed and evaluated for use in the treatment of various neoplastic diseases, viral infections, and bacterial infections. See, e.g., Lasek, et al (2014) Cancer Immunol Immunother (2014) 63:419-435 (Non-Patent Document 7).

[0006] hIL-12 binds to the hIL-12 receptor, a heterodimeric complex of hIL12 receptor subunit beta-1 (IL-12Rβ1, also referred to in the scientific literature as IL-12RB1 or CD212, Uniprot Ref. P42701) and hIL-12 receptor subunit beta-2 (hIL-12Rβ2, also referred to in the scientific literature as hIL-12RB2, Uniprot Ref. Q99665). hIL12Rβ1 and hIL12Rβ2 are members of the class I cytokine receptor family and share homology with gp130. Expression of hIL12Rβ1 and hIL12Rβ2 is upregulated in response to hIL-12, and the majority of hIL12Rβ2 is found in activated T cells.

[0007] hIL12Rβ1 is a constitutively expressed type I transmembrane protein that belongs to the hemopoietin receptor superfamily. hIL12Rβ1 binds hIL-12 with low affinity. hIL12Rβ1 is required for binding to the hIL-12p40 subunit and associates with Tyk-2, a member of the Janus kinase (Jak) family. Binding of the IL12p40 subunit of IL12 to IL12Rβ1 and the IL12p35 subunit to IL12Rβ2 leads to dimerization of IL-12Rβ1 and IL-12Rβ2. In response to dimerization of IL-12Rβ1 and IL-12Rβ2, Jak-2 and Tyk-2 are transphosphorylated, further activating Jak2 and Tyk2 kinase activity, which results in phosphorylation of the IL12Rβ1 and IL12Rβ2 intracellular domains. The phosphorylated intracellular signaling domain of IL12Rβ2 provides a binding site for STAT4. STAT4 binds to phosphorylated IL-12Rβ2 and is subsequently phosphorylated. Phosphorylated STAT4 induces dimerization with another phosphorylated STAT4 molecule. Phosphorylated STAT4 homodimers translocate to the nucleus, resulting in the promotion of IFN-γ gene transcription, among other activities. IFN-γ induces the activity and proliferation of macrophages, NK cells, and T cells, which also secrete IL-12.

[0008] In addition to forming one of the components of the hIL-12 receptor, hIL-12Rβ1 is also a component of the hIL-23 receptor, which is a heterodimer of hIL-23R (Uniprot Ref. Q5VWK5) and hIL-12Rβ1. hIL-23 binds to hIL-23R with an affinity of 44 nanomolar (nM) but to hIL-12Rβ1 with a much lower affinity of 2 micromolar (μM). There is no apparent direct binding of hIL-23R to hIL12Rβ1, and completion of the hIL-23h:IL-23R:hIL-12Rβ1 complex is mediated by the initial formation of the hIL-23:hIL-23R complex, which then binds to IL12Rβ1. IL23 is a heterodimeric cytokine composed of p40 and p19 subunits. IL12 and IL23 receptors share the IL12Rβ1 receptor subunit but induce distinct downstream signaling patterns. IL12 has been implicated as useful in the treatment of various neoplastic diseases, whereas IL23 inhibitors (e.g., ustekinumab, guselkumab, tildrakizumab, and risankizumab) are primarily indicated for treating autoimmune disorders such as psoriatic arthritis and moderate to severe plaque psoriasis.

[0009] In addition to forming a subunit of IL12 and IL23, p40 has important biological activities. It has been reported that p40 exists both as a monomer and as a disulfide-linked homodimer (p80), which has a chemoattractant role for macrophages mediated exclusively by IL12Rβ1 and may act as an IL-12 and IL-23 antagonist by competing for these receptors. Holscher, C. (2004) Med Microbiology and Immunology 193(1)1-17.

[0010] IL12 has many properties that suggested its use in the treatment of cancer, including stimulating IFNγ production by NK cells, enhancing the cytolytic properties of NK cells and cytotoxic T cells, and inhibiting angiogenesis. IL12 has shown significant antitumor activity in animal models, which led to its evaluation in phase I and II clinical trials in the treatment of various cancers in the late 1990s. Lasek, et al., supra. Although beneficial effects were observed, significant adverse events were observed, leading to the termination of these clinical trials. Various approaches have since been evaluated for the use of the hIL12 molecule and gene therapy vectors encoding hIL12, but progress to phase I and II clinical trials has thus far been limited due to the toxicity associated with these agents. There are no commercially available therapeutic agents that contain IL12.

[0011] Because different cell types respond with different sensitivities to binding of a ligand to its cognate receptor, tuning the affinity of the heterodimeric hIL-12 ligand (or its individual components) for the hIL-12 receptor (or its individual components) relative to wild-type hIL-12 (i.e., including wild-type p35 and p40) can stimulate desirable activity on target cells while reducing undesirable activity on non-target cells. In some embodiments, the hIL-12 partial agonists of the present disclosure comprise modified p40 subunit polypeptides that provide intracellular signaling characteristics of wthIL12 on desirable cell types while providing significantly less intracellular signaling on undesirable cell types. This can be achieved, for example, by contacting cells with an IL12 partial agonist that comprises a heterodimeric hIL12Fc mutein with modified binding affinity for hIL-12Rβ1, or a different E polypeptide for hIL-12Rβ1 relative to the binding affinity of the wild-type or parent hIL-12p40 polypeptide for hIL-12Rβ1. max This is accomplished by causing

[0012] Glassman, et al. (2021) Cell 184(4):983-999 (Non-Patent Document 9) describes the crystal structures of the IL12 and IL23 receptors and describes residues of p40 that interact with the IL12Rβ1 receptor. In particular, Glassman et al. describe an IL12 partial agonist containing an altered p40 subunit that preserves IFNγ induction of CD8+ T cells and tumor cell killing, but exhibits reduced activation and cytokine production from NK cells. Stimulation of NK cells is associated with significant systemic side effects such as capillary leak syndrome. IL12 partial agonists that selectively activate CD8+ T cells without significantly upregulating NK cells preserve the beneficial antitumor effects of IL12 while reducing the systemic toxicity associated with NK cell activation. This IL12 partial agonist produced potent antitumor immunity and reduced toxicity compared to IL-12 in preclinical mouse tumor models.

[0013] To maximize the antitumor effect of IL12, it is desirable to provide sustained systemic levels of the cytokine. The in vivo half-life of recombinant human IL12 (rhIL12) is longer than other cytokines such as IL2, but is still relatively short. The half-life of wild-type rhIL-12 was observed to be 5.3 to 10.3 hours after a single bolus intravenous dose of rhIL12 at 500 ng / kg, the maximum tolerated dose in this study. Atkins, et al (1997) Clinical Cancer Research 3:409-417. However, toxicity associated with wt hIL12 treatment has hindered the development of long-term delivery IL12. As previously mentioned, sustained and targeted delivery of IL12 has been evaluated but has not resulted in a successful IL12 therapeutic.

[0014] Fc fusion conjugates have been shown to extend the systemic half-life of biologics, and thus the biologic product does not require frequent administration. Engineered Fc domains have been extensively explored in the context of therapeutic antibodies, particularly bispecific antibodies, and numerous Fc engineered antibodies have been developed and commercialized. See, for example, Czajkowsky, et al. (2012) EMBO Mol Med 4:1015-1028. Fc binds to fetal Fc receptors (FcRn) on the surface of endothelial cells lining blood vessels, and upon binding, the Fc fusion molecule is protected from degradation and released back into circulation, thereby keeping the molecule in circulation longer. These Fc domain properties are believed to be the mechanism by which endogenous IgG retains its long plasma half-life. More recent Fc fusion technology links a copy of the biologic to the Fc region of an antibody to optimize the pharmacokinetic and pharmacodynamic properties of the biologic compared to traditional Fc fusion conjugates. Various modifications to the Fc domain, called Fc engineering, have been developed to provide specific beneficial features to the Fc domain for tuning effector functions (Wang, et al. (2018) Protein Cell 9(1):63-73 (Non-Patent Document 12)). For example, Zalevsky et al. describe the amino acid substitutions M428L and N434S (EU numbering), often referred to as "LS" modifications, to extend half-life. Zalevsky, et al. (2010) Nature Biotechnology 28:157-159 (Non-Patent Document 13).

[0015] Fc conjugates of IL12 have been described in the literature. For example, Gillies et al. described an IL12 Fc conjugate in which wild-type p35 and wild-type P40 subunits were each expressed as a fusion protein with an Fc subunit. Gillies, et al. (1998) J. Immunol. 160:6195-6203 (Non-Patent Document 14) and Gillies et al., U.S. Patent No. 6,838,260 issued January 4, 2005 (Patent Document 2). Similarly, Kim et al. (PCT / KR2017 / 008676 published on February 15, 2018 as WO / 2018 / 030806 (Patent Document 3); U.S. Patent No. 11,078,249 issued on August 3, 2021 (Patent Document 4); U.S. Patent No. 10,696,722 issued on June 30, 2020 (Patent Document 5)) describe an IL12 Fc conjugate in which the p35 and P40 subunits are expressed as fusion proteins with an Fc domain, and the Fc domain is modified to promote heterodimerization. Cheung et al. (PCT International Patent Application No. PCT / US2019 / 057721 (Patent Document 6) published on April 30, 2020 as WO / 2020 / 086758) and Bigelow et al. (PCT International Patent Application No. PCT / US2021 / 028701 (Patent Document 7) published on October 28, 2021 as WO / 2021 / 216916) describe IL12 Fc conjugates. Bernett et al. (PCT International Patent Application PCT / US19 / 54570, published April 9, 2020 as WO / 2020 / 072821; U.S. Patent Application Publication No. US2020 / 0216509, published July 9, 2020; U.S. Patent No. 11,358,999, issued June 14, 2022) describe IL2 Fc conjugates containing wild-type and modified p35 and P40 subunits. See also Epstein et al., Chinese Patent Application No. CN201410597561.4A, published May 4, 2016.

[0016] The present disclosure provides IL12 and IL23 muteins as partial agonists comprising modified human p40 molecules that associate with human p35 (hP35) and human P19 (hP19) to form modified hIL-12 and IL23 partial agonists, where the individual components of the IL12 and IL23 muteins are linked to engineered Fc domains. The IL12 and IL23 agonists of the present disclosure retain many of the beneficial properties of the wild-type parent molecules from which they are derived, while reducing the associated toxicity associated with wild-type IL12 and IL23. [Prior art documents] [Patent documents]

[0017] [Patent Document 1] U.S. Patent No. 6,683,046 [Patent Document 2] U.S. Patent No. 6,838,260 [Patent Document 3] PCT / KR2017 / 008676 [Patent Document 4] U.S. Patent No. 11,078,249 [Patent Document 5] U.S. Patent No. 10,696,722 [Patent Document 6] PCT International Patent Application Number PCT / US2019 / 057721 [Patent Document 7] PCT International Patent Application PCT / US2021 / 028701 [Patent Document 8] PCT International Patent Application PCT / US19 / 54570 [Patent Document 9] U.S. Patent Application Publication No. US2020 / 0216509 [Patent Document 10] U.S. Patent No. 11,358,999 [Patent Document 11] China Patent Application Number CN201410597561.4A [Non-patent literature]

[0018] [Non-licensed document 1] Ihle, et al. (1995) Nature 377(6550):591-4, 1995

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[0019] Disclosure Summary The present disclosure relates to methods and compositions that modulate the multiple effects of hIL-12 binding such that desirable therapeutic signaling occurs, particularly in desirable cell or tissue subtypes, while minimizing undesirable activity and / or intracellular signaling in other cell or tissue subtypes. In some aspects, the present disclosure provides heterodimeric hIL12Fc muteins that have a long duration of action in vivo in a mammalian subject.

[0020] In some aspects, the present disclosure provides a method for producing a method for treating a cancer cell comprising: A heterodimeric hIL12Fc mutein comprising a heterodimeric protein comprising a first fusion protein and a second fusion protein, The first fusion protein is a human p35 polypeptide having 90% or more, alternatively 91% or more, alternatively 92% or more, alternatively 93% or more, alternatively 94% or more, alternatively 95% or more, alternatively 96% or more, alternatively 97% or more, alternatively 98% or more, alternatively 99% or more, alternatively 100% sequence identity to SEQ ID NO:2 (wild-type mature hP35); a first upper hinge region of a human immunoglobulin molecule; the first Fc domain of a human immunoglobulin molecule ("hP35Fc"); Including, The second fusion protein is a human p40 mutein ("hP40M") polypeptide having 90% or more, alternatively 91% or more, alternatively 92% or more, alternatively 93% or more, alternatively 94% or more, alternatively 95% or more, alternatively 96% or more, alternatively 97% or more, alternatively 98% or more, alternatively 99% or more sequence identity to SEQ ID NO:4 (wild-type mature hP40), wherein the human p40 mutein comprises one or more amino acid substitutions at one or more positions selected from the group consisting of W37, P39, D40, A41, K80, E81, F82, K106, E108, D115, H216, K217, L218, and K219 (numbered according to SEQ ID NO:3); a second upper hinge region of a human immunoglobulin molecule; a second Fc domain of a human immunoglobulin molecule ("hP40MFc"); wherein the first Fc domain and the second Fc domain are modified to promote heterodimerization. Heterodimeric hIL12Fc muteins are provided.

[0021] In some aspects, the disclosure provides heterodimeric hIL12Fc muteins comprising a human p40 mutein ("hP40M") polypeptide comprising one or more amino acid substitutions at one or more positions selected from the group consisting of W37, P39, D40, A41, K80, E81, F82, K106, E108, D115, H216, K217, L218, K219, and K282 (numbered according to SEQ ID NO:3). In some aspects, the disclosure provides heterodimeric hIL12Fc muteins comprising a human p40 mutein ("hP40M") polypeptide comprising one or more amino acid substitutions at one or more positions selected from the group consisting of E81, F82, K106, and K217 (numbered according to SEQ ID NO:3). In some aspects, the disclosure provides heterodimeric hIL12Fc muteins comprising a human p40 mutein ("hP40M") polypeptide comprising one or more amino acid substitutions at one or more positions selected from the group consisting of E81, F82, K106, and K217 (numbered according to SEQ ID NO:3). In some aspects, the disclosure provides heterodimeric hIL12Fc muteins comprising a human p40 mutein ("hP40M") polypeptide comprising a set of amino acid substitutions selected from the group consisting of E81A / F82A; E81A / F82A / K106A; and E81A / F82A / K106A / K217A (numbered according to SEQ ID NO:3).

[0022] In some aspects, the disclosure provides heterodimeric hIL12Fc muteins comprising a human p40 mutein ("hP40M") polypeptide comprising one or more amino acid substitutions at one or more positions selected from the group consisting of E81, F82, K106, and K217, and further comprising an amino acid substitution at position K282 (numbered according to SEQ ID NO:3). NO:3)E81A / F82A / K282A;E81A / F82A / K282G;E81A / F82A / K282N;E81A / F82A / K282Q;E81A / F82A / K106A;E81A / F82A / K106A / K282A;E81A / F82A / K106A / K282G;E81A / F82A / K106A / K282N;E81A / F82A / K106A / K282Q;E81 A heterodimeric hIL12Fc mutein is provided, comprising a human p40 mutein ("hP40M") polypeptide comprising a set of amino acid substitutions selected from the group consisting of: A / F82A / K106A / K217A / K282A; E81A / F82A / K106A / K217A / K282Q; E81A / F82A / K106A / K217A / K282N; and E81A / F82A / K106A / K217A / K282G.

[0023] In some aspects, the disclosure provides a heterodimeric hIL12Fc mutein comprising a human p40 mutein ("hP40M") polypeptide, wherein the hP40 mutein is selected from the group consisting of SEQ ID NOs:4, 6, 8, 10, 155, 156, 157, 158, 159, 160, 161, and 162.

[0024] In some embodiments, modifications to the Fc domain of the heterodimeric hIL12Fc mutein to promote heterodimerization. In some embodiments, the modifications to the Fc domain of the heterodimeric hIL12Fc mutein to promote heterodimerization are complementary "knob-into-hole" mutations. In some embodiments, the modifications to the Fc domain of the hP35Fc and hP40MFc domains to promote heterodimerization include the amino acid substitution T366W in the first domain ("knob") and the amino acid substitutions T366S / L368A / Y407V in the second domain ("hole").

[0025] In some embodiments, the present disclosure provides a heterodimeric hIL12Fc mutein in which the hP35Fc and hP40MFc polypeptides of the heterodimeric hIL12Fc mutein are covalently linked via one disulfide bond, optionally two disulfide bonds, optionally three disulfide bonds, or optionally four disulfide bonds. In some embodiments, the hP35Fc and hP40MFc are covalently linked via a disulfide bond between the sulfhydryl group at amino acid C96 of the hP35 domain of hP35Fc and the sulfhydryl group at amino acid C199 of the hP40M domain of hP40MFc. In some embodiments, hP35Fc and hP40MFc are covalently linked via a disulfide bond between the sulfhydryl group at amino acid C226 in the lower hinge domain of hP35Fc and the sulfhydryl group at amino acid C226 in the lower hinge domain of hP40MFc. In some embodiments, hP35Fc and hP40MFc are covalently linked via a disulfide bond between the sulfhydryl group at amino acid C229 in the lower hinge domain of hP35Fc and the sulfhydryl group at amino acid C229 in the lower hinge domain of hP40MFc. In some embodiments, the first Fc domain comprises the amino acid substitution S354C and the second Fc domain comprises the amino acid substitution Y349C. In some embodiments, the heterodimeric hIL12Fc mutein comprises a first Fc domain comprising the amino acid substitution S354C and a second Fc domain comprising the amino acid substitution Y349C, and the hP35Fc and hP40MFc domains are linked via a disulfide bond between S354C of the first Fc domain and Y349C of the second Fc domain.In some embodiments, the hP35Fc and hP40MFc of the heterodimeric hIL12Fc muteins are covalently linked via one or more, optionally two or more, optionally three or more disulfide bonds, optionally four or more disulfide bonds between the side chains of the following cysteine ​​residue pairs: (a) C96 of hP35 and C199 of hP40M; (b) C226 of the first Fc monomer and C226 of the second Fc monomer, (c) C229 of the first Fc monomer and C229 of the second Fc monomer; and (d) S354C of the first Fc domain comprising the S354C amino acid substitution and Y349C of the second Fc domain comprising the Y349C amino acid substitution.

[0026] In some embodiments, the present disclosure provides heterodimeric hIL12Fc muteins, in which one or both of the hP35Fc and hP40MFc subunits of the heterodimeric hIL12Fc mutein comprise one or more amino acid substitutions to reduce effector function. In some embodiments, the hP35Fc and / or hP40MFc polypeptides comprise a set of amino acid substitutions selected from the group consisting of: (a) L234A / L235A / P329A ("LALAPA"); L234A / L235A / P329G ("LALAPG"); L234A / L235E / G237A / A330S / P331S ("AEASS"); and L234F / L235E / P331S ("FES").

[0027] In some embodiments, the present disclosure provides a heterodimeric hIL12Fc mutein, in which one or both of the hP35Fc and hP40MFc subunits of the heterodimeric hIL12Fc mutein comprise an amino acid substitution at position C220 (EU numbering) of the upper hinge domain to eliminate a sulfhydryl side chain. In some embodiments, the substitution at position C220 is a C220S (EU numbering) substitution.

[0028] In some embodiments, the disclosure provides a heterodimeric hIL12Fc mutein, wherein one or both of the hP35Fc and hP40MFc subunits of the heterodimeric hIL12Fc mutein comprise an amino acid substitution at positions M428 and / or N434 (EU numbering) of the Fc domain. In some embodiments, the amino acid substitution at positions M428 and / or N434 is M428L and / or N434S.

[0029] In some aspects, the disclosure provides a heterodimeric hIL12Fc mutein, wherein one or both of the hP35Fc and hP40MFc subunits of the heterodimeric hIL12Fc mutein comprise amino acid deletions at positions G446 and / or K447 (EU numbering) of the Fc domain.

[0030] In some embodiments, the present disclosure provides a heterodimeric hIL12Fc mutein, in which one or both of the hP35Fc and hP40MFc subunits of the heterodimeric hIL12Fc mutein are PEGylated. In some embodiments, one or both of the hP35Fc and hP40MFc subunits are PEGylated via the sulfhydryl side chain of amino acid C220 of the upper hinge.

[0031] In some aspects, the disclosure provides heterodimeric hIL12Fc muteins that (i) induce hIL-12 signaling in CD8+ T cells and (ii) have reduced hIL-12 signaling in NK cells (e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, or 70% reduced) compared to a wild-type hIL-12 comprising a p40 polypeptide lacking one or more amino acid substitutions.

[0032] In some aspects, the disclosure provides heterodimeric hIL12Fc muteins that activate interferon gamma (IFNγ) in CD8+ T cells and have reduced IFNγ signaling in CD8+ T cells, e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, or 70% reduced, compared to wild-type IL12 comprising a p40 subunit lacking such amino acid substitutions.

[0033] In some aspects, the disclosure provides heterodimeric hIL12Fc muteins that have reduced binding affinity to hIL-12Rβ1, e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, or 70% reduced, compared to the binding affinity of wild-type IL12.

[0034] In some aspects, the disclosure provides heterodimeric hIL12Fc muteins that have reduced STAT-4 mediated signaling compared to wild-type hIL12, e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, or 70% reduced, when assessed in a mammalian cell-based assay.

[0035] In some aspects, the disclosure provides a first fusion protein comprising a human p19 polypeptide having 90% or more, alternatively 91% or more, alternatively 92% or more, alternatively 93% or more, alternatively 94% or more, alternatively 95% or more, alternatively 96% or more, alternatively 97% or more, alternatively 98% or more, alternatively 99% or more, or 100% sequence identity to SEQ ID NO:178, a first upper hinge region of a human immunoglobulin molecule, and a first Fc domain of a human immunoglobulin molecule ("hP19Fc"), and having 90% or more, alternatively 91% or more, alternatively 92% or more, alternatively 93% or more, alternatively 94% or more, alternatively 95% or more, alternatively 96% or more, alternatively 97% or more, alternatively 98% or more, alternatively 99% or more sequence identity to SEQ ID NO:4 (wild type mature hP40), (SEQ ID and a second fusion protein comprising a human p40 mutein polypeptide comprising one or more amino acid substitutions at one or more positions selected from the group consisting of W37, P39, D40, A41, Q64, K80, E81, F82, A85, K106, E108, D115, H216, K217, L218, and K219 (numbered according to US Pat. No. 3), a second upper hinge region of a human immunoglobulin molecule, and a second Fc domain of a human immunoglobulin molecule ("hP40MFc"), wherein the first Fc domain and the second Fc domain are modified to promote heterodimerization.

[0036] In some embodiments, the modifications to the Fc domain of the heterodimeric hIL23Fc mutein to promote heterodimerization are complementary "knob-into-hole" mutations. In some embodiments, the modifications to the Fc domain of the hP19Fc and hP40MFc domains to promote heterodimerization comprise the amino acid substitution T366W in the first domain (the "knob") and the amino acid substitutions T366S / L368A / Y407V in the second domain (the "hole").

[0037] In some embodiments, the present disclosure provides a heterodimeric hIL23Fc mutein in which the hP19Fc and hP40MFc polypeptides of hIL23 are covalently linked via one disulfide bond, optionally two disulfide bonds, optionally three disulfide bonds, or optionally four disulfide bonds. In some embodiments, the hP19Fc and hP40MFc are covalently linked via a disulfide bond between the sulfhydryl group at amino acid C76 of the hP19 domain of hP19Fc (numbered according to the pro-hP19 sequence including the signal peptide, Uniprot Reference No. Q9NPF7) and the sulfhydryl group at amino acid C199 of the hP40M domain of hP40MFc. In some embodiments, hP19Fc and hP40MFc are covalently linked via a disulfide bond between the sulfhydryl group at amino acid C226 of hP19Fc and the sulfhydryl group at amino acid C226 of hP40MFc. In some embodiments, hP35Fc and hP40MFc are covalently linked via a disulfide bond between the sulfhydryl group at amino acid C229 of hP19Fc and the sulfhydryl group at amino acid C229 of the hP40M domain. In some embodiments, the first Fc domain comprises the amino acid substitution S354C and the second Fc domain comprises the amino acid substitution Y349C. In some embodiments, the heterodimeric hIL23Fc mutein comprises a first Fc domain comprising the amino acid substitution S354C and a second Fc domain comprising the amino acid substitution Y349C, and the hP19Fc and hP40MFc domains are linked via a disulfide bond between S354C of the first Fc domain and Y349C of the second Fc domain.In some embodiments, the hP19Fc and hP40MFc of the heterodimeric hIL23Fc mutein are covalently linked via one or more, optionally two or more, optionally three or more, optionally four or more disulfide bonds between the side chains of the following groups of cysteine ​​pairs: (a) C76 of hP19 and C199 of hP40M; (b) C226 of the first Fc monomer and C226 of the second Fc monomer, (c) C229 of the first Fc monomer and C229 of the second Fc monomer; and (d) S354C of the first Fc domain comprising an S354C amino acid substitution and Y349C of the second Fc domain comprising an Y349C amino acid substitution.

[0038] In some embodiments, the disclosure provides heterodimeric hIL23Fc muteins, in which one or both of the hP19Fc and hP40MFc subunits of the heterodimeric hIL23Fc mutein comprise one or more amino acid substitutions to reduce effector function. In some embodiments, the hP19Fc and / or hP40MFc polypeptides comprise a set of amino acid substitutions selected from the group consisting of: (a) L234A / L235A / P329A ("LALAPA"); L234A / L235A / P329G ("LALAPG"); L234A / L235E / G237A / A330S / P331S ("AEASS"); and L234F / L235E / P331S ("FES").

[0039] In some aspects, the disclosure provides a heterodimeric hIL23Fc mutein, in which one or both of the hP19Fc and hP40MFc subunits of the heterodimeric hIL23Fc mutein comprise an amino acid substitution at position C220 (EU numbering) of the upper hinge domain to eliminate a sulfhydryl side chain. In some aspects, the substitution at position C220 is a C220S (EU numbering) substitution.

[0040] In some embodiments, the disclosure provides a heterodimeric hIL23Fc mutein, wherein one or both of the hP19Fc and hP40MFc subunits of the heterodimeric hIL23Fc mutein comprise an amino acid substitution at positions M428 and / or N434 (EU numbering) of the Fc domain. In some embodiments, the amino acid substitution at positions M428 and / or N434 is M428L and / or N434S.

[0041] In some embodiments, the present disclosure provides a heterodimeric hIL23Fc mutein, in which one or both of the hP19Fc and hP40MFc subunits of the heterodimeric hIL23Fc mutein are PEGylated. In some embodiments, one or both of the hP35Fc and hP40MFc subunits are PEGylated via the sulfhydryl side chain of amino acid C220 of the upper hinge.

[0042] In some aspects, the disclosure provides a heterodimeric hIL23Fc mutein in which an hP19Fc polypeptide and an hP40MFc polypeptide are covalently linked via one disulfide bond, optionally two disulfide bonds, optionally three disulfide bonds, or optionally four disulfide bonds.

[0043] In some embodiments, the present disclosure provides a compound represented by formula #1: hP40M-L1 a -UH1-Fc1[1] A nucleic acid sequence encoding a polypeptide of During the ceremony, hP40M is a human p40 mutein comprising one or more amino acid substitutions at positions selected from the group consisting of positions W37, P39, D40, A41, K80, E81, F82, K106, E108, D115, H216, K217, L218, and K219, numbered according to wild-type prehuman P40 (SEQ ID NO:3); L1 is a GSA linker; a is selected from 0 (absent) or 1 (present); UH1 is an upper hinge domain of a human immunoglobulin independently selected from the group consisting of IgG1, IgG2, IgG3, and IgG4 upper hinges, optionally comprising the amino acid substitution C220S (EU numbering); and Fc1 is a polypeptide comprising the lower hinge, CH2, and CH3 domains of a human immunoglobulin selected from the group consisting of IgG1, IgG2, IgG3, and IgG4, comprising one or more amino acid substitutions that promote heterodimerization. The nucleic acid sequence is provided.

[0044] In some embodiments, the present disclosure provides a compound represented by formula #2: hP35-L2 b -UH2-Fc2 [2] The nucleic acid sequence of During the ceremony, hP35 is a polypeptide having at least 90%, alternatively at least 91%, alternatively at least 92%, alternatively at least 93%, alternatively at least 94%, alternatively at least 95%, alternatively at least 96%, alternatively at least 97%, alternatively at least 98%, alternatively at least 99%, or alternatively 100% sequence identity to SEQ ID NO:2, L2 is a GSA linker, b is selected from 0 (absent) or 1 (present), UH2 is an upper hinge domain of a human immunoglobulin independently selected from the group consisting of IgG1, IgG2, IgG3, and IgG4 upper hinges, optionally comprising the amino acid substitution C220S (EU numbering), and Fc2 is a polypeptide comprising the lower hinge, CH2, and CH3 domains of a human immunoglobulin selected from the group consisting of IgG1, IgG2, IgG3, and IgG4, comprising one or more amino acid substitutions that promote heterodimerization. The nucleic acid sequence is provided.

[0045] In some aspects, the disclosure provides an expression cassette encoding a heterodimeric hIL12Fc mutein, comprising a nucleic acid sequence encoding a hP35Fc polypeptide and a hP40MFc polypeptide operably linked to one or more heterologous nucleic acid sequences, wherein the nucleic acid sequence encoding the hP35Fc polypeptide and the hP40MFc polypeptide is (a) under the control of one promoter, and (b) linked via an intervening sequence that facilitates co-expression. In some aspects, the nucleic acid sequence encoding the hP35Fc polypeptide and the hP40MFc polypeptide is linked via an intervening sequence that facilitates co-expression, and the nucleic acid sequence encoding the p35Fc polypeptide is 5' to the nucleic acid sequence encoding the hP40MFc polypeptide. In some aspects, the nucleic acid sequence encoding the hP35Fc polypeptide and the hP40MFc polypeptide is linked via an intervening sequence that facilitates co-expression, and the nucleic acid sequence encoding the p40MFc polypeptide is 5' to the nucleic acid sequence encoding the hP35Fc polypeptide. In some embodiments, the intervening sequence that facilitates co-expression is an IRES element or a T2A sequence.

[0046] In some aspects, the disclosure provides an expression cassette encoding a heterodimeric hIL23Fc mutein, comprising a nucleic acid sequence encoding a hP19Fc polypeptide and a hP40MFc polypeptide operably linked to one or more heterologous nucleic acid sequences, wherein the nucleic acid sequence encoding the hP19Fc polypeptide and the hP40MFc polypeptide is (a) under the control of a single promoter, and (b) linked via an intervening sequence that facilitates co-expression. In some aspects, the nucleic acid sequence encoding the hP19Fc polypeptide and the hP40MFc polypeptide is linked via an intervening sequence that facilitates co-expression, and the nucleic acid sequence encoding the p19Fc polypeptide is 5' to the nucleic acid sequence encoding the hP40MFc polypeptide. In some aspects, the nucleic acid sequence encoding the hP19Fc polypeptide and the hP40MFc polypeptide is linked via an intervening sequence that facilitates co-expression, and the nucleic acid sequence encoding the p40MFc polypeptide is 5' to the nucleic acid sequence encoding the hP19Fc polypeptide. In some embodiments, the intervening sequence that facilitates co-expression is an IRES element or a T2A sequence.

[0047] In some aspects, the disclosure provides an expression cassette encoding a heterodimeric hIL23Fc mutein comprising a nucleic acid sequence encoding an hP19Fc polypeptide and an hP40MFc polypeptide operably linked to one or more heterologous nucleic acid sequences, wherein the nucleic acid sequences encoding the hP19Fc polypeptide and the hP40MFc polypeptide are (a) under the control of a single promoter and (b) linked via an intervening sequence facilitating simultaneous expression in a mammalian cell.

[0048] The present disclosure further provides a recombinant vector encoding a heterodimeric hIL12Fc mutein, comprising a first expression cassette encoding a hP35Fc polypeptide, and the same or a second vector, comprising a second expression cassette comprising a nucleic acid sequence encoding a hP40MFc polypeptide. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a non-viral vector.

[0049] The present disclosure further provides a recombinant vector encoding a heterodimeric hIL23Fc mutein, comprising a first expression cassette encoding a hP19Fc polypeptide, and the same or a second vector, comprising a second expression cassette comprising a nucleic acid sequence encoding a hP40MFc polypeptide. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a non-viral vector.

[0050] Further provided is a recombinantly modified cell comprising the nucleic acid molecule or vector of the present disclosure. In some embodiments, the cell is a prokaryotic cell, such as a bacterial cell. In some embodiments, the cell is a eukaryotic cell, such as a mammalian cell. Also provided is a cell culture comprising at least one recombinantly modified cell of the present disclosure and a culture medium.

[0051] In some embodiments, the recombinantly modified cells are transformed with a recombinant vector encoding a heterodimeric hIL12Fc mutein, the recombinant vector comprising a first expression cassette encoding a hP35Fc polypeptide and a second expression cassette comprising a nucleic acid sequence encoding a hP40MFc polypeptide. In some embodiments, the recombinantly modified cells are transformed with a recombinant vector encoding a heterodimeric hIL23Fc mutein, the recombinant vector comprising a first expression cassette encoding a hP19Fc polypeptide and a second expression cassette comprising a nucleic acid sequence encoding a hP40MFc polypeptide.

