Il-12 heterodimeric fc-fusion proteins

Abstract

To provide IL-12 heterodimeric Fc fusion proteins.SOLUTION: The present invention provides novel IL-12 Fc fusion proteins, methods of making and using the same. The IL-12 Fc fusion proteins are useful for treatment of cancer and can be used in combination with checkpoint blockade.SELECTED DRAWING: Figure 8A

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 740,813, filed October 3, 2018, U.S. Provisional Patent Application No. 62 / 810,038, filed February 25, 2019, and U.S. Provisional Patent Application No. 62 / 848,512, filed May 15, 2019, all of which are incorporated by reference herein in their entirety for all purposes. [Background technology]

[0002] For T cells to mount an effective antitumor response, three things must happen: first, T cells must engage antigenic tumor peptides presented by MHC in the tumor environment; second, costimulatory molecules must bind to the T cells; and third, T cells must be induced by cytokines such as IL-12 and IL-2 to produce costimulatory cytokines such as IFNγ that allow differentiation and proliferation. Recognition of tumor peptides alone in the absence of cytokine induction causes T cells to become anergic, thereby resulting in resistance. Thus, a very promising approach in cancer immunotherapy is cytokine-based treatment. In fact, IL-2 has been approved for use in patients with metastatic renal cell carcinoma and malignant melanoma. However, there is currently no approved use of recombinant IL-12 in humans.

[0003] Recombinant IL-12 is a promising cytokine-based therapy due to its broad effects in activating the immune system. However, IL-12 has so far faced hurdles in human clinical trials due to toxicity. As with other cytokines, the short half-life of IL-12 necessitates frequent bolus injections.

[0004] In addition, IL-12 consists of an α chain (p35 subunit, IL-12p35) and a β chain (p40 subunit, IL-12p40) covalently linked to form the biologically active IL-12 heterodimer. IL-12 exerts its cell signaling function through binding to the dimeric IL-12 receptor complex consisting of IL-12 receptor β1 (IL-12Rβ1) and IL-12 receptor β2 (IL-12Rβ2) on T cells and inducing IFNγ secretion. However, the IL-12p40 subunit can also exist as a homodimer, which has been reported to antagonize IL-12 activity by competing for binding to the IL-12 receptor.

[0005] The present invention addresses the short half-life of IL-12 by providing novel IL-12-Fc fusion proteins and novel IL-12 variants with reduced potency. Summary of the Invention

[0006] In one aspect, the invention provides a heterodimeric Fc fusion protein comprising: a) a first fusion protein comprising a variant IL-12p40 subunit domain and a first Fc domain, wherein the IL-12p40 subunit domain is covalently linked to the N-terminus of the first Fc domain; and b) a second fusion protein comprising an IL-12p35 subunit domain and a second Fc domain, wherein the IL-12p35 subunit domain is covalently linked to the N-terminus of the Fc domain, wherein the first and second Fc domains comprise a modification that promotes heterodimerization of the first and second Fc domains. In some embodiments, the invention provides a heterodimeric Fc fusion protein comprising: The present invention provides heterodimeric Fc fusion proteins having one or more amino acid substitutions selected from the group consisting of 18N, D18K, E32Q, E33Q, D34N, D34K, Q42E, S43E, S43K, E45Q, Q56E, D62N, E73Q, D87N, K99E, K99Y, E100Q, N103D, N103Q, N113D, N113Q, Q144E, D161N, R159E, K163E, E187Q, N200D, N200Q, N218Q, Q229E, E235Q, C252S, Q256N, K258E, K260E, E262Q, K264E, N281D, N281Q, and E299Q.

[0007] In some embodiments, the present invention relates to a method for the preparation of IL-12p40 subunits comprising administering to the patient a IL-12p40 subunit comprising administering to the patient a IL-12p40 subunit comprising administering to the patient a IL-12p40 subunit comprising administering to the patient a IL-12p40 subunit comprising administering to the patient a IL-12p40 subunit comprising administering to the patient a IL-12p40 subunit comprising administering to the patient a IL-12p40 subunit comprising administering to the patient a IL-12p40 subunit S43K / E49Q, E59Q / K163E, E59Q / K99E, E59Q / K258E, E59Q / K260E, E59K / K99E , D18K / E59K / K99E, E59K / K99E / K264E, E59K / K99Y, E59Y / K99Y, E59Y / K99E, E 45K / E59K / K99E, E59K / K99E / Q144E, E59K / K99E / Q144K, E59K / K99E / R159E, E59K / K99E / K264E, D18K / E59K / K99E / K264E, DI8K / E59K / K99E / C252S, D18K / E59K / K99E / C252S / K264E, E59K / K99Y / C252S, E59K / K99E / C252S / K264E, E5 9K / K99E / C252S, N103D / N113D, N103D / N200D, N103D / N281D, N113D / N200D, N 113D / N281D, N200D / N281D, N103D / N113D / N200D, N103D / N113D / N281D, N10 3D / N200D / N281D, N113D / N200D / N281D, N103Q / N113Q, N103Q / N200Q, N103Q / N281Q, N113Q / N200Q, N113Q / N281Q, N200Q / N281Q, N103Q / N113Q / N200Q, N1 03Q / N113Q / N281Q, N103Q / N200Q / N281Q, N113Q / N200Q / N281Q, N103Q / N113Q / N200Q / N281Q, E59K / K99E / N103Q / C252S / K264E, E59K / K99E / N113Q / C252S / K264E, E59K / K99E / N200Q / C252S / K264E, E59K / K99E / N281Q / C252S / K264E,E59K / K99E / N103Q / N113Q / C252S / K264E, E59K / K99E / N103Q / N200Q / C252S / K264E, E59K / K99E / N103Q / N281Q / C252S / K264E, E5 9K / K99E / N113Q / N200Q / C252S / K264E, E59K / K99E / N113Q / N281Q / C252S / K264E, E59K / K99E / N200Q / N281Q / C252S / K264E, E59K The present invention provides a heterodimeric Fc fusion protein having amino acid substitutions selected from the group consisting of E59K / K99E / N103Q / N113Q / N200Q / C252S / K264E, E59K / K99E / N103Q / N200Q / N281Q / C252S / K264E, E59K / K99E / N113Q / N200Q / N281Q / C252S / K264E, and E59K / K99E / N103Q / N113Q / N200Q / N281Q / C252S / K264E.

[0008] In some embodiments, the present invention relates to a method for the preparation of IL-12p40 subunits comprising administering to a patient a therapeutically effective amount of the IL-12p40 subunit, the method comprising administering to a patient a therapeutically effective amount of the IL-12p40 subunit, the therapeutically effective amount of the IL-12p40 subunit being: i) SEQ ID NO:57 (IL-12p40(N103D)), ii) SEQ ID NO:58 (IL-12p40(N113D)), iii) SEQ ID NO:59 (IL-12p40(N200D)), iv) SEQ ID NO:60 (IL-12p40(N281D)), v) SEQ ID NO:61 (IL-12p40(N103D / N113D / N200D / N281D)), vi) SEQ ID NO:62 (IL -12p40(Q42E)), vii) SEQ ID NO: 63 (IL-12p40(E45Q)), viii) SEQ ID NO: 64 (IL-12p40(Q56E)), ix) SEQ ID NO: 65 (IL-12p40(E59Q)), x) SEQ ID NO: 66 (IL-12p40(D62N)), xi) SEQ ID NO: 67 (IL-12p40(Q42E / E45Q)), xii) SEQ ID NO: 68 (IL-12p40(E45Q / Q56E)), xiii) SEQ ID NO: 69 (IL-12p40(Q42E / E59Q)), xiv) SEQ ID NO: 70 (IL-12p40(Q56E / E59Q)) , xv) SEQ ID NO: 71 (IL-12p40(Q42E / E45Q / Q56E)), xvi) SEQ ID NO: 72 (IL-12p40(E45Q / Q56E / E59Q)), xvii) SEQ ID NO: 73 (IL-12p40(D161N)), xviii) SEQ ID NO: 74 (IL-12p40(E73Q)), xix) SEQ ID NO: 75 (IL-12p40(Q144E)), xx) SEQ ID NO: 76 (IL-12p40(E262Q)), xxi) SEQ ID NO: 77 (IL-12p40(E100Q)), xxii) SEQ ID NO: 78 (IL-12p40(D18N)), xxiii ) SEQ ID NO: 79 (IL-12p40(E33Q)), xxiv) SEQ ID NO: 80 (IL-12p40(Q229E)), xxv) SEQ ID NO: 81 (IL-12p40(E235Q)), xxvi) SEQ ID NO: 82 (IL-12p40(Q256N)), xxvii) SEQ ID NO: 83 (IL-12p40(E299Q)), xxviii) SEQ ID NO: 84 (IL-12p40(D87N)), xxix) IL-12p40(E32Q), xxx) IL-12p40(D34N), xxxi) IL-12p40(S43E), xxxii) IL-12p40(S43 K), xxxiii) SEQ ID NO: XXX (IL-12p40(E59K / K99E / N103Q / N113Q / N200Q / N281Q / C252S / K264E)), xxxiv) SEQ ID NO: 205 (IL-12p40(E59K)), xxxv) IL-12p40(K99E), xxxvi) IL-12p40(K163E), xxxvii) IL-12p40(E187Q), xxxviii) IL-12p40(K258E), xxxix) IL-12p40(K260E), xl) SEQ ID NO: 206 (IL-12p40(E32Q / E59Q)),xli) SEQ ID NO: 207 (IL-12p40(D34N / E59Q)), xlii) SEQ ID NO: 208 (IL-12p40(E59Q / E187Q)), xliii) SEQ ID NO: 209 (IL-12p40(S43E / E59Q)), xliv) SEQ ID NO: 210 (IL-12p40(S43K / E49Q)), xlv) SEQ ID NO: 211 (IL-12p40(E59Q / K163E)), xlvi) SEQ ID NO: 212 (IL-12p40(E59Q / K99E)), xlvii) SEQ ID NO: 213 (IL-12p40(E59Q / K258E)), xlv iii) SEQ ID NO:214 (IL-12p40(E59Q / K260E)), xlix) SEQ ID NO:XXX (IL-12p40(D34N / E59K)), l) SEQ ID NO:XXX (IL-12p40(E59K / K99E)), li) SEQ ID NO:XXX (IL-12p40(D18K / E59K / K99E)), lii) SEQ ID NO:XXX (IL-12p40(E59K / K99E / K264E)), liii) SEQ ID NO:XXX (IL-12p40(E59K / K99Y)), liv) SEQ ID NO:XXX (IL-12p40(E59Y / K99E)), lv ) SEQ ID NO: XXX(IL-12p40(E45K / E59K / K99E)), lvi) SEQ ID NO: XXX(IL-12p40(E59K / K99E / Q144E)), lvii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / Q144K)), lviii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / R159E)), lix) SEQ ID NO: XXX(IL-12p40(E59K / K99E / K264E)), lx) SEQ ID NO: XXX(IL-12p40(D18K / E59K / K99E / K264E)), lxi) SEQ ID NO: No. XXX(IL-12p40(C252S)), lxii) No. XXX(IL-12p40(DI8K / E59K / K99E / C252S)), lxiii) No. XXX(IL-12p40(D18K / E59K / K99E / C252S / K264E)), lxiv) No. XXX(IL-12p40(E59K / K99Y / C252S)), lxv) No. XXX(IL-12p40(E59K / K99E / C252S / K264E)), lxvi) No. XXX(IL-12p40(E59K / K99E / C252S)),lxvii) SEQ ID NO: XXX (IL-12p40(N103D / N113D, )), lxviii) SEQ ID NO:XXX(IL-12p40(N103D / N200D)), lxix) SEQ ID NO:XXX(IL-12p40(N103D / N281D)), lxx) SEQ ID NO:XXX(IL-12p40(N113D / N200D)), lxxi) SEQ ID NO:XXX(IL-12p40(N113D / N281D)), lxxii) SEQ ID NO:XXX(IL-12p40(N200D / N281D)), lxxiii) SEQ ID NO:XXX(IL-12p50(N103D / N113D / N200D)), lxxiv) SEQ ID NO:XXX (IL-12p40(N103D / N113D / N281D)), lxxv) SEQ ID NO:XXX(IL-12p40(N103D / N200D / N281D)), lxxvi) SEQ ID NO:XXX(IL-12p40(N113D / N200D / N281D)), lxxvii) SEQ ID NO:XXX(IL-12p40(N103Q)), lxxviii) SEQ ID NO:XXX(IL-12p40(N113Q)), lxxix) SEQ ID NO:XXX(IL-12p40(N200Q)), lxxx) SEQ ID NO:XXX(IL-12p40(N281Q)), xxi) SEQ ID NO:XXX(IL-12p40(N103Q / N113Q)), lxxxii) SEQ ID NO:XXX(IL-12p40(N103Q / N200Q)), lxxxiii) SEQ ID NO:XXX(IL-12p40(N103Q / N281Q)), lxxxiv) SEQ ID NO:XXX(IL-12p40(N113Q / N200Q)), lxxxv) SEQ ID NO:XXX(IL-12p40(N113Q / N281Q)), lxxxvi) SEQ ID NO:XXX(IL-12p40(N200Q / N281Q)), lxxxvii) SEQ ID NO:XXX(IL -12p40(N103Q / N113Q / N200Q)), lxxxviii) SEQ ID NO: XXX(IL-12p40(N103Q / N113Q / N281Q)), lxxxix) SEQ ID NO: XXX(IL-12p40(N103Q / N200Q / N281Q)), xc) SEQ ID NO: XXX(IL-12p40(N113Q / N200Q / N281Q)), xci) SEQ ID NO: XXX(IL-12p40(N103Q / N113Q / N200Q / N281Q)), xcii) SEQ ID NO: XXX(IL-12p40(D34N / E59K / K99E)),xciii) SEQ ID NO: XXX (IL-12p40(D34K / E59K / K99E)), xciv) SEQ ID NO: XXX (IL-12p40(E32Q / D34N / E59K / K99E)), xcv) SEQ ID NO: XXX (IL-12p40(E32K / D34N / E59K / K99E)), xcvi) SEQ ID NO: XXX (IL-12p40(E59Y / K99Y)), xcvii) SEQ ID NO: XXX (IL-12p40(E59K / K99E / N103Q / C252S / K264E)), xcviii) SEQ ID NO: XXX (IL-12p40 (E59K / K99E / N113Q / C252S / K264E)), xcix) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N200Q / C252S / K264E)), c) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N281Q / C252S / K264E)), ci) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N103Q / N113Q / C252S / K264E)), cii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N103Q / N200Q / C252S / K 264E)), ciii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N103Q / N281Q / C252S / K264E)), civ) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N113Q / N200Q / C252S / K264E)), cv) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N113Q / N281Q / C252S / K264E)), cvi) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N200Q / N281Q / C252S / K264E)), cv ii) SEQ ID NO:XXX (IL-12p40(E59K / K99E / N103Q / N113Q / N200Q / C252S / K264E)), cviii) SEQ ID NO:XXX (IL-12p40(E59K / K99E / N103Q / N200Q / N281Q / C252S / K264E)), and cix) SEQ ID NO:XXX (IL-12p40(E59K / K99E / N113Q / N200Q / N281Q / C252S / K264E)).

[0009] In some embodiments, the invention provides heterodimeric Fc fusion proteins, wherein said variant IL-12p40 subunit has at least 90% identity to a polypeptide sequence selected from the group consisting of SEQ ID NO:3 (human IL-12 subunit beta (IL-12p40) precursor sequence) and SEQ ID NO:4 (human IL-12 subunit beta (IL-12p40) mature form sequence) and / or said IL-12p35 subunit has a polypeptide sequence selected from the group consisting of SEQ ID NO:1 (human IL-12 subunit alpha (IL-12p35) precursor sequence) and SEQ ID NO:2 (human IL-12 subunit alpha (IL-12p35) mature form sequence).

[0010] In some embodiments, the invention provides a heterodimeric Fc fusion protein, wherein said IL-12p35 subunit is a variant IL-12p35 subunit.

[0011] In some embodiments, the invention provides heterodimeric Fc fusion proteins wherein said IL-12p40 subunit is IL-12 receptor subunit beta-1 (IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2), and / or a variant IL-12p40 subunit with altered affinity for the IL-12 receptor complex, and / or said IL-12p35 subunit is IL-12 receptor subunit beta-1 (IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2), and / or a variant IL-12p35 subunit with altered affinity for the IL-12 receptor complex.

[0012] In some embodiments, the invention provides heterodimeric Fc fusion proteins, wherein the variant IL-12p35 subunit has one or more amino acid substitutions selected from the group consisting of N21D, Q35D, E38Q, D55Q, D55K, N71D, N71Q, L75A, N76D, E79Q, N85D, N85Q, L89A, F96A, M97A, L124A, M125A, Q130E, Q135E, N136D, E143Q, Q146E, N151D, N151K, E153K, E153Q, K158E, E162Q, E163Q, D165N, I171A, N195D, and N195Q.

[0013] In some embodiments, the invention provides heterodimeric Fc fusion proteins, wherein the variant IL-12p35 subunit has amino acid substitutions selected from the group consisting of N71D / N85D / N195D, N151D / E153Q, N151D / D165N, Q130E / N151D, N151D / K158E, E79Q / N151D, D55Q / N151D, N136D / N151D, N21D / N151D, E143Q / N151D, N71Q / N85Q, N71Q / N195Q, N85Q / N195Q, N71Q / N85Q / N195Q, N71D / N85D, N71D / N195D, and N85D / N195D.

[0014] In some embodiments, the present invention relates to a variant IL-12p35 subunit comprising i) SEQ ID NO:113 (IL-12p35(N71D)), ii) SEQ ID NO:114 (IL-12p35(N85D)), iii) SEQ ID NO:115 (IL-12p35(N195D)), iv) SEQ ID NO:116 (IL-12p35(N71D / N85D / N195D)), v) SEQ ID NO:117 (IL-12p35(E153Q)), vi) SEQ ID NO:118 (IL-12p35(E38Q)), vii) SEQ ID NO:119 (IL-12p35(N151 D), viii) SEQ ID NO: 120 (IL-12p35(Q135E)), ix) SEQ ID NO: 121 (IL-12p35(Q35D)), x) SEQ ID NO: 122 (IL-12p35(Q146E)), xi) SEQ ID NO: 123 (IL-12p35(N76D)), xii) SEQ ID NO: 124 (IL-12p35(E162Q)), xiii) SEQ ID NO: 125 (IL-12p35(E163Q)), xiv) IL-12p35(N21D), xv) SEQ ID NO: XXX (IL-12p35(D55Q)), xvi) IL-12p35(E79Q), x vii) IL-12p35(Q130E), xviii) IL-12p35(N136D), xix) IL-12p35(E143Q), xx) SEQ ID NO: 227 (IL-12p35(N151K)), xxi) SEQ ID NO: 226 (IL-12p35(E153K)), xxii) IL-12p35(K158E), xxiii) IL-12p35(D165N), xxiv) SEQ ID NO: 225 (IL-12p35(N151D / E153Q)), xxv) SEQ ID NO: 228 (IL-12p35(N151D / D165N)), xxvi) SEQ ID NO: 229 (IL-12p35(Q130E / N151D)), xxvii) SEQ ID NO: 230 (IL-12p35(N151D / K158E)), xxviii) SEQ ID NO: 231 (IL-12p35(E79Q / N151D)), xxix) SEQ ID NO: 232 (IL-12p35(D55Q / N151D)), xxx) SEQ ID NO: 233 (IL-12p35(N136D / N151D)), xxxi) SEQ ID NO: 234 (IL-12p35(N21D / N151D)), xxxii) SEQ ID NO: 235 (IL-12p35(E143Q / N151D)), xxxiii) SEQ ID NO:XXX(IL-12p35(F96A)), xxxiv) SEQ ID NO:XXX(IL-12p35(M97A)), xxxv) SEQ ID NO:XXX(IL-12p35(L89A)), xxxvi) SEQ ID NO:XXX(IL-12p35(L124A)), xxxvii) SEQ ID NO:XXX(IL-12p35(M125A)), xxxviii) SEQ ID NO:XXX(IL-12p35(L75A)), xxxiv) SEQ ID NO:XXX(IL-12p35(I171A)), xxxv) SEQ ID NO:XXX(IL-12p35(N71Q )), xxxvi) SEQ ID NO:XXX(IL-12p35(N85Q)), xxxvii) SEQ ID NO:XXX(IL-12p35(N195Q)), xxxviii) SEQ ID NO:XXX(IL-12p35(N71Q / N85Q)), xxxix) SEQ ID NO:XXX(IL-12p35(N71Q / N195Q)), xl) SEQ ID NO:XXX(IL-12p35(N85Q / N195Q), xli) SEQ ID NO:XXX(IL-12p35(N71Q / N85Q / N195Q)), xlii) SEQ ID NO:XXX(IL-12p35(N71D / N85D)),xliii) SEQ ID NO:XXX (IL-12p35(N71D / N195D); xliv) SEQ ID NO:XXX (IL-12p35(N85D / N195D)); xlv) SEQ ID NO:XXX (IL-12p35(D55Q)); and xlvi) SEQ ID NO:XXX (IL-12p35(D55K)).