[0052] In some aspects, the recombinantly modified cell is transformed by a first vector comprising a nucleic acid sequence encoding a hP35Fc polypeptide operably linked to one or more expression control sequences, and a second vector comprising an expression cassette comprising a nucleic acid sequence encoding a hP40MFc polypeptide operably linked to one or more expression control sequences. In some aspects, the recombinantly modified cell is transformed by a first vector comprising a nucleic acid sequence encoding a hP19Fc polypeptide operably linked to one or more expression control sequences, and a second vector comprising an expression cassette comprising a nucleic acid sequence encoding a hP40MFc polypeptide operably linked to one or more expression control sequences. In some aspects, the cell is a prokaryotic cell, such as a bacterial cell. In some aspects, the cell is a eukaryotic cell, such as a mammalian cell. Also provided is a cell culture comprising at least one recombinantly modified cell of the present disclosure and a culture medium.

[0053] The present disclosure further provides a method for recombinantly producing, isolating, purifying, and characterizing heterodimeric hIL12Fc muteins or heterodimeric hIL23Fc muteins.Therefore, a method for producing heterodimeric hIL12Fc muteins or heterodimeric hIL23Fc muteins of the present disclosure is provided herein.In some embodiments, the method includes a) providing one or more recombinantly modified cells comprising a nucleic acid molecule or vector comprising a nucleic acid sequence encoding a heterodimeric hIL12Fc mutein or heterodimeric hIL23Fc mutein disclosed herein; and b) culturing one or more cells in a culture medium such that the cells produce the heterodimeric hIL12Fc mutein encoded by the nucleic acid sequence.In some embodiments, the method further includes (c) isolating and / or purifying the modified hIL-12p40 polypeptide.Heterodimeric hIL12Fc muteins produced by the above methods are also provided.

[0054] The present disclosure further provides a pharmaceutical composition comprising the heterodimeric hIL12Fc mutein or heterodimeric hIL23Fc mutein of the present disclosure. In some embodiments, the pharmaceutical composition comprises the heterodimeric hIL12Fc mutein or heterodimeric hIL23Fc mutein of the present disclosure and a pharma- ceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises the nucleic acid molecule or vector of the present disclosure. In some embodiments, the pharmaceutical composition comprises a recombinantly modified cell of the present disclosure. In some embodiments, the recombinantly modified cell is a mammalian cell.

[0055] The present disclosure further provides a method of treating a mammal suffering from a neoplastic disease comprising contacting the mammal with a pharmaceutical formulation of a heterodimeric hIL23Fc mutein disclosed herein.The present disclosure further provides a method of treating a mammal suffering from a neoplastic disease comprising contacting the mammal with a pharmaceutical formulation of a heterodimeric hIL23Fc mutein disclosed herein, wherein the heterodimeric hIL23Fc mutein is a heterodimeric hIL23Fc mutein of Table 8.

[0056] In some aspects, in the method of treating a neoplastic disease with a pharmaceutical formulation of a heterodimeric hIL23Fc mutein, the dose of heterodimeric hIL12Fc mutein provided to the mammal is between 10ug / kg and 500ug / kg, alternatively between 10ug / kg and 250ug / kg, alternatively between 10ug / kg and 80ug / kg, alternatively between 10ug / kg and 40ug / kg, alternatively between 10ug / kg and 30ug / kg, alternatively between 10ug / kg and 20ug / kg, or alternatively about 15ug / kg.

[0057] The present disclosure further provides a method of treating a mammal suffering from a neoplastic disease, comprising contacting the mammal with a pharmaceutical formulation of the heterodimeric hIL23Fc mutein disclosed herein in combination with one or more adjunctive therapeutic agents. In some embodiments, the one or more adjunctive therapeutic agents are selected from the group consisting of checkpoint inhibitors, cytokines, or therapeutic antibodies. In some embodiments of the methods of the present disclosure, the one or more adjunctive therapeutic agents are checkpoint inhibitors selected from the group consisting of anti-PD1 antibodies, anti-PDL1 antibodies, anti-CTLA4 antibodies, and anti-LAG3 antibodies. In some embodiments of the methods of the present disclosure, the one or more adjunctive therapeutic agents are polypeptides having at least 90%, alternatively at least 95%, alternatively at least 97%, alternatively at least 98%, alternatively at least 99%, or alternatively 100% sequence identity to human interleukin-2.

[0058] In some embodiments of the methods of the disclosure, the one or more adjunctive therapeutic agents is a human IL2 mutein comprising one or more amino acid substitutions or deletions at positions 1, 2, 3, 4, 5, 6, 18, 22, 125, and 126, numbered according to mature wild-type human IL2. In some embodiments of the methods of the present disclosure, the one or more adjunctive therapeutic agents are biased IL2 muteins having reduced affinity for the CD132 subunit of the IL2 receptor, as described in Emmerich et al., PCT International Application No. PCT / US2021 / 013456, published July 22, 2021 as WO2021 / 146436A2; Emmerich et al., PCT / US2021 / 013514, published July 22, 2021 as WO2021 / 146481A1, and Garcia et al., PCT / US2018 / 062122, published May 31, 2019 as WO2019 / 104092A1, the entire disclosures of which are incorporated herein by reference.

[0059] In some embodiments of the methods of the present disclosure, the one or more adjunct therapeutic agents are biased human IL2 muteins containing amino acid substitutions at positions 18, 22, and 126 numbered according to mature wild-type human IL2. In some embodiments of the methods of the present disclosure, the one or more adjunct therapeutic agents are biased human IL2 muteins containing amino acid substitutions 18R, Q22E, and Q126K. In some embodiments of the methods of the present disclosure, the one or more adjunct therapeutic agents are biased IL2 muteins containing amino acid substitutions at positions 18, 22, and 126 numbered according to PEGylated mature wild-type human IL2.

[0060] In some aspects, the disclosure provides a method of treating a neoplastic disease using a heterodimeric hIL23Fc mutein alone or in combination with one or more adjuvant agents, wherein the neoplastic disease is characterized by a tumor with T cell infiltration. In some aspects, the disclosure provides a method of treating a neoplastic disease using a heterodimeric hIL23Fc mutein alone or in combination with one or more adjuvant agents, wherein the neoplastic disease is selected from the group consisting of melanoma, renal cell carcinoma (RCC), ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), head and neck cancer, pancreatic cancer, and high microsatellite instability (MSI) cancer.

[0061] In another aspect, the present disclosure provides a method for modulating hIL12-mediated signaling in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition comprising a heterodimeric hIL12Fc mutein as described herein. In some embodiments, the hIL12-mediated signaling comprises STAT4-mediated signaling. In some embodiments, the STAT4-mediated signaling is determined by an assay selected from the group consisting of a gene expression assay, a phospho-flow signaling assay, and an enzyme-linked immunosorbent assay (ELISA). In some embodiments, the STAT4-mediated signaling in the subject is reduced by about 20%, alternatively about 30%, alternatively about 40%, alternatively about 50%, alternatively about 60%, alternatively about 70%, alternatively about 80%, alternatively about 90%, alternatively about 100% compared to a reference level. In some embodiments, the administered composition reduces the ability to induce IFN-γ expression compared to wild-type hIL12.

[0062] The heterodimeric hIL12Fc mutein or heterodimeric hIL23Fc mutein of the present disclosure is useful in the treatment and / or prevention of disease in a mammalian subject.Accordingly, in another aspect, the present disclosure provides a method for treating a health condition in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a composition comprising a heterodimeric hIL12Fc mutein or heterodimeric hIL23Fc mutein; a nucleic acid molecule or vector comprising a nucleic acid sequence encoding a heterodimeric hIL12Fc mutein or heterodimeric hIL23Fc mutein; a recombinantly modified cell comprising a nucleic acid molecule or vector as described herein; or a pharmaceutical composition comprising one or more of the foregoing as described herein.

[0063] In another aspect, the present disclosure provides a method of treating a neoplastic disease, an infectious disease, or an autoimmune disease in a subject in need of such treatment, comprising administering to the subject a therapeutically effective amount of a modified heterodimeric hIL12Fc mutein or heterodimeric hIL23Fc mutein, a nucleic acid molecule or vector comprising a nucleic acid sequence encoding a heterodimeric hIL12Fc mutein or heterodimeric hIL23Fc mutein; a recombinantly modified cell comprising a nucleic acid molecule or vector as described herein, or a pharmaceutical composition as described herein. In some embodiments, the present disclosure provides for the treatment or prevention of an autoimmune disease in a mammalian subject by administering a therapeutically effective amount of a heterodimeric hIL23Fc mutein of the present disclosure. In some embodiments, the present disclosure provides for the treatment or prevention of a neoplastic disease in a mammalian subject by administering a therapeutically effective amount of a heterodimeric hIL12Fc mutein of the present disclosure.

[0064] In some aspects, the present disclosure provides for the treatment or prevention of a neoplastic disease in a mammalian subject by administration of a therapeutically effective amount of a heterodimeric hIL12Fc mutein of the present disclosure in combination with one or more adjunctive therapeutic agents.

[0065] Also provided are kits for modulating hIL-12-mediated signaling or hIL-23 signaling in a subject, or for treating a condition in a subject in need of such treatment. In some embodiments, the kit comprises a heterodimeric hIL12Fc mutein or a heterodimeric hIL23Fc mutein as described herein. In some embodiments, the kit comprises a nucleic acid molecule or vector comprising a nucleic acid sequence encoding a heterodimeric hIL12Fc mutein or a heterodimeric hIL23Fc mutein as described herein, a nucleic acid molecule or vector comprising a nucleic acid sequence encoding a heterodimeric hIL12Fc mutein and / or a heterodimeric hIL23Fc mutein as described herein, or a nucleic acid molecule. In some embodiments, the kit comprises a recombinantly modified cell comprising a nucleic acid molecule or vector as described herein, or a pharmaceutical composition as described herein. [Brief description of the drawings]

[0066] [Figure 1] Figure 1 shows the results of an assessment of interferon gamma induction (vertical axis) for increasing concentrations of test agents (horizontal axis) in CD8, CD4, and NK cells. Panels A, B, and C show the effect of wild-type hIL12 compared to hIL12 muteins containing amino acid substitutions E81A / F82A, indicated by "2xAla," hIL12 muteins containing E81A / F82A / K106A, indicated by "3xAla," and hIL12 muteins containing substitution W37A, on CD8, CD4, and NK cells, respectively. Panels D, E, and F show the results of IFNγ induction of KiH heterodimeric hIL12Fc mutein containing hIL12 compared to KiH heterodimeric hIL12Fc mutein containing amino acid substitutions E81A / F82A, designated "2xAla Fc," KiH heterodimeric hIL12Fc mutein containing E81A / F82A / K106A, designated "3xAla Fc," and KiH heterodimeric hIL12Fc mutein containing substitution W37A (W37A Fc) on CD8 cells, CD4 cells, and NK cells, respectively. [Diagram 2]FIG. 2 shows the results of assessment of interferon-γ and STAT4 induction (vertical axis) for increasing concentrations of the indicated test agents on CD8+ T cells (panel A), CD4+ T cells (panel B), and NK cells (panels C and D) from two different human donors. [Diagram 3] 3 shows a spider plot of tumor volume over time in mice treated with various murine IL12 agents and a murine IL12Fc mutein in an MC38 tumor model study described more fully below. Tumor volume is shown on the Y-axis and time is shown on the X-axis. [Figure 4] FIG. 4 shows the body weight of treated mice (y-axis) over time (x-axis) for mice treated with various murine IL12 agents and a murine IL12 Fc mutein in an MC38 tumor model study, described more fully below. [Diagram 5] FIG. 5 shows survival data for treated mice (probability of survival on the y-axis) over time (x-axis) for mice treated with various murine IL12 agents and a murine IL12 Fc mutein in an MC38 tumor model study, which is described more fully below. [Figure 6] FIG. 6 shows data on tumor volume (y-axis) versus days after initiation of treatment (x-axis) with mIL12 protein in panel A and with IL12 protein subunits in heterodimeric Fc format in panel B. [Figure 7] FIG. 7 shows an amino acid sequence alignment of wild-type mouse and human p40(IL12Ra) proteins with the signal peptide sequences highlighted. [Figure 8] FIG. 8 shows an amino acid sequence alignment of wild-type mouse and human p35(IL12Rb) proteins. [Figure 9]FIG. 9 shows a graphical representation of the concentration of mouse interferon-gamma in picograms per milliliter (pg / mL) (y-axis) measured in serum obtained from blood samples taken over time (x-axis) in the MC38 tumor model. The design is shown in Table 12. Panel A shows the concentration of mouse interferon-gamma in picograms per milliliter (pg / mL) in serum for treatment groups A-E of Table 12 at time 0 (pre-treatment), and 4 hours, 1 day, and 7 days after treatment with a test agent. Panel B relates to the levels of mouse interferon-gamma in serum for treatment groups F, G, and H of Table 12 at time 0 (pre-treatment), and 4 hours, 1 day, and 7 days after treatment with a test agent. [Figure 10] FIG. 10 is a graphical representation of the percent of lymphocytes (y-axis) in spleen and tumor tissues for each of the treatment groups (x-axis) in Table 12, as determined by FACS analysis. [Figure 11] Figure 11 shows the results of phenotypic FACS analysis of NK cells obtained from spleens in the treatment groups and studies described in Table 12. T-bet was measured on the vertical axis and intracellular granzyme B was measured on the horizontal axis. Treatment groups (A, F, G, and H in Table 12) are indicated on the graph. [Figure 12] Figure 12 shows a series of spider plots from the CT26 tumor study described herein. Tumor volume is shown on the vertical axis and time (study day) is shown on the horizontal axis. Each panel of the figure shows the test agent provided and the dosing schedule of the test agent according to the study design shown in Table 13. [Figure 13] 13 shows the results of body weight measurements of mice evaluated in the CT26 tumor study. Percent change in body weight is shown on the vertical axis and time (study days) is shown on the horizontal axis. [Figure 14] FIG. 14 shows the results of FACS analysis of cells obtained from the CT26 tumor research study, sorted by the presence of various markers indicated by arrows and various doses of test agents as indicated by the figure legend. [Figure 15] FIG. 15 shows the efficacy results of various test agents, indicated by the figure legend, in response to NK cell depletion in the studies summarized in Table 14. [Figure 16] Figure 16 shows the results of an assessment of the percent change in body weight (vertical axis) over the course of the study (horizontal axis) in response to various test agent conditions provided in an MC38 tumor study to evaluate the effect of NK and CD8 cell depletion as detailed herein. The study design is summarized in Table 15. [Figure 17] FIG. 17 shows a series of spider plots of anti-tumor efficacy (tumor volume on the vertical axis) over the course of the study (horizontal axis) in response to the various test conditions identified in Table 15. [Figure 18] Figure 18 shows the results of antitumor efficacy of IL12 test agents against MC38 tumors in different types of mice: B6 mice in the first two figures, RAG2 knockout mice in the second two figures, and RAG2 / CD132 double knockout mice in the third column. Tumor volume over the course of the study (horizontal axis) is shown on the vertical axis. The figure legend identifies the various test conditions as shown in Table 15. [Figure 19] Figure 19 shows the results of a study evaluating the antitumor efficacy of heterodimeric mIL12 Fc p40M polypeptides in combination with PD1 inhibitors when treating MC38 tumors in mice. Tumor volume over the course of the study (horizontal axis) is shown on the vertical axis. Figure legends correspond to the treatment groups summarized in Table 16 herein. [Figure 20] Figure 20 shows the results of a study evaluating the antitumor efficacy of heterodimeric mIL12 Fc p40M polypeptides in combination with mIL2 mutein polypeptides in treating MC38 tumors in mice. Tumor volume over the course of the study (horizontal axis) is shown on the vertical axis. Figure legends correspond to the treatment groups summarized in Table 17 herein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0067] Detailed Description of the Invention To further facilitate understanding of this disclosure, certain terms and phrases are defined below and throughout the specification. The definitions provided herein are non-limiting and should be interpreted in light of the knowledge of those of ordinary skill in the art.

[0068] Before the present methods and compositions are described, it is to be understood that this invention is not limited to the particular methods or compositions described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing aspects only and is not intended to be limiting.

[0069] Where a range of values ​​is stated, it is understood that each intervening value between the upper and lower limits of that range is specifically disclosed to the tenth of the unit of the lower limit, unless otherwise clearly indicated by the context. Each narrower range between any stated or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these narrower ranges may be independently included or excluded within the range, and each range that includes one limit, does not include either limit, or includes both limits in the narrower range is also encompassed within the invention, subject to any limits explicitly excluded in the stated range. When a stated range includes one or both limits, ranges excluding either or both of the included limits are also encompassed within the invention.

[0070] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this invention belongs.Although any method and material similar or equivalent to the method and material described herein can be used in the practice or testing of this invention, some possible and preferred methods and materials will now be described.All publications, patents, published patent applications, GenBank accession numbers, and UniProt reference numbers mentioned herein are incorporated by reference to disclose and describe the method and / or material described in the cited publication.

[0071] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to "a cell" includes a plurality of such cells, and a reference to "the peptide" includes a reference to one or more peptides and equivalents thereof known to those skilled in the art, such as, for example, polypeptides.

[0072] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publications by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed. The publications cited herein and the materials for which they are cited are specifically incorporated herein by reference in their entirety.

[0073] Unless otherwise specified, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius (°C), and pressure is at or near atmospheric. Standard abbreviations are used, including: bp = base pairs; kb = kilobases; pl = picoliters; s or sec = seconds; min = minutes; h or hr = hours; AA or aa = amino acid; kb = kilobases; nt = nucleotides; pg = picograms; ng = nanograms; μg = micrograms; mg = milligrams; g = grams; kg = kilograms; dl or dL = deciliters; μl or μL = microliters; ml or mL = milliliters; l or L = liters; μM = micromolar; mM = millimolar. Concentration; M = molar; kDa = kilodaltons; im = intramuscular (into the muscle); ip = intraperitoneal (into the peritoneal cavity); SC or SQ = subcutaneous (under the skin); QD = once daily; BID = twice daily; QW = once weekly; QM = once monthly; HPLC = high performance liquid chromatography; BW = body weight; U = units; ns = not statistically significant; PBS = phosphate buffered saline; PCR = polymerase chain reaction; HSA = human serum albumin; MSA = mouse serum albumin; DMEM = Dulbecco's modified Eagle's medium; EDTA = ethylenediaminetetraacetic acid.

[0074] It will be understood that throughout this disclosure, amino acids will be referred to according to their single letter or three letter code. For the convenience of the reader, the single letter and three letter codes for amino acids are provided in Table 1.

[0075] Table 1. Natural amino acids and their abbreviations TIFF2024539139000002.tif114166

[0076] Standard methods in molecular biology are described in the scientific literature (see, e.g., Sambrook and Russell (2001) Molecular Cloning, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; and Ausubel, et al. (2001) Current Protocols in Molecular Biology, Vols. 1-4, John Wiley and Sons, Inc. New York, NY, which describe cloning and DNA mutagenesis in bacterial cells (Vol. 1), cloning in mammalian cells and yeast (Vol. 2), glycoconjugates and protein expression (Vol. 3), and bioinformatics (Vol. 4)). The scientific literature describes protein purification methods including immunoprecipitation, chromatography, electrophoresis, centrifugation, and crystallization, as well as chemical analysis, chemical modification, post-translational modification, production of fusion proteins, and protein glycosylation (see, e.g., Coligan, et al. (2000) Current Protocols in Protein Science, Vols. 1-2, John Wiley and Sons, Inc., NY).

[0077] Nomenclature of Amino Acid Substitutions and Deletions The present disclosure provides variant polypeptides that contain amino acid substitutions compared to wild-type or parent polypeptides. The following nomenclature is used herein to refer to substitutions, deletions, or insertions. Residues may be designated herein by the single-letter or three-letter amino acid code of the natural amino acid found in the wild-type molecule.

[0078] P19 residue numbering: In this disclosure, the numbering of amino acid residues in human P19 is based on the numbers of the "pro" form of hP19 shown in SEQ ID NO:177.

[0079] P35 residue numbering: In this disclosure, the numbering of amino acid residues in human P35 is based on the numbers in the "pro" form of hP35 shown in SEQ ID NO:1.

[0080] P40 residue numbering: In this disclosure, the numbering of amino acid residues in human P40 is based on the number of the "pro" form of hP40 as shown in (SEQ ID NO:3). Based on hP40 muteins, substitutions are designated herein by the single letter amino acid code followed by the prohp40 (SEQ ID NO:3) amino acid position followed by the single letter amino acid code of the substituted amino acid. For example, an hp40 mutein having the modification "E81A" refers to the substitution of an alanine (A) residue for the glutamic acid (E) residue at position 81 of (SEQ ID NO:3) at this position. Deletions of amino acid residues are indicated by "des" or the symbol "Δ" followed by the amino acid residue and its position.

[0081] Immunoglobulin, upper hinge, and Fc residue numbering: There are various numbering conventions used for numbering amino acid residues of immunoglobulins, including Kabat numbering, Chothia numbering, EU numbering, and IMGT numbering conventions. In the context of the present disclosure, the numbering of amino acid residues of immunoglobulin molecules, including domains of immunoglobulin molecules, including upper hinge and Fc domain (including lower hinge, CH2, and CH3 domain), is done according to the EU numbering convention. The conversion of EU numbering used herein to Kabat numbering, Chothia numbering, or IMGT numbering conventions is easily understood by those skilled in the art. Dondelinger, et al. (2018) Understanding the Significance and Implications of Antibody Numbering and Antigen-Binding Surface / Residue Definition Frontiers in Immunology Volume 9 Article #:2278.

[0082] Additionally, in certain instances herein, the suffix "M" may be added to the polypeptide number to identify such sequences as "mature" molecules lacking a signal sequence (e.g., DR1535M) in order to distinguish the polypeptide from precursor molecules that contain a signal peptide, which precursor forms may be identified by the suffix "P", such as DR1535P.

[0083] definition Unless otherwise specified, the following terms are intended to have the meanings indicated below. Other terms are defined elsewhere throughout the specification.

[0084] The term "about" refers to a value that is plus or minus 10% of a numerical value described herein, for example, plus or minus 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of a numerical value described herein. The term "about" also applies to all numerical ranges described herein. It is understood that all values ​​described herein are modified by the term "about", whether or not the term "about" is explicitly recited with respect to a particular value.

[0085] Activate: As used herein, the term "activate" is used to reflect the biological effect of a receptor or receptor complex, directly and / or by participating in a multi-component signal transduction cascade resulting from the binding of an agonist ligand to the receptor in response to ligand binding. The term activate is also used for cells expressing receptors in which another biological activity of the cell is modulated in response to the binding of a ligand to such receptor (e.g., up-regulation or down-regulation of STAT4 signal transduction).

[0086] Active: As used herein, the term "activity" is used to describe the properties of a molecule in relation to a test system (e.g., an assay), or the biological or chemical properties (e.g., the degree of binding of the molecule to another molecule) or physical properties (e.g., alteration of cell membrane potential) of a material or cell. Examples of such biological functions include, but are not limited to, the catalytic activity of a biological agent, its ability to stimulate intracellular signaling, gene expression, cell proliferation, and its ability to modulate immunological activities, such as inflammatory responses. "Activity" is typically expressed as the level of biological activity per unit of a test agent, such as [catalytic activity] / [mg protein], [immunological activity] / [mg protein], international units of activity (IU), [STAT3 phosphorylation] / [mg protein], [STAT4 phosphorylation] / [mg protein], [proliferation] / [mg protein], plaque forming units (pfu), etc. The term proliferative activity, as used herein, refers to activity that promotes cell growth and replication, including dysregulated cell division, e.g., that observed in neoplastic diseases, inflammatory diseases, fibrosis, metaplasia, cell transformation, metastasis, and angiogenesis.

[0087] Administer / Administer: The terms "administration" and "administering" are used interchangeably herein to refer to the act of contacting a subject's cells, tissues, organs, or biological fluids in vitro, in vivo, or ex vivo, including contacting the subject's cells, tissues, organs, or biological fluids with an agent (e.g., a modified hIL-12p40 polypeptide, a hIL-12 mutein comprising a heterodimeric hIL12Fc mutein, or a hIL-23 mutein comprising a modified hIL-12p40 polypeptide; engineered cells expressing a modified hIL-12p40 polypeptide, engineered cells expressing a hIL-12 mutein comprising a modified hIL-12p40 polypeptide, or engineered cells expressing a hIL-23 mutein comprising a modified hIL-12p40 polypeptide; or a chemotherapeutic agent, an antibody, or a pharmaceutical formulation comprising one or more of the foregoing), alone or in combination with one or more adjuvants. Administration of the agent can be accomplished by any of a variety of methods recognized in the art, including, but not limited to, local administration, intravascular injection (including intravenous or intraarterial infusion), intradermal injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, intracranial injection, intratumoral injection, transdermal delivery, transmucosal delivery, iontophoretic delivery, intralymphatic injection, intragastric injection, intraprostatic injection, intravesical injection (e.g., bladder), inhalation (e.g., respiratory inhaler, including dry powder inhaler), intraocular injection, intraperitoneal injection, intralesional injection, intraovarian injection, intracerebral infusion or injection, intraventricular injection (ICVI), etc. The term "administration" includes contact of the agent with a cell, tissue, or organ, as well as contact of the agent with a fluid in contact with a cell, tissue, or organ.

[0088] Affinity: The term "affinity" as used herein refers to the degree of specific binding between a first molecule (e.g., a ligand) and a second molecule (e.g., a receptor) and is measured by the equilibrium dissociation constant (KD), which is the ratio of the dissociation rate constant (Koff) of the molecule to its target and the association rate constant (Kon) of the molecule to its target.

[0089] Agonists: The term "agonist" as used herein refers to a first agent that specifically binds to a second agent ("target") and interacts with the target to cause or promote increased activation of the target. In some cases, agonists are receptor protein activators that modulate cell activation, enhance activation, increase cell sensitivity to activation by a second agent, or upregulate the expression of one or more genes, proteins, ligands, receptors, biological pathways that may result in cell proliferation or pathway or cell cycle modification. In some embodiments, agonists are modified forms of cognate ligands that bind to their cognate receptors and change the state of the cognate receptor in a biological response that mimics the biological effect of the interaction between the natural cognate ligand and its cognate receptor. The term "agonist" includes partial agonists, full agonists, and superagonists. Agonists may be referred to as "full agonists" or partial agonists when such agonists lead to substantially the complete biological response induced by the receptor under study (i.e., the response associated with the natural ligand / receptor binding interaction). A "superagonist" is a type of agonist that can generate a maximal response that exceeds the endogenous agonist for the target receptor, and thus has more than 100% activity of the native ligand. A superagonist is typically a synthetic molecule that exhibits more than 110%, or more than 120%, or more than 130%, or more than 140%, or more than 150%, or more than 160%, or more than 170% of the response of an assessable quantitative or qualitative parameter of the native molecule when evaluated at similar concentrations in a comparable assay. It should be noted that the biological effects associated with a full agonist may differ in extent and / or type from the biological effects of a partial agonist or a superagonist. In contrast to agonists, antagonists may specifically bind to a receptor but do not trigger a signal cascade, typically one that is initiated by the receptor, and may alter the action of an agonist at that receptor.An inverse agonist is an agent that produces a pharmacological response that is opposite in direction to that of an agonist.

[0090] Antagonists: The term "antagonist" or "inhibitor" as used herein refers to a molecule that opposes the action of an agonist.Antagonists block, reduce, inhibit, or neutralize the activity of agonists, and antagonists can also block, inhibit, or reduce the constitutive activity of targets, such as target receptors, even in the absence of a specified agonist.An inhibitor is a molecule that reduces, blocks, prevents, delays, or inactivates, desensitizes, or downregulates the activation of, for example, genes, proteins, ligands, receptors, biological pathways, including immune checkpoint pathways, or cells.In some cases, antagonists can be muteins of natural ligands, such that binding to the receptor is maintained but downstream signaling is absent.

[0091] Biological samples: As used herein, the term "biological sample" or "sample" refers to a sample obtained (or derived) from a subject. By way of example, a biological sample includes material selected from the group consisting of bodily fluids, blood, whole blood, plasma, serum, mucous secretions, saliva, cerebrospinal fluid (CSF), bronchoalveolar lavage fluid (BALF), ocular fluids (e.g., vitreous humor, aqueous humor), lymphatic fluid, lymph node tissue, spleen tissue, bone marrow, tumor tissue, including immunoglobulin-enriched or cell type-specific enriched fractions derived from one or more of such tissues.

[0092] Equivalent: The term "comparable" as used herein is used to describe the degree of difference between two measurements of an evaluable quantitative or qualitative parameter. For example, two measurements would be considered "comparable" if a first measurement of an evaluable quantitative parameter and a second measurement of an evaluable parameter do not deviate beyond a range that a person skilled in the art would recognize would not result in a statistically significant difference in effect between the two results in this situation. In some cases, measurements may be considered "comparable" if one measurement deviates from another measurement by less than 35%, alternatively less than 30%, alternatively less than 25%, alternatively less than 20%, alternatively less than 15%, alternatively less than 10%, alternatively less than 7%, alternatively less than 5%, alternatively less than 4%, alternatively less than 3%, alternatively less than 2%, or alternatively less than 1%. In certain embodiments, a measurement is comparable to a standard if it deviates from the standard by less than 15%, alternatively less than 10%, or alternatively less than 5%.

[0093] Conservative Amino Acid Substitutions: As used herein, the term "conservative amino acid substitution" refers to an amino acid exchange in which a particular amino acid is changed to another amino acid having similar biochemical properties (e.g., charge, hydrophobicity, and size). For example, amino acids in each of the following groups are considered to be conservative amino acids of each other: (1) hydrophobic amino acids: alanine, isoleucine, leucine, tryptophan, phenylalanine, valine, proline, and glycine; (2) polar amino acids: glutamine, asparagine, histidine, serine, threonine, tyrosine, methionine, and cysteine; (3) basic amino acids: lysine and arginine; and (4) acidic amino acids: aspartic acid and glutamic acid.

[0094] Corresponding to: The term "corresponding" or "corresponding to" as used herein in the context of amino acid or nucleic acid sequences refers to the equivalent position of a reference sequence aligned with one or more other sequences to maximize the percent sequence identity. For example, the "amino acid position corresponding to amino acid position [X]" of a specified hIL-12p40 polypeptide refers to the equivalent position in other hIL-12p40 polypeptides, including structural homologs and variants, based on the alignment. The corresponding position may be based on a reference, wild-type, or parent sequence, e.g., the wild-type mature hp40 amino acid sequence of SEQ ID NO:4.

[0095] Derived from: As used herein, the term "derived from" in the context of an amino acid sequence or nucleic acid is used to indicate that a polypeptide or nucleic acid has a sequence based on a reference polypeptide or nucleic acid sequence, and is not intended to be limiting with respect to the source or method of making the protein or nucleic acid. By way of example, the term "derived from" includes homologs or variants of the reference amino acid or DNA sequence.

[0096] Effective concentration (EC): As used herein, the term "effective concentration" or its abbreviation "EC" are used interchangeably to refer to an agent concentration sufficient to alter a particular parameter in a test system. The abbreviation "E" refers to the magnitude of a particular biological effect observed in a test system when the test system is exposed to a test agent. The abbreviation "EC" is used when the magnitude of the response is expressed as a factor of the concentration ("C") of the test agent. In the context of a biological system, the term Emax refers to the maximum magnitude of a particular biological effect observed in response to a saturating concentration of an activating test agent. When the abbreviation EC is presented with a subscript (e.g., EC 40 , E.C. 50etc.), where the subscript refers to the percent of the Emax of the biological response observed at that concentration. For example, a concentration of a test agent sufficient to induce a measurable biological parameter in a test system that is 30% of the maximum level of such measurable biological parameter in response to such test agent is referred to as the "EC 30 Similarly, it is called "EC 100 The term EC is used to denote the effective concentration of an agent that produces a maximum (100%) response of a measurable parameter in response to such an agent. Similarly, the term EC 50 The term "saturation concentration" refers to the concentration of an agent sufficient to produce a half-maximal (approximately 50%) change in a measurable parameter. The term "saturation concentration" refers to the maximum amount of a test agent that can be dissolved in a standard volume of a particular solvent (e.g., water) under standard conditions of temperature and pressure. In pharmacokinetics, the saturation concentration of a drug is typically used to indicate a sufficient concentration of a drug such that all available receptors are occupied by the drug, and is referred to as the EC 50 is the drug concentration that produces a half-maximal effect.

[0097] Concentrated: The term "enriched" as used herein refers to a sample that contains a species of interest (e.g., a molecule or cell) that is (a) present at a concentration that is higher (e.g., at least 3-fold, alternatively at least 5-fold, alternatively at least 10-fold, alternatively at least 50-fold, alternatively at least 100-fold, or alternatively at least 1000-fold) than the concentration of the species in a starting sample, e.g., a biological sample (e.g., a sample in which the molecule naturally occurs or is present following administration), or (b) that has been non-naturally engineered to be present at a higher concentration than the environment in which the molecule was made (e.g., recombinantly modified bacterial or mammalian cells).