[0015] In some embodiments, the invention provides heterodimeric Fc fusion proteins, wherein the modifications that promote heterodimerization of said first and said second Fc domains are a set of amino acid substitutions selected from the group consisting of: L368D / K370S and S364K; L368D / K370S and S364K / E357L; L368D / K370S and S364K / E357Q; T411E / K360E / Q362E and D401K; L368E / K370S and S364K; K370S and S364K / E357Q; T366S / L368A / Y407V and T366W; T366S / L368A / Y407V / Y349C and T366W / S354C, according to EU numbering.

[0016] In some embodiments, the invention provides heterodimeric Fc fusion proteins in which the variant IL-12p40 subunit domain is linked to the first Fc domain using a first domain linker and / or the IL-12p35 subunit domain is linked to the second Fc domain using a second domain linker.

[0017] In some embodiments, the invention provides heterodimeric Fc fusion proteins, wherein said first and / or said second Fc domain has an additional set of amino acid substitutions comprising Q295E / N384D / Q418E / N421D according to EU numbering.

[0018] In some embodiments, the invention provides a method for the preparation of a medicament for the first and / or second Fc domain. but having an additional set of amino acid substitutions selected from the group consisting of G236R / L328R, E233P / L234V / L235A / G236_ / S239K, E233P / L234V / L235A / G236_ / S239K / A327G, E233P / L234V / L235A / G236_ / S267K / A327G, E233P / L234V / L235A / G236_, and E233P / L234V / L235A / G236_ / S267K, according to EU numbering.

[0019] In some embodiments, the invention provides a heterodimeric Fc fusion protein, wherein said first and second Fc domains further comprise the amino acid substitutions M428L / N424S.

[0020] In some embodiments, the present invention relates to a heterodimeric Fc fusion protein comprising:

[0021] a) i) SEQ ID NO: 47 (XENP27201 chain 1), ii) SEQ ID NO: 85 (XenD24752), iii) SEQ ID NO: 86 (XenD24753), iv) SEQ ID NO: 87 (XenD24754), v) SEQ ID NO: 88 (XenD24755), vi) SEQ ID NO: 89 (XenD24756), vii) SEQ ID NO: 90 (XenD24757), viii) SEQ ID NO: 91 (XenD24758), ix) SEQ ID NO: 92 (XenD24759), x) SEQ ID NO: 93 (XenD24760), xi) SEQ ID NO: 94 (XenD24761), xii) SEQ ID NO: 95 (XenD24762), xiii) SEQ ID NO: 96 (XenD24763), xiv) SEQ ID NO: 97 (XenD24764), xv) SEQ ID NO: 98 (XenD24765), xvi) SEQ ID NO: 99 (XenD24766), xvii) SEQ ID NO: 100 (XenD24767), xviii) SEQ ID NO: 101 (XenD24768), xix) SEQ ID NO: 102 (XenD24769), xx) SEQ ID NO: 103 (XenD24770), xxi) SEQ ID NO: 104 (XenD24771), xxii) SEQ ID NO: 105 (XenD24772), x xiii) SEQ ID NO: 106 (XenD24773), xxiv) SEQ ID NO: 107 (XenD24774), xxv) SEQ ID NO: 108 (XenD24775), xxvi) SEQ ID NO: 109 (XenD24776), xxvii) SEQ ID NO: 110 (XenD24777), xxviii) SEQ ID NO: 111 (XenD24778), xxix) SEQ ID NO: 112 (XenD24792), xxx) SEQ ID NO: 215 (XenD25922), xxxi) SEQ ID NO: 216 (XenD25923), xxxii) SEQ ID NO: 217 (XenD25924), xx xiii) SEQ ID NO: 218 (XenD25925), xxxiv) SEQ ID NO: 219 (XenD25926), xxxv) SEQ ID NO: 220 (XenD25927), xxxvi) SEQ ID NO: 221 (XenD25928), xxxvii) SEQ ID NO: 222 (XenD25929), xxxviii) SEQ ID NO: 223 (XenD25930), xxxix) SEQ ID NO: 224 (XenD25931), xl) SEQ ID NO: XXX (XenD26411), xli) SEQ ID NO: XXX (XenD26412), xlii) SEQ ID NO: XXX (XenD26413),xliii) SEQ ID NO: XXX(XenD26414), xliv) SEQ ID NO: XXX(XenD26415), xlv) SEQ ID NO: XXX(XenD26416), xlvi) SEQ ID NO: XXX(XenD26417), xlvii) SEQ ID NO: XXX(XenD26418), xlviii) SEQ ID NO: XXX(XenD27070), xlix) SEQ ID NO: XXX(XenD27071), l) SEQ ID NO: XXX(XenD27072), li) SEQ ID NO: XXX(XenD27073), lii) SEQ ID NO: XXX(XenD27074, liii) SEQ ID NO: XXX(X enD27075), liv) SEQ ID NO: XXX (XenD27076), lv) SEQ ID NO: XXX (XenD27077), lvi) SEQ ID NO: XXX (XenD27078), lvii) SEQ ID NO: XXX (XenD28173), lviii) SEQ ID NO: XXX (XenD24876), lix) SEQ ID NO: XXX (XenD27162), lx) SEQ ID NO: XXX (XenD27163), lxi) SEQ ID NO: XXX (XenD27164), lxii) SEQ ID NO: XXX (XenD27165) lxiii) SEQ ID NO: XXX (XENP31582 strand 1), lxiv) sequence, Sequence number XXX (XENP31583 strand 1), lxv) sequence number XXX (XENP31584 strand 1), lxvi) sequence number XXX (XENP32187 strand 1), lxvii) sequence number XXX (XENP32188 strand 1), lxviii) sequence number XXX (XENP32189, strand 1), lxix) sequence number XXX (XENP32190 strand 1), lxx) sequence number XXX (XENP32191 strand 1), lxxi) sequence number XXX (XENP32991 strand 1), l xxii) SEQ ID NO: XXX (XENP32992 strand 1), lxxiii) SEQ ID NO: XXX (XENP32993 strand 1), lxxiv) SEQ ID NO: XXX (XENP32994 strand 1), lxxv) SEQ ID NO: XXX (XENP32995 strand 1), lxxvi) SEQ ID NO: XXX (XENP32996 strand 1), lxxvi) SEQ ID NO: XXX (XENP32997 strand 1), lxxvii) SEQ ID NO: XXX (XENP32998 strand 1), lxxvii) SEQ ID NO: XXX (XE NP32999 strand 1), lxxviii) SEQ ID NO: XXX (XENP33000 strand 1), lxxix) SEQ ID NO: XXX (XENP33001 strand 1), lxxx) SEQ ID NO: XXX (XENP33002 strand 1), lxxxi) SEQ ID NO: XXX (XENP33003 strand 1), lxxxii) SEQ ID NO: XXX (XENP33004 strand 1), lxxxiii) SEQ ID NO: XXX (XENP33005 strand 1), lxxxiv) SEQ ID NO: XXX (XENP33006 strand 1), said first fusion protein having a polypeptide sequence selected from the group consisting of: lxxxv) SEQ ID NO:XXX (XENP33007 chain 1); lxxxvi) SEQ ID NO:XXX (XENP33008 chain 1); lxxxvii) SEQ ID NO:XXX (XENP33008 chain 1); lxxxviii) SEQ ID NO:XXX (XENP33009 chain 1); lxxxix) SEQ ID NO:XXX (XENP33010 chain 1); and xc) SEQ ID NO:XXX (XENP33011 chain 1);

[0022] b) i) SEQ ID NO: 48 (XENP27201 strand 2), ii) SEQ ID NO: 126 (XenD24779), iii) SEQ ID NO: 127 (XenD24780), iv) SEQ ID NO: 128 (XenD24781), v) SEQ ID NO: 129 (XenD24782), vi) SEQ ID NO: 130 (XenD24783), vii) SEQ ID NO: 131 (XenD24784), viii) SEQ ID NO: 132 (XenD24785), ix) SEQ ID NO: 133 (XenD24786), x) SEQ ID NO: 134 (XenD24787), xi) SEQ ID NO: 135 (XenD24788), xii) SEQ ID NO: 136 (XenD24789), xiii) SEQ ID NO: 137 (XenD24790), xiv) SEQ ID NO: 138 (XenD24791), xv) SEQ ID NO: 236 (XenD25911), xvi) SEQ ID NO: 237 (XenD25912), xvii) SEQ ID NO: 238 (XenD25913), xviii) SEQ ID NO: 239 (XenD25914), xix) SEQ ID NO: 240 (XenD25915), xx) SEQ ID NO: 241 (XenD25916), xxi) SEQ ID NO: 242 (XenD25917), xxii) SEQ ID NO: 243 (XenD25918) ), xxiii) SEQ ID NO: 244 (XenD25919), xxiv) SEQ ID NO: 245 (XenD25920), xxv) SEQ ID NO: 246 (XenD25921), xxvi) SEQ ID NO: XXX (XenD26427), xxvii) SEQ ID NO: XXX (XenD26428), xxviii) SEQ ID NO: XXX (XenD27089), xxix) SEQ ID NO: XXX (XenD27090), xxx) SEQ ID NO: XXX (XenD27091), xxxi) SEQ ID NO: XXX (XenD27092), xxxii) SEQ ID NO: XXX (XenD27093), xxxii i) SEQ ID NO:XXX (XenD27094), xxxix) SEQ ID NO:XXX (XenD24877), xl) SEQ ID NO:XXX (XenD27166), xli) SEQ ID NO:XXX (XENP31582 strand 2), xlii) SEQ ID NO:XXX (XENP31583 strand 2), xliii) SEQ ID NO:XXX (XENP31584 strand 2), xliv) SEQ ID NO:XXX (XENP32187 strand 2), xlv) SEQ ID NO:XXX (XENP32188 strand 2), xlvi) SEQ ID NO:XXX (XENP32189, strand 2), xlvii) SEQ ID NO:XXX (XENP32190 strand 2),xlviii) SEQ ID NO: XXX (XENP32191 strand 2), xlix) SEQ ID NO: XXX (XENP32991 strand 2), l) SEQ ID NO: XXX (XENP32992 strand 2), li) SEQ ID NO: XXX (XENP32993 strand 2), lii) SEQ ID NO: XXX (XENP3, 2994 strand 2), liii) SEQ ID NO: XXX (XENP32995 strand 2), liv) SEQ ID NO: XXX (XENP32996 strand 2), lv) SEQ ID NO: XXX (XENP32997 strand 2), lvi) SEQ ID NO: XXX (XENP32998 strand 2), lvii) SEQ ID NO: XXX (XENP32999 strand 2), lviii) SEQ ID NO: XXX (XENP33000 strand 2), lix) SEQ ID NO: XXX (XENP33001 strand 2), lx) SEQ ID NO: XXX (XENP33002 strand 2), lxi) SEQ ID NO: XXX (XENP33003 strand 2), lxii) SEQ ID NO: XXX (XENP33004 strand 2), lxiii) SEQ ID NO: and said second fusion protein having a polypeptide sequence selected from the group consisting of SEQ ID NO:XXX (XENP33005 chain 2), lxiv) SEQ ID NO:XXX (XENP33006 chain 2), lxv) SEQ ID NO:XXX (XENP33007 chain 2), lxvi) SEQ ID NO:XXX (XENP33008 chain 2), lxvii) SEQ ID NO:XXX (XENP33008 chain 2), lxviii) SEQ ID NO:XXX (XENP33009 chain 2), lxix) SEQ ID NO:XXX (XENP33010 chain 2), and lxx) SEQ ID NO:XXX (XENP33011 chain 2).

[0023] In some embodiments, the present invention provides heterodimeric Fc fusion proteins, the heterodimeric Fc fusion proteins being selected from the group consisting of XENP27201, XENP28820, XENP28821, XENP28822, XENP28823, XENP28824, XENP28825, XENP28826, XENP28827, XENP28828, XENP28829, XENP28830, XENP28831, XENP28832, XENP28833, XENP28834, XENP28835, XENP28836, XENP28837, XEN P28838, XENP28839, XENP28840, XENP28841, XENP28842, XENP28843, XENP28844, XENP28845, XENP28846, XENP28847, XENP28848, XENP28849, XENP288 50, XENP28851, NP30603, XENP30604, XENP30605, XENP30606, XENP30307, ​​XENP30308, XENP30609, XENP31250, XENP31251, XENP31252, XENP31253, XENP31254, XENP31 255, XENP31256, XENP31257, XENP31258, XENP31259, XENP31260, XENP31261, XENP31262, XENP31263,XENP31264, ENP31143, XENP31144, XENP31145, XENP31146, XENP31582, XENP31583, XENP31584, XENP32186, XENP32187, XENP32188, XENP32189, XENP32190, XENP3 2191, XENP32991, XENP32992, XENP32993, XENP32994, XENP32995, XENP32996, XENP32997, XENP32998, XENP32999, XENP33000,Selected from the group consisting of XENP33003, XENP33004, XENP33005, XENP33006, XENP33007, XENP33008, XENP33008, XENP33009, XENP33010, and XENP33011.

[0024] In some embodiments, the invention provides a heterodimeric Fc fusion protein, wherein the heterodimeric Fc fusion protein is selected from the group consisting of XENP31251, XENP31254, XENP31258, XENP32186, XENP32187, XENP32188, XENP32189, XENP32190, and XENP32191.

[0025] In some embodiments, the invention provides heterodimeric Fc fusion proteins further comprising one or more modifications to the IL-12p40 subunit selected from the group consisting of N103Q, N113Q, N200Q, and N281Q.

[0026] In some embodiments, the present invention provides a composition comprising a heterodimeric Fc-fusion protein for use in treating cancer in a subject.

[0027] In some embodiments, the invention provides one or more nucleic acids encoding a heterodimeric Fc fusion protein.

[0028] In some embodiments, the invention provides host cells comprising one or more of the nucleic acids encoding the heterodimeric Fc fusion proteins.

[0029] In some embodiments, the invention provides a method of making a heterodimeric Fc-fusion protein, the method comprising culturing a host cell under conditions whereby the heterodimeric Fc-fusion protein is produced.

[0030] In some embodiments, the invention provides a method for purifying a heterodimeric Fc-fusion protein, the method comprising: a) providing a composition comprising a heterodimeric Fc-fusion protein; b) loading the composition onto an ion exchange column; and c) collecting fractions containing the heterodimeric Fc-fusion protein.

[0031] In some embodiments, the present invention relates to an IL-12p40 subunit comprising: E3, D7, E12, D14, W15, P17, D18, A19, P20, G21, E22, M23, D29, E32, E33, D34, L40, D41, Q42, S43, E45, L47, T54, I55, Q56, K58, E59, F 60, G61, D62, Q65, Y66, E73, K84, E86, D87, G88, I89, W90, D93, D97, K99, E100, K102, N1 03, K104, F106, E110, N113, Y114, D129, D142, Q144, E156, R159, D161, N162, K163, D16 6, D170, Q172, D174, A176, C177, P178, A179, A180, E181, S183, P185, E187, N200, S204 , F206, R208, D209, D214, N218, Q220, N226, Q229, E231, E235, T242, P243, S245, Y246, Provided are heterodimeric Fc fusion proteins having one or more amino acid modifications at amino acid residues selected from the group consisting of F247, S248, C252, Q256, K258, K260, E262, K264, D265, D270, N281, Q289, D290, R291, Y292, Y293, and E299.

[0032] In some embodiments, the present invention relates to an IL-12 p35 subunit comprising one or more of the following: Q20, N21, Q35, E38, S44, E45, E46, H49, K54, D55, T59, V60, E61, C63, L64, P65, E67, L68, N71, S73, C74, L75, N76, E79, N85, L89, F96, M97, L124, M125, Q130, Q135, N and N195, and A196.

[0033] In another aspect, the present invention provides a first fusion protein comprising: a) a first protein domain and a first Fc domain, said first protein domain being a first Fc domain; and b) a second fusion protein comprising a second protein domain and a second Fc domain, the second protein domain being covalently linked to the C-terminus of the Fc domain, wherein the first and second Fc domains comprise a modification that promotes heterodimerization of the first and second Fc domains, and wherein the first protein domain comprises an IL-12p40 subunit and the second protein domain comprises an IL-12p35 subunit.

[0034] In some embodiments, heterodimeric Fc fusion proteins are provided wherein said modifications that promote heterodimerization of said first and said second Fc domains are a set of amino acid substitutions selected from the group consisting of: L368D / K370S and S364K; L368D / K370S and S364K / E357L; L368D / K370S and S364K / E357Q; T411E / K360E / Q362E and D401K; L368E / K370S and S364K; K370S and S364K / E357Q and T366S / L368A / Y407V:T366W (optionally including a bridging disulfide, T366S / L368A / Y407V / Y349C:T366W / S354C), according to EU numbering.

[0035] In some embodiments, the invention provides heterodimeric Fc-fusion proteins, wherein said first protein domain is linked to said first Fc domain using a first domain linker and / or said second protein domain is linked to said second Fc domain using a second domain linker.

[0036] In some embodiments, the invention provides heterodimeric Fc fusion proteins, wherein said first and / or said second Fc domain has an additional set of amino acid substitutions comprising: Q295E / N384D / Q418E / N421D, according to EU numbering.

[0037] In some embodiments, the invention provides heterodimeric Fc fusion proteins, wherein said first and / or said second Fc domain has an additional set of amino acid substitutions selected from the group consisting of G236R / L328R, E233P / L234V / L235A / G236_ / S239K, E233P / L234V / L235A / G236_ / S239K / A327G, E233P / L234V / L235A / G236_ / S267K / A327G, E233P / L234V / L235A / G236_, and E233P / L234V / L235A / G236_ / S267K, according to EU numbering.

[0038] In some embodiments, the invention provides heterodimeric Fc fusion proteins, wherein said IL-12p40 subunit has a polypeptide sequence selected from the group consisting of SEQ ID NO:3 (human IL-12 subunit beta (IL-12p40) precursor sequence) and SEQ ID NO:4 (human IL-12 subunit beta (IL-12p40) mature form sequence) and / or said IL-12p35 subunit has a polypeptide sequence selected from the group consisting of SEQ ID NO:1 (human IL-12 subunit alpha (IL-12p35) precursor sequence) and SEQ ID NO:2 (human IL-12 subunit alpha (IL-12p35) mature form sequence).

[0039] In some embodiments, the invention provides a heterodimeric Fc fusion protein, wherein said first and second Fc domains further comprise the amino acid substitutions M428L / N424S.

[0040] In some embodiments, the invention relates to a method for treating IL-12p40 infection in a mammalian animal, comprising administering to said mammalian animal an IL-12p40 subunit, a variant IL-12p40 subunit, and / or a variant IL-12p35 subunit. The present invention provides a heterodimeric Fc fusion protein, wherein the subunit is a variant IL-12p35 subunit.

[0041] In some embodiments, the invention provides heterodimeric Fc fusion proteins wherein said IL-12p40 subunit is IL-12 receptor subunit beta-1 (IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2), and / or a variant IL-12p40 subunit with altered affinity for the IL-12 receptor complex, and / or said IL-12p35 subunit is IL-12 receptor subunit beta-1 (IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2), and / or a variant IL-12p35 subunit with altered affinity for the IL-12 receptor complex.

[0042] In some embodiments, the present invention relates to an IL-12p40 subunit comprising: E3, D7, E12, D14, W15, P17, D18, A19, P20, G21, E22, M23, D29, E32, E33, D34, L40, D41, Q42, S43, E45, L47, T54, I55, Q56, K58, E59, F 60, G61, D62, Q65, Y66, E73, K84, E86, D87, G88, I89, W90, D93, D97, K99, E100, K102, N1 03, K104, F106, E110, N113, Y114, D129, D142, Q144, E156, R159, D161, N162, K163, D16 6, D170, Q172, D174, A176, C177, P178, A179, A180, E181, S183, P185, E187, N200, S204 , F206, R208, D209, D214, N218, Q220, N226, Q229, E231, E235, T242, P243, S245, Y246, Provided are heterodimeric Fc fusion proteins having one or more amino acid modifications at amino acid residues selected from the group consisting of F247, S248, C252, Q256, K258, K260, E262, K264, D265, D270, N281, Q289, D290, R291, Y292, Y293, and E299.

[0043] In some embodiments, the present invention relates to a method for the preparation of IL-12p40 subunits comprising administering to the patient a IL-12p40 subunit comprising administering to the patient a IL-12p40 subunit comprising administering to the patient a IL-12p40 subunit comprising administering to the patient a IL-12p40 subunit comprising administering to the patient a IL-12p40 subunit comprising administering to the patient a IL-12p40 subunit , D161N, R159E, K163E, E187Q, N200D, N200Q, N218Q, Q229E, E235Q, C252S, Q256N, K258E, K260E, E262Q, K264E, N281D, N281Q, and E299Q.

[0044] In some embodiments, the present invention relates to a method for the preparation of IL-12p40 subunits comprising administering to the patient a sequence similar to that described herein, comprising administering to the patient a sequence similar to that described herein, wherein the IL-12p40 subunit is N103D / N113D / N200D / N281D, Q42E / E45Q, E45Q / Q56E, Q42E / E59Q, Q56E / E59Q, Q42E / E45Q / Q56E, E45Q / Q56E / E59Q, E32Q / E59Q, D34N / E59K, D34N / E59K / K99E, D34K / E59K / K99E, E32Q / D34N / E59K / K99E, E32K / D34N / E59K / K99E, D34N / E59Q, E59Q / E187Q , S43E / E59Q, S43K / E49Q, E59Q / K163E, E59Q / K99E, E59Q / K258E, E59Q / K2 60E, E59K / K99E, D18K / E59K / K99E, E59K / K99E / K264E, E59K / K99Y, E59Y / K99Y, E59Y / K99E, E45K / E59K / K99E, E59K / K99E / Q144E, E59K / K99E / Q144 K, E59K / K99E / R159E, E59K / K99E / K264E, D18K / E59K / K99E / K264E, DI8K / E59K / K99E / C252S, D18K / E59K / K99E / C252S / K264E, E59K / K99Y / C252S , E59K / K99E / C252S / K264E, E59K / K99E / C252S, N103D / N113D, N103D / N2 00D, N103D / N281D, N113D / N200D, N113D / N281D, N200D / N281D, N103D / N113D / N200D, N103D / N113D / N281D, N103D / N200D / N281D, N113D / N200D / N281D, N103Q / N113Q, N103Q / N200Q, N103Q / N281Q, N113Q / N200Q, N11 3Q / N281Q, N200Q / N281Q, N103Q / N113Q / N200Q, N103Q / N113Q / N281Q, N1 03Q / N200Q / N281Q, N113Q / N200Q / N281Q, N103Q / N113Q / N200Q / N281Q, E59K / K99E / N103Q / C252S / K264E, E59K / K99E / N113Q / C252S / K264E, E59 K / K99E / N200Q / C252S / K264E, E59K / K99E / N281Q / C252S / K264E, E59K / K99E / N103Q / N113Q / C252S / K264E, E59K / K99E / N103Q / N200Q / C252S / K2 64E, E59K / K99E / N103Q / N281Q / C252S / K264E, E59K / K99E / N113Q / N200 Q / C252S / K264E, E59K / K99E / N113Q / N281Q / C252S / K264E, E59K / K99E / N The present invention provides heterodimeric Fc fusion proteins having amino acid substitutions selected from the group consisting of 200Q / N281Q / C252S / K264E, E59K / K99E / N103Q / N113Q / N200Q / C252S / K264E, E59K / K99E / N103Q / N200Q / N281Q / C252S / K264E, E59K / K99E / N113Q / N200Q / N281Q / C252S / K264E, and E59K / K99E / N103Q / N113Q / N200Q / N281Q / C252S / K264E.

[0045] In some embodiments, the present invention relates to a method for the preparation of IL-12p40 subunits comprising administering to the patient a sequence similar to that described herein, comprising administering to the patient a sequence similar to that described herein, and / or administering to the patient a sequence similar to that described herein, , S43K / E49Q, E59Q / K163E, E59Q / K99E, E59Q / K258E, E59Q / K260E, E59K / K99 E, D18K / E59K / K99E, E59K / K99E / K264E, E59K / K99Y, E59Y / K99Y, E59Y / K99E, E45K / E59K / K99E, E59K / K99E / Q144E, E59K / K99E / Q144K, E59K / K99E / R159E , E59K / K99E / K264E, D18K / E59K / K99E / K264E, DI8K / E59K / K99E / C252S, D18K / E59K / K99E / C252S / K264E, E59K / K99Y / C252S, E59K / K99E / C252S / K264E, E 59K / K99E / C252S, N103D / N113D, N103D / N200D, N103D / N281D, N113D / N200D, N113D / N281D, N200D / N281D, N103D / N113D / N200D, N103D / N113D / N281D, N1 03D / N200D / N281D, N113D / N200D / N281D, N103Q / N113Q, N103Q / N200Q, N103Q / N281Q, N113Q / N200Q, N113Q / N281Q, N200Q / N281Q, N103Q / N113Q / N200Q, N 103Q / N113Q / N281Q, N103Q / N200Q / N281Q, N113Q / N200Q / N281Q, N103Q / N113 Q / N200Q / N281Q, E59K / K99E / N103Q / C252S / K264E, E59K / K99E / N113Q / C252 S / K264E, E59K / K99E / N200Q / C252S / K264E, E59K / K99E / N281Q / C252S / K264E , E59K / K99E / N103Q / N113Q / C252S / K264E, E59K / K99E / N103Q / N200Q / C252S / K264E, E59K / K99E / N103Q / N281Q / C252S / K264E, E 59K / K99E / N113Q / N200Q / C252S / K264E, E59K / K99E / N113Q / N281Q / C252S / K264E, E59K / K99E / N200Q / N281Q / C252S / K264E, E59 Heterodimeric Fc fusion proteins are provided having amino acid substitutions selected from the group consisting of K / K99E / N103Q / N113Q / N200Q / C252S / K264E, E59K / K99E / N103Q / N200Q / N281Q / C252S / K264E, E59K / K99E / N113Q / N200Q / N281Q / C252S / K264E, and E59K / K99E / N103Q / N113Q / N200Q / N281Q / C252S / K264E.

[0046] In some embodiments, the present invention relates to a method for the preparation of IL-12p40 subunits comprising administering to a patient a therapeutically effective amount of the IL-12p40 subunit, the method comprising administering to a patient a therapeutically effective amount of the IL-12p40 subunit, the therapeutically effective amount of the IL-12p40 subunit being: i) SEQ ID NO:57 (IL-12p40(N103D)); ii) SEQ ID NO:58 (IL-12p40(N113D)); iii) SEQ ID NO:59 (IL-12p40(N200D)); iv) SEQ ID NO:60 (IL-12p40(N281D)); v) SEQ ID NO:61 (IL-12p40(N103D / N113D / N200D / N281D)); vi) SEQ ID NO:62 (IL-12p40(Q42E)); vii) SEQ ID NO:63 (IL-12p40(E45 Q)), viii) SEQ ID NO:64 (IL-12p40(Q56E)), ix) SEQ ID NO:65 (IL-12p40(E59Q)), x) SEQ ID NO:66 (IL-12p40(D62N)), xi) SEQ ID NO:67 (IL-12p40(Q42E / E45Q)), xii) SEQ ID NO:68 (IL-12p40(E45Q / Q56E)), xiii) SEQ ID NO:69 (IL-12p40(Q42E / E59Q)), xiv) SEQ ID NO:70 (IL-12p40(Q56E / E59Q)), xv) SEQ ID NO:71 (IL-12p40(Q42E / E 45Q / Q56E)), xvi) SEQ ID NO: 72 (IL-12p40(E45Q / Q56E / E59Q)), xvii) SEQ ID NO: 73 (IL-12p40(D161N)), xviii) SEQ ID NO: 74 (IL-12p40(E73Q)), xix) SEQ ID NO: 75 (IL-12p40(Q144E)), xx) SEQ ID NO: 76 (IL-12p40(E262Q)), xxi) SEQ ID NO: 77 (IL-12p40(E100Q)), xxii) SEQ ID NO: 78 (IL-12p40(D18N)), xxiii) SEQ ID NO: 79 (IL-12p 40(E33Q)), xxiv) SEQ ID NO: 80 (IL-12p40(Q229E)), xxv) SEQ ID NO: 81 (IL-12p40(E235Q)), xxvi) SEQ ID NO: 82 (IL-12p40(Q256N)), xxvii) SEQ ID NO: 83 (IL-12p40(E299Q)), xxviii) SEQ ID NO: 84 (IL-12p40(D87N)), xxix) IL-12p40(E32Q), xxx) IL-12p40(D34N), xxxi) IL-12p40(S43E), xxxii) IL-12p40(S43K),xxxiii) SEQ ID NO:XXX (IL-12p40(E59K / K99E / N103Q / N113Q / N200Q / N281Q / C252S / K264E)), xxxiv) SEQ ID NO:205 (IL-12p40(E59K)), xxxv) IL-12p40(K99E), xxxvi) IL-12p40(K163E), xxxvii) IL-12p40(E187Q), xxxviii) IL-12p40(K258E), xxxix) IL-12p40(K260E), xl) SEQ ID NO:206 (IL-12p40(E32Q / E59Q)), xli) SEQ ID NO:207 (IL-12p40(D34N / E59Q)), xlii) SEQ ID NO:2 08 (IL-12p40(E59Q / E187Q)), xliii) SEQ ID NO: 209 (IL-12p40(S43E / E59Q)), xliv) SEQ ID NO: 210 (IL-12p40(S43K / E49Q)), xlv) SEQ ID NO: 211 (IL-12p40(E59Q / K163E)), xlvi) SEQ ID NO: 212 (IL-12p40(E59Q / K99E)), xlvii) SEQ ID NO: 213 (IL-12p40(E59Q / K258E)), xlviii) SEQ ID NO: 214 (IL-12p40(E59Q / K260E)), xlix) SEQ ID NO: XXX (IL-12p40(D34N / E59K)), l) SEQ ID NO: XXX (IL-12p40, (E59K / K99E)), li) SEQ ID NO: XXX(IL-12p40(D18K / E59K / K99E)), lii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / K264E)), liii) SEQ ID NO: XXX(IL-12p40(E59K / K99Y)), liv) SEQ ID NO: XXX(IL-12p40(E59Y / K99E)), lv) SEQ ID NO: XXX(IL-12p40(E45K / E59K / K99E)), lvi) SEQ ID NO: XXX(IL-12p40(E59K / K99E / Q144E)), lvii) SEQ ID NO: XXX(IL- IL-12p40(E59K / K99E / Q144K)), lviii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / R159E)), lix) SEQ ID NO: XXX(IL-12p40(E59K / K99E / K264E)), lx) SEQ ID NO: XXX(IL-12p40(D18K / E59K / K99E / K264E)), lxi) SEQ ID NO: XXX(IL-12p40(C252S)), lxii) SEQ ID NO: XXX(IL-12p40(DI8K / E59K / K99E / C252S)), lxiii) SEQ ID NO: XXX(IL-12p40(D18K / E59K / K99E / C252S / K264E)), lxiv) SEQ ID NO: XXX(IL-12p40(E59K / K99Y / C252S)), lxv) SEQ ID NO: XXX(IL-12p40(E59K / K99E / C252S / K264E)), lxvi) SEQ ID NO: XXX(IL-12p40(E59K / K99E / C252S)), lxvii) SEQ ID NO: XXX(IL-12p40(N103D / N113D)), lxviii) SEQ ID NO: XXX(IL-12p40(N103D / N200D)), lxix) SEQ ID NO: XXX(IL-12p40(N1 lxx) SEQ ID NO: XXX(IL-12p40(N113D / N200D)), lxxi) SEQ ID NO: XXX(IL-12p40(N113D / N281D)), lxxii) SEQ ID NO: XXX(IL-12p40(N200D / N281D)), lxxiii) SEQ ID NO: XXX(IL-12p50(N103D / N113D / N200D)), lxxiv) SEQ ID NO: XXX(IL-12p40(N103D / N113D / N281D)), lxxv) SEQ ID NO: XXX(IL-12p40(N103D / N200D / N281D)),lxxvi) SEQ ID NO:XXX(IL-12p40(N113D / N200D / N281D)), lxxvii) SEQ ID NO:XXX(IL-12p40(N103Q)), lxxviii) SEQ ID NO:XXX(IL-12p40(N113Q)), lxxix) SEQ ID NO:XXX(IL-12p40(N200Q)), lxxx) SEQ ID NO:XXX(IL-12p40(N281Q)), lxxxi) SEQ ID NO:XXX(IL-12p40(N103Q / N113Q)), lxxxii) SEQ ID NO:XXX(IL-12p40(N103Q / N200Q)), lxxxiii) SEQ ID NO: XXX(IL-12p40(N103Q / N281Q)), lxxxiv) SEQ ID NO: XXX(IL-12p40(N113Q / N200Q)), lxxxv) SEQ ID NO: XXX(IL-12p40(N113Q / N281Q)), lxxxvi) SEQ ID NO: XXX(IL-12p40(N200Q / N281Q)), lxxxvii) SEQ ID NO: XXX(IL-12p40(N103Q / N113Q / N200Q)), lxxxviii) SEQ ID NO: XXX(IL-12p40(N103Q / N113Q / N281Q)), xxxix) SEQ ID NO: XXX(IL-12p40(N103Q / N200Q / N281Q)), xc) SEQ ID NO: XXX(IL-12p40(N113Q / N200Q / N281Q)), xci) SEQ ID NO: XXX(IL-12p40(N103Q / N113Q / N200Q / N281Q)), xcii) SEQ ID NO: XXX(IL-12p40(D34N / E59K / K99E)), xciii) SEQ ID NO: XXX(IL-12p40(D34K / E59K / K99E)), xciv) SEQ ID NO: XXX(IL-12p40(E32Q / D34N / E59 K / K99E)), xcv) SEQ ID NO: XXX(IL-12p40(E32K / D34N / E59K / K99E)), xcvi) SEQ ID NO: XXX(IL-12p40(E59Y / K99Y)), xcvii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N103Q / C252S / K264E)), xcviii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N113Q / C252S / K264E)), xcix) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N200Q / C252S / K264E)),c) SEQ ID NO: XXX (IL-12p40(E59K / K99E / , N281Q / C252S / K264E)), ci) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N103Q / N113Q / C252S / K264E)), cii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N103Q / N200Q / C252S / K264E)), ciii) SEQ ID NO: XXX(IL-1 2p40(E59K / K99E / N103Q / N281Q / C252S / K264E)), civ) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N113Q / N200Q / C252S / K264E)), cv) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N113Q / N281Q / C252S / K264E)), E)), cvi) SEQ ID NO:XXX(IL-12p40(E59K / K99E / N200Q / N281Q / C252S / K264E)), cvii) SEQ ID NO:XXX(IL-12p40(E59K / K99E / N103Q / N113Q / N200Q / C252S / K264E)), cviii) SEQ ID NO:XXX(IL-12p40(E59K / K99E / N103Q / N200Q / N281Q / C252S / K264E)), and cix) SEQ ID NO:XXX(IL-12p40(E59K / K99E / N113Q / N200Q / N281Q / C252S / K264E)). In some embodiments, the present invention relates to an IL-12 p35 subunit comprising one or more of the following: Q20, N21, Q35, E38, S44, E45, E46, H49, K54, D55, T59, V60, E61, C63, L64, P65, E67, L68, N71, S73, C74, L75, N76, E79, N85, L89, F96, M97, L124, M125, Q130, Q135, N and N195, and A196.

[0047] In some embodiments, the invention provides heterodimeric Fc fusion proteins, wherein the IL-12p35 subunit has one or more amino acid substitutions selected from the group consisting of N21D, Q35D, E38Q, D55Q, D55K, N71D, N71Q, L75A, N76D, E79Q, N85D, N85Q, L89A, F96A, M97A, L124A, M125A, Q130E, Q135E, N136D, E143Q, Q146E, N151D, N151K, E153K, E153Q, K158E, E162Q, E163Q, D165N, I171A, N195D, and N195Q.

[0048] In some embodiments, the invention provides heterodimeric Fc fusion proteins, wherein the IL-12p35 subunit has amino acid substitutions selected from the group consisting of N71D / N85D / N195D, N151D / E153Q, N151D / D165N, Q130E / N151D, N151D / K158E, E79Q / N151D, D55Q / N151D, N136D / N151D, N21D / N151D, E143Q / N151D, N71Q / N85Q, N71Q / N195Q, N85Q / N195Q, N71Q / N85Q / N195Q, N71D / N85D, N71D / N195D, and N85D / N195D.

[0049] In some embodiments, the present invention relates to a method for the preparation of a IL-12p35 subunit comprising administering to a patient a sequence identical to that of SEQ ID NO: 113 (IL-12p35(N71D)), ii) SEQ ID NO: 114 (IL-12p35(N85D)), iii) SEQ ID NO: 115 (IL-12p35(N195D)), iv) SEQ ID NO: 116 (IL-12p35(N71D / N85D / N195D)), v) the sequence SEQ ID NO: No. 117 (IL-12p35(E153Q)), vi) SEQ ID NO: 118 (IL-12p35(E38Q)), vii) SEQ ID NO: 119 (IL-12p35(N151D)), viii) SEQ ID NO: 120 (IL-12p35(Q135E)), ix) SEQ ID NO: 121 (IL-12p35(Q35D)), x) SEQ ID NO: 122 (IL-12p35(Q146E )), xi) SEQ ID NO: 123 (IL-12p35(N76D)), xii) SEQ ID NO: 124 (IL-12p35(E162Q)), xiii) SEQ ID NO: 125 (IL-12p35(E163Q)), xiv) IL-12p35(N21D), xv) SEQ ID NO: XXX (IL-12p35(D55Q)), xvi) IL-12p35(E79Q), xvii) IL-12p35(Q130E), xviii) IL-12p35(N136D), xix) IL-12p35(E143Q), xx) SEQ ID NO: 227 (IL-12p35(N151K)), x xi) SEQ ID NO: 226 (IL-12p35(E153K)), xxii) IL-12p35(K158E), xxiii) IL-12p35(D165N), xxiv) SEQ ID NO: 225 (IL-12p35(N151D / E153Q)), xxv) SEQ ID NO: 228 (IL-12p35(N151D / D165N)), xxvi) SEQ ID NO: 229 (IL-12p35(Q130E / N151D)), xxvii) SEQ ID NO: 230 (IL-12p35(N151D / K158E)), xxviii) SEQ ID NO: 231 (IL-12p35(E79Q / N1 51D)), xxix) SEQ ID NO:232 (IL-12p35(D55Q / N151D)), xxx) SEQ ID NO:233 (IL-12p35(N136D / N151D)), xxxi) SEQ ID NO:234 ​​(IL-12p35(N21D / N151D)), xxxii) SEQ ID NO:235 (IL-12p35(E143Q / N151D)), xxxiii) SEQ ID NO:XXX(IL-12p35(F96A)), xxxiv) SEQ ID NO:XXX(IL-12p35(M97A)), xxxv) SEQ ID NO:XXX(IL-12p35(L89A)), xxxvi) SEQ ID NO:XXX No. XXX(IL-12p35(L124A)), xxxvii) No. XXX(IL-12p35(M125A)), xxxviii) No. XXX(IL-12p35(L75A)), xxxiv) No. XXX(IL-12p35(I171A)), xxxv) No. XXX(IL-12p35(N71Q)), xxxvi) No. XXX(IL-12p35(N85Q)), xxxvii) No. XXX(IL-12p35(N195Q)), xxxviii) No. XXX(IL-12p35(N71Q / N85Q)),xxxix) SEQ ID NO:XXX(IL-12p35(N71Q / N195Q)), xl) SEQ ID NO:XXX(IL-12p35(N85Q / N195Q), xli) SEQ ID NO:XXX(IL-12p35(N71Q / N85Q / N195Q)), xlii) SEQ ID NO:XXX(IL-12p35(N71D / N85D)), xliii) SEQ ID NO:XXX(IL-12p35(N71D / N195D), xliv) SEQ ID NO:XXX(IL-12p35(N85D / N195D)), xlv) SEQ ID NO:XXX(IL-12p35(D55Q)), and xlvi) SEQ ID NO:XXX(IL-12p35(D55K)).