[0098] Extracellular domain: As used herein, the term "extracellular domain" or its abbreviation "ECD" refers to the portion of a cell surface protein that is outside the plasma membrane of the cell on whose surface it is expressed. Cell surface proteins and ECDs may be transmembrane proteins, cell surface proteins, or membrane-bound proteins that contain a domain that is attached to the cell membrane but lacks an intracellular domain.

[0099] Identity: The term "identity" as used herein with respect to a polypeptide sequence or a DNA sequence refers to the subunit sequence identity between two molecules. If a subunit position of both molecules is occupied by the same amino acid or nucleotide, the molecules are identical at that position. The similarity between two amino acid sequences or between two nucleotide sequences is a linear function of the number of identical positions. Generally, the sequences are aligned to obtain the highest order match. If necessary, identity can be calculated using published techniques and widely available computer programs such as the BLAST 2.0 algorithm described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and Altschul, et al. (1977) Nucleic Acids Res. 25: 3389-3402. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (NCBI) website. The algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that match or match a positive threshold score "T" when aligned with words of the same length in a database sequence. T is called the neighborhood word score threshold (Altschul et.al., supra). These initial neighborhood word hits act as seeds to initiate searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. The cumulative score is calculated using the parameters "M" (reward score for a pair of matching residues; always >0) and "N" (penalty score for mismatching residues; always <0) for nucleotide sequences. For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score.Extension of word hits in each direction stops when: (a) the cumulative alignment score falls by an amount X from its maximum achieved value; when the cumulative score becomes zero or less due to the accumulation of one or more negative-scoring residue alignments; or (b) the end of either sequence is reached. The BLAST algorithm parameters "W", "T", and "X" determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) works similarly, but uses as defaults a word size ("W") of 28, an expectation ("E") of 10, M=1, N=-2, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, (1989) PNAS(USA) 89:10915-10919).

[0100] In sufficient quantity to bring about a response: As used herein, the phrase "in an amount sufficient to elicit a response" refers to an amount of agent sufficient to produce a detectable change in the level of an indicator measured before and after application of the test agent to the test system (e.g., a baseline level). In some embodiments, the test system is a cell, tissue, or organism. In some embodiments, the test system is an in vitro test system, such as a fluorescent assay. In some embodiments, the test system is an in vivo system that involves measuring changes in the level of a parameter of the cell, tissue, or organism that reflects the biological function before and after application of the test agent to the cell, tissue, or organism. In some embodiments, the indicator reflects the biological function or developmental state of the cell evaluated in the assay in response to administration of an amount of the test agent. In some embodiments, the test system involves measuring changes in the level of an indicator of the cell, tissue, or organism that reflects the biological state before and after application of one or more test agents to the cell, tissue, or organism. The term "in an amount sufficient to elicit a response" may be a therapeutically effective amount, but may be more or less than a therapeutically effective amount.

[0101] Needs action: The term "in need of treatment" as used herein refers to the judgment made by a physician or other caregiver that the subject needs or will potentially benefit from treatment. This judgment is based on various factors within the expertise of the physician or caregiver. In some embodiments, the subject in need of treatment has been diagnosed with a disease or condition, such as cancer, an autoimmune disorder, or an infectious disease.

[0102] Precautions required: The term "in need of prevention" as used herein refers to the judgment of a doctor or other caregiver that the subject needs or potentially benefits from preventive care. This judgment is based on various factors within the expertise of the doctor or caregiver. In some embodiments, prevention refers to reducing, forestalling, or delaying the onset of a particular disease, or reducing, forestalling, or delaying the recurrence of a particular disease, for example, after the treatment of the disease. Recurrence does not necessarily have to be after the cure or remission of the disease. It is sufficient that one or more clinical symptoms reappear after a symptom-free period, for example, after the treatment period for cancer, autoimmune disease, or infectious disease.

[0103] Inhibitors: The term "inhibitor" as used herein refers to a molecule that, for example, reduces, blocks, prevents, delays the activation of a gene, protein, ligand, receptor, or cell, or inactivates, desensitizes, or downregulates a gene, protein, ligand, receptor, or cell. An inhibitor can also be defined as a molecule that reduces, blocks, or inactivates a constitutive activity of a cell or organism.

[0104] Intracellular domain: As used herein, the term "intracellular domain" or its abbreviation "ICD" refers to the portion of a cell surface protein (e.g., a cell surface receptor) that is inside the plasma membrane of a cell. A cell surface protein that comprises an ICD can be a transmembrane protein, a cell surface protein that comprises a domain that binds to the cell membrane but lacks an extracellular domain, or a membrane-bound protein. An ICD can comprise the entire cytoplasmic portion of a transmembrane or membrane-bound protein, or it can comprise an intracellular protein. A cell surface protein can be a transmembrane protein, a cell surface protein that comprises a domain that binds to the cell membrane but lacks an intracellular domain, or a membrane-bound protein.

[0105] Isolated: The term "isolated" as used herein is used in reference to a polypeptide of interest that is in an environment different from the environment in which it naturally occurs, if it is naturally occurring. "Isolated" is intended to include a polypeptide in a sample that is substantially enriched for the polypeptide of interest and / or the polypeptide of interest is partially or substantially purified. If the polypeptide is not naturally occurring, "isolated" refers to the polypeptide being separated from the environment in which it was synthesized, for example, being isolated from a recombinant cell culture that includes cells that are engineered to express the polypeptide, or being isolated by a solution resulting from solid phase synthesis means.

[0106] Ligand: The term "ligand" as used herein refers to a molecule that specifically binds to a receptor and induces a change in the receptor that alters the activity of the receptor or the response of a cell expressing the receptor. In one embodiment, the term "ligand" refers to a molecule or complex thereof that can act as an agonist or antagonist of the receptor. A complex of a ligand and a receptor is called a "ligand-receptor complex" (e.g., and hIL-12-hIL-12 receptor complex). In some instances, the terms "cognate ligand" and "cognate receptor" are used to refer to a natural ligand and a receptor to which such ligand shows selective binding in a natural biological system. For example, hIL-12 is a cognate ligand for the hIL-12 receptor. In another example, hIL-23 is a cognate ligand for the hIL-23 receptor.

[0107] Modified: The term "modified" as used herein refers to a molecule, e.g., a polypeptide, that has an altered structure compared to an unmodified parent molecule. A modified polypeptide typically retains one or more activities or functions of the unmodified parent molecule. For example, a modified IL-12 p40 polypeptide, as part of a heterodimer (i.e., a p35 / p40 complex), can activate hIL-12 signaling in cells expressing the hIL-12 receptor, but can have improved properties compared to an unmodified polypeptide. The term modified includes amino acid substitutions that are not present in the parent or wild-type hIL-12, and includes variants and mutants of the hIL-12 p40 polypeptide.

[0108] Adjust: As used herein, the terms "modulate," "modulation," and the like refer to an agent, e.g., a test agent, being capable of causing a positive or negative response, or directly or indirectly causing a response, in a system, including a biological system, or a biochemical pathway. The term modulator includes both agonists (including partial agonists, full agonists, and superagonists) and antagonists.

[0109] Mutein: As used herein, the term "mutein" is used to refer to modified versions of wild-type polypeptides that contain modifications to the primary structure (i.e., amino acid sequence) of such polypeptides. The term mutein may refer to the polypeptide itself, a composition comprising the polypeptide, or a nucleic acid sequence encoding the polypeptide. A mutein may be at least about 99% identical to the parent polypeptide, alternatively at least about 98% identical, alternatively at least about 97% identical, alternatively at least about 96% identical, alternatively at least about 95% identical, alternatively at least about 94% identical, alternatively at least about 93% identical, alternatively at least about 92% identical, alternatively at least about 91% identical, or alternatively at least about 90% identical. In some cases, as used herein, a composition comprises a hP40 mutein, where hP40 comprises an amino acid sequence at least about 99% identical, alternatively at least about 98% identical, alternatively at least about 97% identical, alternatively at least about 96% identical, alternatively at least about 95% identical, alternatively at least about 94% identical, alternatively at least about 93% identical, alternatively at least about 92% identical, alternatively at least about 91% identical, alternatively at least about 90% identical to wthP40 (SEQ ID NO:4). As used herein, the term "mutein" refers to a variant of native hIL-12 (i.e., p35 / p40 complex) or hIL-23 (i.e., p19 / p40 complex), i.e., a heterodimer that retains one or more biological activities of the parent native heterodimer hIL-12 or hIL-23 from which it was derived, including hP40 muteins.

[0110] Nucleic acid: The terms "nucleic acid," "nucleic acid molecule," "polynucleotide," and the like are used interchangeably herein to refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Non-limiting examples of polynucleotides include linear and circular nucleic acids, messenger RNA (mRNA), complementary DNA (cDNA), recombinant polynucleotides, vectors, probes, primers, and the like.

[0111] Functionally linked: The term "operably linked" is used herein to refer to the relationship between molecules, typically polypeptides or nucleic acids, arranged in a construct such that the function of each of the component molecules is retained, but the operably linked may positively or negatively modulate the activity of the individual components of the construct. For example, operably linking a polyethylene glycol (PEG) molecule to a wild-type protein may result in a construct in which the biological activity (e.g., Emax) of that protein is reduced relative to the wild-type molecule. However, the two are nevertheless considered to be operably linked. When the term "operably linked" is applied to the relationship of multiple nucleic acid sequences encoding different functions, the multiple nucleic acid sequences, when combined into a single nucleic acid molecule, provide a nucleic acid capable of transcribing and / or translating a particular nucleic acid sequence in a cell, for example, when the nucleic acid molecule is introduced into a cell using recombinant techniques. For example, a nucleic acid sequence encoding a signal sequence that facilitates secretion of a polypeptide may be considered operably linked to the DNA encoding the polypeptide if it expresses a preprotein. A promoter or enhancer is considered operably linked to a coding sequence if it affects the transcription of the sequence. Alternatively, a sequence is considered operably linked to a coding sequence if the ribosome binding site is positioned to facilitate translation. Generally, in the context of nucleic acid molecules, the term "operably linked" means that the nucleic acid sequences being linked are contiguous, and in the case of a secretory leader or linked subdomain of the molecule, contiguous and in reading phase. However, certain genetic elements, such as enhancers, may function at a distance from the sequence they effect and need not be contiguous with that sequence, but may nevertheless be considered operably linked.

[0112] Parent Polypeptide: As used herein, the term "parent polypeptide" or "parent protein" is used interchangeably to designate the source of a second polypeptide (e.g., a derivative, mutant or variant) that is modified relative to a first "parent" polypeptide. In some cases, the parent polypeptide is a wild-type or naturally occurring protein. In some cases, the parent polypeptide may be a modified version of a naturally occurring protein that has been further modified. The term "parent polypeptide" may refer to the polypeptide itself or a composition that includes the parent polypeptide (e.g., a glycosylated or PEGylated version and / or a fusion protein that includes the parent polypeptide). The term parent polypeptide is also used interchangeably with "reference polypeptide."

[0113] Partial agonists: The term "partial agonist" as used herein refers to a molecule (e.g., a ligand) that specifically binds to and activates a particular receptor, but has only partial activation of the receptor compared to a full agonist. A partial agonist may exhibit both agonistic and antagonistic effects. For example, when both a full agonist and a partial agonist are present, the partial agonist acts as a competitive antagonist by competing with the full agonist for receptor binding, thereby resulting in a net reduction in receptor activation compared to contact of the receptor with the full agonist in the absence of the partial agonist. When an insufficient amount of endogenous ligand is present, a partial agonist can be used to activate the receptor to produce a desired submaximal response in the subject. Alternatively, when an excess amount of endogenous ligand is present, a partial agonist can reduce overstimulation of the receptor. The maximal response (E) produced by a partial agonist can be expressed as a function of the amount of the receptor that is activated by the partial agonist. max) is referred to as its intrinsic activity, and is sometimes expressed on a percentage scale where a full agonist would produce a 100% response. A partial agonist may have more than 10% but less than 100%, alternatively more than 20% but less than 100%, alternatively more than 30% but less than 100%, alternatively more than 40% but less than 100%, alternatively more than 50% but less than 100%, alternatively more than 60% but less than 100%, alternatively more than 70% but less than 100%, alternatively more than 80% but less than 100%, or alternatively more than 90% but less than 100% of the activity of the reference polypeptide when evaluated at similar concentrations in a particular assay system.

[0114] Polypeptides: As used herein, the terms "polypeptide", "peptide" and "protein" are used interchangeably herein and refer to polymeric forms of amino acids of any length, which can include amino acids specified by the genetic code and amino acids not specified by the genetic code, amino acids that have been chemically or biochemically modified or derivatized, and polypeptides with modified polypeptide backbones. The term polypeptide includes fusion proteins, including, but not limited to, fusion proteins with heterologous amino acid sequences; fusion proteins with heterologous and homologous leader sequences; fusion proteins with or without an N-terminal methionine residue; fusion proteins with amino acid sequences that facilitate purification, such as chelating peptides; fusion proteins with immunologically tagged proteins; fusion proteins containing peptides with immunologically active polypeptide fragments (e.g., antigenic diphtheria or tetanus toxins or toxoid fragments), and the like.

[0115] Prevention: As used herein, the terms "prevent", "preventing", "prevention" and the like refer to a course of action that is initiated on a subject prior to the onset of a disease, disorder, condition, or symptoms thereof, to temporarily or permanently prevent, suppress, inhibit, or reduce the subject's risk of developing a disease, disorder, condition, or the like (e.g., as determined by the absence of clinical symptoms), or to delay the onset of the disease, disorder, condition, or the like. A course of action to prevent a disease, disorder, or condition in a subject is typically applied in the context of a subject who is predisposed to developing a disease, disorder, or condition due to genetic, experiential, or environmental factors that lead to the development of a particular disease, disorder, or condition. In certain cases, the terms "prevent", "preventing", "prevention" are also used to refer to delaying the progression of a disease, disorder, or condition from an existing state to a more deleterious state.

[0116] Receptor: The term "receptor" as used herein refers to a polypeptide having a domain that specifically binds to a ligand, where binding of the ligand changes at least one biological property of the polypeptide. In some embodiments, the receptor is a cell membrane-bound protein that includes an extracellular domain (ECD) and a membrane-bound domain that serves to anchor the ECD to the cell surface. In some embodiments of cell surface receptors, the receptor is a transmembrane polypeptide in which the intracellular domain (ICD) and the extracellular domain (ECD) are linked by a cell membrane-spanning domain called the transmembrane domain (TM). Binding of the ligand to the receptor results in a conformational change in the receptor, which results in a measurable biological effect. In some cases, when the receptor is a transmembrane polypeptide that includes an ECD, a TM, and an ICD, binding of the ligand to the ECD results in a measurable intracellular biological effect mediated by one or more domains of the ICD in response to binding of the ligand to the ECD. In some embodiments, the receptor is a component of a multi-component complex to facilitate intracellular signal transduction. For example, a ligand may bind to a cell surface receptor that, alone, is not involved in any intracellular signaling, but upon ligand binding, promotes the formation of a heteromultimeric (including heterodimers, heterotrimers, etc.) or homomultimeric (including homodimers, homotrimers, homotetramers, etc.) complex, which results in a measurable biological effect in the cell, such as activating an intracellular signaling cascade (e.g., the Jak / STAT pathway). In some embodiments, the receptor is a transmembrane single chain polypeptide comprising an ECD, TM and ICD domains, wherein the ECD, TM and ICD domains are derived from the same or different naturally occurring receptor variants or synthetic functional equivalents thereof.

[0117] Recombination: As used herein, the term "recombinant" is used as an adjective to refer to the way in which a polypeptide, nucleic acid, or cell has been modified using recombinant DNA technology. A "recombinant protein" is a protein produced using recombinant DNA technology and is abbreviated with a lower case "r" in front of the protein name to indicate the way in which the protein was produced (e.g., recombinantly produced human growth hormone is usually abbreviated as "rhGH"). Similarly, a cell is called a "recombinant cell" if it has been modified by the incorporation (e.g., transfection, transduction, infection) of an exogenous nucleic acid (e.g., ssDNA, dsDNA, ssRNA, dsRNA, mRNA, viral or non-viral vectors, plasmids, cosmids, etc.) using recombinant DNA technology. Techniques and protocols for recombinant DNA technology, such as those found in Sambrook, et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, NY) and other standard molecular biology laboratory manuals, are well known in the art.

[0118] response: For example, the term "response" of a cell, tissue, organ, or organism encompasses quantitative or qualitative changes in assessable biochemical or physiological parameters (e.g., concentration, density, adhesion, proliferation, activation, phosphorylation, migration, enzyme activity, gene expression level, gene expression rate, energy consumption rate, level or state of differentiation) that correlate with activation, stimulation, or treatment by, or contact with, an internal mechanism, such as an exogenous agent or genetic programming. In certain circumstances, the terms "activation," "stimulation," and the like refer to cell activation as regulated by internal mechanisms as well as by external or environmental factors, whereas the terms "inhibition," "downregulation," and the like refer to the opposite effect. "Response" may be assessed in vitro, for example, by using assay systems, surface plasmon resonance, enzyme activity, mass spectroscopy, amino acid or protein sequencing techniques. "Response" may be quantitatively assessed in vivo by evaluating objective physiological parameters, such as body temperature, body weight, tumor burden, blood pressure, the results of X-ray or other imaging techniques, or qualitatively assessed by changes in reported subjective feelings of happiness, depression, excitement, or pain. In some embodiments, the level of T cell activation in response to administration of a test agent may be determined by flow cytometry. In some methods, the response may be measured by determining the level of STAT (e.g., STAT3, STAT4) phosphorylation, or IFNγ production.

[0119] Significantly reduced binding: As used herein, the term "exhibits significantly reduced binding" is used in reference to a variant of a first molecule (e.g., a ligand) that exhibits a significantly reduced affinity for a second molecule (e.g., a receptor) compared to the parent form of the first molecule. With reference to a variant ligand, e.g., a variant hIL-12p40 polypeptide or hIL-12 mutein described herein, a variant ligand "exhibits significantly reduced binding" if the mutein binds to a receptor with less than 20%, alternatively less than about 10%, alternatively less than about 8%, alternatively less than about 6%, alternatively less than about 4%, alternatively less than about 2%, alternatively less than about 1%, or alternatively less than about 0.5% of the affinity of the parent ligand from which the variant ligand is derived.

[0120] Specific binding to: The term "specifically bind" as used herein refers to the degree of affinity that a first molecule exhibits for a second molecule. In the context of a binding pair (e.g., ligand / receptor), a first molecule of a binding pair is said to specifically bind to a second molecule of the binding pair when the first molecule of the binding pair does not bind in significant amounts to other components present in the sample. A first molecule of a binding pair is said to specifically bind to a second molecule of the binding pair when the affinity of the first molecule for the second molecule is at least 2 times, alternatively at least 5 times, alternatively at least 10 times, alternatively at least 20 times, or alternatively at least 100 times, greater than the affinity of the first molecule for other components present in the sample. Specific binding can be assessed using techniques known in the art, including, but not limited to, competitive ELISA assays, radioactive ligand binding assays (e.g., saturation binding, Scatchard plots, non-linear curve fitting programs, and competitive binding assays); non-radioactive ligand binding assays (e.g., fluorescence polarization (FP), fluorescence resonance energy transfer (FRET); solution-phase ligand binding assays (e.g., real-time polymerase chain reaction (RT-qPCR), and immunoprecipitation); and solid-phase ligand binding assays (e.g., multi-well plate assays, on-bead ligand binding assays, on-column ligand binding assays, and filter assays)), and surface plasmon resonance assays (see, e.g., Drescher et al., (2009) Methods Mol Biol 493:323-343 and commercially available instrumentation, e.g., Biacore 8K, Biacore 8K+, Biacore S200, Biacore T200 (Cytiva, 100 Results Way, Marlborough MA 01752)).

[0121] subject: The terms "recipient," "individual," "subject," and "patient" are used interchangeably herein and refer to any mammalian subject for which diagnosis, treatment, or therapy is desired, particularly humans. For purposes of treatment, "mammal" refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, etc. In some aspects, the mammal is a human.

[0122] Practically pure: As used herein, the term "substantially pure" indicates that a component of a composition comprises more than about 50%, alternatively more than about 60%, alternatively more than about 70%, alternatively more than about 80%, alternatively more than about 90%, alternatively more than about 95% of the total content of the composition. A "substantially pure" protein comprises more than about 50%, alternatively more than about 60%, alternatively more than about 70%, alternatively more than about 80%, alternatively more than about 90%, alternatively more than about 95% of the total content of the composition.

[0123] Suffering from: As used herein, the term "suffering from" refers to a determination made by a physician on a subject based on available objective or subjective information accepted in the field for identifying a disease, disorder, or condition that the subject requires or would benefit from treatment, including, but not limited to, x-rays, CT scans, conventional diagnostic laboratory tests (e.g., blood counts, etc.), genomic data, protein expression data, immunohistochemistry. The term "suffering from" is typically used in conjunction with a specific disease state, for example, "suffering from a neoplastic disease" refers to a subject who has been diagnosed with the presence of a neoplasm.

[0124] T cells: As used herein, the term "T cell" ("T-cell" or "T cell") is used in its conventional sense to refer to lymphocytes that differentiate in the thymus, have specific cell surface antigen receptors, and control the initiation or suppression of cellular and humoral immunity, including those that lyse antigen-bearing cells. In some embodiments, T cells include naive CD8 + T cells, cytotoxic CD8 + T cells, naive CD4 + T cells, helper T cells, e.g., T H 1. T H 2. T H 9. T H 11. T H 22, T FH ; regulatory T cells, e.g. T R 1, Tregs, inducible Tregs; memory T cells, including but not limited to central memory T cells, effector memory T cells, NKT cells, tumor infiltrating lymphocytes (TILs), and engineered variants of such T-cells, including but not limited to CAR-T cells, recombinantly modified TILs, and TCR engineered cells. In some embodiments, the T cells are T cells that express the IL12 receptor, referred to interchangeably as IL12R cells, IL12R+ cells, IL12R T cells, or IL12R+ T cells.

[0125] Terminus / Terminal: As used herein in the context of a polypeptide structure, the terms "N-terminus" (or "amino terminus") and "C-terminus" (or "carboxyl terminus") refer to the extreme amino and carboxyl ends of a polypeptide, respectively. In contrast, the terms "N-terminus" and "C-terminus" refer to the relative position in a polypeptide amino acid sequence relative to the N-terminus and C-terminus, respectively, and may include residues at the N-terminus and C-terminus, respectively. "Immediately N-terminal" refers to the position of a first amino acid residue relative to a second amino acid residue in a contiguous polypeptide sequence, the first amino acid being proximal to the N-terminus of the polypeptide. "Immediately C-terminal" refers to the position of a first amino acid residue relative to a second amino acid residue in a contiguous polypeptide sequence, the first amino acid being proximal to the C-terminus of the polypeptide. As used herein in the context of nucleic acids, the "5' end" (or "five-prime terminus") and the "3' end" (or "carboxyl end") refer to the ends of a nucleic acid sequence, respectively, while the terms "5'" and "3'" refer to the relative position of a polypeptide in a nucleic acid sequence toward the 5' end and toward the 3' end, respectively, and may include residues at the 5' end and the 3' end, respectively.

[0126] Therapeutically Effective Amount: The phrase "therapeutically effective amount" as used herein refers to an amount of an agent that, when administered to a subject alone in a single dose, or as part of a pharmaceutical composition or treatment regimen, or as part of a series of doses, produces a positive effect on any quantitative or qualitative symptoms, aspects, or characteristics of a disease, disorder, or condition.The therapeutically effective amount can be confirmed by measuring the relevant physiological effect, and may be adjusted in conjunction with a dosing regimen and in response to diagnostic analysis of the subject's condition.The parameters for evaluation to determine the therapeutically effective amount of an agent are determined by a physician using diagnostic criteria accepted in the art, including, but not limited to, characteristics such as age, weight, sex, overall physical health, ECOG score, observable physiological parameters, blood levels, blood pressure, electrocardiogram, computed tomography, X-ray, etc. Alternatively, or in addition, to determine whether a therapeutically effective amount of an agent has been administered to a subject, other parameters commonly assessed in a clinical setting may be monitored, such as body temperature, heart rate, normalization of blood chemistry, normalization of blood pressure, normalization of cholesterol levels, or any symptom, aspect, or characteristic of a disease, disorder, or condition, such as biomarkers (e.g., inflammatory cytokines, IFN-γ, granzymes, etc.), reduction in serum tumor markers, improvement in Response Evaluation Criteria in Solid Tumors (RECIST), improvement in Immune Related Response Criteria (irRC), increased survival, increased progression-free survival, increased time to progression, increased time to treatment success, increased recurrence-free survival, increased time to next treatment, improved objective response rate, improved duration of response, reduction in tumor burden, complete remission, partial remission, stable disease, etc., as determined by a clinician in the field to assess improvement in a subject's condition in response to administration of the agent. In one aspect, a therapeutically effective amount is an amount of an agent that, when used alone or in combination with another agent, produces a positive effect on any quantitative or qualitative symptom, aspect, or characteristic of a disease, disorder, or condition, and does not produce irreversible serious adverse events in the course of administration of the agent to a mammalian subject.

[0127] Action: The terms "treat," "treating," "treatment," and the like refer to a course of action (e.g., contacting the subject with a pharmaceutical composition comprising a hIL-12 mutein alone or in combination with adjuvants) undertaken against a subject in response to a diagnosis that the subject is suffering from a disease, disorder, or condition, or a symptom thereof, where the course of action is undertaken to temporarily or permanently eliminate, reduce, inhibit, alleviate, or ameliorate at least one of (a) the underlying cause of such disease, disorder, or condition afflicting the subject; and / or (b) at least one of the symptoms associated with such disease, disorder, or condition. In some embodiments, treating includes a course of action taken against a subject suffering from a disease, where the course of action results in the inhibition of the disease in the subject (e.g., the development of the disease, disorder, or condition is inhibited) or the amelioration of one or more symptoms associated with the presence of the disease in the subject.

[0128] variant: The terms "variant," "protein variant," or "variant protein," or "variant polypeptide" are used interchangeably herein to refer to a polypeptide that differs from a parent polypeptide by at least one amino acid modification, substitution, or deletion. The parent polypeptide may be a native or wild-type (WT) polypeptide or may be a modified version of a WT polypeptide. The term variant polypeptide may refer to the polypeptide itself, a composition comprising the polypeptide, or a nucleic acid sequence encoding it. In some embodiments, a variant polypeptide contains about 1 to about 10, alternatively about 1 to about 8, alternatively about 1 to about 7, alternatively about 1 to about 5, alternatively about 1 to about 4, alternatively about 1 to about 3, alternatively 1 to 2 amino acid modifications, substitutions, or deletions, or alternatively a single amino acid modification, substitution, or deletion, relative to the parent polypeptide from which the variant is derived. A variant may be at least about 99% identical, alternatively at least about 98% identical, alternatively at least about 97% identical, alternatively at least about 95% identical, or alternatively at least about 90% identical to the parent polypeptide from which the variant is derived.

[0129] Wild type: As used herein, "wild-type" or "WT" or "native" refers to an amino acid sequence or nucleotide sequence found in nature, including allelic variations. A wild-type protein, polypeptide, antibody, immunoglobulin, IgG, etc., has an amino acid sequence or nucleotide sequence that has not been modified by the hand of man.

[0130] Each embodiment is described herein separately for clarity and brevity, and it is understood that they can be combined in a non-limiting manner.Thus, the present disclosure includes one or more combinations or all combinations of the embodiments described herein, as if each combination were individually and expressly disclosed.This also applies to any and all subcombinations of the embodiments disclosed herein, as if each subcombination were individually and expressly disclosed, as if the present disclosure includes one or more subcombinations or all subcombinations of the embodiments described herein.

[0131] Wild type hIL12: Wild-type human IL12 (wt hIL12) is a covalently disulfide-linked heterodimeric protein that contains two wild-type subunits, hP40 and hP35. The native form of hIL12 contains an interchain disulfide linkage between residue C96 of p35 (numbered according to SEQ ID NO:1) and residue C199 of p40 (numbered according to SEQ ID NO:3).

[0132] Wild-type human P35: Wild-type human P35 monomer (wt hP35) is expressed as a 219 amino acid proprotein (SEQ ID NO:1) containing a 22 amino acid signal sequence that is post-translationally removed to yield a 197 amino acid mature protein (SEQ ID NO:2). Wild-type hP35 (wt hP35) contains two intrachain disulfide linkages, the first between residues C64 and C196 and the second between residues C85 and C123 (numbered according to SEQ ID NO:1). The classical amino acid sequence of the human proP35 protein (UniProt Reference No. P29459) with the signal sequence (underlined) is: The mature form of wild-type human P35 (wt hP35), which is missing only the 22 amino acid signal sequence, has the amino acid sequence: It is expressed as a 197 amino acid mature protein with TIFF2024539139000004.tif24133.

[0133] Wild-type human P40: Wild-type human P40 (wt hP40) is expressed as a 328 amino acid proprotein (SEQ ID NO:3) that includes a 22 amino acid signal sequence that is post-translationally removed to yield a 306 amino acid mature protein (SEQ ID NO:4). Wild-type hP40 (wt hP40) contains four intrachain disulfides between residues C50 and C90, between C131 and C142, between C170 and C193, and between C300 and C327 (numbered according to SEQ ID NO:3). The classical amino acid sequence of the hP40 proprotein (UniProt Reference No. P29460) with the signal sequence (underlined) is: TIFF2024539139000005.tif44134. The mature form of wild-type human P40 (wt hP40), which is missing only the 22 amino acid signal sequence, is expressed as a 306 amino acid mature protein (SEQ ID NO:4). TIFF2024539139000006.tif34134

[0134] IL12 Receptor: The IL12 receptor contains the IL12Rβ1 and IL12Rβ2 subunits. IL12 receptor activation results from the binding of the IL12 cytokine ligand to both IL12Rβ1 and IL12Rβ2. Binding of the IL12 cytokine ligand to the IL12 receptor complex activates the Janus tyrosine kinases Tyk2, which binds to IL12Rβ1, and Jak2, which binds to IL12Rβ2, phosphorylating the cytoplasmic tail of the receptor. This recruits signal transducer and activator of transcription 4 (STAT4). STAT4 homodimerization releases STAT4 from the receptor, and the phosphorylated STAT4 homodimer translocates to the nucleus, where it binds to the STAT4 binding element of the IFN-γ gene to produce IFN-γ.

[0135] Heterodimeric hIL12Fc mutein: In some aspects, the disclosure provides heterodimeric hIL12Fc muteins, including p40 muteins, with improved pharmacological or therapeutic properties, and methods of using such compositions.

[0136] The present disclosure provides compounds of formula #1: hP40M-L1 a -UH1-Fc1[1] and a first polypeptide of formula #2: hP35-L2 b -UH2-Fc2 [2] A heterodimeric hIL12Fc mutein comprising a second polypeptide of During the ceremony, hP35 is a polypeptide having at least 90%, alternatively at least 91%, alternatively at least 92%, alternatively at least 93%, alternatively at least 94%, alternatively at least 95%, alternatively at least 96%, alternatively at least 97%, alternatively at least 98%, alternatively at least 99%, or alternatively 100% sequence identity to SEQ ID NO:2; hP40M is a human p40 mutein optionally otherwise identical to SEQ ID NO:4, comprising one or more amino acid substitutions at positions selected from the group consisting of positions W37, P39, D40, A41, K80, E81, F82, K106, E108, D115, H216, K217, L218, and K219 numbered according to wild-type prehuman P40 (SEQ ID NO:3), or having at least 90%, alternatively at least 91%, alternatively at least 92%, alternatively at least 93%, alternatively at least 94%, alternatively at least 95%, alternatively at least 96%, alternatively at least 97%, alternatively at least 98%, or alternatively at least 99% sequence identity to SEQ ID NO:4; L1 and L2 are GSA linkers, and a and b are independently selected from 0 (absent) or 1 (present); UH1 and UH2 are each an upper hinge domain of a human immunoglobulin independently selected from the group consisting of IgG1, IgG2, IgG3, and IgG4 upper hinges, optionally containing the amino acid substitution C220S (EU numbering); Fc1 is a polypeptide comprising the lower hinge, CH2, and CH3 domains of a human immunoglobulin selected from the group consisting of IgG1, IgG2, IgG3, and IgG4, comprising one or more amino acid substitutions that promote heterodimerization with Fc2; Fc2 is a polypeptide comprising the lower hinge, CH2, and CH3 domains of a human immunoglobulin selected from the group consisting of IgG1, IgG2, IgG3, and IgG4, comprising one or more amino acid substitutions that promote heterodimerization with Fc1; wherein the polypeptide of formula 1 and the polypeptide of formula 2 are linked by at least one interchain disulfide bond; Heterodimeric hIL12Fc muteins are provided.

[0137] In some embodiments, the polypeptide of Formula 1 is selected from the group consisting of SEQ ID NOs:80, 83, 85, 86, 88, 90, 92, 121, 129, 132, 135, 138, 141, 144, 147, 150, and 153, or any P40M-Fc sequence in the informal sequence listing.

[0138] In some embodiments, the polypeptide of Formula 2 comprises any one of SEQ ID NOs:81, 82, 84, 87, 89, 91, 93, and 124, or any hp35-Fc sequence in the informal sequence listing.

[0139] In some embodiments, L1 and L2 are independently selected from the group consisting of SEQ ID NOs: 27-79. In some embodiments, L1 and L2 are independently selected from the group consisting of SEQ ID NOs: 36, 37, and 65.

[0140] In some embodiments, UH1 and UH2 are selected from the group consisting of SEQ ID NO:11 and SEQ ID NO:12.

[0141] In some aspects, the disclosure provides a heterodimeric hIL12Fc mutein comprising a first polypeptide selected from the group consisting of SEQ ID NOs:80, 83, 85, 86, 88, 90, 92, 121, 129, 132, 135, 138, 141, 144, 147, 150, and 153, and a second polypeptide selected from the group consisting of SEQ ID NOs:81, 82, 84, 87, 89, 91, 93, and 124.

[0142] In some aspects, the disclosure provides a heterodimeric hIL12Fc mutein comprising a polypeptide of SEQ ID NO:80 and a polypeptide of SEQ ID NO:81.

[0143] In some aspects, the disclosure provides a heterodimeric hIL12Fc mutein comprising a polypeptide of SEQ ID NO:121 and a polypeptide of SEQ ID NO:124.

[0144] In some aspects, the disclosure provides a heterodimeric hIL12Fc mutein comprising a polypeptide of SEQ ID NO:83 and a polypeptide of SEQ ID NO:82.

[0145] In some aspects, the disclosure provides a heterodimeric hIL12Fc mutein comprising a polypeptide of SEQ ID NO:141 and a polypeptide of SEQ ID NO:124.

[0146] In some aspects, the disclosure provides a heterodimeric hIL12Fc mutein comprising a polypeptide of SEQ ID NO:144 and a polypeptide of SEQ ID NO:124.

[0147] In some aspects, the disclosure provides a heterodimeric hIL12Fc mutein comprising a polypeptide of SEQ ID NO:129 and a polypeptide of SEQ ID NO:124.

[0148] In some aspects, the disclosure provides a heterodimeric hIL12Fc mutein comprising a polypeptide of SEQ ID NO:147 and a polypeptide of SEQ ID NO:82.

[0149] In some aspects, the disclosure provides a heterodimeric hIL12Fc mutein comprising a polypeptide of SEQ ID NO:150 and a polypeptide of SEQ ID NO:82.

[0150] In some aspects, the disclosure provides a heterodimeric hIL12Fc mutein comprising a polypeptide of SEQ ID NO:153 and a polypeptide of SEQ ID NO:82.

[0151] In some aspects, the disclosure provides a heterodimeric hIL12Fc mutein comprising a polypeptide of SEQ ID NO:135 and a polypeptide of SEQ ID NO:124.

[0152] In some aspects, the disclosure provides a heterodimeric hIL12Fc mutein comprising a polypeptide of SEQ ID NO:138 and a polypeptide of SEQ ID NO:124.

[0153] In some aspects, the disclosure provides a recombinant mammalian host cell comprising a first nucleic acid sequence encoding a polypeptide of SEQ ID NO:80 and a second nucleic acid sequence encoding a polypeptide of SEQ ID NO:81.

[0154] In some aspects, the disclosure provides a recombinant mammalian host cell comprising a first nucleic acid sequence encoding a polypeptide of SEQ ID NO:121 and a second nucleic acid sequence encoding a polypeptide of SEQ ID NO:124.

[0155] In some aspects, the disclosure provides a recombinant mammalian host cell comprising a first nucleic acid sequence encoding a polypeptide of SEQ ID NO:83 and a second nucleic acid sequence encoding a polypeptide of SEQ ID NO:82.

[0156] In some aspects, the disclosure provides a recombinant mammalian host cell comprising a first nucleic acid sequence encoding a polypeptide of SEQ ID NO:141 and a polypeptide of SEQ ID NO:124.

[0157] In some aspects, the disclosure provides a recombinant mammalian host cell comprising a first nucleic acid sequence encoding a polypeptide of SEQ ID NO:144 and a second nucleic acid sequence encoding a polypeptide of SEQ ID NO:124.

[0158] In some aspects, the disclosure provides a recombinant mammalian host cell comprising a first nucleic acid sequence encoding a polypeptide of SEQ ID NO: and a second nucleic acid sequence encoding a polypeptide of SEQ ID NO:124.

[0159] In some aspects, the disclosure provides a recombinant mammalian host cell comprising a first nucleic acid sequence encoding a polypeptide of SEQ ID NO:147 and a second nucleic acid sequence encoding a polypeptide of SEQ ID NO:82.

[0160] In some aspects, the disclosure provides a recombinant mammalian host cell comprising a first nucleic acid sequence encoding a polypeptide of SEQ ID NO:150 and a second nucleic acid sequence encoding a polypeptide of SEQ ID NO:82.

[0161] In some aspects, the disclosure provides a recombinant mammalian host cell comprising a first nucleic acid sequence encoding a polypeptide of SEQ ID NO:153 and a second nucleic acid sequence encoding a polypeptide of SEQ ID NO:82.

[0162] In some aspects, the disclosure provides a recombinant mammalian host cell comprising a first nucleic acid sequence encoding a polypeptide of SEQ ID NO:135 and a second nucleic acid sequence encoding a polypeptide of SEQ ID NO:124.

[0163] In some aspects, the disclosure provides a recombinant mammalian host cell comprising a first nucleic acid sequence encoding a polypeptide of SEQ ID NO:138 and a second nucleic acid sequence encoding a polypeptide of SEQ ID NO:124.

[0164] Human P40 mutein The heterodimeric hIL12Fc muteins of the present disclosure comprise a modified human P40 polypeptide or "hP40 mutein" (also abbreviated as "hp40M" or "hP40M") that comprises one or more amino acid substitutions, modifications and / or deletions at the interface with the extracellular domain of IL12Rβ1 that result in a reduced binding affinity of hp40M to IL12Rβ1 compared to the mature form of wt hP40 (SEQ ID NO:4). In some embodiments, the binding affinity of the hP40 mutein to the extracellular domain of hIL12Rβ1 is reduced by about 10%, alternatively about 20%, alternatively about 30%, alternatively about 40%, alternatively about 50%, alternatively about 60%, alternatively about 60%, alternatively about 70%, alternatively about 80%, alternatively about 900%, alternatively by up to about 100%, as compared to the binding affinity of the reference polypeptide (wt hP40) as determined by surface plasmon resonance (SPR) spectroscopy. In some embodiments, hP40M is a modified wild-type human hp40 polypeptide having at least 70% sequence identity to SEQ ID NO:4 (e.g., at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:4). In some embodiments, hP40M comprises one or more amino acid substitutions at residues selected from the group consisting of W37, P39, D40, A41, K80, E81, F82, K106, E108, D115, H216, K217, L218, and K219, numbered according to SEQ ID NO:3. In some embodiments, the hP40M comprises one or more amino acid substitutions at residues selected from the group consisting of E81, F82, K106, and K217, numbered according to SEQ ID NO: 3. In some embodiments, the one or more amino acid substitutions at positions W37, P39, D40, A41, K80, E81, F82, K106, E108, D115, H216, K217, L218, and K219 are selected from the group consisting of P39A, D40A, E81A, F82A, K106A, D109A, K217A, K219A.In some embodiments, the hP40 mutein comprises two or more amino acid substitutions at residues selected from the group consisting of W37, P39, D40, A41, K80, E81, F82, K106, E108, D115, H216, K217, L218, and K219, numbered according to SEQ ID NO:3. In some embodiments, the hP40M comprises two or more amino acid substitutions at residues selected from the group consisting of E81, F82, K106, and K217, numbered according to SEQ ID NO:3. In some embodiments, the hP40 mutein comprises three or more amino acid substitutions at residues selected from the group consisting of W37, P39, D40, A41, K80, E81, F82, K106, E108, D115, H216, K217, L218, and K219, numbered according to SEQ ID NO:3. In some embodiments, the hP40M comprises two or more amino acid substitutions at W37, P39, D40, A41, K80, E81, F82, K106, E108, D115, H216, K217, L218, and K219, wherein the two or more substitutions comprise a set of amino acid substitutions selected from the group consisting of the following sets of amino acid substitutions: E81A / F82A, E81K / F82A, E81L / F82A, E81H / F82A, and E81S / F82A. In some embodiments, the hP40 mutein comprises three or more amino acid substitutions at residues selected from the group consisting of E81, F82, K106, and K217 numbered according to SEQ ID NO:3. In some embodiments, the hP40M comprises three or more amino acid substitutions at residues selected from the group consisting of W37, P39, D40, A41, K80, E81, F82, K106, E108, D115, H216, K217, L218, and K219, numbered according to SEQ ID NO:3.In some embodiments, the hP40 mutein comprises three or more amino acid substitutions at W37, P39, D40, A41, K80, E81, F82, K106, E108, D115, H216, K217, L218, and K219, wherein the three or more substitutions comprise a set of amino acid substitutions selected from the group consisting of the following sets of amino acid substitutions: W37A / E81A / F82A; E81A / F82A / K106A; E81A / F82A / K106A / K219A, E81A / F82A / K106N, E81A / F82A / K106Q, E81A / F82A / K106T, and E81A / F82A / K106R. In some embodiments, the hP40 mutein comprises four or more amino acid substitutions at residues selected from the group consisting of W37, P39, D40, A41, K80, E81, F82, K106, E108, D115, H216, K217, L218, and K219, numbered according to SEQ ID NO:3. In some embodiments, the hP40 mutein comprises four or more amino acid substitutions at W37, P39, D40, A41, K80, E81, F82, K106, E108, D115, H216, K217, L218, and K219, wherein the four or more substitutions comprise a set of amino acid substitutions selected from the group consisting of the following sets of amino acid substitutions: E81A / F82A / K106A / K217A, 81A / F82A / K106A / E108A / D115A, and P39A / D40A / E81A / F82A.

[0165] In some embodiments, hP40M comprises the set of amino acid substitutions E81A / F82A, referred to herein as "2xAla" (SEQ ID NO:6). In some embodiments, hP40M comprises the set of amino acid substitutions E81A / F82A / K106A, referred to herein as "3xAla" (SEQ ID NO:8). In some embodiments, hP40M comprises the set of amino acid substitutions E81A / F82A / K106A / K217A, referred to herein as "4xAla" (SEQ ID NO:10).

[0166] In some embodiments, the binding affinity of a heterodimeric hIL12Fc mutein of the disclosure comprising one or more, optionally two or more, optionally three or more, or optionally four or more amino acid substitutions at residues selected from the group consisting of W37, P39, D40, A41, K80, E81, F82, K106, E108, D115, H216, K217, L218, and K219 (numbered according to SEQ ID NO:3) to the extracellular domain (ECD) of IL12Rβ1 is reduced by at least 5%, optionally at least 10%, optionally at least 20%, optionally at least 30%, optionally at least 40%, optionally at least 50%, optionally at least 60%, or optionally at least 70% compared to the binding affinity of wild-type hP40 (SEQ ID NO:4) to the extracellular domain (ECD) of IL12Rβ1 as determined by surface plasmon resonance.

[0167] Characterization of heterodimeric hIL12Fc muteins In some aspects, the heterodimeric hIL12Fc muteins described herein provide cell type biased signaling of downstream signaling mediated by the IL12 receptor compared to a reference polypeptide (e.g., wild type hIL12). In some aspects, the reduced binding affinity of the hP40 mutein of the heterodimeric hIL12Fc mutein for IL12Rβ1 results in reduced signaling mediated by STAT4 compared to the reference polypeptide (wt hIL12). In some aspects, the heterodimeric hIL12Fc muteins of the present disclosure are partial agonists. In some aspects, the heterodimeric hIL12Fc muteins described herein are partial agonists of signaling mediated by STAT3 ("STAT3 signaling") and / or signaling mediated by STAT4 ("STAT4 signaling"). In some aspects, the heterodimeric hIL12Fc muteins have reduced signaling mediated by STAT3 compared to a reference polypeptide (wt hIL12). In some embodiments, STAT3 signaling and / or STAT4 signaling is determined by an assay selected from the group consisting of a gene expression assay, a phospho-flow signaling assay, and an enzyme-linked immunosorbent assay (ELISA).

[0168] The heterodimeric hIL12Fc muteins comprising the hP40 muteins described herein provide beneficial properties, such as anti-inflammatory properties, and / or selective activation of certain cell types with reduced undesirable properties, such as proinflammatory side effects, compared to wt hIL-12. In some embodiments, the heterodimeric hIL12Fc muteins comprising the hP40 muteins described herein provide cell type-biased signaling of downstream signaling mediated by the IL-12 receptor compared to a reference polypeptide (e.g., wild-type hIL12). For example, the heterodimeric hIL12Fc muteins of the present disclosure retain the properties of wild-type hIL12 to stimulate or activate IL-12 signaling in CD8+ T cells, but show reduced signaling mediated by IFNγ and / or STAT4 in natural killer (NK) cells. In some aspects, the cell type biased signaling of the heterodimeric hIL12Fc muteins comprising hP40 muteins described herein of the present disclosure includes the ability to provide substantial IL12 signaling (e.g., at least 30%, alternatively at least 40%, alternatively at least 50%, alternatively at least 60%, alternatively at least 70%, alternatively at least 80%, alternatively at least 90%) of wt hIL12 activity in CD8+ T cells. In some aspects, the heterodimeric hIL12Fc muteins described herein increase STAT4 signaling in CD8+ T cells by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400% or more and decrease STAT4 signaling in NK cells, e.g., by at least about 10%, 20%, 30%, 40%, 50%, 60%, or 70%, compared to a reference polypeptide (wt hIL12).In some embodiments, the heterodimeric hIL12Fc muteins described herein activate interferon gamma (IFNγ) in CD8+ T cells by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400% and reduce IFNγ signaling in NKT cells by at least 10%, 20%, 30%, 40%, 50%, 60%, or 70% compared to a reference polypeptide (wt hIL12). Thus, the heterodimeric hIL12Fc muteins including the hP40 muteins described herein exhibit reduced activation of NK cells while retaining the ability to stimulate CD8+ T cells.

[0169] GSA Linker: In the polypeptides of formulas [1] and [2], the Fc domain fusion incorporating p40 mutein and / or p35 may optionally contain a GSA linker molecule between the p40 mutein and the upper hinge. As used herein, the term "GSA linker" refers to a polypeptide having 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids composed of amino acids selected from the group consisting of glycine, serine, and alanine. In some embodiments, the polypeptide linker has the structure (GGGGS m ) n , (GGGS m ) n , (GGGA m ) n , and (GGGGGA m ) nand combinations thereof, where m, n, and o are each independently selected from 1, 2, 3, or 4. In constructing such polymers, it may be desirable to avoid repeating "GSG" sequences, which could potentially introduce non-native glycosylation sites. Exemplary glycine-serine linkers include, but are not limited to, the monomers: GGGS (referred to as "G4S"), GGGGA (referred to as "G4A"), GGGS (referred to as "G3S"), and GGGA (referred to as "G3A"), or homopolymers (e.g., "GGGGSGGGGS", also referred to as (G4S)2) or heteropolymers thereof. Exemplary GSA linkers are shown in Table 2 below.

[0170] Table 2: Exemplary GSA Linkers TIFF2024539139000007.tif85150TIFF2024539139000008.tif202150

[0171] Top Hinge: The heterodimeric hIL12Fc mutein of the present disclosure is a heterodimer comprising the polypeptides of formulas [1] and [2], each of which incorporates an upper hinge region of a human immunoglobulin. The term "upper hinge" or "UH" refers to the amino acid sequence corresponding to amino acid residues 216-220 (EU numbering) of a human immunoglobulin molecule. In some embodiments, the upper hinge region is a native upper hinge region of a human immunoglobulin selected from the LH region of a human IgG1, human IgG2, human IgG3, and human IgG4 upper hinge domain. In some embodiments, the upper hinge region is an upper hinge region of a human IgG1 immunoglobulin. In some embodiments, the upper hinge region is an upper hinge region of a human IgG1 immunoglobulin comprising the pentameric amino acid sequence: EPKSC (SEQ ID NO:11).

[0172] In some embodiments, the upper hinge region contains an unpaired cysteine ​​residue at position 220 (EU numbering), which typically binds to a cysteine ​​on the light chain in a complete immunoglobulin molecule. If only the Fc domain, including the hinge domain, is used, the unpaired cysteine ​​in the hinge domain creates the potential for improper disulfide bond formation. As a result, in some embodiments, the cysteine ​​at position 220 (C220, numbered according to EU numbering) is substituted with an amino acid that does not promote disulfide bonding. In some embodiments, the Fc domain comprises a C220S mutation having the amino acid sequence EPKSS (SEQ ID NO:12).

[0173] Fc1 and Fc2: The heterodimeric hIL12Fc muteins of the present disclosure are heterodimers comprising polypeptides of formulas [1] and [2], each incorporating an Fc region (Fc1 and Fc2) of a human immunoglobulin molecule modified to promote heterodimerization.

[0174] As used herein, the terms "Fc" and "Fc monomer" are used interchangeably herein to designate a monomeric polypeptide subunit of an Fc dimer. Fc comprises an amino acid sequence comprising (from amino terminus to carboxy terminus) the lower hinge domain and the CH2 and CH3 domains of a human immunoglobulin molecule. In some embodiments, an Fc monomer is a polypeptide comprising the lower hinge domain and the CH2 and CH3 domains of a human immunoglobulin molecule domain of human IgG1, human IgG2, human IgG3, and human IgG4 hinge domain. The CH2 domain of hIgG1 corresponds to amino acid residues 231-340 (EU numbering) and is set forth in SEQ ID NO:14. The CH3 domain of hIgG1 corresponds to amino acid residues 341-447 (EU numbering) and is set forth in SEQ ID NO:15.

[0175] The polypeptides of formula [1] and [2] each incorporate a lower hinge region of a human immunoglobulin. As used herein, the term "lower hinge" or "LH" refers to an amino acid sequence corresponding to amino acid residues 221-229 (EU numbering) of a human immunoglobulin molecule. In some embodiments, the lower hinge region is a native lower hinge region of a human immunoglobulin selected from the LH region of an IgG1, IgG2, IgG3, and IgG4 lower hinge domain. In some embodiments, the lower hinge region is a lower hinge region of a human IgG1 immunoglobulin. In some embodiments, the lower hinge region is a lower hinge region of a human IgG1 immunoglobulin comprising the decameric amino acid sequence: DKTHTCPPCP (SEQ ID NO:13).

[0176] In some embodiments, Fc1 and Fc2 have the amino acid sequence (EU numbering shown, SEQ ID NO:16): It is derived from a polypeptide corresponding to amino acids 221-447 (EU numbering) of human IgG1 immunoglobulin, having TIFF2024539139000009.tif61128.

[0177] As shown in the sequence above, the wild-type C-terminal residue of the IgG1 Fc domain is a lysine, designated K447 according to EU numbering. K447 is inconsistently removed by the producing cells in the recombinant product. As a result, the population of recombinant Fc monomers may be heterogeneous in that some fraction of recombinantly produced Fc monomers contain K447 and others do not. Thus, such inconsistent proteolytic processing by the producing cells may result in a heterogeneous hIL12Fc population. Typically, such heterogeneity of active pharmaceutical ingredients must be avoided, especially in the context of human pharmaceutical agents. Consequently, in addition to modifications to the Fc monomer sequence that promote heterodimerization, the disclosure provides nucleic acid sequences encoding Fc monomers that further comprise a deletion of the C-terminal K447 or a deletion of G446 and K447, as well as Fc monomers that comprise (a) a deletion of the lysine residue at position 447 (K447, EU numbering, abbreviated as ΔK447 or des-K447), or (b) a deletion of the glycine at position 456 (G446 EU numbering, abbreviated as des-G446) and a deletion of K447 (this double deletion of G446 and K447 is referred to herein as des-G446 / des-K447 or ΔG446 / ΔK447).

[0178] Modification of hp40 K282 to avoid proteolytic cleavage In some aspects, a heterodimeric IL12Fc mutein of the present disclosure comprises an amino acid substitution of a lysine (K) residue (K260) at position 260 (SEQ ID NO:4, corresponding to position 282 of the human p40 precursor polypeptide SEQ ID NO:3) of the mature form of the human p40 polypeptide. As described in Webster et al. (U.S. Patent No. 7,872,107, issued Jan. 18, 2011), the substitution at position 260 of the mature human p40 polypeptide renders the human p40 polypeptide resistant to proteolytic cleavage. In some aspects, a human p40 polypeptide of a heterodimeric IL12Fc mutein of the present disclosure comprises a substitution of a lysine at position K282 (numbered according to SEQ ID NO:3) of the polypeptide with a non-basic amino acid. In some aspects, the non-basic amino acid is selected from the group consisting of alanine, glycine, asparagine, or glutamine. In some aspects, the p40 polypeptide of a heterodimeric IL12Fc mutein of the present disclosure comprises a mutation at position K282 (numbered according to SEQ ID NO:3) selected from the group consisting of K282G, K282A, K282N, K282Q (numbered according to SEQ ID NO:3).

[0179] In some embodiments, a heterodimeric IL12Fc mutein of the disclosure comprises a human p40 polypeptide comprising a set of amino acid substitutions selected from the group consisting of E81A / F82A / K106A / K282G, E81A / F82A / K106A / K282A, E81A / F82A / K106A / K282N, and E81A / F82A / K106A / K282Q (numbered according to SEQ ID NO:3). In some embodiments, a heterodimeric IL12Fc mutein of the disclosure comprises a human p40 polypeptide comprising a set of amino acid substitutions selected from the group consisting of E81A / F82A / K282G, E81A / F82A / K282A, E81A / F82A / K282N, and E81A / F82A / K282Q (numbered according to SEQ ID NO:3).

[0180] In one aspect, the present disclosure provides a method for the preparation of a peptide having the amino acid sequence: The present invention provides a heterodimeric IL12Fc mutein comprising a human p40 mutein having TIFF2024539139000010.tif32132.

[0181] In one aspect, the present disclosure provides a method for the preparation of a peptide having the amino acid sequence: The present invention provides a heterodimeric IL12Fc mutein comprising a human p40 mutein having TIFF2024539139000011.tif32132.

[0182] In one aspect, the present disclosure provides a method for the preparation of a peptide having the amino acid sequence: The present invention provides a heterodimeric IL12Fc mutein comprising a human p40 mutein having TIFF2024539139000012.tif32132.

[0183] In one aspect, the present disclosure provides a method for the preparation of a peptide having the amino acid sequence: The present invention provides a heterodimeric IL12Fc mutein comprising a human p40 mutein having TIFF2024539139000013.tif32132.

[0184] In one aspect, the present disclosure provides a method for the preparation of a peptide having the amino acid sequence: The present invention provides a heterodimeric IL12Fc mutein comprising a human p40 mutein having TIFF2024539139000014.tif32132.

[0185] In one aspect, the present disclosure provides a method for the preparation of a peptide having the amino acid sequence: The present invention provides a heterodimeric IL12Fc mutein comprising a human p40 mutein having TIFF2024539139000015.tif32132.

[0186] In one aspect, the present disclosure provides a method for the preparation of a peptide having the amino acid sequence: The present invention provides a heterodimeric IL12Fc mutein comprising a human p40 mutein having TIFF2024539139000016.tif32132.

[0187] In one aspect, the present disclosure provides a method for the preparation of a peptide having the amino acid sequence: The present invention provides a heterodimeric IL12Fc mutein comprising a human p40 mutein having TIFF2024539139000017.tif32132.

[0188] Engineering the Fc subunit to promote heterodimerization As shown in the above formulas [1] and [2], the Fc1 and Fc2 monomers of the dimeric Fc contain amino acid substitutions that promote heterodimerization between Fc1 and Fc2. Various techniques have been established to promote heterodimerization of Fc domains. See, for example, Kim et al., U.S. Patent No. 11,087,249, issued August 3, 2021. In some embodiments, the modification that promotes heterodimerization of Fc1 and Fc2 monomers is HF-TA mutation and HA-TF mutation as described in Moore, et al (2011) mAbs 3(6):546-557. The HF-TA method uses S364H / T394F substitutions on one Fc monomer and Y349T / F405A substitutions on the complementary Fc monomer. The (HA-TF) method uses S364H / F405A substitutions on one Fc monomer and Y349T / T394F substitutions on the complementary Fc monomer. Alternatively, Fc1 and Fc2 monomers are modified to promote heterodimerization by the ZW1 heterodimerization method, which uses T350V / L351Y / F405A / Y407V substitutions on one Fc monomer and T350V / T366L / K392L / T394W substitutions on the complementary Fc monomer. Von Kreudenstein, et al (2013) mAbs, 5(5):646-654. Alternatively, the Fc1 and Fc2 monomers are engineered to promote heterodimerization by the EW-RVT heterodimerization method, which uses K360E / K409W substitutions on one Fc monomer and Q347R / D399V / F405T substitutions on the complementary Fc monomer. Choi, et al (2015) Molecular Immunology 65(2):377-83.

[0189] In one embodiment, Fc1 and Fc2 are modified to promote heterodimerization by the use of "knobs-into-holes" (abbreviated KiH) modifications as exemplified herein. KiH modifications include one or more amino acid substitutions in a first Fc monomer (e.g., Fc1) that create a bulky "knob" domain on the first Fc, and one or more amino acid substitutions on a second Fc monomer (e.g., Fc2) to accept the "knob" of the first Fc monomer, creating a complementary pocket or "hole" on the first Fc.

[0190] Various amino acid substitutions have been established to generate complementary knob and hole Fc monomers. See, e.g., Ridgway, et al (1996) Protein Engineering 9(7):617-921; Atwell, et al (1997) J. Mol. Biol. 270:26-35; Carter et al., U.S. Patent No. 5,807,706 issued September 15, 1998; Carter et al., U.S. Patent No. 7,695,936 issued April 13, 2010; Zhao et al. "A new approach to produce IgG4-like bispecific antibodies", Scientific Reports 11: 18630 ​​(2021); Cao et al. "Characterization and Monitoring of a Novel Light-heavy-light Chain Mispair in a Therapeutic Bispecific Antibody", and Liu et al. "Fc Engineering for Developing Therapeutic Bispecific Antibodies and Novel Scaffolds". Frontiers in Immunology. 8: 38. See doi:10.3389 / fimmu.2017.00038 (2017).

[0191] In some embodiments, the Fc domain comprises two Fc monomers in which the threonine at position 366 (EU numbering) has been altered with a bulky residue (e.g., T366W), the CH3 domain of a first Fc monomer provides a "knob" and substitution, and a second Fc monomer that includes one or more substitutions at a complementary residue in the CH3 domain of the second Fc monomer, e.g., by amino acid substitutions such as T366S, L368A, and / or Y407V, providing a pocket or "hole" to accommodate the bulky residue.

[0192] In one embodiment, the Fc1 monomer of formula 1 is a "knob" modified Fc monomer comprising the amino acid substitution T366W, and the Fc2 monomer of formula 2 is a "hole" modified Fc comprising the set of amino acid substitutions T366S / L368A / Y407V.

[0193] Alternatively, the Fc1 monomer of formula 1 is a "hole" modified Fc monomer comprising the set of amino acid substitutions T366S / L368A / Y407V, and the Fc2 monomer of formula 2 is a "knob" modified Fc monomer comprising the amino acid substitution T366W.

[0194] An example of an engineered Fc heterodimer pair containing complementary KiH modifications is shown in Table 3 below.

[0195] Table 3. Amino acid substitution sets for complementary IgG1 KiH heterodimer pairs TIFF2024539139000018.tif31169

[0196] As mentioned, the heterodimeric hIL12Fc mutein of the present disclosure is provided as a complementary heterodimeric pair of polypeptides of formula [1] and [2], where the first and second polypeptides are linked by at least one disulfide bond. In some embodiments, the incorporation of a disulfide bond between the polypeptides of formula [1] and [2] may be achieved by cysteine ​​substitution at specific locations in the Fc1 and Fc2 domains. In one embodiment, the Fc1 domain of the polypeptide of formula [1] is derived from the Fc domain of hIgG1 containing the amino acid substitution S354C (EU numbering), and the Fc2 domain of the polypeptide of formula [2] is derived from the Fc domain of hIgG1 containing the amino acid substitution Y349C (EU numbering), such that the disulfide bond is formed between S354C of Fc1 and Y349C of Fc2. Alternatively, the Fc1 domain of the polypeptide of formula [1] is derived from the Fc domain of hIgG1 containing the amino acid substitution Y349C (EU numbering), such that a disulfide bond is formed between S354C of Fc1 and Y349C of Fc2, and the Fc2 domain of the polypeptide of formula [2] is derived from the Fc domain of hIgG1 containing the amino acid substitution S354C (EU numbering).

[0197] In some embodiments, the hP35Fc and hP40MFc of the heterodimeric hIL12Fc mutein are covalently linked via one or more, optionally two or more, optionally three or more, optionally four or more disulfide bonds between the side chains of the following groups of cysteine ​​pairs: (a) C96 of hP35 and C199 of hP40M; (b) C226 of the first Fc monomer and C226 of the second Fc monomer, (c) C229 of the first Fc monomer and C229 of the second Fc monomer; and (d) S354C of the first Fc domain comprising an S354C amino acid substitution and Y349C of the second Fc domain comprising an Y349C amino acid substitution.

[0198] Further examples of complementary KiH engineered heterodimeric Fc pairs that can be used in the practice of the present disclosure are shown in Table 4 below.

[0199] Table 4. Knob-into-hole Fc dimer pairs TIFF2024539139000019.tif76166 * wt=wt hIgG1;C220S refers to wt hIgG1 with a C to S mutation at position 220 (EU numbering).

[0200] Modifications to reduce effector function In some embodiments, the amino acid sequence of the Fc1 and / or Fc2 monomers modified to promote heterodimerization may be further modified to reduce effector function. In some embodiments, the Fc domain may be modified to substantially reduce binding to Fc receptors (FcyR and FcR), reducing or eliminating antibody directed cytotoxicity (ADCC) effector function. Modifications of Fc domains to reduce effector function are well known in the art. See, for example, Wang, et al. (2018) IgG Fc engineering to modulate antibody effector functions, Protein Cell 9(1):63-73. For example, mutation of the lysine residue at position 235 (EU numbering) from leucine (L) to glutamic acid (E) is known to reduce effector function by reducing FcgR and C1q binding. Alegre, et al. (1992) J. Immunology 148:3461-3468. Furthermore, substitution of two leucine (L) residues at positions 234 and 235 (EU numbering) in the IgG1 hinge region with alanine (A), i.e., L234A and L235A, results in reduced complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC). Hezereh et al., (2001) J. Virol 75(24):12161-68. Furthermore, mutation of proline at position 329 (EU numbering) with alanine (P329A) or glycine (P329G) reduces effector function and can be combined with the L234A and L235A substitutions. In some embodiments, the Fc domains (Fc1 and Fc2) of the compositions of the invention may contain amino acid substitutions L234A / L235A / P329A (EU numbering), referred to as "LALAPA" substitutions, or L234A / L235A / P329G (EU numbering), referred to as "LALAPG" substitutions.In some embodiments, the Fc domains (Fc1 and Fc2) of the compositions of the disclosure may contain the amino acid substitutions E233P / L234V / L235A / ΔG237 (referred to in the scientific literature as PVAdelG mutations).

[0201] In some embodiments, the Fc domains (Fc1 and Fc2) of the compositions of the disclosure are derived from hIgG4. In such cases where the Fc domains of the heterodimeric IL12 and IL23 muteins are derived from hIgG4, attenuation of effector function may be achieved by introduction of S228P and / or L235E mutations (EU numbering).

[0202] Examples of paired KiH Fc dimer constructs that can be incorporated into the hIL12 and heterodimeric hIL23Fc muteins of the present disclosure are shown in Table 5 below.

[0203] Table 5. Set of amino acid substitutions for complementary IgG1 KiH UH / Fc heterodimer pairs, including mutations to reduce effector function. TIFF2024539139000020.tif181167

[0204] Fc sequence modification to extend duration of action In some embodiments, the amino acid sequence of the Fc1 monomer and / or Fc2 monomer modified to promote heterodimerization may be further modified to incorporate amino acid substitutions that extend the duration of action of the molecule and prevent clearance. In some embodiments, such modifications to the Fc monomer include amino acid substitutions M428L and N434S (EU numbering), referred to as "LS" modifications. The LS modifications may be optionally combined with amino acid substitutions to reduce effector function and create disulfide bonds between Fc1 and Fc2. Table 6 below shows exemplary Fc1 and Fc1 heterodimer pairs with complementary sequence modifications to promote heterodimerization that may be used in designing the Fc1 and Fc2 polypeptides of formulas [1] and [2].

[0205] Table 6 below shows exemplary Fc heterodimer pairs that may be used in preparing the Fc1 and Fc2 polypeptides of heterodimeric hIL12Fc muteins of the disclosure.