[0050] In some embodiments, the invention provides a heterodimeric Fc fusion protein comprising a) a first fusion protein having the polypeptide sequence of SEQ ID NO: 49 (XENP27202 chain 1), and b) a second fusion protein having the polypeptide sequence of SEQ ID NO: 50 (XENP27202 chain 2).

[0051] In some embodiments, the invention provides a heterodimeric Fc-fusion protein, wherein the heterodimeric Fc-fusion protein is XENP27202.

[0052] In some embodiments, the present invention provides a composition comprising a heterodimeric Fc-fusion protein for use in treating cancer in a subject.

[0053] In some embodiments, the invention provides one or more nucleic acids encoding a heterodimeric Fc fusion protein.

[0054] In some embodiments, the invention provides host cells comprising one or more of the nucleic acids encoding the heterodimeric Fc fusion proteins.

[0055] In some embodiments, the invention provides a method of making a heterodimeric Fc-fusion protein, the method comprising culturing a host cell under conditions whereby the heterodimeric Fc-fusion protein is produced.

[0056] In some embodiments, the invention provides a method for purifying a heterodimeric Fc-fusion protein, the method comprising: a) providing a composition comprising a heterodimeric Fc-fusion protein; b) loading the composition onto an ion exchange column; and c) collecting fractions containing the heterodimeric Fc-fusion protein.

[0057] In another aspect, the invention provides a heterodimeric Fc fusion protein comprising: a) a fusion protein comprising a first protein domain, a second protein domain, and a first Fc domain, wherein the first protein domain is covalently linked to the second protein domain and the second protein domain is covalently linked to the N-terminus of the first Fc domain; and b) a second Fc domain, wherein the first and second Fc domains comprise a modification that promotes heterodimerization of the first and second Fc domains, wherein the first protein domain comprises an IL-12p40 subunit and the second protein domain comprises an IL-12p35 subunit.

[0058] In some embodiments, heterodimeric Fc fusion proteins are provided wherein said modifications that promote heterodimerization of said first and said second Fc domains are a set of amino acid substitutions selected from the group consisting of: L368D / K370S and S364K; L368D / K370S and S364K / E357L; L368D / K370S and S364K / E357Q; T411E / K360E / Q362E and D401K; L368E / K370S and S364K; K370S and S364K / E357Q and T366S / L368A / Y407V:T366W (optionally including a bridging disulfide, T366S / L368A / Y407V / Y349C:T366W / S354C), according to EU numbering.

[0059] In some embodiments, the invention provides heterodimeric Fc-fusion proteins, wherein said first protein domain is linked to said second protein domain using a first domain linker and / or said second protein domain is linked to said first Fc domain using a second domain linker.

[0060] In some embodiments, the invention provides heterodimeric Fc fusion proteins, wherein said first and / or said second Fc domain has an additional set of amino acid substitutions comprising: Q295E / N384D / Q418E / N421D, according to EU numbering.

[0061] In some embodiments, the invention provides heterodimeric Fc fusion proteins, wherein said first and / or said second Fc domain has an additional set of amino acid substitutions selected from the group consisting of G236R / L328R, E233P / L234V / L235A / G236_ / S239K, E233P / L234V / L235A / G236_ / S239K / A327G, E233P / L234V / L235A / G236_ / S267K / A327G, E233P / L234V / L235A / G236_, and E233P / L234V / L235A / G236_ / S267K, according to EU numbering.

[0062] In some embodiments, the present invention relates to a method for the preparation of a human IL-12p40 subunit comprising the steps of: The present invention provides a heterodimeric Fc fusion protein having a polypeptide sequence selected from the group consisting of SEQ ID NO:1 (human IL-12 subunit alpha (IL-12p35) precursor sequence) and / or wherein said IL-12p35 subunit has a polypeptide sequence selected from the group consisting of SEQ ID NO:2 (human IL-12 subunit alpha (IL-12p35) mature form sequence).

[0063] In some embodiments, the invention provides a heterodimeric Fc fusion protein, wherein said first and second Fc domains further comprise the amino acid substitutions M428L / N424S.

[0064] In some embodiments, the invention provides heterodimeric Fc fusion proteins, wherein said IL-12p40 subunit is a variant IL-12p40 subunit and / or said IL-12p35 subunit is a variant IL-12p35 subunit.

[0065] In some embodiments, the invention provides heterodimeric Fc fusion proteins wherein said IL-12p40 subunit is IL-12 receptor subunit beta-1 (IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2), and / or a variant IL-12p40 subunit with altered affinity for the IL-12 receptor complex, and / or said IL-12p35 subunit is IL-12 receptor subunit beta-1 (IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2), and / or a variant IL-12p35 subunit with altered affinity for the IL-12 receptor complex.

[0066] In some embodiments, the present invention relates to an IL-12p40 subunit comprising: E3, D7, E12, D14, W15, P17, D18, A19, P20, G21, E22, M23, D29, E32, E33, D34, L40, D41, Q42, S43, E45, L47, T54, I55, Q56, K58, E59, F 60, G61, D62, Q65, Y66, E73, K84, E86, D87, G88, I89, W90, D93, D97, K99, E100, K102, N1 03, K104, F106, E110, N113, Y114, D129, D142, Q144, E156, R159, D161, N162, K163, D16 6, D170, Q172, D174, A176, C177, P178, A179, A180, E181, S183, P185, E187, N200, S204 , F206, R208, D209, D214, N218, Q220, N226, Q229, E231, E235, T242, P243, S245, Y246, Provided are heterodimeric Fc fusion proteins having one or more amino acid modifications at amino acid residues selected from the group consisting of F247, S248, Q256, K158, C252, K260, E262, K264, D265, D270, N281, Q289, D290, R291, Y292, Y293, and E299.

[0067] In some embodiments, the present invention relates to a method for the preparation of IL-12p40 subunits comprising administering to the patient a IL-12p40 subunit comprising administering to the patient a IL-12p40 subunit comprising administering to the patient a IL-12p40 subunit comprising administering to the patient a IL-12p40 subunit comprising administering to the patient a IL-12p40 subunit comprising administering to the patient a IL-12p40 subunit , D161N, R159E, K163E, E187Q, N200D, N200Q, N218Q, Q229E, E235Q, C252S, Q256N, K258E, K260E, E262Q, K264E, N281D, N281Q, and E299Q.

[0068] In some embodiments, the present invention relates to a method for the preparation of IL-12p40 subunits comprising administering to the patient a IL-12p40 subunit comprising administering to the patient a IL-12p40 subunit comprising administering to the patient a IL-12p40 subunit comprising administering to the patient a IL-12p40 subunit comprising administering to the patient a IL-12p40 subunit comprising administering to the patient a IL-12p40 subunit comprising administering to the patient a IL-12p40 subunit comprising administering to the patient a IL-12p40 subunit S43K / E49Q, E59Q / K163E, E59Q / K99E, E59Q / K258E, E59Q / K260E, E59K / K99E , D18K / E59K / K99E, E59K / K99E / K264E, E59K / K99Y, E59Y / K99Y, E59Y / K99E, E 45K / E59K / K99E, E59K / K99E / Q144E, E59K / K99E / Q144K, E59K / K99E / R159E, E59K / K99E / K264E, D18K / E59K / K99E / K264E, DI8K / E59K / K99E / C252S, D18K / E59K / K99E / C252S / K264E, E59K / K99Y / C252S, E59K / K99E / C252S / K264E, E5 9K / K99E / C252S, N103D / N113D, N103D / N200D, N103D / N281D, N113D / N200D, N 113D / N281D, N200D / N281D, N103D / N113D / N200D, N103D / N113D / N281D, N10 3D / N200D / N281D, N113D / N200D / N281D, N103Q / N113Q, N103Q / N200Q, N103Q / N281Q, N113Q / N200Q, N113Q / N281Q, N200Q / N281Q, N103Q / N113Q / N200Q, N1 03Q / N113Q / N281Q, N103Q / N200Q / N281Q, N113Q / N200Q / N281Q, N103Q / N113Q / N200Q / N281Q, E59K / K99E / N103Q / C252S / K264E, E59K / K99E / N113Q / C252S / K264E, E59K / K99E / N200Q / C252S / K264E, E59K / K99E / N281Q / C252S / K264E,E59K / K99E / N103Q / N113Q / C252S / K264E, E59K / K99E / N103Q / N200Q / C252S / K264E, E59K / K99E / N103Q / N281Q / C252S / K264E, E5 9K / K99E / N113Q / N200Q / C252S / K264E, E59K / K99E / N113Q / N281Q / C252S / K264E, E59K / K99E / N200Q / N281Q / C252S / K264E, E59K The present invention provides a heterodimeric Fc fusion protein having amino acid substitutions selected from the group consisting of E59K / K99E / N103Q / N113Q / N200Q / C252S / K264E, E59K / K99E / N103Q / N200Q / N281Q / C252S / K264E, E59K / K99E / N113Q / N200Q / N281Q / C252S / K264E, and E59K / K99E / N103Q / N113Q / N200Q / N281Q / C252S / K264E.

[0069] In some embodiments, the present invention relates to a method for the preparation of IL-12p40 subunits comprising administering to a patient a therapeutically effective amount of the IL-12p40 subunit, the method comprising administering to a patient a therapeutically effective amount of the IL-12p40 subunit, the method comprising administering to a patient a therapeutically effective amount of the IL-12p40 subunit, the method comprising administering to a patient a therapeutically effective amount of the IL-12p40 subunit, 0(Q56E)), ix) SEQ ID NO: 65 (IL-12p40(E59Q)), x) SEQ ID NO: 66 (IL-12p40(D62N)), xi) SEQ ID NO: 67 (IL-12p40(Q42E / E45Q)), xii) SEQ ID NO: 68 (IL-12p40(E45Q / Q56E)), xiii) SEQ ID NO: 69 (IL-12p40(Q42E / E59Q)), xiv) SEQ ID NO: 70 (IL-12p40(Q56E / E59Q)), xv) SEQ ID NO: 71 (IL-12p40(Q42E / E45Q / Q56E)), xvi) SEQ ID NO: 72 (IL-12p40(E45Q / Q56E / E59Q)) , xvii) SEQ ID NO: 73 (IL-12p40(D161N)), xviii) SEQ ID NO: 74 (IL-12p40(E73Q)), xix) SEQ ID NO: 75 (IL-12p40(Q144E)), xx) SEQ ID NO: 76 (IL-12p40(E262Q)), xxi) SEQ ID NO: 77 (IL-12p40(E100Q)), xxii) SEQ ID NO: 78 (IL-12p40(D18N)), xxiii) SEQ ID NO: 79 (IL-12p40(E33Q)), xxiv) SEQ ID NO: 80 (IL-12p40(Q229E)), xxv) SEQ ID NO: 81 (IL-12p 40(E235Q)), xxvi) SEQ ID NO: 82 (IL-12p40(Q256N)), xxvii) SEQ ID NO: 83 (IL-12p40(E299Q)), xxviii) SEQ ID NO: 84 (IL-12p40(D87N)), xxix) IL-12p40(E32Q), xxx) IL-12p40(D34N), xxxi) IL-12p40(S43E), xxxii) IL-12p40(S43K), xxxiii) SEQ ID NO: XXX (IL-12p40(E59K / K99E / N103Q / N113Q / N200Q / N281Q / C252S / K2 64E), xxxiv) SEQ ID NO: 205 (IL-12p40(E59K)), xxxv) IL-12p40(K99E), xxxvi) IL-12p40(K163E), xxxvii) IL-12p40(E187Q), xxxviii) IL-12p40(K258E), xxxix) IL-12p40(K260E), xl) SEQ ID NO: 206 (IL-12p40(E32Q / E59Q)), xli) SEQ ID NO: 207 (IL-12p40(D34N / E59Q)), xlii) SEQ ID NO: 208 (IL-12p40(E59Q / E187Q) ), xliii) SEQ ID NO: 209 (IL-12p40(S43E / E59Q)), xliv) SEQ ID NO: 210 (IL-12p40(S43K / E49Q)), xlv) SEQ ID NO: 211 (IL-12p40(E59Q / K163E)), xlvi) SEQ ID NO: 212 (IL-12p40(E59Q / K99E)), xlvii) SEQ ID NO: 213 (IL-12p40(E59Q / K258E)), xlviii) SEQ ID NO: 214 (IL-12p40(E59Q / K260E)), xlix) SEQ ID NO: XXX (IL-12p40(D34N / E59K)),l) SEQ ID NO:XXX(IL-12p40(E59K / K99E)), li) SEQ ID NO:XXX(IL-12p40(D18K / E59K / K99E)), lii) SEQ ID NO:XXX(IL-12p40(E59K / K99E / K264E)), liii) SEQ ID NO:XXX(IL-12p40(E59K / K99Y)), liv) SEQ ID NO:XXX(IL-12p40(E59Y / K99E)), lv) SEQ ID NO:XXX(IL-12p40(E45K / E59K / K99E)), lvi) SEQ ID NO:XXX(IL-12p40(E59K / K99E / Q144E)), lvii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / Q144K)), lviii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / R159E)), lix) SEQ ID NO: XXX(IL-12p40(E59K / K99E / K264E)), lx) SEQ ID NO: XXX(IL-12p40(D18K / E59K / K99E / K264E)), lxi) SEQ ID NO: XXX(IL-12p40(C252S)), lxii) SEQ ID NO: XXX(IL-12p40(DI8K / E59K / K99E / C252S) ), lxiii) SEQ ID NO: XXX(IL-12p40(D18K / E59K / K99E / C252S / K264E)), lxiv) SEQ ID NO: XXX(IL-12p40(E59K / K99Y / C252S)), lxv) SEQ ID NO: XXX(IL-12p40(E59K / K99E / C252S / K264E)), lxvi) SEQ ID NO: XXX(IL-12p40(E59K / K99E / C252S)), lxvii) SEQ ID NO: XXX(IL-12p40(N103D / N113D)), lxviii) SEQ ID NO: XXX(IL-12p40(N1 03D / N200D)), lxix) SEQ ID NO: XXX(IL-12p40(N103D / N281D)), lxx) SEQ ID NO: XXX(IL-12p40(N113D / N200D)), lxxi) SEQ ID NO: XXX(IL-12p40(N113D / N281D)), lxxii) SEQ ID NO: XXX(IL-12p40(N200D / N281D)), lxxiii) SEQ ID NO: XXX(IL-12p50(N103D / N113D / N200D)), lxxiv) SEQ ID NO: XXX(IL-12p40(N103D / N113D / N281D)),lxxv) SEQ ID NO: XXX (IL-12p40 (N103D / N200D / N281D)), lxxvi); SEQ ID NO:XXX(IL-12p40(N113D / N200D / N281D)), lxxvii) SEQ ID NO:XXX(IL-12p40(N103Q)), lxxviii) SEQ ID NO:XXX(IL-12p40(N113Q)), lxxix) SEQ ID NO:XXX(IL-12p40(N200Q)), lxxx) SEQ ID NO:XXX(IL-12p40(N281Q)), lxxxi) SEQ ID NO:XXX(IL-12p40(N103Q / N113Q)), lxxxii) SEQ ID NO:XXX(IL-12p40(N103Q / N200Q)), lxxx iii) SEQ ID NO: XXX(IL-12p40(N103Q / N281Q)), lxxxiv) SEQ ID NO: XXX(IL-12p40(N113Q / N200Q)), lxxxv) SEQ ID NO: XXX(IL-12p40(N113Q / N281Q)), lxxxvi) SEQ ID NO: XXX(IL-12p40(N200Q / N281Q)), lxxxvii) SEQ ID NO: XXX(IL-12p40(N103Q / N113Q / N200Q)), lxxxviii) SEQ ID NO: XXX(IL-12p40(N103Q / N113Q / N281Q)), lxxx ix) SEQ ID NO: XXX(IL-12p40(N103Q / N200Q / N281Q)), xc) SEQ ID NO: XXX(IL-12p40(N113Q / N200Q / N281Q)), xci) SEQ ID NO: XXX(IL-12p40(N103Q / N113Q / N200Q / N281Q)), xcii) SEQ ID NO: XXX(IL-12p40(D34N / E59K / K99E)), xciii) SEQ ID NO: XXX(IL-12p40(D34K / E59K / K99E)), xciv) SEQ ID NO: XXX(IL-12p40(E32Q / D34N / E59K / K99E)), xcv) SEQ ID NO: XXX(IL-12p40(E32K / D34N / E59K / K99E)), xcvi) SEQ ID NO: XXX(IL-12p40(E59Y / K99Y)), xcvii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N103Q / C252S / K264E)), xcviii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N113Q / C252S / K264E)), xcix) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N200Q / C252S / K264E)),c) SEQ ID NO: XXX (IL-12p40(E59K / K99E / N281Q / C252S / K264E)), ci) SEQ ID NO: XXX (IL-12p40(E59K / K99E / N103Q / N113Q / C252S / K264E)), cii) SEQ ID NO: XXX (IL-12p40(E59K / K99E / N103Q / N200Q / C252S / K26 4E)), ciii) SEQ ID NO: XXX (IL-12p40(E59K / K99E / N103Q / N281Q / C252S / K264E)), civ) SEQ ID NO: XXX (IL-12p40(E59K / K99E / N113Q / N200Q / C252S / K264E)), cv) SEQ ID NO: XXX (IL-12p40(E59K / K99E / N113Q / N2 cvi) SEQ ID NO: XXX (IL-12p40(E59K / K99E / N200Q / N281Q / C252S / K264E)), cvii) SEQ ID NO: XXX (IL-12p40(E59K / K99E / N103Q / N113Q / N200Q / C252S / K264E)), cviii) SEQ ID NO: XXX (IL-1 The present invention provides a heterodimeric Fc fusion protein having a polypeptide sequence selected from the group consisting of SEQ ID NO: XXX (IL-12p40(E59K / K99E / N103Q / N200Q / N281Q / C252S / K264E)), and SEQ ID NO: XXX (IL-12p40(E59K / K99E / N113Q / N200Q / N281Q / C252S / K264E)).