[0206] Table 6. Set of amino acid substitutions for complementary IgG1 KiH UH / Fc heterodimer pairs, including mutations for reduced effector function and extended LS half-life. TIFF2024539139000021.tif184170

[0207] In some embodiments, the Fc domains (Fc1 and Fc2) of the compositions of the present disclosure are derived from hIgG4. In such cases where the Fc domains of the heterodimeric IL12 and IL23 muteins are derived from hIgG4, heterodimerization of the Fc1 and Fc2 domains is achieved by introduction of the mutations K370E, K409W and E357N, D399V, F405T (EU numbering) in the complementary Fc sequences that constitute the heterodimeric Fc domain.

[0208] Fc modification to eliminate glycosylation sites In some embodiments, the amino acid sequence of the Fc1 and / or Fc2 monomers modified to promote heterodimerization may be further modified to eliminate N- or O-linked glycosylation sites. Aglycosylated variants of Fc domains, particularly those of the IgG1 subclass, are known to be poor mediators of effector function. Jefferies et al. 1998, Immol. Rev., vol. 163, 50-76). Glycosylation at position 297 (EU numbering) has been shown to contribute to effector function. Edelman, et al (1969) PNAS (USA) 63:78-85. In some embodiments, the Fc domain of the composition of the present disclosure comprises one or more modifications to eliminate N- or O-linked glycosylation sites. Examples of modifications at N297 to eliminate glycosylation sites in the Fc domain include the amino acid substitutions N297Q and N297G.

[0209] PEGylation In some embodiments, the polypeptides of formula [1] and / or [2] of the heterodimeric hIL12Fc muteins of the present disclosure may be conjugated with one or more polyethylene glycol molecules, i.e., "PEGylated." The method or site for attaching PEG to the binding molecule may vary, but in certain embodiments, PEGylation does not change, or only minimally changes, the activity of the binding molecule.

[0210] The present disclosure provides a PEGylated heterodimeric hIL12Fc mutein. In some embodiments, the hP35Fc polypeptide of the heterodimeric hIL12Fc mutein is PEGylated. In some embodiments, the hP40MFc polypeptide of the heterodimeric hIL12Fc mutein is PEGylated. In some embodiments, both the hP35Fc and hP40MFc of the heterodimeric hIL12Fc mutein are PEGylated.

[0211] The present disclosure provides a PEGylated heterodimeric hIL23Fc mutein. In some embodiments, the hP19Fc polypeptide of the heterodimeric hIL23Fc mutein is PEGylated. In some embodiments, the hP40MFc polypeptide of the heterodimeric hIL23Fc mutein is PEGylated. In some embodiments, both the hP19Fc and hP40MFc of the heterodimeric hIL23Fc mutein are PEGylated.

[0212] In some embodiments, the PEG moiety may be conjugated via a sulfhydryl (-SH) group of a cysteine ​​residue. In some embodiments, PEGylation of the heterodimeric hIL12Fc mutein is provided at one or both of the native cysteine ​​residues at position 220 (C220, EU numbering) of the upper hinge region of the hP35Fc and / or hP40MFc heterodimeric hIL12Fc mutein. In some embodiments, PEGylation of the heterodimeric hIL23Fc mutein is provided at one or both of the native cysteine ​​residues at position 220 (C220, EU numbering) of the upper hinge region of the hP19Fc and / or hP40MFc heterodimeric hIL23Fc mutein. When preparing a PEGylated heterodimeric hIL12Fc mutein or a PEGylated heterodimeric hIL23Fc mutein, in which a conjugate of a PEG molecule is provided at position C220, the above-referenced C220S modification of the upper hinge region is not used.

[0213] PEGs suitable for conjugation to polypeptide sequences are generally water soluble at room temperature and have the general formula R(O-CH2-CH2) n OR where R is hydrogen or a protecting group, such as an alkyl or alkanol group, and n is an integer from 1 to 1000. When R is a protecting group, it generally has from 1 to 8 carbons. PEG can be linear or branched. Branched PEG derivatives, "star PEGs," and multi-armed PEGs are contemplated by the present disclosure.

[0214] The PEGylation of the heterodimeric hIL12Fc muteins and heterodimeric hIL23Fc muteins of the present disclosure may be modified by incorporating one or more unnatural amino acids with side chains that facilitate selective PEG conjugation. Specific PEGylation sites can be selected such that PEGylation of the binding molecule does not affect binding of the binding molecule to the target receptor.

[0215] In certain embodiments, the increase in half-life is greater than any decrease in biological activity. PEGs suitable for conjugation to polypeptide sequences are generally soluble in water at room temperature and have the general formula R(O-CH2-CH2)nO-R, where R is hydrogen or a protecting group, e.g., an alkyl or alkanol group, and n is an integer between 1 and 1000. When R is a protecting group, it generally has 1 to 8 carbons. PEGs for conjugation to polypeptide sequences can be linear or branched. Branched PEG derivatives, "star PEGs," and multi-armed PEGs are contemplated by the present disclosure.

[0216] The molecular weight of PEG used in the present disclosure is not limited to any particular range. The molecular weight of the PEG component of the binding molecule may be greater than about 5 kDa, greater than about 10 kDa, greater than about 15 kDa, greater than about 20 kDa, greater than about 30 kDa, greater than about 40 kDa, or greater than about 50 kDa. In some embodiments, the molecular weight is about 5 kDa to about 10 kDa, about 5 kDa to about 15 kDa, about 5 kDa to about 20 kDa, about 10 kDa to about 15 kDa, about 10 kDa to about 20 kDa, about 10 kDa to about 25 kDa, or about 10 kDa to about 30 kDa. A linear or branched PEG molecule having a molecular weight of about 2,000 to about 80,000 daltons, alternatively about 2,000 to about 70,000 daltons, alternatively about 5,000 to about 50,000 daltons, alternatively about 10,000 to about 50,000 daltons, alternatively about 20,000 to about 50,000 daltons, alternatively about 30,000 to about 50,000 daltons, alternatively about 20,000 to about 40,000 daltons, or alternatively about 30,000 to about 40,000 daltons. In one embodiment of the present disclosure, the PEG is a 40 kD branched PEG comprising two 20 kD arms.

[0217] The present disclosure also contemplates compositions of conjugates in which PEG has different n values, and thus various different PEGs are present in specific ratios. For example, some compositions include compositions of conjugates where n=1, 2, 3, and 4. In some compositions, the percentage of conjugates where n=1 is 18-25%, the percentage of conjugates where n=2 is 50-66%, the percentage of conjugates where n=3 is 12-16%, and the percentage of conjugates where n=4 is up to 5%. Such compositions can be produced by reaction conditions and purification methods known in the art. Chromatography may be used to separate the conjugate fractions, and then, for example, fractions containing conjugates with a desired number of PEGs attached are identified and purified free of unmodified protein sequences and free of conjugates with other numbers of PEGs attached.

[0218] PEG suitable for conjugation to a polypeptide sequence is generally soluble in water at room temperature and has the general formula R(O-CH2-CH2)nO-R, where R is hydrogen or a protecting group, such as an alkyl or alkanol group, and n is an integer from 1 to 1000. When R is a protecting group, it generally has from 1 to 8 carbons.

[0219] Two widely used first generation activated monomethoxy PEGs (mPEGs) are succinimidyl carbonate PEG (SC-PEG; see, e.g., Zalipsky, et al. (1992) Biotehnol. Appl. Biochem 15:100-114) and benzotriazole carbonate PEG (BTC-PEG; see, e.g., Dolence et al., U.S. Pat. No. 5,650,234), which react preferentially with the side chains of lysine residues to form carbamate linkages, but are also known to react with histidine and tyrosine residues. The use of PEG-aldehyde linkers targets a single site at the polypeptide N-terminus via reductive amination.

[0220] PEGylation is frequently performed at the α-amino group at the N-terminus of a polypeptide, the ε-amino group at the side chain of a lysine residue, and the imidazole group at the side chain of a histidine residue. Most recombinant polypeptides have one α-amino group and many ε-amino groups and imidazole groups, so a large number of positional isomers can be generated depending on the linker chemistry. General PEGylation strategies known in the art can be applied herein.

[0221] PEG can be attached to the binding molecules of the present disclosure via a terminal reactive group ("spacer") that mediates a bond between one or more free amino or carboxyl groups of the polypeptide sequence and polyethylene glycol. PEGs having a spacer that can be attached to a free amino group include N-hydroxysuccinimide polyethylene glycol, which can be prepared by activating the succinate ester of polyethylene glycol with N-hydroxysuccinimide.

[0222] The PEG conjugated to the polypeptide sequence can be linear or branched. Branched PEG derivatives, "star PEGs," and multi-armed PEGs are contemplated by the present disclosure.Specific embodiments of PEG useful in the practice of the present disclosure include 10 kDa linear PEG-aldehyde (e.g., Sunbright® ME-100AL, NOF America Corporation, One North Broadway, White Plains, NY 10601 USA), 10 kDa linear PEG-NHS ester (e.g., Sunbright® ME-100CS, Sunbright® ME-100AS, Sunbright® ME-100GS, Sunbright® ME-100HS, NOF), 20 kDa linear PEG-aldehyde (e.g., Sunbright® ME-200AL, NOF), 20 kDa linear PEG-NHS ester (e.g., Sunbright® ME-200CS, Sunbright® ME-200AS, Sunbright® ME-200GS, Sunbright® ME-200HS, NOF), 20kDa 2-arm branched PEG-aldehyde, 20kDA PEG-aldehyde containing two 10kDA linear PEG molecules (e.g., Sunbright® GL2-200AL3, NOF), 20kDa 2-arm branched PEG-NHS ester, 20kDA PEG-NHS ester containing two 10kDA linear PEG molecules (e.g., Sunbright® GL2-200TS, Sunbright® GL200GS2, NOF), 40kDa 2-arm branched PEG-aldehyde, 40kDA PEG-aldehyde containing two 20kDA linear PEG molecules (e.g., Sunbright® GL2-400AL3), 40kDa 2-arm branched PEG-NHS ester, 40kDA containing two 20kDA linear PEG molecules These include PEG-NHS esters (e.g., Sunbright® GL2-400AL3, Sunbright® GL2-400GS2, NOF), linear 30 kDa PEG-aldehydes (e.g., Sunbright® ME-300AL), and linear 30 kDa PEG-NHS esters.

[0223] In some embodiments, a linker can be used to connect the PEG molecule to the heterodimeric hIL12Fc mutein or heterodimeric hIL23Fc mutein. Suitable linkers generally include "flexible linkers" of sufficient length to allow some movement between the modified polypeptide sequence and the linked components and molecules. The linker molecule is generally about 6-50 atoms long. The linker molecule can be, for example, an aryl acetylene, an ethylene glycol oligomer containing 2-10 monomer units, a diamine, a dibasic acid, an amino acid, or a combination thereof. Suitable linkers can be readily selected and can be of any suitable length, for example, a linker of 1 amino acid (e.g., Gly), 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-20, 20-30, 30-50, or more than 50 amino acids. Examples of flexible linkers are described in Section IV. Furthermore, multimers (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-20, 20-30, or 30-50) of these linker sequences may be linked together to provide a flexible linker that can be used to conjugate two molecules. In some embodiments, the linker is a GSA linker as described above. As an alternative to a polypeptide linker, the linker may be a chemical linker, e.g., a PEG-aldehyde linker. In some embodiments, the binding molecule is acetylated at the N-terminus by enzymatic reaction with an N-terminal acetyltransferase, e.g., acetyl-CoA. Alternatively, or in addition to N-terminal acetylation, the binding molecule may be acetylated at one or more lysine residues, e.g., by enzymatic reaction with a lysine acetyltransferase. See, e.g., Choudhary et al. (2009) Science 325 (5942):834 840.

[0224] In some aspects, the disclosure provides PEGylated heterodimeric hIL12Fc muteins and heterodimeric hIL23Fc muteins, wherein the PEG is conjugated to the heterodimeric hIL12Fc or hIL23Fc mutein and has a molecular weight of about 2,000 to about 80,000 daltons, alternatively about 2,000 to about 70,000 daltons, alternatively about 5,000 to about 50,000 daltons, alternatively about 10,000 to about 50,000 daltons. The PEGylated heterodimeric hIL12Fc muteins and heterodimeric hIL23Fc muteins are provided, the PEGylated heterodimeric hIL12Fc muteins being linear or branched PEG molecules having a molecular weight of about 0,000 daltons, alternatively about 20,000 to about 50,000 daltons, alternatively about 30,000 to about 50,000 daltons, alternatively about 20,000 to about 40,000 daltons, or alternatively about 30,000 to about 40,000 daltons. In one embodiment of the disclosure, the PEG is a 40 kD branched PEG comprising two 20 kD arms. In some embodiments, the PEG is conjugated to the N-terminus of the hP35Fc and / or hP40MFc polypeptides.

[0225] Exemplary heterodimeric hIL12Fc muteins: The following table summarizes certain compositions of the present disclosure that include KiH heterodimerization, as shown in Table 7 below.

[0226] Table 7. Exemplary KiH p35 hole and hP40M knob constructs TIFF2024539139000022.tif133161

[0227] In some embodiments, the present disclosure provides a compound represented by formula #1: hP40M-L1 a -UH1-Fc1[1] and a first polypeptide of formula #2: hP35-L2 b -UH2-Fc2 [2] and a second polypeptide of formula #2 selected from the group consisting of SEQ ID NOs: 81, 82, 84, 87, 89, 91, 93, and 124.

[0228] In some embodiments, the hIL12Fc muteins of the disclosure are heterodimeric hIL12Fc muteins shown in Table 8 below.

[0229] Table 8. Exemplary heterodimeric hIL12 Fc muteins TIFF2024539139000023.tif104164

[0230] 1. STK-021 (DR1442M / DR1535M) In one aspect, the present disclosure provides a heterodimeric hIL12Fc mutein having formula #1: hP40M-L1 a -UH1-Fc1[1] and a first polypeptide of formula #2: hP35-L2 b -UH2-Fc2 [2] and a second polypeptide of Wherein a=1 and b=1, The polypeptide of formula #1 has the amino acid sequence: Contains TIFF2024539139000024.tif40134, The polypeptide of formula #2 has the amino acid sequence: Heterodimeric hIL12Fc muteins, including TIFF2024539139000025.tif35134, are provided.

[0231] 2. STK-022(1947 / DR1948M) In one aspect, the present disclosure provides a heterodimeric hIL12Fc mutein having formula #1: hP40M-L1a -UH1-Fc1[1] and a first polypeptide of formula #2: hP35-L2 b -UH2-Fc2 [2] and a second polypeptide of Wherein a=1 and b=1, The polypeptide of formula #1 has the amino acid sequence: Contains TIFF2024539139000026.tif36128, The polypeptide of formula #2 has the amino acid sequence: Heterodimeric hIL12Fc muteins are provided, including TIFF2024539139000027.tif32128.

[0232] 3. STK-023 (DR1537M / DR1536M) In one aspect, the present disclosure provides a heterodimeric hIL12Fc mutein having formula #1: hP40M-L1 a -UH1-Fc1[1] and a first polypeptide of formula #2: hP35-L2 b -UH2-Fc2 [2] and a second polypeptide of Wherein a=1 and b=1, The polypeptide of formula #1 has the amino acid sequence: Contains TIFF2024539139000028.tif36128, The polypeptide of formula #2 has the amino acid sequence: Heterodimeric hIL12Fc muteins are provided, including TIFF2024539139000029.tif32128.

[0233] 4. DR2086M / DR1948M In one aspect, the present disclosure provides a heterodimeric hIL12Fc mutein having formula #1: hP40M-L1 a -UH1-Fc1[1] and a first polypeptide of formula #2: hP35-L2b -UH2-Fc2 [2] and a second polypeptide of Wherein a=1 and b=1, The polypeptide of formula #1 has the amino acid sequence: Contains TIFF2024539139000030.tif44128, The polypeptide of formula #2 has the amino acid sequence: Heterodimeric hIL12Fc muteins are provided, including TIFF2024539139000031.tif35128.

[0234] 5. DR2087M / DR1948M In one aspect, the present disclosure provides a heterodimeric hIL12Fc mutein having formula #1: hP40M-L1 a -UH1-Fc1[1] and a first polypeptide of formula #2: hP35-L2 b -UH2-Fc2 [2] and a second polypeptide of Wherein a=1 and b=1, The polypeptide of formula #1 has the amino acid sequence: Contains TIFF2024539139000032.tif44128, The polypeptide of formula #2 has the amino acid sequence: Heterodimeric hIL12Fc muteins are provided, including TIFF2024539139000033.tif35128.

[0235] 6. STK-026 (DR2088M / DR1948M) In one aspect, the present disclosure provides a heterodimeric hIL12Fc mutein having formula #1: hP40M-L1 a -UH1-Fc1[1] and a first polypeptide of formula #2: hP35-L2 b -UH2-Fc2 [2] and a second polypeptide of Wherein a=1 and b=1, The polypeptide of formula #1 has the amino acid sequence: Contains TIFF2024539139000034.tif36128, The polypeptide of formula #2 has the amino acid sequence: Heterodimeric hIL12Fc muteins are provided, including TIFF2024539139000035.tif35128.

[0236] 7. DR2090M / DR1536M In one aspect, the present disclosure provides a heterodimeric hIL12Fc mutein having formula #1: hP40M-L1 a -UH1-Fc1[1] and a first polypeptide of formula #2: hP35-L2 b -UH2-Fc2 [2] and a second polypeptide of Wherein a=1 and b=1, The polypeptide of formula #1 has the amino acid sequence: Contains TIFF2024539139000036.tif49128, The polypeptide of formula #2 has the amino acid sequence: Heterodimeric hIL12Fc muteins are provided, including TIFF2024539139000037.tif32128.

[0237] 8. DR2091M / DR1536M In one aspect, the present disclosure provides a heterodimeric hIL12Fc mutein having formula #1: hP40M-L1 a -UH1-Fc1[1] and a first polypeptide of formula #2: hP35-L2 b -UH2-Fc2 [2] and a second polypeptide of Wherein a=1 and b=1, The polypeptide of formula #1 has the amino acid sequence: Contains TIFF2024539139000038.tif44128, The polypeptide of formula #2 has the amino acid sequence: Heterodimeric hIL12Fc muteins are provided, including TIFF2024539139000039.tif32128.

[0238] 9. STK-027 (DR2092M / DR1536M) In one aspect, the present disclosure provides a heterodimeric hIL12Fc mutein having formula #1: hP40M-L1 a -UH1-Fc1[1] and a first polypeptide of formula #2: hP35-L2 b -UH2-Fc2 [2] and a second polypeptide of Wherein a=1 and b=1, The polypeptide of formula #1 has the amino acid sequence: Contains TIFF2024539139000040.tif35128, The polypeptide of formula #2 has the amino acid sequence: Heterodimeric hIL12Fc muteins are provided, including TIFF2024539139000041.tif28128.

[0239] 10. STK-028 (DR2455M / DR1948M): In one aspect, the present disclosure provides a heterodimeric hIL12Fc mutein having formula #1: hP40M-L1 a -UH1-Fc1[1] and a first polypeptide of formula #2: hP35-L2 b -UH2-Fc2 [2] and a second polypeptide of Wherein a=1 and b=1, The polypeptide of formula #1 has the amino acid sequence: Contains TIFF2024539139000042.tif35128, The polypeptide of formula #2 has the amino acid sequence: Heterodimeric hIL12Fc muteins are provided, including TIFF2024539139000043.tif31128.

[0240] 11. STK-029 (DR2456M / DR1948M): In one aspect, the present disclosure provides a heterodimeric hIL12Fc mutein having formula #1: hP40M-L1 a -UH1-Fc1[1] and a first polypeptide of formula #2: hP35-L2 b -UH2-Fc2 [2] and a second polypeptide of Wherein a=1 and b=1, The polypeptide of formula #1 has the amino acid sequence: Contains TIFF2024539139000044.tif32128, The polypeptide of formula #2 has the amino acid sequence: Heterodimeric hIL12Fc muteins are provided, including TIFF2024539139000045.tif31128.

[0241] Evaluation of the activity of heterodimeric hIL12Fc muteins As discussed above, the heterodimeric hIL12Fc muteins of the present disclosure provide cell type biased signaling of downstream signaling mediated by the IL12 receptor compared to a reference polypeptide (e.g., wild-type hIL12). In particular, the heterodimeric hIL12Fc muteins of the present disclosure retain significant hIL-12 signaling in CD8+ T cells and reduce hIL-12 signaling in NK cells compared to wild-type hIL-12. Selective activation of CD8+ T cells versus NK cells can be assessed by reduced STAT4 signaling in NK cells concomitant with interferon gamma (IFNγ) activation.

[0242] Enhancement of IFNγ in CD8+ T cells versus NK cells: The heterodimeric hIL12Fc muteins of the present disclosure activate interferon gamma (IFNγ) in CD8+ T cells and have reduced IFNγ signaling in CD8+ T cells compared to wild-type IL12. A series of experiments were performed to evaluate the IFNγ-inducing effects of wt hIL12 and hIL12 proteins containing the p40 subunit E81A / F82A, indicated as "2xAla", E81A / F82A / K106A, indicated as "3xAla", and substitution W37A, as well as KiH heterodimeric hIL12Fc containing the same mutations E81A / F82A, indicated as "2xAla Fc", E81A / F82A / K106A, indicated as "3xAla Fc", and substitution W37A (W37A Fc) on CD8, CD4, and NK cells. These molecules were recombinantly produced generally as disclosed in Example 1 and evaluated for IFNγ generally as disclosed in Example 3 herein. As illustrated by the data presented in Figure 1, the heterodimeric hIL12Fc muteins of the present disclosure activate IFNγ in CD8+ T cells and reduce IFNγ signaling in CD8+ T cells compared to wild-type IL12.

[0243] Reducing STAT4 signaling: In some embodiments, the heterodimeric hIL12Fc muteins described herein increase STAT4 signaling in CD8+ T cells and decrease STAT4 signaling in NK cells compared to the reference polypeptide (wt hIL12). A series of experiments were performed to evaluate the effect of various IL12 muteins, including hP40 muteins as proteins and as Fc fusions, on STAT4 signaling in CD8+ T cells, CD4+ T cells, compared to STAT4 signaling in NK cells. STAT4 evaluation was performed generally as disclosed in Example 2. The test articles evaluated were wt hIL12, hIL12 proteins comprising a p40 subunit with substitutions E81A / F82A, indicated as "2xAla", hIL12 proteins comprising a p40 subunit with substitutions E81A / F82A / K106A, indicated as "3xAla", and wild type hP35 and KiH heterodimeric hIL12Fc constructs comprising hP40(wt Fc), E81A / F82A, indicated as "2xAla Fc", and E81A / F82A / K106A, indicated as "3xAla Fc", on CD8, CD4 and NK cells. The results of these experiments are shown in Figure 2 of the accompanying drawings. As illustrated by the results shown in Figure 2, the heterodimeric hIL12Fc muteins of the present disclosure provide differential STAT4 signaling to CD8+ T cells compared to NK cells. Furthermore, the data presented in FIG. 2, particularly panels A and B, demonstrate that the heterodimeric hIL12Fc muteins of the present disclosure act as IL12 partial agonists on T cells.

[0244] Use in the treatment of neoplastic diseases: The heterodimeric hIL12Fc muteins described herein are useful in the treatment of neoplastic disease.To prove the activity of heterodimeric hIL12Fc muteins, surrogate mouse IL12Fc muteins containing similar mutations to human molecules were made and evaluated for their efficacy in MC38 mouse tumor model.The sequence alignment of natural human and mouse p40 and p35 polypeptides is shown in Figure 7 and Figure 8 of the attached drawings, respectively.The description of the heterodimeric mIL12Fc test agent used in MC38 tumor study is summarized in Table 9 and Table 10 below.

[0245] Table 9. Mouse IL12Fc polypeptide sequence TIFF2024539139000046.tif26155

[0246] Table 10. Description of mouse IL12Fc heterodimer constructs TIFF2024539139000047.tif38155

[0247] MC38 Tumor Study 1: Briefly, approximately 1x10 cells in Matrigel 6 MC38 cells were subcutaneously implanted into 6- to 8-week-old C57BL / 6 mice, and the average tumor volume at the start of treatment was approximately 100 mm 3 ~120mm 3 The mice were divided into individual treatment groups. The mice were treated by intraperitoneal administration of various test agents at the doses and dosing schedules shown in Table 11 below. In this study, body weight (BW), an indicator of toxicity, and tumor volume (TV), an indicator of antitumor efficacy, were measured twice weekly.

[0248] Table 11. MC38 Tumor Model Study #1 Design TIFF2024539139000048.tif90164

[0249] The data generated from the above studies are presented in the accompanying drawings in Figure 3 (tumor volume), Figure 4 (body weight), and Figure 5 (survival). Note that the panel designations in Figures 3 and 4 correspond to the treatment groups in Table 11. In each figure, tumor volume and body weight are shown on the y-axis and time (study days) is shown on the x-axis.

[0250] Figure 3 shows a spider plot summary of the effect on tumor volume for each animal in each study group. As evidenced by the data presented, the mouse surrogate of the heterodimeric IL12Fc mutein described herein was effective in managing tumor growth in this study.

[0251] Figure 4 shows the average body weight of the animals over the course of the study. Although the wild-type IL12 Fc test agents evaluated in this study showed inhibition of tumor growth, the data shown in Figure 4 show that such wild-type IL12 Fc fusions are associated with significant toxicity as indicated by significant body weight loss (see, e.g., Figure 4, panels B, C, and E). In contrast, the heterodimeric IL12Fc mutein evaluated in group H, which contains a 2xAla mutation in the mP40 domain, did not suggest significant toxicity. This is particularly noteworthy since the IL12Fc mutein containing the 2xAla P40 mutation was administered at a dose more than 50-fold higher than the other wild-type IL12Fc conjugates evaluated. The ability of the IL12Fc mutein containing the 2xAla mutation to manage tumor growth in the absence of significant toxicity is further supported by the data shown in Figure 5. The data shown in Figure 5 show that all animals treated with IL12Fc muteins containing 2xAla survived, whereas the test group receiving wild-type IL12Fc conjugates had a significant negative effect on survival, despite the clear anti-tumor effects of these other molecules. As a result, these data demonstrate that the heterodimeric IL12Fc muteins of the present disclosure are useful in the treatment of neoplastic disease and exhibit significantly less toxicity than wild-type IL12Fc conjugates that do not have a mutation in the p40 domain of the IL12Fc conjugate.

[0252] MC38 Tumor Study 2: A second MC38 tumor study was conducted generally following the study design described above and shown in Table 12. In this study, body weight (BW), an indicator of toxicity, and tumor volume (TV), an indicator of antitumor efficacy, were measured twice weekly. Mice were bled prior to the start of the study (0 hours), and 4 hours, 1 day, and 7 days after administration of the test agent. Some animals were sacrificed 2 and 24 days after administration of the test agent for immunohistochemical evaluation and FACS analysis.

[0253] Table 12. MC38 Tumor Model Study #2 Design TIFF2024539139000049.tif76163 * This wt mIL12 was produced by baculovirus expression in insect cells.

[0254] The effect of these test agents on tumor growth and data for the studies described in Table 12 are shown in Figure 6 of the accompanying drawings. Data for the IL12 protein (not conjugated to an Fc dimer) is shown in Figure 6 Panel A, and the Fc conjugated IL12 protein is shown in Figure 6 Panel B. Comparison of the data in Figure 6 Panels A and B demonstrates that IL12 molecules conjugated to a dimeric Fc domain provide significantly improved anti-tumor efficacy compared to their non-Fc conjugated counterparts.

[0255] For treatment groups A-H, blood samples obtained at 0 hours and 4 hours, 1 day, and 7 days after administration of the test agent were evaluated for serum levels of murine interferon gamma (mIFNg) as determined by enzyme-linked immunosorbent assay (ELISA). The data obtained are shown in Figure 9 of the accompanying drawings. Panel A of Figure 9 shows the results from treatment groups A-E (i.e., IL12 molecules not conjugated to Fc), and Panel B shows the results from treatment groups F, G, and H (i.e., IL12 molecules conjugated to Fc). As can be seen from the data shown in Figure 9, Fc-conjugated heterodimeric mIL12-Fc molecules containing amino acid substitutions (i.e., "2xAla" and "3xAla") showed a significant delay in interferon gamma induction compared to the other treatment groups, and especially compared to IL12 Fc containing the wild-type p40 sequence. This delayed IFNg induction reduces the acute toxicity associated with IL12 treatment.

[0256] As mentioned, samples from this study were subjected to FACS analysis to sort NK cells from spleen and tumor tissues. The percentage of lymphocytes in each tissue in response to each treatment group in Table 12 is shown in Figure 10 of the attached drawings. As shown, Fc-conjugated molecules resulted in weaker NK cell induction compared to non-Fc-conjugated molecules in each tissue type. However, as shown in Figure 10, panels B and C (zoomed view of NK frequency in tumor tissue), Fc-conjugated heterodimeric mIL12-Fc molecules containing P40 amino acid substitutions (i.e., "IL12 2xAla Fc" and "IL12 3xAla Fc") showed a lower frequency of intratumoral NK cells compared to heterodimeric mIL12 Fc containing wild-type p40 sequence ("IL12 WT Fc").

[0257] Additionally, the phenotype of NK cells derived from the spleens in the above studies was evaluated for T-bet compared to intracellular granzyme B. T-bet is required for NK cell effector function and NK cell cytolytic activity. The results of this analysis are shown in FIG. 11 of the accompanying drawings. T-bet is shown on the ordinate and granzyme B on the abscissa. As can be seen from the data shown in FIG. 10, Tbet+ NK cells are lost with heterodimeric mIL12 Fc containing the wild type p40 sequence ("IL12 WT Fc"), but not with Fc-conjugated heterodimeric mIL12-Fc molecules containing p40 amino acid substitutions (i.e., "IL12 2xAla Fc" and "IL12 3xAla Fc").

[0258] Dose titration of mIL12 Fc constructs in the CT26 model: The effect of dosing with various levels of mIL12 Fc heterodimer was evaluated in the CT26 tumor model. Briefly, 6-8 week old BALB / c mice were administered approximately 0.3x10 6 CT26 mice were implanted subcutaneously with 100 cells of each tumor. The average tumor volume was 118 mm. 3 Mice were randomized into multiple groups at the time of admission. Treatment groups and study design are summarized in Table 13. Body weight and tumor volume were measured twice weekly. Some mice were sacrificed on study days 8 and 29 for FACS, IHC, and serum analysis. Serum PK was measured on study days 1, 8, 15, 22, and 28.

[0259] Table 13. CT26 tumor model study design TIFF2024539139000050.tif78166

[0260] Data on the effect on tumor growth in the above studies are shown in Figures 12, 13, and 14 of the accompanying drawings. As shown in Figure 12, wild type IL-12WT Fc shows strong antitumor activity at 0.8ug (0.5ug IL-12) / dose in the CT26 model. IL-12 3xAla Fc causes tumor regression with a delay of 5-7 days compared to IL-12WT Fc. The CT26 model appears to be more sensitive to IL-12 treatment than our in-house MC38. In Figure 13, neither IL-12WT Fc nor IL-12 3xAla Fc reduces body weight. Considering the toxicity observed with the test agents in the MC38 study performed in C57BL / 6 mice, BALB / c mice appeared to be more resistant to IL-12WT Fc toxicity. FIG. 14 shows FACS analysis demonstrating that wild type IL12 Fc degranulates NK cells, whereas 3xAla IL12 Fc does not substantially induce NK cell degranulation.

[0261] Evaluation of toxicity in combination with NK cell depletion study #1S6-21-005 Studies were performed in mice to evaluate the effect of IL12 Fc in combination with test agents on NK cell depletion. NK1.1 antibody was used to deplete NK cells. 6-8 week old C57BL / 6 were treated with NK cell depleting antibody (αNK1.1 / IL12) followed by IL-12. PBS or αNK1.1 was administered on days -3, 0, 3, and 7. IL12 was administered on days 0, 4, and 8 (see Table 14). Moribund mice were sacrificed for serum analysis and IHC. BW was measured daily. Blood was drawn from some mice and assessed for the presence or absence of NK cells to confirm that the anti-NK1.1 antibody was depleting NK cells. These assessments confirmed by FACS that the antibody was indeed depleting NK cells. Surviving mice were sacrificed on day 13.

[0262] Table 14. NK depletion toxicity model study design TIFF2024539139000051.tif50164

[0263] PBS / αNK1.1 was administered on days -3, 0, 3, and 7. IL12 was administered on days 0, 4, and 8. Animals were monitored for viable body weight. Animals were sacrificed on day 13. Results of the study are shown in FIG. 15. As can be seen from the data shown, NK depletion reduces toxicity as assessed by body weight loss and prevents mortality following 1.6ug IL-12WT Fc treatment. NK cell depletion also significantly reduces mortality and morbidity at the 4.8ug dose. Furthermore, NK cell depletion is transient, with NK cells returning on d6 after the last NK depletion dose.

[0264] Evaluation of antitumor efficacy and toxicity from NK / CD8 T cell depletion study S6-21-006 A second study was conducted to evaluate the effect of combining IL-Fc test agents on NK and CD8 T cell depletion. In this study, approximately 1x10 6 MC38 cells were subcutaneously implanted into 6- to 8-week-old C57BL / 6 mice. The average tumor volume was approximately 85 mm. 3 Mice were randomized into groups when the tumor reached tumor size. NK cells were also depleted using NK1.1 antibody. CD8 T cells were depleted. Antibodies and test agents were administered according to the study design shown in Table 15 below. Mice were bled at various time points to confirm depletion efficiency. Tumor volume and body weight were measured twice a week, and animals were sacrificed at the end of the study for FACS analysis.

[0265] Table 15. Antitumor efficacy and toxicity assessment by NK / CD8 T cell depletion study design TIFF2024539139000052.tif89166

[0266] The results of the study are shown in Figures 16 and 17. As shown by the data shown in Figure 16, depletion of NK cells reduces toxicity. This indicates that NK cells contribute to the toxicity observed with IL12 agents. However, as shown in Figure 17, NK cells contribute relatively minimally to anti-tumor efficacy. This data indicates that IL12 Fc agents (e.g., heterodimeric hIL12Fc containing hP40M polypeptide) with biased activation of CD8 T cells and reduced activation of NK cells are effective in treating cancer and have significantly reduced toxicity compared to that observed with IL12 agents containing wild-type p40 polypeptide.

[0267] Evaluation of IL12 agents in B6, RAG2 KO, and RAG2 / CD132 double KO mice To further evaluate the activity of hIL12Fc muteins with respect to T cell versus NK cell activation, antitumor efficacy studies were performed in B6 mice, RAG 2 knockout mice, and RAG2 / CD132 double knockout mice with IL12 and control test agents evaluated above. B6 mice were used as a control group for RAG2 knockout (KO) mice, which lack T and B cells, and RAG2 / CD132 double knockout mice, which lack T, B, and NK cells. Briefly, 11 days prior to the start of treatment (day -11), approximately 1x10 6 MC38 cells were placed in Matrigel and implanted subcutaneously, with the tumor volume reaching approximately 120 mm 3 When mice reached 18 days of age, they were randomized into treatment groups as described below in Table 16. Test articles and controls were administered according to the schedules described below in Table 16. Mice were assessed twice weekly for weight loss and tumor volume.

[0268] Table 16. Evaluation of IL12 test agents in Rag2 and Rag2 / CD132 knockout mice TIFF2024539139000053.tif66166

[0269] The results of this study are illustrated in Figure 18. As observed from the data, genetic loss of T cells significantly reduces IL-12-mediated tumor control, while genetic loss of T, NK, and ILCs renders mice completely resistant to IL-12. Combined with the aforementioned data, these studies demonstrate that the antitumor effect of IL12 is not dependent on the presence of NK cells, and that heterodimeric IL12 Fc muteins with reduced NK cell activation retain antitumor efficacy.

[0270] Combination studies: To evaluate the activity of the heterodimeric hIL12Fc muteins of the present disclosure in combination with adjunctive therapeutic agents in the treatment of neoplastic disease, two studies were conducted to evaluate the heterodimeric mIL12Fc surrogate muteins in combination with interleukin-2 and anti-PD1 checkpoint inhibitor molecules in the MC38 tumor model as previously described herein. The study design is shown in Table 17 below.

[0271] Table 17. Evaluation of mIL12 p40M heterodimer in combination with anti-PD1 and IL2 muteins in the MC38 tumor model TIFF2024539139000054.tif118166

[0272] For human IL2 muteins containing amino acid substitutions at positions 18, 22, and 126 numbered according to mature wild-type hIL2, specifically, a hIL2 mutein containing the amino acid substitutions L18R / Q22E / Q126K, a murine IL2 mutein was developed for in vivo studies in mice to correlate activity between rodent (mouse) and primate (human) environments. The amino acid sequence of the murine IL2 (mIL2) polypeptide used in this study is TIFF2024539139000055.tif26131, which is N-terminally PEGylated with a 40 kD branched PEG using a linker and referred to as PEG-mREH. The results of this study are shown in Figure 19 (PD1) and Figure 20 (mIL2 mutein). As can be seen from the data shown in these figures, the combination of heterodimeric Fc P40M mutein enhances the anti-tumor effect in this model. In particular, the combination of heterodimeric Fc P40M mutein with anti-PD1 antibody shows significantly enhanced effect leading to complete remission. Although not shown in the figures, the combination of heterodimeric wild-type IL12 Fc with anti-PD1 antibody was observed to reduce the toxicity previously observed with heterodimeric wild-type IL12 Fc in this model. The foregoing data demonstrate that the heterodimeric hIL2 IL12 muteins of the present disclosure are useful in treating neoplastic diseases in combination with adjunctive therapeutic agents, particularly IL2, IL2 muteins, and checkpoint inhibitors, such as anti-PD1 antibodies.

[0273] Synthesis of heterodimeric hIL12Fc muteins and heterodimeric hIL23Fc muteins The heterodimeric hIL12Fc mutein and heterodimeric hIL23Fc mutein of the present disclosure comprise a polypeptide.However, in some embodiments, the heterodimeric hIL12Fc mutein and heterodimeric hIL23Fc mutein of the present disclosure comprise a non-peptidyl component such as a PEG molecule.The process for PEGylating a protein is discussed elsewhere herein.The following relates to the synthesis of the polypeptide component of the heterodimeric hIL12Fc mutein and heterodimeric hIL23Fc mutein of the present disclosure, including hP40MFc, hP19Fc, and hP35Fc polypeptide subunits, and the recombinant production of the heterodimeric hIL12Fc mutein and heterodimeric hIL23Fc mutein of the present disclosure.

[0274] solid phase synthesis The following provides guidance to enable solid phase and recombinant synthesis of the polypeptide hP40MFc, hP19Fc, and hP35Fc domains of the heterodimeric hIL12Fc muteins and heterodimeric hIL23Fc muteins of the present disclosure. In embodiments where only a portion of the heterodimeric hIL12Fc muteins or heterodimeric hIL23Fc muteins is a polypeptide, it is understood that the hP40MFc, hP19Fc, and hP35Fc polypeptidyl domains of the heterodimeric hIL12Fc muteins or heterodimeric hIL23Fc muteins are process intermediates that may undergo further processing to complete the synthesis of the desired heterodimeric hIL12Fc muteins or heterodimeric hIL23Fc muteins.

[0275] The hP40MFc, hP19Fc, and hP35Fc polypeptide domains of the heterodimeric hIL12Fc muteins and the heterodimeric hIL23Fc muteins may be produced by conventional methodologies for polypeptide construction, including recombinant synthesis or solid phase synthesis, as described in further detail below.

[0276] chemical synthesis In addition to producing mutant polypeptides through expression of nucleic acid molecules that have been altered by recombinant molecular biology techniques, the hP40MFc, hP19Fc, and hP35Fc polypeptide domains of heterodimeric hIL12Fc muteins and heterodimeric hIL23Fc muteins can be chemically synthesized. Chemically synthesized polypeptides are routinely produced by those skilled in the art. Chemical synthesis includes direct peptide synthesis by chemical means of the hP40MFc, hP19Fc, and hP35Fc polypeptide domains of heterodimeric hIL12Fc muteins and heterodimeric hIL23Fc muteins that exhibit the described properties. This method can incorporate natural and non-natural amino acids at desired positions that facilitate the linkage of certain molecules (e.g., PEG).

[0277] In some embodiments, the hP40MFc, hP19Fc, and hP35Fc polypeptide domains of the heterodimeric hIL12Fc muteins and heterodimeric hIL23Fc muteins of the present disclosure can be prepared by chemical synthesis.The chemical synthesis of the hP40MFc, hP19Fc, and hP35Fc polypeptide domains of the heterodimeric hIL12Fc muteins and heterodimeric hIL23Fc muteins can proceed via liquid phase or solid phase.The use of solid phase peptide synthesis (SPPS) allows the incorporation of unnatural amino acids and / or peptide / protein backbone modifications. Various types of SPPS can be utilized to synthesize the hP40MFc, hP19Fc, and hP35Fc polypeptide domains of the heterodimeric hIL12Fc muteins and heterodimeric hIL23Fc muteins of the present disclosure and are known in the art (e.g., Ganesan A. (2006) Mini Rev. Med. Chem. 6:3-10; and Camarero JA et al., (2005) Protein Pept Lett. 12:723-8). During chemical synthesis, the alpha functional group and any reactive side chains may be protected by acid- or base-labile groups that are stable under conditions that link amide bonds, but can be easily cleaved without compromising the formed peptide chain.

[0278] In solid phase synthesis, either the N-terminal or C-terminal amino acid can be coupled to a suitable support material. A suitable support material is one that is inert to the reagents and reaction conditions for the stepwise condensation and cleavage reactions of the synthesis process and is not soluble in the reaction medium used. Examples of commercially available support materials include styrene / divinylbenzene copolymers modified with reactive groups and / or polyethylene glycol; chloromethylated styrene / divinylbenzene copolymers; hydroxymethylated or aminomethylated styrene / divinylbenzene copolymers, etc. Peptide synthesis, typically in an automated peptide synthesizer, can be performed according to conventional methods to sequentially couple protected amino acids.

[0279] At the end of solid phase synthesis, the peptide is cleaved from the support material while the side chain protecting groups are simultaneously cleaved. The resulting peptide can be purified by a variety of chromatographic methods, including, but not limited to, hydrophobic adsorption chromatography, ion exchange chromatography, distribution chromatography, high pressure liquid chromatography (HPLC), and reverse phase HPLC.

[0280] Recombinant production The hP40MFc and hp35Fc subunits of the heterodimeric hIL12Fc mutein, or the hP40MFc and hp19Fc subunits of the heterodimeric hIL23Fc mutein, or the complete heterodimeric hIL12Fc mutein and heterodimeric hIL23Fc mutein of the present disclosure may be produced by recombinant DNA technology. In a typical implementation of recombinant production of a polypeptide, a nucleic acid sequence encoding the desired polypeptide is incorporated into an expression vector suitable for the host cell in which expression will take place, and the nucleic acid sequence is operably linked to one or more expression control sequences encoded by the vector and functional in the target host cell. The recombinant protein may be recovered by disrupting the host cell, or from the cell culture medium if a secretory leader sequence (signal peptide) is incorporated into the polypeptide. The recombinant protein may be purified and concentrated for further use, including incorporation.

[0281] Synthesis of nucleic acid sequences encoding the hP40MFc, hP19Fc, and hP35Fc domains of heterodimeric hIL12Fc muteins or heterodimeric hIL23Fc muteins In some embodiments, the hP40MFc, hP19Fc, and hP35Fc polypeptide domains of the heterodimeric hIL12Fc mutein or heterodimeric hIL23Fc mutein are produced by recombinant methods using a nucleic acid sequence encoding the hP40MFc, hP19Fc, and hP35Fc polypeptide domains of the heterodimeric hIL12Fc mutein or heterodimeric hIL23Fc mutein (or a fusion protein comprising the hP40MFc, hP19Fc, and hP35Fc polypeptide domains of the heterodimeric hIL12Fc mutein or heterodimeric hIL23Fc mutein). Nucleic acid sequences encoding the desired hP40MFc, hP19Fc, and hP35Fc polypeptide domains of the heterodimeric hIL12Fc mutein or heterodimeric hIL23Fc mutein can be synthesized by chemical means using an oligonucleotide synthesizer.

[0282] The nucleic acid molecule of the present disclosure is not limited to the sequence that codes for a polypeptide. It may also include some or all of the non-coding sequence upstream or downstream from the coding sequence (e.g., the coding sequence of the hP40MFc, hP19Fc, and hP35Fc polypeptide domains of heterodimeric hIL12Fc mutein or heterodimeric hIL23Fc mutein). Those skilled in the art of molecular biology are familiar with routine procedures for isolating nucleic acid molecules. For example, nucleic acid molecules can be produced by treating genomic DNA with restriction endonucleases or by carrying out polymerase chain reaction (PCR). If the nucleic acid molecule is ribonucleic acid (RNA), the molecule can be produced, for example, by in vitro transcription.

[0283] Nucleic acid molecules encoding the hP40MFc, hP19Fc, and hP35Fc polypeptide domains (and fusions thereof) of heterodimeric hIL12Fc muteins or heterodimeric hIL23Fc muteins may comprise natural sequences or may comprise sequences that differ from those occurring in nature but that, due to the degeneracy of the genetic code, encode the same polypeptide. These nucleic acid molecules may consist of RNA or DNA (e.g., genomic DNA, cDNA, or synthetic DNA, e.g., produced by phosphoramidite-based synthesis), or combinations or modifications of nucleotides found within these types of nucleic acids. Furthermore, the nucleic acid molecules may be double-stranded or single-stranded (i.e., either the sense or antisense strand).

[0284] Nucleic acid sequences encoding the hP40MFc, hP19Fc, and hP35Fc polypeptide domains of heterodimeric hIL12Fc muteins or heterodimeric hIL23Fc muteins may be obtained from various commercial suppliers that provide custom synthesis of nucleic acid sequences. The amino acid sequence variants of the hP40MFc, hP19Fc, and hP35Fc polypeptide subunits of heterodimeric hIL12Fc muteins and heterodimeric hIL23Fc muteins of the present disclosure are prepared by introducing appropriate nucleotide changes into the coding sequence based on the genetic code well known in the art. Such variations are insertions, substitutions, and / or deletions of residues as mentioned. Any combination of insertions, substitutions, and / or deletions can be added to arrive at the final construct. Provided that the final construct has the desired biological activity as defined herein.

[0285] In some embodiments, the nucleic acid sequence encoding the wild-type human p40 signal peptide and the hP40MFc polypeptide is selected from the group consisting of SEQ ID NOs:94, 97, 99, 100, 102, 105, 118, 120, 126, 128, 131, 134, 137, 140, 143, 146, 149, and 152.

[0286] In some embodiments, the nucleic acid sequence encoding the wild-type human p35 signal peptide and the hP40MFc polypeptide is selected from the group consisting of SEQ ID NOs:95, 96, 98, 103, 104, 106, and 123.

[0287] Methods for constructing DNA sequences encoding the hP40MFc, hP19Fc, and hP35Fc polypeptide subunits of heterodimeric hIL12Fc muteins and heterodimeric hIL23Fc muteins, and for expressing these sequences in a suitable transformed host, include, but are not limited to, using PCR-assisted mutagenesis.Mutations consisting of deletion or addition of amino acid residues to the hP40MFc, hP19Fc, and hP35Fc polypeptide subunits of heterodimeric hIL12Fc muteins or heterodimeric hIL23Fc muteins can also be added using standard recombinant methods.In the case of deletion or addition, the nucleic acid molecule encoding the hP40MFc, hP19Fc, and hP35Fc polypeptide subunits of heterodimeric hIL12Fc muteins or heterodimeric hIL23Fc muteins is optionally digested with a suitable restriction endonuclease. The resulting fragments may be expressed directly or may be further manipulated, for example, by ligating to a second fragment. Ligation may be facilitated if the two ends of the nucleic acid molecule contain overlapping complementary nucleotides, although blunt-ended fragments may also be ligated. Nucleic acids generated by PCR may also be used to generate a variety of mutant sequences.

[0288] The hP40MFc, hP19Fc, and hP35Fc polypeptide subunits of the heterodimeric hIL12Fc mutein and heterodimeric hIL23Fc mutein of the present disclosure may be produced recombinantly directly or as a fusion polypeptide with a heterologous polypeptide, such as a signal peptide or other polypeptide having a specific cleavage site at the N-terminus or C-terminus of the hP40MFc, hP19Fc, and hP35Fc polypeptide subunit of the heterodimeric hIL12Fc mutein or heterodimeric hIL23Fc mutein. In general, the nucleic acid sequence encoding the signal sequence peptide may be a component of the vector or may be part of the coding sequence inserted into the vector. If a heterologous signal sequence is used, it is preferably a signal peptide that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell.

[0289] In some embodiments, the signal peptide is selected from the group consisting of human serum albumin signal peptide, prolactin albumin signal peptide, human IL2 signal peptide, human trypsinogen-2, human CD-5, human immunoglobulin kappa light chain, human azurocidin, Gaussia luciferase, and functional derivatives thereof. Specific amino acid substitutions for increasing secretion efficiency using signal peptides are described in Stern, et al. (2007) Trends in Cell and Molecular Biology 2:1-17 and Kober, et al. (2013) Biotechnol Bioeng. 1110(4):1164-73. Alternatively, the signal peptide may be a synthetic sequence prepared according to established principles. See, e.g., Nielsen, et al. (1997) Protein Engineering 10(1):1-6 (Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites); Bendtsen, et al (2004) J. Mol. Biol 340(4):783-795 (Improved Prediction of Signal Peptides SignalP 3.0); Petersen, et al (2011) Nature Methods 8:785-796 (Signal P 4.0; discriminating signal peptides from transmembrane regions).

[0290] In some embodiments, the signal peptides of the hP40MFc, hP19Fc, and hP35Fc polypeptide subunits of the heterodimeric hIL12Fc mutein or the heterodimeric IL23Fc mutein are the naturally occurring hP40, hP19, and hP35 signal peptides, respectively (i.e., human hP40, hP19, and hP35 signal sequences). In some embodiments, the signal peptide of the hP35Fc sequence has the amino acid sequence TIFF2024539139000056.tif4128 is a naturally occurring wild-type human p35 sequence. In some embodiments, the signal peptide of the hP40MFc sequence is The native wild-type human p40 sequence has the sequence TIFF2024539139000057.tif4128.

[0291] The incorporation of a signal peptide depends on whether it is desired to secrete the heterodimeric hIL12Fc mutein or the heterodimeric hIL23Fc mutein from the recombinant cell in which it is produced.If the selected cell is prokaryotic, it is generally preferred that the DNA sequence does not code for a signal sequence.If the host cell for expressing the heterodimeric hIL12Fc mutein or the heterodimeric hIL23Fc mutein is eukaryotic, the signal peptides of the hP40MFc, hP19Fc, and hP35Fc polypeptide subunits are the natural hP40, hP19, and hP35 signal peptides, respectively (i.e., human hP40, hP19, and hP35 signal sequences). Alternatively, heterologous mammalian signal sequences, such as signal sequences derived from secreted polypeptides of the same or related species, as well as viral secretory leaders, such as herpes simplex gD signal peptide, may be suitable. When the recombinant host cell is a yeast cell, such as Saccharomyces cerevisiae, the alpha-mating factor secretion signal sequence may be used for extracellular secretion of the heterodimeric hIL12Fc mutein or heterodimeric hIL23Fc mutein, or one or more of its hP40MFc, hP19Fc, and hP35Fc polypeptide subunits, into the culture medium, as described in Singh, U.S. Patent No. 7,198,919B1.

[0292] When one or more of the heterodimeric hIL12Fc mutein or heterodimeric hIL23Fc mutein or its hP40MFc, hP19Fc, and hP35Fc polypeptide subunits are expressed as a chimera (e.g., a fusion protein comprising the heterodimeric hIL12Fc mutein or heterodimeric hIL23Fc mutein or its hP40MFc, hP19Fc, and hP35Fc polypeptide subunits and a heterologous polypeptide sequence), the chimeric protein may be encoded by a hybrid nucleic acid molecule comprising a first sequence encoding all or a portion of the polypeptide domains of the heterodimeric hIL12Fc mutein or heterodimeric hIL23Fc mutein or its hP40MFc, hP19Fc, and hP35Fc polypeptide subunits, and a second sequence encoding all or a portion of the heterologous polypeptide. For example, the polypeptide domains of the heterodimeric hIL12Fc muteins or heterodimeric hIL23Fc muteins or their hP40MFc, hP19Fc, and hP35Fc polypeptide subunits described herein may be fused to a chelating peptide. Incorporation of a chelating peptide facilitates purification by immobilized metal affinity chromatography (IMAC), as described in U.S. Pat. No. 4,569,794, issued Feb. 11, 1986 to Smith et al. Examples of chelating peptides useful in the practice of the present disclosure are described in Smith et al., supra, and U.S. Pat. No. 5,320,663, issued May 10, 1995 to Dobeli et al. A particular transition metal chelating peptide useful in the practice of the present disclosure is a polypeptide containing 3-6 consecutive histidine residues, e.g., a 6-histidine (His) 6 peptide, often referred to in the art as a "His-tag." Alternatively, a hemagglutinin tag may be incorporated into the chimeric protein to facilitate purification of the protein expressed in eukaryotic cells. The first and second should not be understood as limitations on the orientation of the elements of the fusion protein, and the heterologous polypeptide may be linked to the N-terminus and / or C-terminus of the polypeptide domain of the heterodimeric hIL12Fc mutein or heterodimeric hIL23Fc mutein.For example, the N-terminus may be linked to a targeting domain and the C-terminus may be linked to a hexahistidine tag purification handle.

[0293] The amino acid sequences of the P40MFc, hP19Fc, and hP35Fc polypeptide subunits (or fusions / chimeras) of the heterodimeric hIL12Fc muteins and heterodimeric hIL23Fc muteins to be expressed can be used to construct back-translated genes. DNA oligomers can be synthesized that contain nucleotide sequences that code for the P40MFc, hP19Fc, and hP35Fc polypeptide subunits of the heterodimeric hIL12Fc muteins or heterodimeric hIL23Fc muteins. For example, several small oligonucleotides that code for portions of the desired polypeptide can be synthesized and then ligated. Each oligonucleotide typically contains a 5' or 3' overhang for complementary assembly.

[0294] In some embodiments, the nucleic acid sequences encoding the hP40MFc, hP19Fc, and hP35Fc polypeptide subunits of the heterodimeric hIL12Fc muteins and heterodimeric hIL23Fc muteins of the present disclosure may be "codon-optimized" to facilitate expression in a particular host cell type. Techniques for codon optimization in a wide variety of expression systems, including mammalian host cells, yeast host cells, and bacterial host cells, are well known in the art, and there are online tools to provide codon-optimized sequences for expression in various host cell types. See, for example, Hawash, et al., (2017) 9:46-53 and Mauro and Chappell in Recombinant Protein Expression in Mammalian Cells: Methods and Protocols , edited by David Hacker (Human Press New York). Additionally, there are a variety of web-based online software packages freely available to aid in the preparation of codon-optimized nucleic acid sequences.

[0295] Control Elements The nucleic acid sequence encoding the hP40MFc and hp35Fc subunits of the heterodimeric hIL12Fc mutein (or the hP40MFc and hp19Fc subunits of the heterodimeric hIL23Fc mutein), prepared as provided above, is operably linked to suitable genetic control elements that allow the expression of the polypeptide in a host cell to be transformed by the expression vector. The term "operably linked" refers to the linkage of polynucleotide elements in a functional relationship. A nucleic acid sequence is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if it controls the transcription of the polypeptide. A ribosome binding site is operably linked to a coding sequence if it is positioned to allow translation, and a nucleic acid encoding a signal peptide is operably linked to a nucleic acid sequence encoding such a polypeptide if it is expressed as a fusion protein and is involved in directing the fusion protein to the cell membrane or in the secretion of the polypeptide. Typically, operably linked nucleotide sequences are contiguous. However, because enhancers generally function when separated by several kilobases from the promoter, and intron sequences can vary in length, some polynucleotide elements may be operably linked but physically separate and may also function in trans from different alleles or chromosomes.

[0296] The specific type of control element required to induce expression depends on the cell type to be transformed. In the practice of the present invention, the cell to be transformed is a mammalian T cell. The term control element refers collectively to promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, replication origins, internal ribosome entry sites ("IRES"), enhancers, transcription enhancers to increase mRNA expression levels, sequences that code for suitable ribosome binding sites, and sequences that terminate transcription and translation to affect the replication, transcription, and translation of polypeptide coding sequences in recipient cells. Expression vectors also usually contain a replication origin that allows this vector to replicate independently of host cells.

[0297] promoter In one embodiment, the nucleic acid sequence to be expressed (e.g., encoding hP40MFc and / or hP35Fc) is operably linked to a promoter sequence. The term "promoter" is used in the conventional sense to refer to a nucleotide sequence at which the initiation and rate of transcription of a coding sequence are controlled. A promoter contains a site for binding of RNA polymerase and also contains a site for binding of a regulatory factor (e.g., a repressor or a transcription factor). A promoter may be natural or synthetic. A promoter may be constitutively active, may be activated in response to an external stimulus (inducible), may be active in a particular cell type or cell state (tissue-specific or tumor-specific) promoter, and / or may be regulatable. The term "inducible promoter" refers to a promoter that facilitates transcription of a bioactive polypeptide preferably (or exclusively) under certain conditions and / or in response to an external chemical or other stimulus. Examples of inducible promoters are known in the scientific literature (see, e.g., Yoshida et al., Biochem. Biophys. Res. Comm., 230:426-430 (1997); Iida et al., J. Virol., 70(9): 6054-6059 (1996); Hwang et al., J. Virol., 71(9): 7128-7131 (1997); Lee et al., Mol. Cell. Biol., 17(9): 5097-5105 (1997); and Dreher et al., J. Biol. Chem., 272(46): 29364-29371 (1997)). Examples of radiation-inducible promoters include the EGR-1 promoter. Boothman et al., volume 138, supplement pages S68-S71 (1994).

[0298] For example, in some embodiments in which a nucleic acid sequence (or a vector comprising the same) encoding a heterodimeric hIL12 mutein (or a heterodimeric hIL23 mutein) of the present disclosure is administered to a subject, the nucleic acid sequence encoding the heterodimeric hIL12 mutein (or a heterodimeric hIL23 mutein) is operably linked to a tissue-specific promoter. The use of a tissue-specific promoter enhances expression in a particular tissue or cell type. In some embodiments, the promoter is a tumor-specific promoter. Tissue-specific and tumor-specific promoters, such as pancreatic (Palmiter et al., Cell, 50:435 (1987)), liver-specific (Rovet et al., J. Biol. Chem., 267:20765 (1992); Lemaigne et al., J. Biol. Chem., 268:19896 (1993); Nitsch et al., Mol. Cell. Biol., 13:4494 (1993)), stomach-specific (Kovarik et al., J. Biol. Chem., 268:9917 (1993)), pituitary-specific (Rhodes et al., Genes Dev., 7:913 (1993)), and the like. (1993), and prostate-specific promoters (Henderson et al., U.S. Patent No. 5,698,443, issued December 16, 1997) are well known in the art. In some embodiments of the invention, the nucleic acid sequence encoding the hp35Fc and / or hP40MFc sequence is operably linked to a human cytomegalovirus (CMV) promoter.

[0299] Multicistronic expression constructs When expressing a multi-subunit protein in the practice of the invention, each polypeptide subunit may be operably linked to an expression control sequence (monocistronic), or multiple polypeptides may be encoded by a polycistronic construct in which multiple polypeptides are expressed under the control of a single expression control sequence. An example of an element that can be used to facilitate polycistronic expression is an internal ribosome entry site (IRES) element or the foot and mouth disease virus protein 2A (FMVD2A) system. A wide variety of IRES sites are known (see, for example, Doudna JA, Sarnow P. Translation initiation by viral internal ribosome entry sites. In: Translational Control in Biology and Medicine(See, Mathews et al, Ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 2007. pp. 129-154; http: / / www.IRESite.org). Examples of IRES elements include picornavirus IRES from poliovirus, rhinovirus, encepahlomyocardits virus, aphthovirus IRES from foot and mouth disease virus, IRES cricket paralysis virus (CrPV), hepatitis A IRES from hepatitis A virus, hepatitis C IRES from hepatitis C virus, pestivirus IRES from swine fever or bovine diarrhea virus, cripavirus IRES, and mammalian IRES elements such as fibroblast growth factor-1 IRES, fibroblast growth factor-2 IRES, PDGF IRES, VEGF IRES, IGF-2 IRES. The use of an IRES element typically results in significantly lower expression of the second protein of the polycistronic message. The use of the FMDV2A system results in more efficient production of downstream proteins because multiple proteins are first expressed as fusion proteins containing an autoproteolytic FMDV2A domain that cleaves the polyprotein into functional subunits. Ryan and Drew (1994) EMBO J. 13(4):928-933. Depending on the construction of the polycistronic coding sequence, particularly to facilitate restriction endonuclease sites, the use of the FMDV2A system can frequently result in the addition of a few amino acids to the carboxy terminus of the upstream protein.

[0300] In preparing a bicistronic nucleic acid sequence encoding a heterodimeric hIL23Fc mutein or a heterodimeric hIL12Fc mutein, the nucleic acid sequences encoding the hP40MFc and hP19Fc subunits of a heterodimeric hIL23Fc mutein of the present disclosure, or the nucleic acid sequences encoding the hP40MFc and hp35Fc subunits of a heterodimeric hIL12Fc mutein of the present disclosure, may be provided in a bicistronic expression cassette for co-expression of the subunits in a mammalian host cell. In some aspects, the present disclosure provides bicistronic nucleic acids arranged as shown below: 5'-hP40MFc-P2A-hP35Fc-3' 5'-hP35Fc-P2A-hP40MFc-3' 5'-hP40Fc-IRES-hP35Fc-3' 5'-hP35Fc-IRES-hP40MFc-3' 5'-hP40MFc-T2A-hP35Fc-3' 5'-hP35Fc-T2A-hP40MFc-3' 5'-hP40MFc-P2A-hP19Fc-3' 5'-hP19Fc-P2A-hP40MFc-3' 5'-hP40Fc-IRES-hP19Fc-3' 5'-hP35Fc-IRES-hP40MFc-3' 5'-hP40Fc-T2A-hP19Fc-3', and 5'-hP35Fc-T2A-hP40MFc-3'.

[0301] vector Once assembled (by synthesis, site-directed mutagenesis, or another method), the nucleic acid sequence encoding the expression cassette of the P40MFc, hP19Fc, and hP35Fc polypeptide subunits of the polypeptide domain of the heterodimeric hIL12Fc mutein or heterodimeric hIL23Fc mutein is inserted into a vector. A variety of expression vectors are available for use in a variety of host cells and are typically selected based on the host cell for expression. Expression vectors typically include, but are not limited to, one or more of the following: an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. Vectors include viral vectors, plasmid vectors, integrating vectors, and the like. Plasmids are an example of non-viral vectors. To facilitate efficient expression of the recombinant polypeptide, the nucleic acid sequence encoding the polypeptide sequence to be expressed is operably linked to transcriptional and translational regulatory control sequences that function in the selected expression host.

[0302] Expression vectors typically contain a selection gene, also called a selection marker. This gene encodes a protein necessary for the survival or growth of transformed host cells grown in a selective culture medium. Host cells not transformed with a vector containing the selection gene will not survive in the culture medium. Typical selection genes encode (a) a protein that confers resistance to antibiotics or other toxins, such as ampicillin, neomycin, methotrexate, or tetracycline, (b) a protein that complements an auxotrophic defect, or (c) a protein that supplies a vital nutrient unavailable from a complex medium.

[0303] The expression vectors for the hP40MFc, hP19Fc, and hP35Fc polypeptide subunits of the heterodimeric hIL12Fc muteins and heterodimeric hIL23Fc muteins of the present disclosure contain a regulatory sequence that is recognized by the host organism and is operably linked to the nucleic acid sequence encoding the hP40MFc, hP19Fc, and hP35Fc polypeptide subunits of the heterodimeric hIL12Fc muteins and heterodimeric hIL23Fc muteins. The terms "regulatory control sequence", "regulatory sequence", or "expression control sequence" are used interchangeably herein to refer to promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). See, e.g., Goeddel (1990) in Gene Expression Technology: Methods in Enzymology 185 (Academic Press, San Diego CA USA). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of a nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of an expression vector may depend on factors such as the choice of the host cell to be transformed, the level of expression of protein desired, and the like. In selecting an expression control sequence, various factors that will be appreciated by those skilled in the art must be considered. These include, for example, the relative strength of the sequence, its controllability, and, particularly in terms of potential secondary structures, compatibility with the actual DNA sequences encoding the hP40MFc, hP19Fc, or hP35Fc polypeptide subunits of the subject heterodimeric hIL12Fc muteins and heterodimeric hIL23Fc muteins.

[0304] In some embodiments, the regulatory sequence is a promoter, which is selected, for example, based on the cell type in which expression is desired. A promoter is a non-translated sequence located upstream (5') of the start codon of a structural gene (generally within about 100-1000 bp) that controls the transcription and translation of a particular nucleic acid sequence operably linked to it. Such promoters are typically divided into two classes, inducible promoters and constitutive promoters. An inducible promoter is a promoter that initiates high levels of transcription from DNA under its control in response to some change in culture conditions, e.g., the presence or absence of a nutrient or a change in temperature. A large number of promoters recognized by a variety of potential host cells are well known.

[0305] T7 promoter can be used in bacteria, polyhedrin promoter can be used in insect cells, and cytomegalovirus or metallothionein promoter can be used in mammalian cells.Similarly, for higher eukaryotes, tissue-specific promoters and cell type-specific promoters are widely available.These promoters are named after their ability to induce the expression of nucleic acid molecules in certain tissues or cell types in the body.Those skilled in the art are familiar with a great number of promoters and other regulatory elements that can be used to induce nucleic acid expression.

[0306] Transcription from the vector in mammalian host cells may be controlled by promoters derived from the genomes of viruses, such as polyoma virus, fowlpox virus, adenovirus (e.g., human adenovirus serotype 5), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retroviruses (e.g., murine stem cell virus), hepatitis B virus, and most preferably simian virus 40 (SV40), heterologous mammalian promoters, such as the actin promoter, PGK (phosphoglycerate kinase), or immunoglobulin promoters, heat shock promoters, provided such promoters are compatible with the host cell system. Conveniently, the early and late promoters of the SV40 virus are obtained as an SV40 restriction fragment, which also contains the SV40 viral origin of replication.