[0070] In some embodiments, the present invention relates to an IL-12 p35 subunit comprising one or more of the following: Q20, N21, Q35, E38, S44, E45, E46, H49, K54, D55, T59, V60, E61, C63, L64, P65, E67, L68, N71, S73, C74, L75, N76, E79, N85, L89, F96, M97, L124, M125, Q1 30, Q135, N136, E143, Q146, N151, E153, K158, E162, E163, D165, I171, R181, I182, R183, V185, T186, D188, R189, V190, S192, Y193, N195, and A196. Dimeric Fc fusion proteins are provided.

[0071] In some embodiments, the invention provides heterodimeric Fc fusion proteins, wherein the IL-12p35 subunit has one or more amino acid substitutions selected from the group consisting of N21D, Q35D, E38Q, D55Q, D55K, N71D, N71Q, L75A, N76D, E79Q, N85D, N85Q, L89A, F96A, M97A, L124A, M125A, Q130E, Q135E, N136D, E143Q, Q146E, N151D, N151K, E153K, E153Q, K158E, E162Q, E163Q, D165N, I171A, N195D, and N195Q.

[0072] In some embodiments, the invention provides heterodimeric Fc fusion proteins, wherein the IL-12p35 subunit has amino acid substitutions selected from the group consisting of N71D / N85D / N195D, N151D / E153Q, N151D / D165N, Q130E / N151D, N151D / K158E, E79Q / N151D, D55Q / N151D, N136D / N151D, N21D / N151D, E143Q / N151D, N71Q / N85Q, N71Q / N195Q, N85Q / N195Q, N71Q / N85Q / N195Q, N71D / N85D, N71D / N195D, and N85D / N195D.

[0073] In some embodiments, the present invention relates to a method for the preparation of a IL-12p35 subunit comprising administering to a patient a sequence identical to that of SEQ ID NO:113 (IL-12p35(N71D)), ii) SEQ ID NO:114 (IL-12p35(N85D)), iii) SEQ ID NO:115 (IL-12p35(N195D)), iv) SEQ ID NO:116 (IL-12p35(N71D / N85D / N195D)), v) SEQ ID NO:117 (IL-12p35(E153Q)), vi) SEQ ID NO:118 (IL-12p35(E38Q)), vii) SEQ ID NO:119 (IL-12p35(N151D)), viii) SEQ ID NO:120 (IL-12p35(N151D)), or viii) SEQ ID NO: 120 (IL-12p35(Q135E)), ix) SEQ ID NO: 121 (IL-12p35(Q35D)), x) SEQ ID NO: 122 (IL-12p35(Q146E)), xi) SEQ ID NO: 123 (IL-12p35(N76D)), xii) SEQ ID NO: 124 (IL-12p35(E162Q)), xiii) SEQ ID NO: 125 (IL-12p35(E163Q)), xiv) IL-12p35(N21D), xv) SEQ ID NO: XXX (IL-12p35(D55Q)), xvi) IL-12p35(E79Q), xvii) IL-12p35(Q130E), x viii) IL-12p35(N136D), xix) IL-12p35(E143Q), xx) SEQ ID NO: 227 (IL-12p35(N151K)), xxi) SEQ ID NO: 226 (IL-12p35(E153K)), xxii) IL-12p35(K158E), xxiii) IL-12p35(D165N), xxiv) SEQ ID NO: 225 (IL-12p35(N151D / E153Q)), xxv) SEQ ID NO: 228 (IL-12p35(N151D / D165N)), xxvi) SEQ ID NO: 229 (IL-12p35(Q130E / N151D)) , xxvii) SEQ ID NO: 230 (IL-12p35(N151D / K158E)), xxviii) SEQ ID NO: 231 (IL-12p35(E79Q / N151D)), xxix) SEQ ID NO: 232 (IL-12p35(D55Q / N151D)), xxx) SEQ ID NO: 233 (IL-12p35(N136D / N151D)), xxxi) SEQ ID NO: 234 (IL-12p35(N21D / N151D)), xxxii) SEQ ID NO: 235 (IL-12p35(E143Q / N151D)), xxxiii) SEQ ID NO: XXX (IL-12p35(F96A)),xxxiv) SEQ ID NO:XXX(IL-12p35(M97A)), xxxv) SEQ ID NO:XXX(IL-12p35(L89A)), xxxvi) SEQ ID NO:XXX(IL-12p35(L124A)), xxxvii) SEQ ID NO:XXX(IL-12p35(M125A)), xxxviii) SEQ ID NO:XXX(IL-12p35(L75A)), xxxiv) SEQ ID NO:XXX(IL-12p35(I171A)), xxxv) SEQ ID NO:XXX(IL-12p35(N71Q)), xxxvi) SEQ ID NO:XXX(IL-12p35(N85Q)), xxxvi, i) SEQ ID NO:XXX(IL-12p35(N195Q)), xxxviii) SEQ ID NO:XXX(IL-12p35(N71Q / N85Q)), xxxix) SEQ ID NO:XXX(IL-12p35(N71Q / N195Q)), xl) SEQ ID NO:XXX(IL-12p35(N85Q / N195Q), xli) SEQ ID NO:XXX(IL-12p35(N71Q / N85Q / N195Q)), xlii) SEQ ID NO:XXX(IL-12p xlii) SEQ ID NO:XXX(IL-12p35(N71D / N85D)), xliv) SEQ ID NO:XXX(IL-12p35(N85D / N195D)), xlv) SEQ ID NO:XXX(IL-12p35(D55Q)), and xlvi) SEQ ID NO:XXX(IL-12p35(D55K)).

[0074] In some embodiments, the invention provides a heterodimeric Fc fusion protein comprising: a) the fusion protein having the polypeptide sequence of SEQ ID NO:51 (XENP27203 chain 1); and b) the second Fc domain having the polypeptide sequence of SEQ ID NO:52 (XENP27203 chain 2).

[0075] In some embodiments, the invention provides a heterodimeric Fc-fusion protein, wherein the heterodimeric Fc-fusion protein is XENP27203.

[0076] In some embodiments, the invention provides a heterodimeric Fc-fusion protein, wherein the heterodimeric Fc-fusion protein is XENP31290.

[0077] In some embodiments, the present invention provides a composition comprising a heterodimeric Fc-fusion protein for use in treating cancer in a subject.

[0078] In some embodiments, the invention provides one or more nucleic acids encoding a heterodimeric Fc fusion protein.

[0079] In some embodiments, the invention provides host cells comprising one or more of the nucleic acids encoding the heterodimeric Fc fusion proteins.

[0080] In some embodiments, the invention provides a method of making a heterodimeric Fc-fusion protein, the method comprising culturing a host cell under conditions whereby the heterodimeric Fc-fusion protein is produced.

[0081] In some embodiments, the invention provides a method for purifying a heterodimeric Fc-fusion protein, the method comprising: a) providing a composition comprising a heterodimeric Fc-fusion protein; b) loading the composition onto an ion exchange column; and c) collecting fractions containing the heterodimeric Fc-fusion protein.

[0082] In another aspect, the invention provides a heterodimeric Fc fusion protein comprising: a) a first protein domain, a second protein domain, and a first Fc domain, wherein the first protein domain is covalently linked to the second protein domain and the second protein domain is covalently linked to the N-terminus of the first Fc domain; and b) a second Fc domain, wherein the first and second Fc domains comprise a modification that promotes heterodimerization of the first and second Fc domains, and wherein the first protein domain comprises an IL-12p35 subunit and the second protein domain comprises an IL-12p40 subunit.

[0083] In some embodiments, heterodimerization of the first and second Fc domains is The facilitating modifications are a set of amino acid substitutions selected from the group consisting of: L368D / K370S and S364K; L368D / K370S and S364K / E357L; L368D / K370S and S364K / E357Q; T411E / K360E / Q362E and D401K; L368E / K370S and S364K; K370S and S364K / E357Q and T366S / L368A / Y407V:T366W (optionally including a bridging disulfide, T366S / L368A / Y407V / Y349C:T366W / S354C), according to EU numbering.

[0084] In some embodiments, the invention provides heterodimeric Fc-fusion proteins, wherein said first protein domain is linked to said second protein domain using a first domain linker and / or said second protein domain is linked to said first Fc domain using a second domain linker.

[0085] In some embodiments, the invention provides heterodimeric Fc fusion proteins, wherein said first and / or said second Fc domain has an additional set of amino acid substitutions comprising: Q295E / N384D / Q418E / N421D, according to EU numbering.

[0086] In some embodiments, the invention provides heterodimeric Fc fusion proteins, wherein said first and / or said second Fc domain has an additional set of amino acid substitutions selected from the group consisting of G236R / L328R, E233P / L234V / L235A / G236_ / S239K, E233P / L234V / L235A / G236_ / S239K / A327G, E233P / L234V / L235A / G236_ / S267K / A327G, E233P / L234V / L235A / G236_, and E233P / L234V / L235A / G236_ / S267K, according to EU numbering.

[0087] In some embodiments, the invention provides heterodimeric Fc fusion proteins, wherein the IL-12p40 subunit has a polypeptide sequence selected from the group consisting of SEQ ID NO:3 (human IL-12 subunit beta (IL-12p40) precursor sequence) and / or SEQ ID NO:4 (human IL-12 subunit beta (IL-12p40) mature form sequence), and the IL-12p35 subunit has a polypeptide sequence selected from the group consisting of SEQ ID NO:1 (human IL-12 subunit alpha (IL-12p35) precursor sequence) and SEQ ID NO:2 (human IL-12 subunit alpha (IL-12p35) mature form sequence).

[0088] In some embodiments, the invention provides a heterodimeric Fc fusion protein, wherein said first and second Fc domains further comprise the amino acid substitutions M428L / N424S.

[0089] In some embodiments, the invention provides heterodimeric Fc fusion proteins, wherein said IL-12p40 subunit is a variant IL-12p40 subunit and / or said IL-12p35 subunit is a variant IL-12p35 subunit.

[0090] In some embodiments, the invention relates to a method for treating IL-12p40 deficiencies, comprising administering to a patient a therapeutically effective amount of IL-12p40 subunit that is an IL-12 receptor subunit beta-1 (IL-12Rβ1), an IL-12 receptor subunit beta-2 (IL-12Rβ2), and / or a variant IL-12p40 subunit having altered affinity for the IL-12 receptor complex; and / or wherein the IL-12p35 subunit is an IL-12 receptor subunit beta-1 (IL-12Rβ1), an IL-12 receptor subunit beta-2 (IL-12Rβ2), and / or a variant IL-12p40 subunit having altered affinity for the IL-12 receptor complex. and / or variant IL-12p35 subunits with altered affinity for the IL-12 receptor complex.

[0091] In some embodiments, the present invention relates to an IL-12p40 subunit comprising: E3, D7, E12, D14, W15, P17, D18, A19, P20, G21, E22, M23, D29, E32, E33, D34, L40, D41, Q42, S43, E45, L47, T54, I55, Q56, K58, E59, F 60, G61, D62, Q65, Y66, E73, K84, E86, D87, G88, I89, W90, D93, D97, K99, E100, K102, N1 03, K104, F106, E110, N113, Y114, D129, D142, Q144, E156, R159, D161, N162, K163, D16 6, D170, Q172, D174, A176, C177, P178, A179, A180, E181, S183, P185, E187, N200, S204 , F206, R208, D209, D214, N218, Q220, N226, Q229, E231, E235, T242, P243, S245, Y246, Provided are heterodimeric Fc fusion proteins having one or more amino acid modifications at amino acid residues selected from the group consisting of F247, S248, C252, Q256, K258, K260, E262, K264, D265, D270, N281, Q289, D290, R291, Y292, Y293, and E299.

[0092] In some embodiments, the present invention relates to a method for the preparation of IL-12p40 subunits comprising administering to the patient a IL-12p40 subunit comprising administering to the patient a IL-12p40 subunit comprising administering to the patient a IL-12p40 subunit comprising administering to the patient a IL-12p40 subunit comprising administering to the patient a IL-12p40 subunit comprising administering to the patient a IL-12p40 subunit , D161N, R159E, K163E, E187Q, N200D, N200Q, N218Q, Q229E, E235Q, C252S, Q256N, K258E, K260E, E262Q, K264E, N281D, N281Q, and E299Q.

[0093] In some embodiments, the present invention relates to a method for the preparation of IL-12p40 subunits comprising administering to the patient a sequence similar to that described above, comprising administering to the patient a sequence similar to that described above, wherein the IL-12p40 subunit is N103D / N113D / N200D / N281D, Q42E / E45Q, E45Q / Q56E, Q42E / E59Q, Q56E / E59Q, Q42E / E45Q / Q56E, E45Q / Q56E / E59Q, E32Q / E59Q, D34N / E59K, D34N / E59K / K99E, D34K / E59K / K99E, E32Q / D34N / E59K / K99E, E32K / D34N / E59K / K99E, D34N / E59Q, E59Q / E187Q, S43E / E59Q, S43K / E49Q, E59Q / K163E, E59Q / K99E, E59Q / K258E, E59Q / K260E, E59K / K99E, D 18K / E59K / K99E, E59K / K99E / K264E, E59K / K99Y, E59Y / K99Y, E59Y / K99E, E45K / E59K / K99E, E59K / K99E / Q144E, E59K / K99E / Q144 K, E59K / K99E / R159E, E59K / K99E / K264E, D18K / E59K / K99E / K264E, DI8K / E59K / K99E / C252S, D18K / E59K / K99E / C252S / K264E, E5 9K / K99Y / C252S, E59K / K99E / C252S / K264E, E59K / K99E / C252S, N103D / N113D, N103D / N200D, N103D / N281D, N113D / N200D, N113D / N281D, N200D / N281D, N103D / N113D / N200D, N103D / N113D / N281D, N103D / N200D / N281D, N113D / N200D / N281D, N103Q / N113Q, N1 03Q / N200Q, N103Q / N281Q, N113Q / N200Q, N113Q / N281Q, N200Q / N281Q, N103Q / N113Q / N200Q, N103Q / N113Q / N281Q, N103Q / N200Q / N281Q, N113Q / N200Q / N281Q, N103Q / N113Q / N200Q / N281Q, E59K / K99E / N103Q / C252S / K 264E, E59K / K99E / N113Q / C252S / K264E, E59K / K99E / N200Q / C252S / K264E, E59K / K99E / N 281Q / C252S / K264E, E59K / K99E / N103Q / N113Q / C252S / K264E, E59K / K99E / N103Q / N200Q / C252S / K264E, E59K / K99E / N103Q / N281Q / C252S / K264E, E59K / K99E / N113Q / N200Q / C25 2S / K264E, E59K / K99E / N113Q / N281Q / C252S / K264E, E59K / K99E / N200Q / N281Q / C252S / K 264E, E59K / K99E / N103Q / N113Q / N200Q / C252S / K264E, E59K / K99E / N103Q / N200Q / N281Q The present invention provides heterodimeric Fc fusion proteins having amino acid substitutions selected from the group consisting of E59K / K99E / N103Q / N113Q / N200Q / N281Q / C252S / K264E, ... and E59K / K99E / N103Q / N113Q / N200Q / N281Q / C252S / K264E.

[0094] In some embodiments, the present invention relates to a method for the preparation of IL-12p40 subunits comprising administering to a patient a therapeutically effective amount of the IL-12p40 subunit, the method comprising administering to a patient a therapeutically effective amount of the IL-12p40 subunit, the therapeutically effective amount of the IL-12p40 subunit being: i) SEQ ID NO:57 (IL-12p40(N103D)); ii) SEQ ID NO:58 (IL-12p40(N113D)); iii) SEQ ID NO:59 (IL-12p40(N200D)); iv) SEQ ID NO:60 (IL-12p40(N281D)); v) SEQ ID NO:61 (IL-12p40(N103D / N113D / N200D / N281D)); vi) SEQ ID NO:62 (IL-12p40(Q42E)); vii) SEQ ID NO:63 (IL-12p40(E45 Q)), viii) SEQ ID NO:64 (IL-12p40(Q56E)), ix) SEQ ID NO:65 (IL-12p40(E59Q)), x) SEQ ID NO:66 (IL-12p40(D62N)), xi) SEQ ID NO:67 (IL-12p40(Q42E / E45Q)), xii) SEQ ID NO:68 (IL-12p40(E45Q / Q56E)), xiii) SEQ ID NO:69 (IL-12p40(Q42E / E59Q)), xiv) SEQ ID NO:70 (IL-12p40(Q56E / E59Q)), xv) SEQ ID NO:71 (IL-12p40(Q42E / E 45Q / Q56E)), xvi) SEQ ID NO: 72 (IL-12p40(E45Q / Q56E / E59Q)), xvii) SEQ ID NO: 73 (IL-12p40(D161N)), xviii) SEQ ID NO: 74 (IL-12p40(E73Q)), xix) SEQ ID NO: 75 (IL-12p40(Q144E)), xx) SEQ ID NO: 76 (IL-12p40(E262Q)), xxi) SEQ ID NO: 77 (IL-12p40(E100Q)), xxii) SEQ ID NO: 78 (IL-12p40(D18N)), xxiii) SEQ ID NO: 79 (IL-12p 40(E33Q)), xxiv) SEQ ID NO: 80 (IL-12p40(Q229E)), xxv) SEQ ID NO: 81 (IL-12p40(E235Q)), xxvi) SEQ ID NO: 82 (IL-12p40(Q256N)), xxvii) SEQ ID NO: 83 (IL-12p40(E299Q)), xxviii) SEQ ID NO: 84 (IL-12p40(D87N)), xxix) IL-12p40(E32Q), xxx) IL-12p40(D34N), xxxi) IL-12p40(S43E), xxxii) IL-12p40(S43K),xxxiii) SEQ ID NO: XXX (IL-12p40(E59K / K99E / N103Q / N113Q / N200Q / N281Q / C252S / K264E)), xxxiv) SEQ ID NO: 205 (IL-12p40(E59K)), xxxv) IL-12p40(K99E), xxxvi) IL-12p40(K163E), xxxvii) IL-12p40(E187Q), xxxviii) IL-12p40(K258E), xxxix) IL-12p40(K2 60E), xl) SEQ ID NO: 206 (IL-12p40(E32Q / E59Q)), xli) SEQ ID NO: 207 (IL-12p40(D34N / E59Q)), xlii) SEQ ID NO: 208 (IL-12p40(E59Q / E187Q)), xliii) SEQ ID NO: 209 (IL-12p40(S43E / E59Q)), xliv) SEQ ID NO: 210 (IL-12p40(S43K / E49Q)), xlv) SEQ ID NO: 211 (IL-12p40(E59Q / K163E)), , xlvi) SEQ ID NO: 212 (IL-12p40(E59Q / K99E)), xlvii) SEQ ID NO: 213 (IL-12p40(E59Q / K258E)), xlviii) SEQ ID NO: 214 (IL-12p40(E59Q / K260E)), xlix) SEQ ID NO: XXX (IL-12p40(D34N / E59K)), l) SEQ ID NO: XXX (IL-12p40(E59K / K99E)), li) SEQ ID NO: XXX (IL-12p40(D18K / E59K / K99E)), lii) SEQ ID NO: XXX (IL-12p40(E59K / K99E / K264E) ), liii) SEQ ID NO: XXX(IL-12p40(E59K / K99Y)), liv) SEQ ID NO: XXX(IL-12p40(E59Y / K99E)), lv) SEQ ID NO: XXX(IL-12p40(E45K / E59K / K99E)), lvi) SEQ ID NO: XXX(IL-12p40(E59K / K99E / Q144E)), lvii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / Q144K)), lviii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / R159E)), lix) SEQ ID NO: XXX(IL-12p40( E59K / K99E / K264E)), lx) SEQ ID NO: XXX(IL-12p40(D18K / E59K / K99E / K264E)), lxi) SEQ ID NO: XXX(IL-12p40(C252S)), lxii) SEQ ID NO: XXX(IL-12p40(DI8K / E59K / K99E / C252S)), lxiii) SEQ ID NO: XXX(IL-12p40(D18K / E59K / K99E / C252S / K264E)), lxiv) SEQ ID NO: XXX(IL-12p40(E59K / K99Y / C252S)), lxv) SEQ ID NO: XXX(IL-12p40 (E59K / K99E / C252S / K264E)), lxvi) SEQ ID NO: XXX(IL-12p40(E59K / K99E / C252S)), lxvii) SEQ ID NO: XXX(IL-12p40(N103D / N113D)), lxviii) SEQ ID NO: XXX(IL-12p40(N103D / N200D)), lxix) SEQ ID NO: XXX(IL-12p40(N103D / N281D)), lxx) SEQ ID NO: XXX(IL-12p40(N113D / N200D)), lxxi) SEQ ID NO: XXX(IL-12p40(N113D / N281D)),lxxii) SEQ ID NO: XXX(IL-12p40(N200D / N281D)), lxxiii) SEQ ID NO: XXX(IL-12p50(N103D / N113D / N200D)), lxxiv) SEQ ID NO: XXX(IL-12p40(N103D / N113D / N281D)), lxxv) SEQ ID NO: XXX(IL-12p40(N103D / N200D / N281D)), lxxvi) SEQ ID NO: XXX(IL-12p40(N113D / N200D / N281D)), lxxvii) SEQ ID NO: XXX(IL-12p40(N103Q)), lxxvi ii) SEQ ID NO:XXX(IL-12p40(N113Q)), lxxix) SEQ ID NO:XXX(IL-12p40(N200Q)), lxxx) SEQ ID NO:XXX(IL-12p40(N281Q)), lxxxi) SEQ ID NO:XXX(IL-12p40(N103Q / N113Q)), lxxxii) SEQ ID NO:XXX(IL-12p40(N103Q / N200Q)), lxxxiii) SEQ ID NO:XXX(IL-12p40(N103Q / N281Q)), lxxxiv) SEQ ID NO:XXX(IL-12p40(N113Q / N200Q)), lxxxv ) SEQ ID NO: XXX(IL-12p40(N113Q / N281Q)), lxxxvi) SEQ ID NO: XXX(IL-12p40(N200Q / N281Q)), lxxxvii) SEQ ID NO: XXX(IL-12p40(N103Q / N113Q / N200Q)), lxxxviii) SEQ ID NO: XXX(IL-12p40(N103Q / N113Q / N281Q)), lxxxix) SEQ ID NO: XXX(IL-12p40(N103Q / N200Q / N281Q)), xc) SEQ ID NO: XXX(IL-12p40(N113Q / N200Q / N281Q)), x ci) SEQ ID NO: XXX(IL-12p40(N103Q / N113Q / N200Q / N281Q)), xcii) SEQ ID NO: XXX(IL-12p40(D34N / E59K / K99E)), xciii) SEQ ID NO: XXX(IL-12p40(D34K / E59K / K99E)), xciv) SEQ ID NO: XXX(IL-12p40(E32Q / D34N / E59K / K99E)), xcv) SEQ ID NO: XXX(IL-12p40(E32K / D34N / E59K / K99E)), xcvi) SEQ ID NO: XXX(IL-12p40(E59Y / K99Y)),xcvii) SEQ ID NO: XXX (IL-12p40(E59K / , K99E / N103Q / C252S / K264E)), xcviii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N113Q / C252S / K264E)), xcix) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N200Q / C252S / K264E)), c) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N281Q / C252S / K264E)), ci) SEQ ID NO: XXX (IL-12p40(E59K / K99E / N103Q / N113Q / C252S / K264E)), cii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N103Q / N200Q / C252S / K264E)), ciii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N103Q / N281Q / C252S / K264E)), civ) SEQ ID NO: XXX(IL-12p40(E59K / K99 cv) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N113Q / N281Q / C252S / K264E)), cvi) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N200Q / N281Q / C252S / K264E)), cvii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N103Q / N113Q / N200Q XXX) SEQ ID NO:XXX (IL-12p40(E59K / K99E / N103Q / N200Q / N281Q / C252S / K264E)), cviii) SEQ ID NO:XXX (IL-12p40(E59K / K99E / N113Q / N200Q / N281Q / C252S / K264E)), and cix) SEQ ID NO:XXX (IL-12p40(E59K / K99E / N113Q / N200Q / N281Q / C252S / K264E)).

[0095] In some embodiments, the present invention relates to an IL-12 p35 subunit comprising one or more of the following: Q20, N21, Q35, E38, S44, E45, E46, H49, K54, D55, T59, V60, E61, C63, L64, P65, E67, L68, N71, S73, C74, L75, N76, E79, N85, L89, F96, M97, L124, M125, Q130, Q135, N and N195, and A196.

[0096] In some embodiments, the invention provides heterodimeric Fc fusion proteins, wherein the IL-12p35 subunit has one or more amino acid substitutions selected from the group consisting of N21D, Q35D, E38Q, D55Q, D55K, N71D, N71Q, L75A, N76D, E79Q, N85D, N85Q, L89A, F96A, M97A, L124A, M125A, Q130E, Q135E, N136D, E143Q, Q146E, N151D, N151K, E153K, E153Q, K158E, E162Q, E163Q, D165N, I171A, N195D, and N195Q.

[0097] In some embodiments, the invention provides heterodimeric Fc fusion proteins, wherein the IL-12p35 subunit has amino acid substitutions selected from the group consisting of N71D / N85D / N195D, N151D / E153Q, N151D / D165N, Q130E / N151D, N151D / K158E, E79Q / N151D, D55Q / N151D, N136D / N151D, N21D / N151D, E143Q / N151D, N71Q / N85Q, N71Q / N195Q, N85Q / N195Q, N71Q / N85Q / N195Q, N71D / N85D, N71D / N195D, and N85D / N195D.

[0098] In some embodiments, the present invention relates to a method for the preparation of IL-12p35 subunits comprising the steps of: i) SEQ ID NO: 113 (IL-12p35(N71D)); ii) SEQ ID NO: 114 (IL-12p3 5(N85D)), iii) SEQ ID NO:115 (IL-12p35(N195D)), iv) SEQ ID NO:116 (IL-12p35(N71D / N85D / N195D)), v) SEQ ID NO:117 (IL-12p35(E153Q)), vi) SEQ ID NO:118 (IL-12p35(E38Q)), vii) SEQ ID NO:119 (IL-12p35(N151D)), viii) SEQ ID NO:120 (IL-12p35(Q135E)), ix) SEQ ID NO:121 (IL-12p35(Q35D)), x) SEQ ID NO:122 (IL-12p35(Q146E)), xi) SEQ ID NO: 123 (IL-12p35(N76D)), xii) SEQ ID NO: 124 (IL-12p35(E162Q)), xiii) SEQ ID NO: 125 (IL-12p35(E163Q)), xiv) IL-12p35(N21D), xv) SEQ ID NO: XXX (IL-12p35(D55Q)), xvi) IL-12p35(E79Q), xvii) IL-12p35(Q130E), xviii) IL-12p35(N136D), xix) IL-12p35(E143Q), xx) SEQ ID NO: 227 (IL-12p35(N151K)), xxi) SEQ ID NO: 226 (IL- 227) IL-12p35(E153K)), xxii) IL-12p35(K158E), xxiii) IL-12p35(D165N), xxiv) SEQ ID NO: 225 (IL-12p35(N151D / E153Q)), xxv) SEQ ID NO: 228 (IL-12p35(N151D / D165N)), xxvi) SEQ ID NO: 229 (IL-12p35(Q130E / N151D)), xxvii) SEQ ID NO: 230 (IL-12p35(N151D / K158E)), xxviii) SEQ ID NO: 231 (IL-12p35(E79Q / N151D)), xxix) SEQ ID NO: 232 (IL-12p35(D55Q / N151D)), xxx) SEQ ID NO: 233 (IL-12p35(N136D / N151D)), xxxi) SEQ ID NO: 234 (IL-12p35(N21D / N151D)), xxxii) SEQ ID NO: 235 (IL-12p35(E143Q / N151D)), xxxiii) SEQ ID NO: XXX (IL-12p35(F96A)), xxxiv) SEQ ID NO: XXX (IL-12p35(M97A)), xxxv) SEQ ID NO: XXX (IL-12p35(L89A)), xxxvi) SEQ ID NO: XXX (IL-12p35(L124A)),xxxvii) SEQ ID NO:XXX(IL-12p35(M125A)), xxxviii) SEQ ID NO:XXX(IL-12p35(L75A)), xxxiv) SEQ ID NO:XXX(IL-12p35(I171A)), xxxv) SEQ ID NO:XXX(IL-12p35(N71Q)), xxxvi) SEQ ID NO:XXX(IL-12p35(N85Q)), xxxvii) SEQ ID NO:XXX(IL-12p35(N195Q)), xxxviii) SEQ ID NO:XXX(IL-12p35(N71Q / N85Q)), xxxix) SEQ ID NO:XXX(IL-12p35(N71Q / N195Q)), xl) SEQ ID NO:XXX (IL-12p35(N85Q / N195Q), xli) SEQ ID NO:XXX(IL-12p35(N71Q / N85Q / N195Q)), xlii) SEQ ID NO:XXX(IL-12p35(N71D / N85D)), xliii) SEQ ID NO:XXX(IL-12p35(N71D / N195D), xliv) SEQ ID NO:XXX(IL-12p35(N85D / N195D)), xlv) SEQ ID NO:XXX(IL-12p35(D55Q)), and xlvi) SEQ ID NO:XXX(IL-12p35(D55K)).

[0099] In some embodiments, the invention provides a heterodimeric Fc fusion protein comprising: a) the fusion protein having the polypeptide sequence of SEQ ID NO: 53 (XENP27204 chain 1); and b) the second Fc domain having the polypeptide sequence of SEQ ID NO: 54 (XENP27204 chain 2).

[0100] In some embodiments, the invention provides a heterodimeric Fc-fusion protein, wherein the heterodimeric Fc-fusion protein is XENP27204.

[0101] In some embodiments, the present invention provides a composition comprising a heterodimeric Fc-fusion protein for use in treating cancer in a subject.

[0102] In some embodiments, the invention provides one or more nucleic acids encoding a heterodimeric Fc fusion protein.

[0103] In some embodiments, the invention provides host cells comprising one or more of the nucleic acids encoding the heterodimeric Fc fusion proteins.

[0104] In some embodiments, the invention provides a method of making a heterodimeric Fc-fusion protein, the method comprising culturing a host cell under conditions whereby the heterodimeric Fc-fusion protein is produced.

[0105] In some embodiments, the invention provides a method for purifying a heterodimeric Fc-fusion protein, the method comprising: a) providing a composition comprising a heterodimeric Fc-fusion protein; b) loading the composition onto an ion exchange column; and c) collecting fractions containing the heterodimeric Fc-fusion protein.

[0106] In another aspect, the invention provides a heterodimeric Fc fusion protein comprising: a) a first protein domain, a second protein domain, and a first Fc domain, wherein the first protein domain is linked to the C-terminus of the first Fc domain and the second protein domain is covalently linked to the first protein domain; and b) a second Fc domain, wherein the first and second Fc domains comprise a modification that promotes heterodimerization of the first and second Fc domains, and wherein the first protein domain comprises an IL-12p40 subunit and the second protein domain comprises an IL-12p35 subunit.

[0107] In some embodiments, heterodimeric Fc fusion proteins are provided wherein said modifications that promote heterodimerization of said first and said second Fc domains are a set of amino acid substitutions selected from the group consisting of: L368D / K370S and S364K; L368D / K370S and S364K / E357L; L368D / K370S and S364K / E357Q; T411E / K360E / Q362E and D401K; L368E / K370S and S364K; K370S and S364K / E357Q and T366S / L368A / Y407V:T366W (optionally including a bridging disulfide, T366S / L368A / Y407V / Y349C:T366W / S354C), according to EU numbering.

[0108] In some embodiments, the invention provides heterodimeric Fc-fusion proteins, wherein said first protein domain is linked to said second protein domain using a first domain linker and / or said second protein domain is linked to said first Fc domain using a second domain linker.

[0109] In some embodiments, the invention provides heterodimeric Fc fusion proteins, wherein said first and / or said second Fc domain has an additional set of amino acid substitutions comprising: Q295E / N384D / Q418E / N421D, according to EU numbering.

[0110] In some embodiments, the invention relates to a method for the preparation of a polypeptide comprising a polypeptide having an amino acid substitution selected from the group consisting of: G236R / L328R, E233P / L234V / L235A / G236_ / S239K, E233P / L234V / L235A / G236_ / S239K / A327G, E233P / L234V / L235A / G236_ / S267K / A327G, E233P / L234V / L235A / G236_, and E233P / L234V / L235A / G236_ / S267K, according to EU numbering. Heterodimeric Fc fusion proteins having an additional set are provided.

[0111] In some embodiments, the invention provides heterodimeric Fc fusion proteins, wherein said IL-12p40 subunit has a polypeptide sequence selected from the group consisting of SEQ ID NO:3 (human IL-12 subunit beta (IL-12p40) precursor sequence) and SEQ ID NO:4 (human IL-12 subunit beta (IL-12p40) mature form sequence) and / or said IL-12p35 subunit has a polypeptide sequence selected from the group consisting of SEQ ID NO:1 (human IL-12 subunit alpha (IL-12p35) precursor sequence) and SEQ ID NO:2 (human IL-12 subunit alpha (IL-12p35) mature form sequence).

[0112] In some embodiments, the invention provides a heterodimeric Fc fusion protein, wherein said first and second Fc domains further comprise the amino acid substitutions M428L / N424S.

[0113] In some embodiments, the invention provides heterodimeric Fc fusion proteins, wherein said IL-12p40 subunit is a variant IL-12p40 subunit and / or said IL-12p35 subunit is a variant IL-12p35 subunit.

[0114] In some embodiments, the invention provides heterodimeric Fc fusion proteins wherein said IL-12p40 subunit is IL-12 receptor subunit beta-1 (IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2), and / or a variant IL-12p40 subunit with altered affinity for the IL-12 receptor complex, and / or said IL-12p35 subunit is IL-12 receptor subunit beta-1 (IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2), and / or a variant IL-12p35 subunit with altered affinity for the IL-12 receptor complex.

[0115] In some embodiments, the present invention relates to an IL-12p40 subunit comprising: E3, D7, E12, D14, W15, P17, D18, A19, P20, G21, E22, M23, D29, E32, E33, D34, L40, D41, Q42, S43, E45, L47, T54, I55, Q56, K58, E59, F 60, G61, D62, Q65, Y66, E73, K84, E86, D87, G88, I89, W90, D93, D97, K99, E100, K102, N1 03, K104, F106, E110, N113, Y114, D129, D142, Q144, E156, R159, D161, N162, K163, D16 6, D170, Q172, D174, A176, C177, P178, A179, A180, E181, S183, P185, E187, N200, S204 , F206, R208, D209, D214, N218, Q220, N226, Q229, E231, E235, T242, P243, S245, Y246, Provided are heterodimeric Fc fusion proteins having one or more amino acid modifications at amino acid residues selected from the group consisting of F247, S248, C252, Q256, K258, K260, E262, K264, D265, D270, N281, Q289, D290, R291, Y292, Y293, and E299.

[0116] In some embodiments, the present invention relates to a method for the preparation of IL-12p40 subunits comprising administering to the patient a IL-12p40 subunit comprising administering to the patient a IL-12p40 subunit comprising administering to the patient a IL-12p40 subunit comprising administering to the patient a IL-12p40 subunit comprising administering to the patient a IL-12p40 subunit comprising administering to the patient a IL-12p40 subunit Heterodimeric Fc fusion proteins are provided having one or more amino acid substitutions selected from the group consisting of 18Q, Q229E, E235Q, C252S, Q256N, K258E, K260E, E262Q, K264E, N281D, N281Q, and E299Q.

[0117] In some embodiments, the present invention relates to a method for the preparation of IL-12p40 subunits comprising administering to the patient a IL-12p40 subunit comprising administering to the patient a IL-12p40 subunit comprising administering to the patient a IL-12p40 subunit comprising administering to the patient a IL-12p40 subunit comprising administering to the patient a IL-12p40 subunit comprising administering to the patient a IL-12p40 subunit comprising administering to the patient a IL-12p40 subunit comprising administering to the patient a IL-12p40 subunit S43K / E49Q, E59Q / K163E, E59Q / K99E, E59Q / K258E, E59Q / K260E, E59K / K99E , D18K / E59K / K99E, E59K / K99E / K264E, E59K / K99Y, E59Y / K99Y, E59Y / K99E, E 45K / E59K / K99E, E59K / K99E / Q144E, E59K / K99E / Q144K, E59K / K99E / R159E, E59K / K99E / K264E, D18K / E59K / K99E / K264E, DI8K / E59K / K99E / C252S, D18K / E59K / K99E / C252S / K264E, E59K / K99Y / C252S, E59K / K99E / C252S / K264E, E5 9K / K99E / C252S, N103D / N113D, N103D / N200D, N103D / N281D, N113D / N200D, N 113D / N281D, N200D / N281D, N103D / N113D / N200D, N103D / N113D / N281D, N10 3D / N200D / N281D, N113D / N200D / N281D, N103Q / N113Q, N103Q / N200Q, N103Q / N281Q, N113Q / N200Q, N113Q / N281Q, N200Q / N281Q, N103Q / N113Q / N200Q, N1 03Q / N113Q / N281Q, N103Q / N200Q / N281Q, N113Q / N200Q / N281Q, N103Q / N113Q / N200Q / N281Q, E59K / K99E / N103Q / C252S / K264E, E59K / K99E / N113Q / C252S / K264E, E59K / K99E / N200Q / C252S / K264E, E59K / K99E / N281Q / C252S / K264E,E59K / K99E / N103Q / N113Q / C252S / K264E, E59K / K99E / N103Q / N200Q / C252S / K264E, E59K / K99E / N103Q / N281Q / C252S / K264E, E5 9K / K99E / N113Q / N200Q / C252S / K264E, E59K / K99E / N113Q / N281Q / C252S / K264E, E59K / K99E / N200Q / N281Q / C252S / K264E, E59K The present invention provides a heterodimeric Fc fusion protein having amino acid substitutions selected from the group consisting of E59K / K99E / N103Q / N113Q / N200Q / C252S / K264E, E59K / K99E / N103Q / N200Q / N281Q / C252S / K264E, E59K / K99E / N113Q / N200Q / N281Q / C252S / K264E, and E59K / K99E / N103Q / N113Q / N200Q / N281Q / C252S / K264E.