[0307] Transcription by higher eukaryotes is often increased by inserting enhancer sequences into the vector. Enhancers are cis-acting DNA elements, usually about 10-300 bp, that act on promoters to increase transcription. Enhancers are relatively orientation and position independent, and have been found 5' and 3' to the transcription unit, in introns, and within the coding sequence itself. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, alpha-fetoprotein, and insulin). However, enhancers from eukaryotic viruses are usually used. Examples include the SV40 enhancer on the late side of the replication origin, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. Enhancers may be spliced ​​into the expression vector at a position 5' or 3' to the coding sequence, and are preferably located at a site 5' from the promoter. Expression vectors used in eukaryotic host cells also contain sequences required for transcription termination and sequences required for stabilizing mRNA.Such sequences can generally be obtained from the 5' untranslated region, or sometimes the 3' untranslated region, of eukaryotic or viral DNA or cDNA.Construction of suitable vectors containing one or more of the components listed above uses standard techniques.

[0308] In addition to sequences that facilitate transcription of the inserted nucleic acid molecule, vectors may contain origins of replication and other genes that code for selection markers. For example, the neomycin resistance (neoR) gene confers G418 resistance to cells in which it is expressed, thus allowing phenotypic selection of transfected cells. Further examples of marker or reporter genes include β-lactamase, chloramphenicol acetyltransferase (CAT), adenosine deaminase (ADA), dihydrofolate reductase (DHFR), hygromycin-B-phosphotransferase (HPH), thymidine kinase (TK), lacZ (encoding β-galactosidase), and xanthine guanine phosphoribosyltransferase (XGPRT). Those skilled in the art can easily determine whether a particular regulatory element or selection marker is suitable for use in a particular experimental situation. Correct assembly of an expression vector can be confirmed by nucleotide sequencing, restriction enzyme mapping, and expression of a biologically active polypeptide in a suitable host.

[0309] In some embodiments of the present disclosure, an expression cassette comprising a CMV promoter and a nucleic acid sequence encoding hP35Fc and hP40MFc polypeptides is inserted into a pCDNA3.4 mammalian expression vector (Life Technologies, Carlsbad, Calif.). In some embodiments of the present disclosure, an expression cassette comprising a CMV promoter and a nucleic acid sequence encoding hP35Fc and hP40MFc polypeptides is inserted into the multiple cloning site of a pExSyn2.0 expression vector, as prepared according to the disclosure of Example 1.

[0310] host cell Furthermore, the present disclosure provides prokaryotic or eukaryotic cells that contain and express the nucleic acid molecules that code for the hP40MFc, hP19Fc, and hP35Fc polypeptide subunits of the heterodimeric hIL12Fc muteins and the heterodimeric hIL23Fc muteins.The cells of the present disclosure are transfected cells, i.e., cells into which nucleic acid molecules, such as the nucleic acid molecules that code for the hP40MFc, hP19Fc, and hP35Fc polypeptide subunits of the heterodimeric hIL12Fc muteins and the heterodimeric hIL23Fc muteins, are introduced by recombinant DNA techniques.The progeny of such cells are also considered to be within the scope of the present disclosure.

[0311] Host cells are typically selected according to compatibility with the selected expression vector, toxicity of the product encoded by the DNA sequence, secretion characteristics, ability to correctly fold the polypeptide, fermentation or culture requirements, and ease of purification of the product encoded by the DNA sequence. Suitable host cells for cloning or expressing the DNA in the vector herein are prokaryotic cells, yeast cells, or higher eukaryotic cells.

[0312] In some embodiments, the hP40MFc, hP19Fc, and hP35Fc polypeptide subunits of the heterodimeric hIL12Fc muteins and heterodimeric hIL23Fc muteins and biologically active variants and fragments thereof can also be produced in eukaryotes, such as yeast or human cells. Suitable eukaryotic host cells include insect cells (examples of baculovirus vectors available for protein expression in cultured insect cells, such as Sf9 cells, include the pAc series (Smith et al. (1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170:31-39)); yeast cells (examples of vectors for expression in the yeast S. cerevisiae include pYepSecl (Baldari et al. (1987) EMBO J. 6:229-234), pMFa (Kurjan and Herskowitz (1982) Cell 30:933-943), pJRY88 (Schultz et al. (1987) Gene 54:113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), and pPicZ (Invitrogen Corporation, San Diego, Calif.); or mammalian cells (mammalian expression vectors include pCDM8 (Seed (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6:187:195)).

[0313] Examples of useful mammalian host cell lines include mouse L cells (LM[TK-], ATCC#CRL-2648), Expi293 cells, SV40 transformed monkey kidney CV1 line (COS-7, ATCC CRL 1651); human embryonic kidney cells (HEK293 cells or HEK293 cells subcloned for growth in suspension culture; baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO); mouse Sertoli cells (TM4); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1 587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells; MRC5 cells; FS4 cells; and human hepatoma line (HepG2). In mammalian cells, control functions of expression vectors are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, and simian virus 40.

[0314] In some embodiments, the recombinant hP40MFc, hP19Fc, and hP35Fc polypeptide subunits of the heterodimeric hIL12Fc muteins and heterodimeric hIL23Fc muteins may be glycosylated or aglycosylated depending on the host organism used to produce the hP40MFc, hP19Fc, and hP35Fc polypeptides.

[0315] Transfection The expression construct can be introduced into host cells to produce the recombinant polypeptide domain of heterodimeric hIL12Fc mutein or heterodimeric hIL23Fc mutein disclosed herein or produce its biologically active mutein.Vector DNA can be introduced into prokaryotic or eukaryotic cells by conventional transformation or transfection methods.Suitable methods for transforming or transfecting host cells can be found in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, NY) and other standard molecular biology laboratory manuals.

[0316] To facilitate the transfection of target cells, target cells may be directly exposed to non-viral vectors under conditions that facilitate the uptake of non-viral vectors.Examples of conditions that facilitate the uptake of foreign nucleic acid by mammalian cells are well known in the art, and include, but are not limited to, chemical means (e.g., Lipofectamine®, Thermo-Fisher Scientific), high salt, and magnetic field (electroporation).

[0317] In some embodiments, the nucleic acid sequence encoding hP35Fc and the nucleic acid sequence encoding hP40MFc are each provided as separate expression vectors, and then introduced into a host cell by co-transfection. In some embodiments, a first recombinant expression vector comprising a nucleic acid sequence encoding hp35Fc operably linked to a promoter functional in mammalian cells, and a second recombinant expression vector comprising a nucleic acid sequence encoding hP40MFc operably linked to a promoter functional in mammalian cells, are introduced into a mammalian host cell by co-transfection. In some embodiments, the promoter functional in mammalian cells of the first recombinant expression vector and the second recombinant expression vector is a CMV promoter. In some embodiments, the first recombinant expression vector and the second recombinant expression vector are pCDNA3.4 mammalian expression vector (Life Technologies, Carlsbad, CA). In some embodiments, the first recombinant expression vector and the second recombinant expression vector are pExSyn2.0 expression vectors, as prepared according to the disclosure of Example 1.

[0318] In some aspects, the disclosure provides a recombinant mammalian host cell comprising a first recombinant expression vector comprising a nucleic acid sequence encoding hp35Fc operably linked to a promoter functional in a recombinant mammalian host cell, and a second recombinant expression vector comprising a nucleic acid sequence encoding hP40MFc operably linked to a promoter functional in a recombinant mammalian host cell. In some aspects, the nucleic acid sequence encoding hp35Fc and the nucleic acid sequence encoding hP40MFc further encode a signal peptide. In some aspects, the signal peptide of the hp35Fc polypeptide is wild-type human p35 signal peptide (SEQ ID NO:179). In some aspects, the signal peptide of the hP40MFc polypeptide is wild-type human p40 signal peptide (SEQ ID NO:180). In some aspects, the nucleic acid sequences encoding hp35Fc and hP40MFc signal peptide. In some aspects, the recombinant mammalian host cell is selected from the group consisting of CHO cells and 293 cells. In some aspects, the disclosure provides a recombinant CHO cell comprising a first recombinant expression vector comprising a nucleic acid sequence encoding hp35Fc operably linked to a promoter functional in a CHO cell, and a second recombinant expression vector comprising a nucleic acid sequence encoding hP40MFc operably linked to a promoter functional in a CHO cell. In some aspects, the disclosure provides a recombinant CHO cell comprising a first recombinant expression vector comprising a nucleic acid sequence encoding hp35Fc operably linked to a CMV promoter, and a second recombinant expression vector comprising a nucleic acid sequence encoding hP40MFc operably linked to a CMV promoter. In some aspects, the first recombinant expression vector and the second recombinant expression vector are pCDNA3.4 mammalian expression vector (Life Technologies, Carlsbad, CA). In some aspects, the first recombinant expression vector and the second recombinant expression vector are pExSyn2.0 expression vectors as prepared according to the disclosure of Example 1.In some aspects, the disclosure provides a recombinant mammalian host cell transformed with a first expression vector comprising a nucleic acid sequence encoding hP40MFc selected from the group consisting of SEQ ID NOs:80, 83, 121, 141, 144, 129, 135, 138, 147, 150, and 153, and a second expression vector comprising a nucleic acid sequence encoding hP35 Fc selected from the group consisting of SEQ ID NOs:81, 82, and 124.

[0319] In some aspects, the disclosure provides a recombinant mammalian host cell transformed with a first expression vector comprising a nucleic acid sequence encoding SEQ ID NO:80 and a second expression vector comprising a nucleic acid sequence encoding SEQ ID NO:81.

[0320] In some aspects, the disclosure provides a recombinant mammalian host cell transformed with a first expression vector comprising a nucleic acid sequence encoding SEQ ID NO:121 and a second expression vector comprising a nucleic acid sequence encoding SEQ ID NO:124.

[0321] In some aspects, the disclosure provides a recombinant mammalian host cell transformed with a first expression vector comprising a nucleic acid sequence encoding SEQ ID NO:83 and a second expression vector comprising a nucleic acid sequence encoding SEQ ID NO:82.

[0322] In some aspects, the disclosure provides a recombinant mammalian host cell transformed with a first expression vector comprising a nucleic acid sequence encoding SEQ ID NO:141 and a second expression vector comprising a nucleic acid sequence encoding SEQ ID NO:124.

[0323] In some aspects, the disclosure provides a recombinant mammalian host cell transformed with a first expression vector comprising a nucleic acid sequence encoding SEQ ID NO:144 and a second expression vector comprising a nucleic acid sequence encoding SEQ ID NO:124.

[0324] In some aspects, the disclosure provides a recombinant mammalian host cell transformed with a first expression vector comprising a nucleic acid sequence encoding SEQ ID NO:129 and a second expression vector comprising a nucleic acid sequence encoding SEQ ID NO:124.

[0325] In some aspects, the disclosure provides a recombinant mammalian host cell transformed with a first expression vector comprising a nucleic acid sequence encoding SEQ ID NO:147 and a second expression vector comprising a nucleic acid sequence encoding SEQ ID NO:82.

[0326] In some aspects, the disclosure provides a recombinant mammalian host cell transformed with a first expression vector comprising a nucleic acid sequence encoding SEQ ID NO:150 and a second expression vector comprising a nucleic acid sequence encoding SEQ ID NO:82.

[0327] In some aspects, the disclosure provides a recombinant mammalian host cell transformed with a first expression vector comprising a nucleic acid sequence encoding SEQ ID NO:153 and a second expression vector comprising a nucleic acid sequence encoding SEQ ID NO:82.

[0328] In some aspects, the disclosure provides a recombinant mammalian host cell transformed with a first expression vector comprising a nucleic acid sequence encoding SEQ ID NO:135 and a second expression vector comprising a nucleic acid sequence encoding SEQ ID NO:124.

[0329] In some aspects, the disclosure provides a recombinant mammalian host cell transformed with a first expression vector comprising a nucleic acid sequence encoding SEQ ID NO:138 and a second expression vector comprising a nucleic acid sequence encoding SEQ ID NO:124.

[0330] In some aspects, the disclosure provides a recombinant mammalian host cell transformed with a first expression vector comprising the nucleic acid sequence of SEQ ID NO:94 and a second expression vector comprising the nucleic acid sequence of SEQ ID NO:95.

[0331] In some aspects, the disclosure provides a recombinant mammalian host cell transformed with a first expression vector comprising the nucleic acid sequence of SEQ ID NO:120 and a second expression vector comprising the nucleic acid sequence of SEQ ID NO:123.

[0332] In some aspects, the disclosure provides a recombinant mammalian host cell transformed with a first expression vector comprising the nucleic acid sequence of SEQ ID NO:97 and a second expression vector comprising the nucleic acid sequence of SEQ ID NO:96.

[0333] In some aspects, the disclosure provides a recombinant mammalian host cell transformed with a first expression vector comprising the nucleic acid sequence of SEQ ID NO:140 and a second expression vector comprising the nucleic acid sequence of SEQ ID NO:123.

[0334] In some aspects, the disclosure provides a recombinant mammalian host cell transformed with a first expression vector comprising the nucleic acid sequence of SEQ ID NO:143 and a second expression vector comprising the nucleic acid sequence of SEQ ID NO:123.

[0335] In some aspects, the disclosure provides a recombinant mammalian host cell transformed with a first expression vector comprising the nucleic acid sequence of SEQ ID NO:128 and a second expression vector comprising the nucleic acid sequence of SEQ ID NO:123.

[0336] In some aspects, the disclosure provides a recombinant mammalian host cell transformed with a first expression vector comprising the nucleic acid sequence of SEQ ID NO:146 and a second expression vector comprising the nucleic acid sequence of SEQ ID NO:96.

[0337] In some aspects, the disclosure provides a host cell transformed with a first expression vector comprising the nucleic acid sequence of SEQ ID NO:149 and a second expression vector comprising the nucleic acid sequence of SEQ ID NO:96.

[0338] In some aspects, the disclosure provides a recombinant mammalian host cell transformed with a first expression vector comprising the nucleic acid sequence of SEQ ID NO:152 and a second expression vector comprising the nucleic acid sequence of SEQ ID NO:96.

[0339] In some aspects, the disclosure provides a recombinant mammalian host cell transformed with a first expression vector comprising the nucleic acid sequence of SEQ ID NO:134 and a second expression vector comprising the nucleic acid sequence of SEQ ID NO:123.

[0340] In some aspects, the disclosure provides a recombinant mammalian host cell transformed with a first expression vector comprising the nucleic acid sequence of SEQ ID NO:137 and a second expression vector comprising the nucleic acid sequence of SEQ ID NO:123.

[0341] cell culture The cells may be cultured in conventional nutrient media, modified as appropriate for inducing promoters, selecting transformants, or amplifying genes encoding the desired sequences. Mammalian host cells can be cultured in a variety of media. Commercially available media, such as Ham's F10 (Sigma), Minimum Essential Medium ((MEM), Sigma), RPMI 1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma), are suitable for culturing host cells. Any of these media may be supplemented with hormones and / or other growth factors (e.g., insulin, transferrin, or epidermal growth factor), salts (e.g., sodium chloride, calcium, magnesium, and phosphates), buffers (e.g., HEPES), nucleosides (e.g., adenosine and thymidine), antibiotics, trace elements, and glucose or an equivalent energy source, as required. Any other necessary supplements may also be included at appropriate concentrations known to those of skill in the art. Culture conditions, such as temperature, pH, etc., are those previously used with the host cell selected for expression and will be apparent to those of skill in the art.

[0342] Recombinant protein recovery If a secretory leader sequence is used, the recombinantly produced polypeptide can be recovered from the culture medium as a secreted polypeptide. Alternatively, the recombinant polypeptide can also be recovered from host cell lysates. Protease inhibitors such as phenylmethylsulfonyl fluoride (PMSF) may be used during recovery from cell lysates to inhibit proteolysis during purification, and antibiotics may be included to prevent the growth of adventitious contaminants.

[0343] purification Various purification steps, such as affinity chromatography, are known and used in the art. Affinity chromatography typically utilizes highly specific binding sites present on biological macromolecules to separate molecules capable of binding to a specific ligand. The ligand is covalently attached to an insoluble porous support medium in such a way that it is clearly presented to the protein sample, thereby using the natural specific binding of one molecular species to separate and purify a second species from the mixture. Antibodies are commonly used in affinity chromatography. A size selection step may also be used, for example, gel filtration chromatography (also known as size exclusion or molecular sieve chromatography) is used to separate proteins according to size. In gel filtration, a protein solution is passed through a column packed with a semipermeable porous resin. The semipermeable resin has a range of pore sizes that determine the size of proteins that can be separated by the column.

[0344] The recombinant polypeptide domains of heterodimeric hIL12Fc muteins or heterodimeric hIL23Fc muteins produced by the transformed host can be purified according to any suitable method. Heterodimeric hIL12Fc muteins and heterodimeric hIL23Fc muteins may be isolated from inclusion bodies generated in E. coli using cation exchange, gel filtration, and / or reversed-phase liquid chromatography, or from conditioned medium derived from either mammalian or yeast cultures producing a particular heterodimeric hIL12Fc mutein or heterodimeric hIL23Fc mutein. In some embodiments where the recombinant protein is expressed with a chelating peptide, a purification tag, as discussed above, this purification handle may be used to isolate the modified recombinant protein from cell lysates or cell culture media. When the purification tag is a chelating peptide, methods for isolating such molecules using immobilized metal affinity chromatography are well known in the art. See, for example, Smith et al., U.S. Pat. No. 4,569,794.

[0345] The heterodimeric hIL12Fc mutein or heterodimeric hIL23Fc mutein in substantially purified form can be used as a therapeutic agent, for example, as described herein. The biological activity of the heterodimeric hIL12Fc mutein or heterodimeric hIL23Fc mutein produced as described above can be determined by competitive ELISA, radioligand binding assays (e.g., saturation binding, Scatchard plots, non-linear curve fitting programs, and competitive binding assays); non-radioligand binding assays (e.g., fluorescence polarization (FP), fluorescence resonance energy transfer (FRET), and surface plasmon resonance assays (see, for example, Drescher et al., Methods Mol Biol 493:323-343 (2009)) and commercially available instrumentation from GE Healthcare Bio-Sciences, such as Biacore 8+, Biacore S200, Biacore T200 (GE Healthcare Bio-Sciences, 100 Results Way, Marlborough MA). 01752)); liquid phase ligand binding assays (e.g., real-time polymerase chain reaction (RT-qPCR), and immunoprecipitation); and solid phase ligand binding assays (e.g., multi-well plate assays, on-bead ligand binding assays, on-column ligand binding assays, and filter assays).

[0346] Pharmaceutical preparations In some embodiments, the heterodimeric hIL12Fc mutein or heterodimeric hIL23Fc mutein (and / or the nucleic acid encoding the heterodimeric hIL12Fc mutein or heterodimeric hIL23Fc mutein and / or the recombinant cell incorporating the nucleic acid sequence and modified to express the heterodimeric hIL12Fc mutein or heterodimeric hIL23Fc mutein) can be incorporated into a composition, including a pharmaceutical composition. In some embodiments, the pharmaceutical composition comprises the heterodimeric hIL12Fc mutein or heterodimeric hIL23Fc mutein described herein. Such compositions typically comprise the polypeptide or nucleic acid molecule and a pharma- ceutically acceptable carrier. The pharmaceutical composition is formulated to be compatible with its intended route of administration and is compatible with therapeutic applications in which the heterodimeric hIL12Fc mutein or heterodimeric hIL23Fc mutein is administered to a subject in need of treatment or prophylaxis.

[0347] Carrier Carriers include sterile diluents, such as water for injection, saline, fixed oils, polyethylene glycol, glycerin, propylene glycol or other synthetic solvents. Carriers can be, for example, a solvent or dispersion medium containing water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of dispersions, and by using surfactants, such as sodium dodecyl sulfate. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ), or phosphate buffered saline (PBS).

[0348] buffer solution The term buffer includes buffers such as acetates, citrates, or phosphates, and agents for adjusting tonicity, such as sodium chloride or dextrose. The pH can be adjusted (e.g., to a pH of about 7.2 to 7.8, e.g., 7.5) with an acid or base, such as sodium dihydrogen phosphate and / or sodium hydrogen phosphate, hydrochloric acid, or sodium hydroxide.

[0349] dispersion liquid Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0350] Preservatives Pharmaceutical preparations for parenteral administration to subjects must be sterile and fluid to facilitate easy syringability. They must be stable under the conditions of manufacture and storage and preserved to prevent contamination. Microbial activity can be prevented by various antibacterial and antifungal agents, such as agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. Sterile solutions can be prepared by incorporating the required amount of active compound in a suitable solvent with one or a combination of the above-listed ingredients as required, followed by sterilization filtration.

[0351] Tonicity Agent In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition.

[0352] Route of administration In some embodiments, the therapeutic methods of the present disclosure involve administration to a subject in need of treatment of a pharmaceutical formulation comprising a heterodimeric hIL12Fc mutein or a heterodimeric hIL23Fc mutein, as well as a nucleic acid, a vector comprising the same, or a transformed cell. In other embodiments, the therapeutic methods of the present disclosure involve administration of a pharmaceutical formulation comprising a heterodimeric hIL12Fc mutein or a heterodimeric hIL23Fc mutein as described herein. Any pharmaceutical composition of the present disclosure can be administered to a subject in need of treatment or prevention by a variety of administration routes, including parenteral, oral, topical, or inhalation routes.

[0353] Parenteral Administration In some embodiments, the methods of the disclosure involve parenteral administration of a pharmaceutical formulation comprising a heterodimeric hIL12Fc mutein or a heterodimeric hIL23Fc mutein (and / or a nucleic acid encoding a heterodimeric hIL12Fc mutein or a heterodimeric hIL23Fc mutein, or a recombinantly engineered host cell expressing a heterodimeric hIL12Fc mutein or a heterodimeric hIL23Fc mutein) to a subject in need of treatment. In some embodiments, the methods of the disclosure involve parenteral administration of a pharmaceutical formulation comprising a heterodimeric hIL12Fc mutein or a heterodimeric hIL23Fc mutein to a subject in need of treatment. Examples of parenteral routes of administration include, for example, intravenous administration, intradermal administration, subcutaneous administration, transdermal (topical) administration, transmucosal administration, and rectal administration. Parenteral formulations include solutions or suspensions used for parenteral use and may include vehicles, carriers, and buffers. Pharmaceutical preparations for parenteral administration include sterile aqueous solutions (if water soluble) or dispersions, and sterile powders for extemporaneous preparation of sterile injectable solutions or dispersions. Parenteral preparations can be contained in ampoules, disposable syringes, or multi-dose vials made of glass or plastic. In one embodiment, the formulation is provided in a pre-filled syringe.

[0354] Oral route In some embodiments, the disclosed method involves oral administration of a pharmaceutical formulation comprising a heterodimeric hIL12Fc mutein or a heterodimeric hIL23Fc mutein (and / or a nucleic acid encoding a heterodimeric hIL12Fc mutein or a heterodimeric hIL23Fc mutein, or a recombinantly modified host cell expressing a heterodimeric hIL12Fc mutein or a heterodimeric hIL23Fc mutein) to a subject in need of treatment. In some embodiments, the disclosed method involves oral administration of a pharmaceutical formulation comprising a heterodimeric hIL12Fc mutein or a heterodimeric hIL23Fc mutein to a subject in need of treatment. If an oral composition is used, it generally includes an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound can be incorporated with an excipient and used in the form of a tablet, a troche, or a capsule, for example, a gelatin capsule. Oral compositions can also be prepared with a fluid carrier for use as a mouthwash. Pharmaceutically compatible binders and / or adjuvant materials can be included as part of the composition.Tablet, pill, capsule, troche etc. can contain any of the following components: binder, such as microcrystalline cellulose, tragacanth gum or gelatin; excipient, such as starch or lactose; disintegrant, such as alginic acid, Primogel (trademark) or corn starch; lubricant, such as magnesium stearate or Sterotes (trademark); flow agent, such as colloidal silicon dioxide; sweetener, such as sucrose or saccharin; or flavoring, such as peppermint, methyl salicylate or orange flavor, or compounds of similar nature.

[0355] Inhalation formulations In some embodiments, the methods of the disclosure involve inhalation administration of a pharmaceutical formulation comprising a heterodimeric hIL12Fc mutein or a heterodimeric hIL23Fc mutein (and / or a nucleic acid encoding a heterodimeric hIL12Fc mutein or a heterodimeric hIL23Fc mutein, or a recombinantly modified host cell expressing a heterodimeric hIL12Fc mutein or a heterodimeric hIL23Fc mutein) to a subject in need of treatment. In some embodiments, the methods of the disclosure involve inhalation administration of a pharmaceutical formulation comprising a heterodimeric hIL12Fc mutein or a heterodimeric hIL23Fc mutein to a subject in need of treatment. When administered by inhalation, the heterodimeric hIL12Fc mutein or the heterodimeric hIL23Fc mutein, or a nucleic acid encoding the same, is delivered in the form of an aerosol spray from a pressurized container or dispenser containing a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer. Such methods include those described in US Pat. No. 6,468,798.

[0356] Mucosal and transdermal preparations In some embodiments, the methods of the disclosure involve mucosal or transdermal administration of a pharmaceutical formulation comprising a heterodimeric hIL12Fc mutein or a heterodimeric hIL23Fc mutein (and / or a nucleic acid encoding a heterodimeric hIL12Fc mutein or a heterodimeric hIL23Fc mutein, or a recombinantly modified host cell expressing a heterodimeric hIL12Fc mutein or a heterodimeric hIL23Fc mutein) to a subject in need of treatment. In some embodiments, the methods of the disclosure involve mucosal or transdermal administration of a pharmaceutical formulation comprising a heterodimeric hIL12Fc mutein or a heterodimeric hIL23Fc mutein to a subject in need of treatment. For transmucosal or transdermal administration, a penetrant appropriate to the barrier to be permeated is used in the formulation. Such penetrants are generally known in the art and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished using nasal sprays, or for rectal delivery, suppositories (e.g., those using conventional suppository bases, such as cocoa butter and other glycerides) or retention enemas. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art, and may incorporate penetration enhancers, such as ethanol or lanolin.

[0357] Extended release and depot formulations In some embodiments of the disclosed method, the modified hIL-12p40 polypeptide is administered to a subject in need of treatment in the form of a formulation for long-term release of the heterodimeric hIL12Fc mutein or heterodimeric hIL23Fc mutein. An example of a long-term release formulation of an injectable composition can be caused by including an agent that delays absorption, such as aluminum monostearate and gelatin, in the composition. In one embodiment, the subject heterodimeric hIL12Fc mutein or heterodimeric hIL23Fc mutein or nucleic acid is prepared with a carrier that protects the heterodimeric hIL12Fc mutein or heterodimeric hIL23Fc mutein from rapid elimination from the body, such as a sustained release formulation including implants and microencapsulated delivery systems. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Such formulations can be prepared using standard techniques. This material is also commercially available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells using monoclonal antibodies against viral antigens) can also be used as pharma- ceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.

[0358] Administration of nucleic acids encoding modified hIL-12p40 polypeptides In some embodiments of the methods of the present disclosure, delivery of a heterodimeric hIL12Fc mutein or a heterodimeric hIL23Fc mutein to a subject in need of treatment is accomplished by administration of a nucleic acid encoding a modified heterodimeric hIL12Fc mutein or a heterodimeric hIL23Fc mutein. Methods for administering to a subject a nucleic acid encoding a modified heterodimeric hIL12Fc mutein or a heterodimeric hIL23Fc mutein are accomplished by transfection or infection using methods known in the art, including, but not limited to, those described in McCaffrey et al. (Nature (2002) 418:6893), Xia et al. (Nature Biotechnol. (2002) 20:1006-1010), or Putnam (Am. J. Health Syst. Pharm. (1996) 53: 151-160 erratum at Am. J. Health Syst. Pharm. (1996) 53:325). In some embodiments, the heterodimeric hIL12Fc mutein or heterodimeric hIL23Fc mutein is administered to the subject by administering a pharma- ceutically acceptable formulation of a recombinant expression vector comprising a nucleic acid sequence encoding the heterodimeric hIL12Fc mutein or heterodimeric hIL23Fc mutein operably linked to one or more expression control sequences operable in a mammalian subject. In some embodiments, an expression control sequence operable in a limited range of cell types (or a single cell type) can be selected to facilitate selective expression of the modified hIL heterodimeric hIL12Fc mutein or heterodimeric hIL23Fc mutein in a particular target cell type. In one embodiment, the recombinant expression vector is a viral vector. In some embodiments, the recombinant vector is a recombinant viral vector. In some embodiments, the recombinant viral vector is a recombinant adeno-associated virus (rAAV) or recombinant adenovirus (rAd), in particular a replication-deficient adenovirus derived from human adenovirus serotypes 3 and / or 5.In some embodiments, the replication-deficient adenovirus has one or more modifications to the E1 region, which prevents the virus from initiating cell cycle and / or apoptosis pathway in human cells.The replication-deficient adenovirus vector can optionally contain deletion in the E3 domain.In some embodiments, the adenovirus is a replication-competent adenovirus.In some embodiments, the adenovirus is a replication-competent recombinant virus that is engineered to selectively replicate in target cell type.

[0359] In particular, in some embodiments for administering heterodimeric hIL23Fc muteins to a subject, particularly for treating intestinal disease or bacterial infection in a subject, nucleic acid encoding heterodimeric hIL23Fc muteins can be delivered to a subject by administration of a recombinantly modified bacteriophage vector encoding heterodimeric hIL23Fc muteins. As used herein, the terms "prokaryotic virus", "bacteriophage" and "phage" are used interchangeably herein to describe any of a variety of bacterial viruses that infect and replicate within bacteria. Bacteriophages selectively infect prokaryotic cells, thus restricting expression of heterodimeric hIL23Fc muteins to prokaryotic cells in a subject while avoiding expression in mammalian cells. A wide variety of bacteriophages capable of selecting a wide range of bacterial cells have been identified and extensively characterized in the scientific literature. In some embodiments, the phage is modified to remove adjacent motifs (PAM). Elimination of the Cas9 sequence from the phage genome reduces the ability of the Cas9 endonuclease of the target prokaryotic cell to disable invading phage encoding the heterodimeric hIL23Fc muteins.

[0360] Administration of recombinantly modified cells expressing modified hIL-12p40 polypeptide In some embodiments of the disclosed methods, delivery of modified hIL-12p40 polypeptides to a subject in need of treatment is accomplished by administration of a recombinant host modified to express a heterodimeric hIL12Fc mutein or a heterodimeric hIL23Fc mutein, which may be administered in therapeutic and prophylactic applications as described herein. In some embodiments, the recombinant host cell is a mammalian cell, e.g., a human cell.

[0361] In some embodiments, the nucleic acid sequence encoding the heterodimeric hIL12Fc mutein or the heterodimeric hIL23Fc mutein (or a vector containing the same) may be maintained extrachromosomally in the recombinantly modified host cell for administration. In other embodiments, the nucleic acid sequence encoding the heterodimeric hIL12Fc mutein or the heterodimeric hIL23Fc mutein may be integrated into the genome of the host cell to be administered using at least one endonuclease to facilitate the insertion of the nucleic acid sequence into the genomic sequence of the cell. As used herein, the term "endonuclease" is used to refer to a wild-type or variant enzyme capable of catalyzing the cleavage of an internucleic acid bond in a DNA or RNA molecule, preferably a DNA molecule. When such an endonuclease has a polynucleotide recognition site greater than about 12 base pairs (bp) in length, more preferably 14-55 bp, the endonuclease is referred to as a "rare-cutting" endonuclease. Rare-cutting endonucleases can be used to inactivate genes at a locus or to integrate transgenes by homologous recombination (HR), i.e., by inducing a DNA double-strand break (DSB) at the locus and inserting exogenous DNA at this locus by gene repair mechanisms. Examples of rare-cutting endonucleases include homing endonucleases (Grizot, et al (2009) Nucleic Acids Research 37(16):5405-5419), chimeric zinc finger nucleases (ZFNs) resulting from the fusion of engineered zinc finger domains (Porteus M and Carroll D., Gene targeting using zinc finger nucleases (2005) Nature Biotechnology 23(3):967-973), TALEN-nucleases, the Cas9 endonuclease derived from the CRISPR system, or modified restriction endonucleases for extended sequence specificity (Eisenschmidt, et al. 2005; 33(22): 7039-7047).

[0362] How to use Treatment of Neoplastic Disease The present disclosure provides methods of using the heterodimeric hIL12Fc muteins of the present disclosure in treating a subject suffering from a neoplastic disease disorder or condition by administration of a therapeutically effective amount of the heterodimeric hIL12Fc mutein (or nucleic acids encoding the heterodimeric hIL12Fc muteins, including recombinant vectors encoding the heterodimeric hIL12Fc muteins, and eukaryotic and prokaryotic cells modified to express the heterodimeric hIL12Fc muteins) as described herein.

[0363] Neoplasms to be treated: The compositions and methods of the present disclosure are useful in treating neoplastic diseases characterized by the presence of a neoplasm, including benign and malignant neoplasms, as well as subjects suffering from neoplastic diseases.