[0118] In some embodiments, the present invention relates to a method for the preparation of IL-12p40 subunits comprising administering to a patient a therapeutically effective amount of the IL-12p40 subunit, the method comprising administering to a patient a therapeutically effective amount of the IL-12p40 subunit, the therapeutically effective amount of the IL-12p40 subunit, the therapeutically effective amount of the IL-12p40 subunit, 0 (N103D / N113D / N200D / N281D)), vi) SEQ ID NO: 62 (IL-12p40(Q42E)), vii) SEQ ID NO: 63 (IL-12p40(E45Q)), viii) SEQ ID NO: 64 (IL-12p40(Q56E)), ix) SEQ ID NO: 65 (IL-12p40(E59Q)), x) SEQ ID NO: 66 (IL-12p40(D62N)), xi) SEQ ID NO: 67 (IL-12p40(Q42E / E45Q)), xii) SEQ ID NO: 68 (IL-12p40(E45Q / Q56E)), xiii) SEQ ID NO: 69 (IL-12p40(Q42E / E59Q)), xiv) SEQ ID NO: 70 (IL-12p40(Q56E / E59Q)), xv) SEQ ID NO: 71 (IL-12p40(Q42E / E45Q / Q56E)), xvi) SEQ ID NO: 72 (IL-12p40(E45Q / Q56E / E59Q)), xvii) SEQ ID NO: 73 (IL-12p40(D161N)), xviii) SEQ ID NO: 74 ( IL-12p40(E73Q)), xix) SEQ ID NO: 75 (IL-12p40(Q144E)), xx) SEQ ID NO: 76 (IL-12p40(E262Q)), xxi) SEQ ID NO: 77 (IL-12p40(E100Q)), xxii) SEQ ID NO: 78 (IL-12p40(D18N)), xxiii) SEQ ID NO: 79 (IL-12p40(E33Q)), xxiv) SEQ ID NO: 80 (IL-12p40(Q229E)), xxv) SEQ ID NO: 81 (IL-12p40(E235Q)), xxvi) SEQ ID NO: 82 (IL-12p40(Q256N)), xvii) SEQ ID NO: 83 (IL-12p40(E299Q)), xxviii) SEQ ID NO: 84 (IL-12p40(D87N)), xxix) IL-12p40(E32Q), xxx) IL-12p40(D34N), xxxi) IL-12p40(S43E), xxxii) IL-12p40(S43K), xxxiii) SEQ ID NO: XXX (IL-12p40(E59K / K99E / N103Q / N113Q / N200Q / N281Q / C252S / K264E)), xxxiv) SEQ ID NO: 205 (IL-12p40(E59K)), xx xv) IL-12p40(K99E), xxxvi) IL-12p40(K163E), xxxvii) IL-12p40(E187Q), xxxviii) IL-12p40(K258E), xxxix) IL-12p40(K260E), xl) SEQ ID NO: 206 (IL-12p40(E32Q / E59Q)), xli) SEQ ID NO: 207 (IL-12p40(D34N / E59Q)), xlii) SEQ ID NO: 208 (IL-12p40(E59Q / E187Q)), xliii) SEQ ID NO: 209 (IL-12p40(S43E / E59Q)),xliv) SEQ ID NO: 210 (IL-12p40(S43K / E49Q)), xlv) SEQ ID NO: 211 (IL-12p40(E59Q / K163E)), xlvi) SEQ ID NO: 212 (IL-12p40(E59Q / K99E)), xlvii) SEQ ID NO: 213 (IL-12p40(E59Q / K258E)), xlviii) SEQ ID NO: 214 (IL-12p40(E59Q / K260E)), xlix) SEQ ID NO: XXX (IL-12p40(D34N / E59K)), l) SEQ ID NO: XXX (IL-12p40(E59K / K99E)), li) SEQ ID NO: No. XXX(IL-12p40(D18K / E59K / K99E)), lii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / K264E)), liii) SEQ ID NO: XXX(IL-12p40(E59K / K99Y)), liv) SEQ ID NO: XXX(IL-12p40(E59Y / K99E)), lv) SEQ ID NO: XXX(IL-12p40(E45K / E59K / K99E)), lvi) SEQ ID NO: XXX(IL-12p40(E59K / K99E / Q144E)), lvii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / Q 144K), lviii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / R159E)), lix) SEQ ID NO: XXX(IL-12p40(E59K / K99E / K264E)), lx) SEQ ID NO: XXX(IL-12p40(D18K / E59K / K99E / K264E)), lxi) SEQ ID NO: XXX(IL-12p40(C252S)), lxii) SEQ ID NO: XXX(IL-12p40(DI8K / E59K / K99E / C252S)), lxiii) SEQ ID NO: XXX(IL-12p40(D18K / E59K / K99E / C252 S / K264E)), lxiv) SEQ ID NO: XXX(IL-12p40(E59K / K99Y / C252S)), lxv) SEQ ID NO: XXX(IL-12p40(E59K / K99E / C252S / K264E)), lxvi) SEQ ID NO: XXX(IL-12p40(E59K / K99E / C252S)), lxvii) SEQ ID NO: XXX(IL-12p40(N103D / N113D)), lxviii) SEQ ID NO: XXX(IL-12p40(N103D / N200D)), lxix) SEQ ID NO: XXX(IL-12p40(N103D / N281D)),lxx) SEQ ID NO: XXX (IL-12p40 (N113D / N200D)), lxxi) SEQ ID NO: No. XXX(IL-12p40(N113D / N281D)), lxxii) No. XXX(IL-12p40(N200D / N281D)), lxxiii) No. XXX(IL-12p50(N103D / N113D / N200D)), lxxiv) No. XXX(IL-12p40(N103D / N113D / N281D)), lxxv) No. XXX(IL-12p40(N103D / N200D / N281D)), lxxvi) No. XXX(IL-12p40(N113D / N200D / N281D)), lxxvii) SEQ ID NO:XXX(IL-12p40(N103Q)), lxxviii) SEQ ID NO:XXX(IL-12p40(N113Q)), lxxix) SEQ ID NO:XXX(IL-12p40(N200Q)), lxxx) SEQ ID NO:XXX(IL-12p40(N281Q)), lxxxi) SEQ ID NO:XXX(IL-12p40(N103Q / N113Q)), lxxxii) SEQ ID NO:XXX(IL-12p40(N103Q / N200Q)), lxxxiii) SEQ ID NO:XXX(IL-12p40(N103Q / N281Q)), lxxxiv) SEQ ID NO:X XX(IL-12p40(N113Q / N200Q)), lxxxv) SEQ ID NO:XXX(IL-12p40(N113Q / N281Q)), lxxxvi) SEQ ID NO:XXX(IL-12p40(N200Q / N281Q)), lxxxvii) SEQ ID NO:XXX(IL-12p40(N103Q / N113Q / N200Q)), lxxxviii) SEQ ID NO:XXX(IL-12p40(N103Q / N113Q / N281Q)), lxxxix) SEQ ID NO:XXX(IL-12p40(N103Q / N200Q / N281Q)), xc) SEQ ID NO:X XX(IL-12p40(N113Q / N200Q / N281Q)), xci) SEQ ID NO: XXX(IL-12p40(N103Q / N113Q / N200Q / N281Q)), xcii) SEQ ID NO: XXX(IL-12p40(D34N / E59K / K99E)), xciii) SEQ ID NO: XXX(IL-12p40(D34K / E59K / K99E)), xciv) SEQ ID NO: XXX(IL-12p40(E32Q / D34N / E59K / K99E)), xcv) SEQ ID NO: XXX(IL-12p40(E32K / D34N / E59K / K99E)),xcvi) SEQ ID NO: XXX (IL-12p40(E59Y / K99Y)), xcvii) SEQ ID NO: XXX (IL-12p40(E59K / K99E / N103Q / C252S / K264E)), xcviii) SEQ ID NO: XXX (IL-12p40(E59K / K99E / N113Q / C252S / K264E)), xcix) SEQ ID NO: XXX (IL-12p40(E59K / K99E / N200Q / C252S / K264E)), c) SEQ ID NO: XXX (IL -12p40(E59K / K99E / N281Q / C252S / K264E)), ci) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N103Q / N113Q / C252S / K264E)), cii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N103Q / N200Q / C252S / K264E)), ciii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N103Q / N281Q / C252S / K264E) ), civ) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N113Q / N200Q / C252S / K264E)), cv) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N113Q / N281Q / C252S / K264E)), cvi) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N200Q / N281Q / C252S / K264E)), cvii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N1 cviii) SEQ ID NO: XXX (IL-12p40(E59K / K99E / N103Q / N200Q / N281Q / C252S / K264E)), cviii) SEQ ID NO: XXX (IL-12p40(E59K / K99E / N113Q / N200Q / N281Q / C252S / K264E)), and cix) SEQ ID NO: XXX (IL-12p40(E59K / K99E / N113Q / N200Q / N281Q / C252S / K264E)).

[0119] In some embodiments, the present invention relates to an IL-12 p35 subunit comprising one or more of the following amino acids: Q20, N21, Q35, E38, S44, E45, E46, H49, K54, D55, T59, V60, E61, C63, L64, P65, E67, L68, N71, S73, C74, Heterodimeric Fc fusion proteins are provided, comprising one or more amino acid modifications at an amino acid residue selected from the group consisting of L75, N76, E79, N85, L89, F96, M97, L124, M125, Q130, Q135, N136, E143, Q146, N151, E153, K158, E162, E163, D165, I171, R181, I182, R183, V185, T186, D188, R189, V190, S192, Y193, N195, and A196.

[0120] In some embodiments, the invention provides heterodimeric Fc fusion proteins, wherein the IL-12p35 subunit has one or more amino acid substitutions selected from the group consisting of N21D, Q35D, E38Q, D55Q, D55K, N71D, N71Q, L75A, N76D, E79Q, N85D, N85Q, L89A, F96A, M97A, L124A, M125A, Q130E, Q135E, N136D, E143Q, Q146E, N151D, N151K, E153K, E153Q, K158E, E162Q, E163Q, D165N, I171A, N195D, and N195Q.

[0121] In some embodiments, the invention provides heterodimeric Fc fusion proteins, wherein the IL-12p35 subunit has amino acid substitutions selected from the group consisting of N71D / N85D / N195D, N151D / E153Q, N151D / D165N, Q130E / N151D, N151D / K158E, E79Q / N151D, D55Q / N151D, N136D / N151D, N21D / N151D, E143Q / N151D, N71Q / N85Q, N71Q / N195Q, N85Q / N195Q, N71Q / N85Q / N195Q, N71D / N85D, N71D / N195D, and N85D / N195D.

[0122] In some embodiments, the present invention relates to a method for the preparation of a IL-12p35 subunit comprising administering to a patient a sequence identical to that of SEQ ID NO:113 (IL-12p35(N71D)), ii) SEQ ID NO:114 (IL-12p35(N85D)), iii) SEQ ID NO:115 (IL-12p35(N195D)), iv) SEQ ID NO:116 (IL-12p35(N71D / N85D / N195D)), v) SEQ ID NO:117 (IL-12p35(E153Q)), vi) SEQ ID NO:118 (IL-12p35(E38Q)), vii) SEQ ID NO:119 (IL-12p35(N151D)), viii) SEQ ID NO:120 (IL-12p35(N151D)), or viii) SEQ ID NO: 120 (IL-12p35(Q135E)), ix) SEQ ID NO: 121 (IL-12p35(Q35D)), x) SEQ ID NO: 122 (IL-12p35(Q146E)), xi) SEQ ID NO: 123 (IL-12p35(N76D)), xii) SEQ ID NO: 124 (IL-12p35(E162Q)), xiii) SEQ ID NO: 125 (IL-12p35(E163Q)), xiv) IL-12p35(N21D), xv) SEQ ID NO: XXX (IL-12p35(D55Q)), xvi) IL-12p35(E79Q), xvii) IL-12p35(Q130E), x viii) IL-12p35(N136D), xix) IL-12p35(E143Q), xx) SEQ ID NO: 227 (IL-12p35(N151K)), xxi) SEQ ID NO: 226 (IL-12p35(E153K)), xxii) IL-12p35(K158E), xxiii) IL-12p35(D165N), xxiv) SEQ ID NO: 225 (IL-12p35(N151D / E153Q)), xxv) SEQ ID NO: 228 (IL-12p35(N151D / D165N)), xxvi) SEQ ID NO: 229 (IL-12p35(Q130E / N151D)) , xxvii) SEQ ID NO: 230 (IL-12p35(N151D / K158E)), xxviii) SEQ ID NO: 231 (IL-12p35(E79Q / N151D)), xxix) SEQ ID NO: 232 (IL-12p35(D55Q / N151D)), xxx) SEQ ID NO: 233 (IL-12p35(N136D / N151D)), xxxi) SEQ ID NO: 234 (IL-12p35(N21D / N151D)), xxxii) SEQ ID NO: 235 (IL-12p35(E143Q / N151D)), xxxiii) SEQ ID NO: XXX (IL-12p35(F96A)),xxxiv) SEQ ID NO: XXX (IL-12p35(M97A)), xxxv) SEQ ID NO: XXX (IL-12p, 35(L89A)), xxxvi) SEQ ID NO:XXX(IL-12p35(L124A)), xxxvii) SEQ ID NO:XXX(IL-12p35(M125A)), xxxviii) SEQ ID NO:XXX(IL-12p35(L75A)), xxxiv) SEQ ID NO:XXX(IL-12p35(I171A)), xxxv) SEQ ID NO:XXX(IL-12p35(N71Q)), xxxvi) SEQ ID NO:XXX(IL-12p35(N85Q)), xxxvii) SEQ ID NO:XXX(IL-12p35(N195Q)), xxxviii) SEQ ID NO:XXX(IL-12p35(N71Q / N85Q)), xxxix) SEQ ID NO:XXX(IL-12p35(N71 The present invention provides a heterodimeric Fc fusion protein having a polypeptide sequence selected from the group consisting of: xl) SEQ ID NO:XXX(IL-12p35(N85Q / N195Q), xli) SEQ ID NO:XXX(IL-12p35(N71Q / N85Q / N195Q)), xlii) SEQ ID NO:XXX(IL-12p35(N71D / N85D)), xliii) SEQ ID NO:XXX(IL-12p35(N71D / N195D), xliv) SEQ ID NO:XXX(IL-12p35(N85D / N195D)), xlv) SEQ ID NO:XXX(IL-12p35(D55Q)), and xlvi) SEQ ID NO:XXX(IL-12p35(D55K)).

[0123] In some embodiments, the present invention provides a composition comprising a heterodimeric Fc-fusion protein for use in treating cancer in a subject.

[0124] In some embodiments, the invention provides one or more nucleic acids encoding a heterodimeric Fc fusion protein.

[0125] In some embodiments, the invention provides host cells comprising one or more of the nucleic acids encoding the heterodimeric Fc fusion proteins.

[0126] In some embodiments, the invention provides a method of making a heterodimeric Fc-fusion protein, the method comprising culturing a host cell under conditions whereby the heterodimeric Fc-fusion protein is produced.

[0127] In some embodiments, the invention provides a method for purifying a heterodimeric Fc-fusion protein, the method comprising: a) providing a composition comprising a heterodimeric Fc-fusion protein; b) loading the composition onto an ion exchange column; and c) collecting fractions containing the heterodimeric Fc-fusion protein.

[0128] In another aspect, the invention provides a heterodimeric Fc fusion protein comprising: a) a first protein domain, a second protein domain, and a first Fc domain, wherein the first protein domain is linked to the C-terminus of the first Fc domain and the second protein domain is covalently linked to the first protein domain; and b) a second Fc domain, wherein the first and second Fc domains comprise a modification that promotes heterodimerization of the first and second Fc domains, and wherein the first protein domain comprises an IL-12p35 subunit and the second protein domain comprises an IL-12p40 subunit.

[0129] In some embodiments, the modification that promotes heterodimerization of the first and second Fc domains is selected from the group consisting of: L368D / K370S and S364K; L368D / K370S and S364K / E357L; L368D / K370S and S364K / E357Q; T411E / K360E / Q362E and D401K; L368E / K370S and S364K; K370S and S364K / E357Q and T366S / L368A / Y407V:T366W (optionally including the bridging disulfide, T366S / L368A / Y407V / Y349C:T366W / S354C), according to EU numbering. The present invention provides a heterodimeric Fc fusion protein, wherein the set of amino acid substitutions is selected from the group consisting of:

[0130] In some embodiments, the invention provides heterodimeric Fc-fusion proteins, wherein said first protein domain is linked to said second protein domain using a first domain linker and / or said second protein domain is linked to said first Fc domain using a second domain linker.

[0131] In some embodiments, the invention provides heterodimeric Fc fusion proteins, wherein said first and / or said second Fc domain has an additional set of amino acid substitutions comprising: Q295E / N384D / Q418E / N421D, according to EU numbering.

[0132] In some embodiments, the invention provides heterodimeric Fc fusion proteins, wherein said first and / or said second Fc domain has an additional set of amino acid substitutions selected from the group consisting of G236R / L328R, E233P / L234V / L235A / G236_ / S239K, E233P / L234V / L235A / G236_ / S239K / A327G, E233P / L234V / L235A / G236_ / S267K / A327G, E233P / L234V / L235A / G236_, and E233P / L234V / L235A / G236_ / S267K, according to EU numbering.

[0133] In some embodiments, the invention provides heterodimeric Fc fusion proteins, wherein said IL-12p40 subunit has a polypeptide sequence selected from the group consisting of SEQ ID NO:3 (human IL-12 subunit beta (IL-12p40) precursor sequence) and SEQ ID NO:4 (human IL-12 subunit beta (IL-12p40) mature form sequence) and / or said IL-12p35 subunit has a polypeptide sequence selected from the group consisting of SEQ ID NO:1 (human IL-12 subunit alpha (IL-12p35) precursor sequence) and SEQ ID NO:2 (human IL-12 subunit alpha (IL-12p35) mature form sequence).

[0134] In some embodiments, the invention provides a heterodimeric Fc fusion protein, wherein said first and second Fc domains further comprise the amino acid substitutions M428L / N424S.

[0135] In some embodiments, the invention provides heterodimeric Fc fusion proteins, wherein said IL-12p40 subunit is a variant IL-12p40 subunit and / or said IL-12p35 subunit is a variant IL-12p35 subunit.

[0136] In some embodiments, the invention provides heterodimeric Fc fusion proteins wherein said IL-12p40 subunit is IL-12 receptor subunit beta-1 (IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2), and / or a variant IL-12p40 subunit with altered affinity for the IL-12 receptor complex, and / or said IL-12p35 subunit is IL-12 receptor subunit beta-1 (IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2), and / or a variant IL-12p35 subunit with altered affinity for the IL-12 receptor complex.

[0137] In some embodiments, the present invention relates to a method for the preparation of IL-12p40 subunits comprising administering to the patient a IL-12 p40 subunit ... 7, T54, I55, Q56, K58, E59, F60, G61, D62, Q65, Y66, E73, K84, E86, D87, G88, I89, W90, D93, D97, K99, E100, K102, N103, K104, F106, E110, N113, Y114, D129, D142, Q144, E156, R159, D161, N162, K163, D166, D170, Q172, D174, A176, C177, P178, A179, A180, E181, S183, P185, E18 7, N200, S204, F206, R208, D209, D214, N218, Q220, N226, Q229, E231, E235, T242, P243, S245, Y246, F247, S248, C252, Q256, K258, K260, E262, K264, D265, D270, N281, Q289, D290, R291, Y292, Y293, and E299.

[0138] In some embodiments, the present invention relates to a method for the preparation of IL-12p40 subunits comprising administering to the patient a IL-12p40 subunit comprising administering to the patient a IL-12p40 subunit comprising administering to the patient a IL-12p40 subunit comprising administering to the patient a IL-12p40 subunit comprising administering to the patient a IL-12p40 subunit comprising administering to the patient a IL-12p40 subunit , D161N, R159E, K163E, E187Q, N200D, N200Q, N218Q, Q229E, E235Q, C252S, Q256N, K258E, K260E, E262Q, K264E, N281D, N281Q, and E299Q.

[0139] In some embodiments, the present invention relates to a method for the preparation of IL-12p40 subunits comprising administering to the patient a IL-12p40 subunit comprising administering to the patient a IL-12p40 subunit comprising administering to the patient a IL-12p40 subunit comprising administering to the patient a IL-12p40 subunit comprising administering to the patient a IL-12p40 subunit comprising administering to the patient a IL-12p40 subunit comprising administering to the patient a IL-12p40 subunit comprising administering to the patient a IL-12p40 subunit S43K / E49Q, E59Q / K163E, E59Q / K99E, E59Q / K258E, E59Q / K260E, E59K / K99E , D18K / E59K / K99E, E59K / K99E / K264E, E59K / K99Y, E59Y / K99Y, E59Y / K99E, E 45K / E59K / K99E, E59K / K99E / Q144E, E59K / K99E / Q144K, E59K / K99E / R159E, E59K / K99E / K264E, D18K / E59K / K99E / K264E, DI8K / E59K / K99E / C252S, D18K / E59K / K99E / C252S / K264E, E59K / K99Y / C252S, E59K / K99E / C252S / K264E, E5 9K / K99E / C252S, N103D / N113D, N103D / N200D, N103D / N281D, N113D / N200D, N 113D / N281D, N200D / N281D, N103D / N113D / N200D, N103D / N113D / N281D, N10 3D / N200D / N281D, N113D / N200D / N281D, N103Q / N113Q, N103Q / N200Q, N103Q / N281Q, N113Q / N200Q, N113Q / N281Q, N200Q / N281Q, N103Q / N113Q / N200Q, N1 03Q / N113Q / N281Q, N103Q / N200Q / N281Q, N113Q / N200Q / N281Q, N103Q / N113Q / N200Q / N281Q, E59K / K99E / N103Q / C252S / K264E, E59K / K99E / N113Q / C252S / K264E, E59K / K99E / N200Q / C252S / K264E, E59K / K99E / N281Q / C252S / K264E,E59K / K99E / N103Q / N113Q / C252S / K264E, E59K / K99E / N103Q / N200Q / C252S / K264E, E59K / K99E / N, 103Q / N281Q / C252S / K264E, E59K / K99E / N113Q / N200Q / C252S / K264E, E59K / K99E / N113Q / N281Q / C252S / K264E, E59K / K99E / N200Q / N281Q / C252S / K264E, E59K / K99E / N103Q / N113Q / N200Q / C252S / K264E, E59 Heterodimeric Fc fusion proteins are provided having amino acid substitutions selected from the group consisting of K / K99E / N103Q / N200Q / N281Q / C252S / K264E, E59K / K99E / N113Q / N200Q / N281Q / C252S / K264E, and E59K / K99E / N103Q / N113Q / N200Q / N281Q / C252S / K264E.