[0364] Examples of benign neoplasms that can be treated with the compositions and methods of the present disclosure include, but are not limited to, adenomas, fibromas, hemangiomas, and lipomas. Examples of premalignant neoplasms that can be treated with the compositions and methods of the present disclosure include, but are not limited to, hyperplasia, atypia, metaplasia, and dysplasia. Examples of malignant neoplasms that can be treated with the compositions and methods of the present disclosure include, but are not limited to, carcinomas (cancers that arise from epithelial tissues, such as the skin or tissues that line internal organs), leukemias, lymphomas, and sarcomas that typically originate from bone, fat, muscle, blood vessels, or connective tissue. The term neoplasm also includes virus-induced neoplasms, such as warts, and EBV-induced diseases (i.e., infectious mononucleosis), scar formation, hyperproliferative vascular diseases including intimal smooth muscle cell hyperplasia, restenosis, and vascular occlusion.

[0365] The term "neoplastic disease" includes cancers characterized by solid and non-solid tumors, including, but not limited to, breast cancer; sarcoma (including, but not limited to, osteosarcoma and angiosarcoma and fibrosarcoma), leukemia, lymphoma, genitourinary cancer (including, but not limited to, ovarian, urethral, ​​bladder, and prostate cancer); gastrointestinal cancer (including, but not limited to, colon, esophageal, and gastric cancer); lung cancer; myeloma; pancreatic cancer; liver cancer; renal cancer; endocrine cancer; skin cancer; and tumors, malignant or benign, of the brain or central and peripheral nervous system (CNS) including gliomas and neuroblastomas, astrocytomas, myelodysplastic disorders; cervical intraepithelial neoplasia; intestinal polyposis; oral leukoplakia; histiocytosis, hyperproliferative scars including keloid scars, hemangiomas; hyperproliferative arterial stenosis, psoriasis, inflammatory arthritis; hyperkeratosis, and papular scaly eruptions including arthritis.

[0366] The term neoplastic disease includes carcinoma. The term "carcinoma" refers to malignant tumors of epithelial or endocrine tissues, including cancers of the respiratory system, gastrointestinal system, genitourinary system, testicular cancer, breast cancer, prostate cancer, endocrine system cancer, and melanoma. The term neoplastic disease includes adenocarcinoma. "Adenocarcinoma" refers to carcinomas derived from glandular tissue or in which the tumor cells form recognizable glandular structures.

[0367] As used herein, the term "hematopoietic neoplastic disorder" refers to a neoplastic disease involving hyperplasia / neoplastic cells arising from hematopoietic origin, e.g., myeloid, lymphoid, or erythroid lineages, or precursor cells thereof.

[0368] Myeloid neoplasms include, but are not limited to, myeloproliferative neoplasms, myeloid and lymphoid disorders with eosinophilia, myeloproliferative / myelodysplastic neoplasms, myelodysplastic syndromes, acute myeloid leukemia and related precursor neoplasms, and acute leukemia of ill-defined lineage. Exemplary myeloid disorders amenable to treatment according to the present disclosure include, but are not limited to, acute promyeloid leukemia (APML), acute myeloid leukemia (AML), and chronic myeloid leukemia (CML).

[0369] Lymphoid neoplasms include, but are not limited to, precursor lymphoid neoplasms, mature B-cell neoplasms, mature T-cell neoplasms, Hodgkin's lymphoma, and immunodeficiency-associated lymphoproliferative disorders. Exemplary lymphoid disorders amenable to treatment according to the present disclosure include, but are not limited to, acute lymphoblastic leukemia (ALL), including B-lineage ALL and T-lineage ALL, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), hairy cell leukemia (HLL), and Waldenstrom's macroglobulinemia (WM).

[0370] In some cases, hematopoietic neoplastic disorders result from poorly differentiated acute leukemias (e.g., erythroblastic leukemia and acute megakaryoblastic leukemia). As used herein, the term "hematopoietic neoplastic disorders" refers to malignant lymphomas, including, but not limited to, non-Hodgkin's lymphoma and its variants, peripheral T-cell lymphoma, adult T-cell leukemia / lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), large granular lymphocytic leukemia (LGF), Hodgkin's disease, and Reed-Sternberg disease.

[0371] A determination of whether a subject is "suffering from a neoplastic disease" refers to a determination made by a physician about a subject based on available information accepted in the field for identifying a disease, disorder, or condition, including, but not limited to, x-rays, CT scans, conventional clinical diagnostic tests (e.g., blood counts, etc.), genomic data, protein expression data, immunohistochemistry, that the subject requires or would benefit from treatment.

[0372] Combination of hIL12 muteins with adjunctive anti-neoplastic agents: The present disclosure provides for the use of the hIL12 muteins of the present disclosure in combination with one or more additional active anti-neoplastic agents ("adjuncts") to treat neoplastic disease. Such additional combinations are referred to interchangeably as "antineoplastic adjunct combinations" or "antineoplastic adjunct combination therapies," and the therapeutic agents used in combination with the hIL12 muteins of the present disclosure are referred to as "antineoplastic adjuncts." As used herein, the term "antineoplastic adjunct" includes anti-neoplastic agents that can be administered or introduced separately, e.g., that can be formulated separately for separate administration (e.g., as may be provided in a kit), and / or therapies that can be administered or introduced in combination with the hIL12 muteins.

[0373] Chemotherapy agents: In some embodiments, the antineoplastic adjuvant agent is a chemotherapeutic agent. In some embodiments, the adjuvant agent is a "cocktail" of multiple chemotherapeutic agents. In some embodiments, the chemotherapeutic agent or cocktail is administered in combination with one or more physical methods (e.g., radiation therapy). The term "chemotherapeutic agent" includes alkylating agents, such as thiotepa and cyclophosphamide; alkyl sulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines, including altretamine, methylameramine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphaolamide, and trimethylolmelamine. Nitrogen mustards, for example, thiolambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembicine, phenesterine, prednimustine, trofosfamide, uracil mustard; Nitrosoureas, for example, carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; Antibiotics, for example, aclacinomycin, acla Tinomycin, ausramycin, azaserine, bleomycins, e.g. bleomycin A2, cactinomycin, calicheamicin, carabicin, caminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin and derivatives, e.g. demethoxy-daunomycin, 11-deoxydaunorubicin, 13-deoxydaunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, epirubicin, Sorubicin, idarubicin, marcelomycin, mitomycins, e.g., mitomycin C, N-methylmitomycin C; mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfilomycin, puromycin, queramycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites, e.g., methotrexate and 5-fluorouracil (5-FU);Folic acid analogues, such as denopterin, methotrexate, pteropterin, trimetrexate, dideazatetrahydrofolic acid, and folic acid; purine analogues, such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogues, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens, such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane, testosterone, Lactones;Antiadrenal drugs, e.g., Aminoglutethimide, Mitotane, Trilostane;Folic acid supplements, e.g., Floline;Aceglatone;Aldophosphamide glycosides;Aminolevulinic acid;Amsacrine;Bestravcil;Bisantrene;Edatrexate;Defofamine;Demecolcine;Diaziquone;Elformitine;Elliptinium acetate;Etoglucide;Gallium nitrate;Hydroxyurea;Lentinan;Lonidamine;Mitoguazone;Mitoxantrone;Mopidamol;Nitracrine;Pentostatin;Phenamet;Pirarubicin;Podophyllinic cis acid;2-ethylhydrazide;procarbazine;razoxane;sizofiran;spirogermanium;tenuazonic acid;triaziquone;2,2',2''-trichlorotriethylamine;urethane;vindesine;dacarbazine;mannomustine;mitobronitol;mitolactol;pipobroman;gacitosine;arabinoside (Ara-C);cyclophosphamide;thiotepa;taxoids, such as paclitaxel, nab-paclitaxel, and doxetaxel;chlorambucil;gemcitabine;6-thioguanine;mercaptopurine;methotrexate platinum and platinum coordination complexes, such as cisplatin, oxaplatin, and carboplatin;vinblastine;etoposide (VP-16);ifosfamide;mitomycin C;mitoxantrone;vincristine;vinorelbine;navelbine;novantrone;teniposide;daunomycin;aminopterin;xeloda;ibandronate;CPT11;topoisomerase inhibitors;difluoromethylornithine (DMFO);retinoic acid;esperamicin;capecitabine;taxanes, such as paclitaxel, docetaxel, cabazitaxel;Carminomycin, adriamycin, e.g., 4'-epidriamycin, 4-adriamycin-14-benzoate, adriamycin-14-octanoate, adriamycin-14-naphthalene acetate; colchicine, as well as pharmaceutically acceptable salts, acids, or derivatives of any of the above;

[0374] The term "chemotherapeutic agent" also includes antihormonal agents that act to regulate or inhibit hormone action on tumors, such as antiestrogens including tamoxifen, raloxifene, aromatase-inhibiting 4(5)-imidazole, 4-hydroxytamoxifen, trioxyphene, ketoxifene, onapristone, and toremifene, and antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin, as well as pharmaceutical acceptable salts, acids, or derivatives of any of the above.

[0375] In some embodiments, the antineoplastic adjuvant is a cytokine or cytokine antagonist, e.g., IL-12, INFα, or anti-epidermal growth factor receptor, irinotecan; a tetrahydrofolate antimetabolite, e.g., pemetrexed; an antibody against a tumor antigen, a monoclonal antibody-toxin conjugate, a T-cell adjuvant, bone marrow transplant, or an antigen-presenting cell (e.g., dendritic cell therapy), an antitumor vaccine, a replication-competent virus, a signal transduction inhibitor (e.g., Gleevec® or Herceptin®) or an immunomodulatory agent to achieve additive or synergistic inhibition of tumor growth, a nonsteroidal anti-inflammatory drug (NSAID), a cyclooxygenase-2 (COX-2) inhibitor, a steroid, a TNF antagonist (e.g., Remica One or more chemical or biological agents identified in the art as useful in the treatment of neoplastic diseases, including, but not limited to, interferon-β1a (Avonex®) and interferon-β1b (Betaseron®), as well as combinations of one or more of the foregoing as implemented in known chemotherapy treatment regimens, including, but not limited to, TAC, FOLFOX, TPC, FEC, ADE, FOLFOX-6, EPOCH, CHOP, CMF, CVP, BEP, OFF, FLOX, CVD, TC, FOLFIRI, PCV, FOLFOXIRI, ICE-V, XELOX, and others that will be readily appreciated by one of skill in the art.

[0376] In some embodiments, the hIL12 mutein is administered in combination with a BRAF / MEK inhibitor, a kinase inhibitor such as sunitinib, a PARP inhibitor such as olaparib, an EGFR inhibitor such as osimertinib (Ahn, et al. (2016) J Thorac Oncol 11:S115), an IDO inhibitor such as epacadostat, and an oncolytic virus such as talimogene laherparepvec (T-VEC).

[0377] Antitumor antigen-antibody therapeutic agent as an adjuvant In some embodiments, the "antineoplastic adjuvant" is a therapeutic antibody (including bispecific and trispecific antibodies that bind to one or more tumor-associated antigens, including but not limited to bispecific T cell engagers (BITEs), dual affinity retargeting (DART) constructs, and trispecific killer engager (TriKE) constructs).

[0378] In some aspects, the therapeutic antibody is selected from the group consisting of HER2 (e.g., trastuzumab, pertuzumab, adotrastuzumab emtansine), nectin-4 (e.g., enfortumab), CD79 (e.g., polatuzumab vedotin), CTLA4 (e.g., ipilumumab), CD22 (e.g., moxetumomab pasudotox), CCR4 (e.g., magamuizumab), IL23p19 (e.g., tildra), and the like. kizumab), PDL1 (e.g., durvalumab, avelumab, atezolizumab), IL17a (e.g., ixekizumab), CD38 (e.g., daratumumab), SLAMF7 (e.g., elotuzumab), CD20 (e.g., rituximab, tositumomab, ibritumomab, and ofatumumab), CD30 (e.g., brentuximab vedotin), CD33 (e.g., gemtuzumab The antibody binds to at least one tumor antigen selected from the group consisting of ozogamicin), CD52 (e.g., alemtuzumab), EpCam, CEA, fpA33, TAG-72, CAIX, PSMA, PSA, folate binding protein, GD2 (e.g., dinutuximab), GD3, IL6 (e.g., siltuximab) GM2, Ley, VEGF (e.g., bevacizumab), VEGFR, VEGFR2 (e.g., ramucirumab), PDGFR□ (e.g., orartumumab), EGFR (e.g., cetuximab, panitumumab, and necitumumab), ERBB2 (e.g., trastuzumab), ERBB3, MET, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKLRAP, tenascin, integrin□V□3, and integrin□4□1.

[0379] In some embodiments, the therapeutic antibody is an immune checkpoint modulator for treating and / or preventing neoplastic disease in a subject, as well as diseases, disorders, or conditions associated with neoplastic disease. The term "immune checkpoint pathway" refers to a biological response triggered by a first molecule (e.g., a protein such as PD1) expressed on an antigen-presenting cell (APC) binding to a second molecule (e.g., a protein such as PDL1) expressed on an immune cell (e.g., T-cell) that regulates the immune response, thereby stimulating (e.g., upregulating T-cell activity) or inhibiting (e.g., downregulating T-cell activity) the immune response. The molecules involved in the formation of a binding pair that regulates the immune response are commonly referred to as "immune checkpoints." In one embodiment, the immune checkpoint pathway modulator is a negative immune checkpoint pathway antagonist ("PD1 pathway inhibitor") that inhibits binding of PD1 to PDL1 and / or PDL2. The term PD1 pathway inhibitor includes monoclonal antibodies that interfere with the binding of PD1 to PDL1 and / or PDL2. Examples of commercially available PD1 pathway inhibitors useful as adjuncts in neoplastic disease treatment include nivolumab (Opdivo®, BMS-936558, MDX1106, available from BristolMyers Squibb, Princeton NJ), pembrolizumab (Keytruda® MK-3475, lambrolizumab, available from Merck and Company, Kenilworth NJ), and atezolizumab (Tecentriq®, Genentech / Roche, South San Francisco Antibodies that interfere with the binding of PD1 to PDL1 and / or PDL2, including, but not limited to, antibodies ...Additional PD1 pathway inhibitor antibodies are in clinical development, including, but not limited to, durvalumab (MEDI4736, Mediimmune / AstraZeneca), pidilizumab (CT-011, CureTech), PDR001 (Novartis), BMS-936559 (MDX1105, BristolMyers Squibb), and avelumab (MSB0010718C, Merck Serono / Pfizer), and SHR-1210 (Incyte). Additional antibody PD1 pathway inhibitors are described in U.S. Patent No. 8,217,149, issued July 10, 2012 (Genentech, Inc); U.S. Patent No. 8,168,757, issued May 1, 2012 (Merck Sharp and Dohme Corp.), U.S. Patent No. 8,008,449, issued August 30, 2011 (Medarex), and U.S. Patent No. 7,943,743, issued May 17, 2011 (Medarex, Inc).

[0380] Examples of antibody therapeutics that have been approved by the FDA and may be used as adjuncts for use in the treatment of neoplastic diseases include atezolizumab, olaratumab, ixekizumab, trastuzumab, infliximab, rituximab, edrecolomab, daratumumab, elotuzumab, necitumumab, dinutuximab, nivolumab, blinatumomab, pembrolizumab, pertuzumab, brentuximab vedotin, ipilimumab, ofatumumab, certolizumab pegol, catumaxomab, panitumumab. , bevacizumab, ramucirumab, siltuximab, enfortumab vetotin, polatuzumab vedotin, [fam]-trastuzumab deruxtecan, cemiplimab, moxetumomab pasudotox, mogamuizumab, tildrakizumab, ibalizumab, durvalumab, inotuzumab, ozogamicin, avelumab, obinutuzumab, ado-trastuzumab emtansine, cetuximab, tositumomab-I131, ibritumomab tiuxetan, gemtuzumab, and ozogamicin.

[0381] physical method In some embodiments, the antineoplastic adjuvant is one or more non-pharmacological modalities (e.g., local or total body radiation therapy or surgery). By way of example, the present disclosure contemplates a treatment regimen in which a treatment with a treatment regimen comprising a hIL12 mutein and one or more antineoplastic adjuvant is administered before or after a radiation step. In some embodiments, the present disclosure further contemplates the use of a hIL12 mutein in combination with surgery (e.g., tumor resection). In some embodiments, the present disclosure further contemplates the use of a hIL12 mutein in combination with bone marrow transplantation, peripheral blood stem cell transplantation, or other types of transplantation therapy.

[0382] In some embodiments, the methods of the present disclosure may include the combination of administration of a hIL12 mutein and an adjunct in the form of a cell therapy to treat a neoplastic, autoimmune, or inflammatory disease. Examples of cell therapies of interest for use in combination with the methods of the present disclosure include, but are not limited to, engineered T cell products, including one or more activated CAR-T cells, engineered TCR cells, tumor infiltrating lymphocytes (TILs), and engineered Treg cells.

[0383] CARs useful in the practice of the present invention are prepared according to principles well known in the art.See, for example, Eshhaar et al., U.S. Patent No. 7,741,465 B1, issued June 22, 2010; Sadelain, et al. (2013) Cancer Discovery 3(4):388-398; Jensen and Riddell (2015) Current Opinions in Immunology 33:9-15; Gross, et al. (1989) PNAS(USA) 86(24):10024-10028; Curran, et al. (2012) J Gene Med 14(6):405-15. Examples of commercially available CAR-T cell products include axicabtagenecilloreucel (commercially available as Yescarta® from Gilead Pharmaceuticals) and tisagenlecleucel (commercially available as Kymriah® from Novartis). In some embodiments, the CAR-T has a CAR that specifically binds to a cell surface molecule associated with tumor cells selected from the group consisting of GD2, BCMA, CD19, CD33, CD38, CD70, GD2, IL3R□2, CD19, mesothelin, Her2, EpCam, Muc1, ROR1, CD133, CEA, EGRFRVIII, PSCA, GPC3, Pan-ErbB, and FAP.

[0384] Physical method: In some embodiments, the antineoplastic adjuvant is an antineoplastic physical method, including, but not limited to, radiation therapy, cryotherapy, hyperthermia, surgery, laser ablation, and proton therapy.

[0385] Methods for modulating hIL-12 signaling In another aspect, the disclosure provides a method for modulating IL-12-mediated signal transduction in a subject. In some embodiments, the method comprises administering to the subject an effective amount of a pharmaceutical composition, the pharmaceutical composition comprising a heterodimeric hIL12Fc mutein as described herein, a nucleic acid molecule encoding a heterodimeric hIL12Fc mutein as described herein, a nucleic acid molecule encoding a heterodimeric hIL12Fc mutein as described herein, or a recombinantly modified cell comprising a nucleic acid molecule encoding a heterodimeric hIL12Fc mutein as described herein. In some embodiments, the pharmaceutical composition comprises a pharma- ceutically acceptable carrier.

[0386] In some embodiments, the method for modulating IL-12-mediated signaling in a subject includes determining STAT4-mediated signaling in one or more cells obtained from the subject. In some embodiments, the STAT4-mediated signaling is determined by an assay selected from the group consisting of a gene expression assay, a phosphoflow signaling assay, and an enzyme-linked immunosorbent assay (ELISA). In some embodiments, the STAT4-mediated signaling in the subject is reduced by about 20% to about 100% compared to a reference level. In some embodiments, the administered composition reduces the ability to induce IFN-γ expression.

[0387] kit Also provided is a kit comprising the heterodimeric hIL12Fc mutein or heterodimeric hIL23Fc mutein of the present disclosure. In some embodiments, the kit comprises one or more components for modulating IL-12-mediated signal transduction in a subject or for treating a health condition in a subject in need of such treatment, the components being selected from the hIL12Fc mutein or heterodimeric hIL23Fc mutein described herein, a nucleic acid molecule encoding the hIL12Fc mutein or heterodimeric hIL23Fc mutein, a recombinantly modified cell comprising the nucleic acid molecule encoding the hIL12Fc mutein or heterodimeric hIL23Fc mutein described herein, or a pharmaceutical composition comprising one or more of the components. In some embodiments, the pharmaceutical composition comprises a pharma- ceutically acceptable carrier.

[0388] Further Aspects 1. Formula #1: hP40M-L1 a -UH1-Fc1[1] and a first polypeptide of formula #2: hP35-L2 b -UH2-Fc2 [2] A heterodimeric hIL12Fc mutein comprising a second polypeptide of During the ceremony, hP35 is a polypeptide having at least 90%, alternatively at least 91%, alternatively at least 92%, alternatively at least 93%, alternatively at least 94%, alternatively at least 95%, alternatively at least 96%, alternatively at least 97%, alternatively at least 98%, or alternatively at least 99% sequence identity to SEQ ID NO:2; hP40M is a human p40 mutein comprising one or more amino acid substitutions at positions selected from the group consisting of positions W37, P39, D40, A41, K80, E81, F82, K106, E108, D115, H216, K217, L218, and K219, numbered according to wild-type prehuman P40 (SEQ ID NO:3); L1 and L2 are GSA linkers, and a and b are independently selected from 0 (absent) or 1 (present); UH1 and UH2 are each an upper hinge domain of a human immunoglobulin independently selected from the group consisting of IgG1, IgG2, IgG3, and IgG4 upper hinges, optionally comprising the amino acid substitution C220S (EU numbering); Fc1 is a polypeptide comprising the lower hinge, CH2, and CH3 domains of a human immunoglobulin selected from the group consisting of IgG1, IgG2, IgG3, and IgG4, containing one or more amino acid substitutions that promote heterodimerization with Fc2; FC2 is a polypeptide comprising the lower hinge, CH2, and CH3 domains of a human immunoglobulin selected from the group consisting of IgG1, IgG2, IgG3, and IgG4, comprising one or more amino acid substitutions that promote heterodimerization with Fc1; wherein the polypeptide of formula 1 and the polypeptide of formula 2 are linked by at least one interchain disulfide bond; The heterodimeric hIL12Fc mutein. 2. The heterodimeric hIL12Fc mutein of embodiment 1, wherein hP40M has at least 70% sequence identity to SEQ ID NO:4 (e.g., at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:4). 3. The heterodimeric hIL12Fc mutein of embodiment 1 or 2, wherein hP40M comprises one or more amino acid substitutions at residues selected from the group consisting of W37, P39, D40, A41, K80, E81, F82, K106, E108, D115, H216, K217, L218, and K219, numbered according to SEQ ID NO:3. 4. The heterodimeric hIL12Fc mutein of embodiment 3, wherein hP40M comprises one or more amino acid substitutions selected from the group consisting of P39A, D40A, E81A, F82A, K106A, D109A, K217A, K219A. 5. The heterodimeric hIL12Fc mutein of embodiment 3, wherein hP40M comprises one or more amino acid substitutions at residues selected from the group consisting of E81, F82, K106, and K217, numbered according to SEQ ID NO:3. 6. The heterodimeric hIL12Fc mutein of embodiment 1, wherein hP40M comprises two or more amino acid substitutions at residues selected from the group consisting of E81, F82, K106, and K217, numbered according to SEQ ID NO:3. 7. The heterodimeric hIL12Fc mutein of embodiment 6, wherein hP40M comprises a set of amino acid substitutions selected from the group consisting of the following sets of amino acid substitutions: E81A / F82A, E81K / F82A, E81L / F82A, E81H / F82A, and E81S / F82A. 8. The heterodimeric hIL12Fc mutein of embodiment 1, wherein hP40M comprises three or more amino acid substitutions at residues selected from the group consisting of W37, P39, D40, A41, K80, E81, F82, K106, E108, D115, H216, K217, L218, and K219, numbered according to SEQ ID NO:3. 9. The heterodimeric hIL12Fc mutein of embodiment 1, wherein hP40M comprises three or more amino acid substitutions at W37, P39, D40, A41, K80, E81, F82, K106, E108, D115, H216, K217, L218, and K219. 10. The heterodimeric hIL12Fc mutein of embodiment 9, wherein the three or more substitutions comprise a set of amino acid substitutions selected from the group consisting of the following sets of amino acid substitutions: W37A / E81A / F82A; E81A / F82A / K106A; E81A / F82A / K106A / K219A, E81A / F82A / K106N, E81A / F82A / K106Q, E81A / F82A / K106T, and E81A / F82A / K106R. 11. The heterodimeric hIL12Fc mutein of embodiment 1, wherein hP40M comprises four or more amino acid substitutions at: W37, P39, D40, A41, K80, E81, F82, K106, E108, D115, H216, K217, L218, and K219. 12. The heterodimeric hIL12Fc mutein of embodiment 11, wherein the four or more substitutions comprise a set of amino acid substitutions selected from the group consisting of the following sets of amino acid substitutions: E81A / F82A / K106A / K217A, 81A / F82A / K106A / E108A / D115A, and P39A / D40A / E81A / F82A. 13. The heterodimeric hIL12Fc mutein of embodiment 1, wherein hP40M comprises the set of amino acid substitutions E81A / F82A (SEQ ID NO:6). 14. The heterodimeric hIL12Fc mutein of embodiment 1, wherein hP40M comprises the set of amino acid substitutions E81A / F82A / K106A (SEQ ID NO:8). 15. The heterodimeric hIL12Fc mutein of embodiment 1, wherein hP40M comprises the set of amino acid substitutions E81A / F82A / K106A / K217A (SEQ ID NO:10). 16. The heterodimeric hIL12Fc mutein according to any one of aspects 1 to 15, wherein the binding affinity of the heterodimeric hIL12Fc to the extracellular domain (ECD) of IL12Rβ1 is reduced by at least 5%, optionally at least 10%, optionally at least 20%, optionally at least 30%, optionally at least 40%, optionally at least 50%, optionally at least 60%, optionally at least 70% compared to the binding affinity of wild-type hP40 (SEQ ID NO:4) to the extracellular domain (ECD) of IL12Rβ1 as determined by surface plasmon resonance. 17. The heterodimeric hIL12Fc mutein according to any one of aspects 1 to 16, wherein the GSA linker is a polypeptide having 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids composed of amino acids selected from the group consisting of glycine, serine, and alanine. 18. The GSA linker has the structure (GGGGS m ) n , (GGGS m ) n , (GGGA m ) n , and (GGGGGA m ) n and combinations thereof, wherein m, n, and o are each independently selected from 1, 2, 3, or 4. 19. The heterodimeric hIL12Fc mutein according to any one of aspects 1 to 18, wherein the GSA linker is a polypeptide selected from the group consisting of SEQ ID NOs: 27 to 79. 20. The heterodimeric hIL12Fc mutein according to any one of aspects 1 to 19, wherein Fc1 and Fc2 are native upper hinge regions of a human immunoglobulin selected from the UH region of a human IgG1, human IgG2, human IgG3, and human IgG4 upper hinge domain. 21. The heterodimeric hIL12Fc mutein of embodiment 20, wherein the upper hinge region comprises the amino acid sequence: EPKSC (SEQ ID NO:11). 22. The heterodimeric hIL12Fc mutein according to embodiment 20, wherein the upper hinge region comprises the amino acid sequence: EPKSS (SEQ ID NO:12). 23. The heterodimeric hIL12Fc mutein according to any one of aspects 1 to 22, further comprising: (a) a deletion of the lysine residue at position 447; or (b) a deletion of both the glycine at position 446 and the lysine residue at position 447. 24. The heterodimeric hIL12Fc mutein of embodiment 1, wherein Fc1 and Fc2 comprise amino acid substitutions that promote heterodimerization between Fc1 and Fc2. 25. The heterodimeric hIL12Fc mutein of embodiment 24, wherein one of Fc1 and Fc2 comprises the amino acid substitutions S364H / T394F, and the other comprises the amino acid substitutions Y349T / F405A. 26. The heterodimeric hIL12Fc mutein of embodiment 24, wherein one of Fc1 and Fc2 comprises the amino acid substitutions T350V / L351Y / F405A / Y407V, and the other comprises the amino acid substitutions T350V / T366L / K392L / T394W. 27. The heterodimeric hIL12Fc mutein of embodiment 1, wherein one of Fc1 and Fc2 comprises the amino acid substitutions K360E / K409W, and the other comprises the amino acid substitutions Q347R / D399V / F405T. 28. The heterodimeric hIL12Fc mutein of embodiment 1, wherein one of Fc1 and Fc2 comprises amino acid substitutions to provide a knob, and the other of Fc1 and Fc2 comprises amino acid substitutions to provide a hole. 29. The heterodimeric hIL12Fc mutein of embodiment 1, wherein the acid substitution to provide a knob is T366W and the acid substitution to provide a hole is the set of amino acid substitutions T366S / L368A / Y407V. 30. The heterodimeric hIL12Fc mutein of embodiment 1, wherein Fc1 and Fc2 are covalently linked via one or more, optionally two or more, optionally three or more, optionally four or more disulfide bonds between the side chains of the following groups of cysteine ​​pairs: (a) C96 of hP35 and C199 of hP40M; (b) C226 of the first Fc monomer and C226 of the second Fc monomer, (c) C229 of the first Fc monomer and C229 of the second Fc monomer; and (d) S354C of the first Fc domain comprising an S354C amino acid substitution and Y349C of the second Fc domain comprising an Y349C amino acid substitution. 31. The heterodimeric hIL12Fc mutein of embodiment 1, wherein Fc1 and Fc2 comprise one or more amino acid substitutions to reduce effector function. 32. The heterodimeric hIL12Fc mutein according to embodiment 31, wherein one or both of Fc1 and Fc2 comprise mutations selected from the group consisting of: L234E; L234A / L235A; L234A / L235A / P329A; and L234A / L235A / P329G. 33. The heterodimeric hIL12Fc mutein according to any one of aspects 1 to 32, wherein Fc1 and Fc2 comprise the amino acid substitutions M428L and N434S. 34. The heterodimeric hIL12Fc mutein according to any one of aspects 1-32, wherein Fc1 and / or Fc2 comprises one or more modifications to eliminate N- o...

Claims

1. A first polypeptide having any one of SEQ ID NOs: 129, 80, 83, 85, 86, 88, 90, 92, 121, 132, 135, 138, 141, 144, 147, 150, and 153; and A second polypeptide having the sequence of any one of SEQ ID NOs: 124, 81, 82, 84, 87, 89, 91, and 93. A heterodimeric hIL12Fc mutein comprising:

2. A heterodimeric hIL12Fc mutein as described in claim 1, wherein the first polypeptide has the sequence of SEQ ID NO: 129 and the second polypeptide has the sequence of SEQ ID NO:

124.

3. A heterodimeric hIL12Fc mutein as described in claim 1, wherein the first polypeptide has the sequence of SEQ ID NO: 80 and the second polypeptide has the sequence of SEQ ID NO:

81.

4. A heterodimeric hIL12Fc mutein as described in claim 1, wherein the first polypeptide has the sequence of SEQ ID NO: 121 and the second polypeptide has the sequence of SEQ ID NO:

124.

5. A heterodimeric hIL12Fc mutein as described in claim 1, wherein the first polypeptide has the sequence of SEQ ID NO: 83 and the second polypeptide has the sequence of SEQ ID NO:

82.

6. A heterodimeric hIL12Fc mutein as described in claim 1, wherein the first polypeptide has the sequence of SEQ ID NO: 141 and the second polypeptide has the sequence of SEQ ID NO:

124.

7. A heterodimeric hIL12Fc mutein as described in claim 1, wherein the first polypeptide has the sequence of SEQ ID NO: 144 and the second polypeptide has the sequence of SEQ ID NO:

124.

8. A heterodimeric hIL12Fc mutein as described in claim 1, wherein the first polypeptide has the sequence of SEQ ID NO: 147 and the second polypeptide has the sequence of SEQ ID NO:

82.

9. A heterodimeric hIL12Fc mutein as described in claim 1, wherein the first polypeptide has the sequence of SEQ ID NO: 150 and the second polypeptide has the sequence of SEQ ID NO:

82.

10. The heterodimeric hIL12Fc mutein of claim 1, wherein the first polypeptide has the sequence of SEQ ID NO: 153 and the second polypeptide has the sequence of SEQ ID NO:

82.

11. A heterodimeric hIL12Fc mutein as described in claim 1, wherein the first polypeptide has the sequence of SEQ ID NO: 135 and the second polypeptide has the sequence of SEQ ID NO:

124.

12. A heterodimeric hIL12Fc mutein as described in claim 1, wherein the first polypeptide has the sequence of SEQ ID NO: 138 and the second polypeptide has the sequence of SEQ ID NO:

124.

13. The heterodimeric hIL12Fc mutein of claim 1, wherein the first polypeptide and the second polypeptide are linked by at least one interchain disulfide bond.

14. A heterodimeric hIL12Fc mutein according to claim 1, which is PEGylated.

15. A pharmaceutically acceptable formulation comprising the heterodimeric hIL12Fc mutein of claim 1 as an active ingredient.

16. Use of the heterodimeric hIL12Fc mutein of claim 1 for the manufacture of a medicament for the treatment of a neoplastic disease.

17. A nucleic acid encoding the first polypeptide of claim 1.

18. A nucleic acid encoding the second polypeptide of claim 1.

19. A vector comprising a nucleic acid according to claim 17 or 18, or both claims 17 and 18.

20. 20. A recombinant host cell transformed with the vector of claim 19.

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