[0140] In some embodiments, the present invention relates to a method for the preparation of IL-12p40 subunits comprising administering to a patient a therapeutically effective amount of the IL-12p40 subunit, the method comprising administering to a patient a therapeutically effective amount of the IL-12p40 subunit, the therapeutically effective amount of the IL-12p40 subunit being: i) SEQ ID NO:57 (IL-12p40(N103D)); ii) SEQ ID NO:58 (IL-12p40(N113D)); iii) SEQ ID NO:59 (IL-12p40(N200D)); iv) SEQ ID NO:60 (IL-12p40(N281D)); v) SEQ ID NO:61 (IL-12p40(N103D / N113D / N200D / N281D)); vi) SEQ ID NO:62 (IL-12p40(Q42E)); vii) SEQ ID NO:63 (IL-12p40(E45 Q)), viii) SEQ ID NO:64 (IL-12p40(Q56E)), ix) SEQ ID NO:65 (IL-12p40(E59Q)), x) SEQ ID NO:66 (IL-12p40(D62N)), xi) SEQ ID NO:67 (IL-12p40(Q42E / E45Q)), xii) SEQ ID NO:68 (IL-12p40(E45Q / Q56E)), xiii) SEQ ID NO:69 (IL-12p40(Q42E / E59Q)), xiv) SEQ ID NO:70 (IL-12p40(Q56E / E59Q)), xv) SEQ ID NO:71 (IL-12p40(Q42E / E 45Q / Q56E)), xvi) SEQ ID NO: 72 (IL-12p40(E45Q / Q56E / E59Q)), xvii) SEQ ID NO: 73 (IL-12p40(D161N)), xviii) SEQ ID NO: 74 (IL-12p40(E73Q)), xix) SEQ ID NO: 75 (IL-12p40(Q144E)), xx) SEQ ID NO: 76 (IL-12p40(E262Q)), xxi) SEQ ID NO: 77 (IL-12p40(E100Q)), xxii) SEQ ID NO: 78 (IL-12p40(D18N)), xxiii) SEQ ID NO: 79 (IL-12p 40(E33Q)), xxiv) SEQ ID NO: 80 (IL-12p40(Q229E)), xxv) SEQ ID NO: 81 (IL-12p40(E235Q)), xxvi) SEQ ID NO: 82 (IL-12p40(Q256N)), xxvii) SEQ ID NO: 83 (IL-12p40(E299Q)), xxviii) SEQ ID NO: 84 (IL-12p40(D87N)), xxix) IL-12p40(E32Q), xxx) IL-12p40(D34N), xxxi) IL-12p40(S43E), xxxii) IL-12p40(S43K),xxxiii) SEQ ID NO: XXX (IL-12p40(E59K / K99E / N103Q / N113Q / N200Q / N281Q / C252S / K264E)), xxxiv) SEQ ID NO: 205 (IL-12p40(E59K)), xxxv) IL-12p40(K99E), xxxvi) IL-12p40(K163E), xxxvii) IL-12p40(E187 Q), xxxviii) IL-12p40(K258E), xxxix) IL-12p40(K260E), xl) SEQ ID NO: 206 (IL-12p40(E32Q / E59Q)), xli) SEQ ID NO: 207 (IL-12p40(D34N / E59Q)), xlii) SEQ ID NO: 208 (IL-12p40(E59Q / E187Q)), xliii) SEQ ID NO: 209 (IL-12p40(I L-12p40(S43E / E59Q)), xliv) SEQ ID NO: 210 (IL-12p40(S43K / E49Q)), xlv) SEQ ID NO: 211 (IL-12p40(E59Q / K163E)), xlvi) SEQ ID NO: 212 (IL-12p40(E59Q / K99E)), xlvii) SEQ ID NO: 213 (IL-12p40(E59Q / K258E)), xlviii) SEQ ID NO: 214 (IL-12p40(E59Q / K258E)), ) SEQ ID NO: 214 (IL-12p40(E59Q / K260E)), xlix) SEQ ID NO: XXX (IL-12p40(D34N / E59K)), l) SEQ ID NO: XXX (IL-12p40(E59K / K99E)), li) SEQ ID NO: XXX (IL-12p40(D18K / E59K / K99E)), lii) SEQ ID NO: XXX (IL-12p40(E59K / K99E, / K264E)), liii) SEQ ID NO: XXX(IL-12p40(E59K / K99Y)), liv) SEQ ID NO: XXX(IL-12p40(E59Y / K99E)), lv) SEQ ID NO: XXX(IL-12p40(E45K / E59K / K99E)), lvi) SEQ ID NO: XXX(IL-12p40(E59K / K99E / Q144E)), lvii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / Q144K)), lviii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / R159E)), lix) SEQ ID NO: XXX(IL -12p40(E59K / K99E / K264E)), lx) SEQ ID NO: XXX(IL-12p40(D18K / E59K / K99E / K264E)), lxi) SEQ ID NO: XXX(IL-12p40(C252S)), lxii) SEQ ID NO: XXX(IL-12p40(DI8K / E59K / K99E / C252S)), lxiii) SEQ ID NO: XXX(IL-12p40(D18K / E59K / K99E / C252S / K264E)), lxiv) SEQ ID NO: XXX(IL-12p40(E59K / K99Y / C252S)), lxv) SEQ ID NO: XXX(IL L-12p40(E59K / K99E / C252S / K264E)), lxvi) SEQ ID NO: XXX(IL-12p40(E59K / K99E / C252S)), lxvii) SEQ ID NO: XXX(IL-12p40(N103D / N113D)), lxviii) SEQ ID NO: XXX(IL-12p40(N103D / N200D)), lxix) SEQ ID NO: XXX(IL-12p40(N103D / N281D)), lxx) SEQ ID NO: XXX(IL-12p40(N113D / N200D)), lxxi) SEQ ID NO: XXX(IL-12p40(N113D / N 281D)), lxxii) SEQ ID NO: XXX(IL-12p40(N200D / N281D)), lxxiii) SEQ ID NO: XXX(IL-12p50(N103D / N113D / N200D)), lxxiv) SEQ ID NO: XXX(IL-12p40(N103D / N113D / N281D)), lxxv) SEQ ID NO: XXX(IL-12p40(N103D / N200D / N281D)), lxxvi) SEQ ID NO: XXX(IL-12p40(N113D / N200D / N281D)), lxxvii) SEQ ID NO: XXX(IL-12p40(N103Q)),lxxviii) SEQ ID NO:XXX(IL-12p40(N113Q)), lxxix) SEQ ID NO:XXX(IL-12p40(N200Q)), lxxx) SEQ ID NO:XXX(IL-12p40(N281Q)), lxxxi) SEQ ID NO:XXX(IL-12p40(N103Q / N113Q)), lxxxii) SEQ ID NO:XXX(IL-12p40(N103Q / N200Q)), lxxxiii) SEQ ID NO:XXX(IL-12p40(N103Q / N281Q)), lxxxiv) SEQ ID NO:XXX(IL-12p40(N113Q / N200Q)), lxxxv) SEQ ID NO: XXX(IL-12p40(N113Q / N281Q)), lxxxvi) SEQ ID NO: XXX(IL-12p40(N200Q / N281Q)), lxxxvii) SEQ ID NO: XXX(IL-12p40(N103Q / N113Q / N200Q)), lxxxviii) SEQ ID NO: XXX(IL-12p40(N103Q / N113Q / N281Q)), lxxxix) SEQ ID NO: XXX(IL-12p40(N103Q / N200Q / N281Q)), xc) SEQ ID NO: XXX(IL-12p40 (N113Q / N200Q / N281Q)), xci) SEQ ID NO: XXX(IL-12p40(N103Q / N113Q / N200Q / N281Q)), xcii) SEQ ID NO: XXX(IL-12p40(D34N / E59K / K99E)), xciii) SEQ ID NO: XXX(IL-12p40(D34K / E59K / K99E)), xciv) SEQ ID NO: XXX(IL-12p40(E32Q / D34N / E59K / K99E)), xcv) SEQ ID NO: XXX(IL-12p40(E32K / D34N / E59K / K99E)), xcvi ) SEQ ID NO: XXX(IL-12p40(E59Y / K99Y)), xcvii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N103Q / C252S / K264E)), xcviii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N113Q / C252S / K264E)), xcix) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N200Q / C252S / K264E)), c) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N281Q / C252S / K264E)),ci) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N103Q / N113Q / C252S / K264E)), cii); SEQ ID NO:XXX(IL-12p40(E59K / K99E / N103Q / N200Q / C252S / K264E)), ciii) SEQ ID NO:XXX(IL-12p40(E59K / K99E / N103Q / N281Q / C252S / K264E)), civ) SEQ ID NO:XXX(IL-12p40(E59K / K99E / N113Q / N200Q / C252S / K264E)), cv) SEQ ID NO:XXX(IL-12p40(E59K / K99E / N113Q / N281Q / C252S / K264E)), cvi) SEQ ID NO:XXX(IL-12p40(E59K / K99E / N200Q 103Q / N200Q / N281Q / C252S / K264E)), cvii) SEQ ID NO:XXX(IL-12p40(E59K / K99E / N103Q / N113Q / N200Q / C252S / K264E)), cviii) SEQ ID NO:XXX(IL-12p40(E59K / K99E / N103Q / N200Q / N281Q / C252S / K264E)), and cix) SEQ ID NO:XXX(IL-12p40(E59K / K99E / N113Q / N200Q / N281Q / C252S / K264E)).

[0141] In some embodiments, the present invention relates to an IL-12 p35 subunit comprising one or more of the following: Q20, N21, Q35, E38, S44, E45, E46, H49, K54, D55, T59, V60, E61, C63, L64, P65, E67, L68, N71, S73, C74, L75, N76, E79, N85, L89, F96, M97, L124, M125, Q130, Q135, N and N195, and A196.

[0142] In some embodiments, the invention provides heterodimeric Fc fusion proteins, wherein the IL-12p35 subunit has one or more amino acid substitutions selected from the group consisting of N21D, Q35D, E38Q, D55Q, D55K, N71D, N71Q, L75A, N76D, E79Q, N85D, N85Q, L89A, F96A, M97A, L124A, M125A, Q130E, Q135E, N136D, E143Q, Q146E, N151D, N151K, E153K, E153Q, K158E, E162Q, E163Q, D165N, I171A, N195D, and N195Q.

[0143] In some embodiments, the invention provides heterodimeric Fc fusion proteins, wherein the IL-12p35 subunit has amino acid substitutions selected from the group consisting of N71D / N85D / N195D, N151D / E153Q, N151D / D165N, Q130E / N151D, N151D / K158E, E79Q / N151D, D55Q / N151D, N136D / N151D, N21D / N151D, E143Q / N151D, N71Q / N85Q, N71Q / N195Q, N85Q / N195Q, N71Q / N85Q / N195Q, N71D / N85D, N71D / N195D, and N85D / N195D.

[0144] In some embodiments, the present invention relates to a method for the preparation of a IL-12p35 subunit comprising administering to a patient a sequence identical to that of SEQ ID NO: 113 (IL-12p35(N71D)), ii) SEQ ID NO: 114 (IL-12p35(N85D)), iii) SEQ ID NO: 115 (IL-12p35(N195D)), iv) SEQ ID NO: 116 (IL-12p35(N71D / N85D / N195D)), v) the sequence SEQ ID NO: No. 117 (IL-12p35(E153Q)), vi) SEQ ID NO: 118 (IL-12p35(E38Q)), vii) SEQ ID NO: 119 (IL-12p35(N151D)), viii) SEQ ID NO: 120 (IL-12p35(Q135E)), ix) SEQ ID NO: 121 (IL-12p35(Q35D)), x) SEQ ID NO: 122 (IL-12p35(Q146E )), xi) SEQ ID NO: 123 (IL-12p35(N76D)), xii) SEQ ID NO: 124 (IL-12p35(E162Q)), xiii) SEQ ID NO: 125 (IL-12p35(E163Q)), xiv) IL-12p35(N21D), xv) SEQ ID NO: XXX (IL-12p35(D55Q)), xvi) IL-12p35(E79Q), xvii) IL-12p35(Q130E), xviii) IL-12p35(N136D), xix) IL-12p35(E143Q), xx) SEQ ID NO: 227 (IL-12p35(N151K)), x xi) SEQ ID NO: 226 (IL-12p35(E153K)), xxii) IL-12p35(K158E), xxiii) IL-12p35(D165N), xxiv) SEQ ID NO: 225 (IL-12p35(N151D / E153Q)), xxv) SEQ ID NO: 228 (IL-12p35(N151D / D165N)), xxvi) SEQ ID NO: 229 (IL-12p35(Q130E / N151D)), xxvii) SEQ ID NO: 230 (IL-12p35(N151D / K158E)), xxviii) SEQ ID NO: 231 (IL-12p35(E79Q / N1 51D)), xxix) SEQ ID NO:232 (IL-12p35(D55Q / N151D)), xxx) SEQ ID NO:233 (IL-12p35(N136D / N151D)), xxxi) SEQ ID NO:234 ​​(IL-12p35(N21D / N151D)), xxxii) SEQ ID NO:235 (IL-12p35(E143Q / N151D)), xxxiii) SEQ ID NO:XXX(IL-12p35(F96A)), xxxiv) SEQ ID NO:XXX(IL-12p35(M97A)), xxxv) SEQ ID NO:XXX(IL-12p35(L89A)), xxxvi) SEQ ID NO:XXX No. XXX(IL-12p35(L124A)), xxxvii) No. XXX(IL-12p35(M125A)), xxxviii) No. XXX(IL-12p35(L75A)), xxxiv) No. XXX(IL-12p35(I171A)), xxxv) No. XXX(IL-12p35(N71Q)), xxxvi) No. XXX(IL-12p35(N85Q)), xxxvii) No. XXX(IL-12p35(N195Q)), xxxviii) No. XXX(IL-12p35(N71Q / N85Q)),xxxix) SEQ ID NO:XXX(IL-12p35(N71Q / N195Q)), xl) SEQ ID NO:XXX(IL-12p35(N85Q / N195Q), xli) SEQ ID NO:XXX(IL-12p35(N71Q / N85Q / N195Q)), xlii) SEQ ID NO:XXX(IL-12p35(N71D / N85D)), xliii) SEQ ID NO:XXX(IL-12p35(N71D / N195D), xliv) SEQ ID NO:XXX(IL-12p35(N85D / N195D)), xlv) SEQ ID NO:XXX(IL-12p35(D55Q)), and xlvi) SEQ ID NO:XXX(IL-12p35(D55K)).

[0145] In some embodiments, the present invention provides a composition comprising a heterodimeric Fc-fusion protein for use in treating cancer in a subject.

[0146] In some embodiments, the invention provides one or more nucleic acids encoding a heterodimeric Fc fusion protein.

[0147] In some embodiments, the invention provides host cells comprising one or more of the nucleic acids encoding the heterodimeric Fc fusion proteins.

[0148] In some embodiments, the invention provides a method of making a heterodimeric Fc-fusion protein, the method comprising culturing a host cell under conditions whereby the heterodimeric Fc-fusion protein is produced.

[0149] In some embodiments, the invention provides a method for purifying a heterodimeric Fc-fusion protein, the method comprising: a) providing a composition comprising a heterodimeric Fc-fusion protein; b) loading the composition onto an ion exchange column; and c) purifying the heterodimeric Fc-fusion protein. and collecting the fraction containing the fusion protein.

[0150] In another aspect, the present invention provides a heterodimeric complex comprising: a) an IL-12p40 subunit, and b) an IL-12p35 subunit.

[0151] In some embodiments, the invention provides a heterodimeric complex, wherein the IL-12p40 subunit is covalently linked to the IL-12p35 subunit using a domain linker.

[0152] In some embodiments, the invention provides a heterodimeric complex, wherein the IL-12p40 subunit has a polypeptide sequence selected from the group consisting of SEQ ID NO:3 (human IL-12 subunit beta (IL-12p40) precursor sequence) and SEQ ID NO:4 (human IL-12 subunit beta (IL-12p40) mature form sequence) and / or the IL-12p35 subunit has a polypeptide sequence selected from the group consisting of SEQ ID NO:1 (human IL-12 subunit alpha (IL-12p35) precursor sequence) and SEQ ID NO:2 (human IL-12 subunit alpha (IL-12p35) mature form sequence).

[0153] In some embodiments, the invention provides a heterodimeric complex, wherein said first and second Fc domains further comprise the amino acid substitutions M428L / N424S.

[0154] In some embodiments, the invention provides heterodimeric complexes, wherein said IL-12p40 subunit is a variant IL-12p40 subunit and / or said IL-12p35 subunit is a variant IL-12p35 subunit.

[0155] In some embodiments, the invention provides heterodimeric complexes wherein the IL-12p40 subunit is an IL-12 receptor subunit beta-1 (IL-12Rβ1), an IL-12 receptor subunit beta-2 (IL-12Rβ2), and / or a variant IL-12p40 subunit with altered affinity for the IL-12 receptor complex, and / or the IL-12p35 subunit is an IL-12 receptor subunit beta-1 (IL-12Rβ1), an IL-12 receptor subunit beta-2 (IL-12Rβ2), and / or a variant IL-12p35 subunit with altered affinity for the IL-12 receptor complex.

[0156] In some embodiments, the present invention relates to a method for the preparation of IL-12p40 subunits comprising administering to the patient a sequence selected from the group consisting of E3, D7, E12, D14, W15, P17, D18, A19, P20, G21, E22, M23, D29, E32, E33, D34, L40, D41, Q42, S43, E45, L47, T54, I55, Q56, K58, E59, and the like. , F60, G61, D62, Q65, Y66, E73, K84, E86, D87, G88, I89, W90, D93, D97, K99, E100, K102 , N103, K104, F106, E110, N113, Y114, D129, D142, Q144, E156, R159, D161, N162, K163, D166, D170, Q172, D174, A176, C177, P178, A179, A180, E181, S183, P185, E187, N200, S204, F206, R208, D209, D214, N218, Q220, N226, Q229, E231, E235, T242, P243, S245, Y and E299.

[0157] In some embodiments, the present invention relates to an IL-12p40 subunit comprising D18N, D18K, E32Q, E33Q, D34N, D34K, Q42E, S43E, S43K, E45Q, Q56E, E59Q, E59K, D62N, E73Q, D87N, K99E, K99Y, E100Q, N103D, N103Q, N113D, N113Q, Q1 The heterodimeric complexes have one or more amino acid substitutions selected from the group consisting of 44E, D161N, R159E, K163E, E187Q, N200D, N200Q, N218Q, Q229E, E235Q, C252S, Q256N, K258E, K260E, E262Q, K264E, N281D, N281Q, and E299Q.

[0158] In some embodiments, the present invention relates to a method for the preparation of IL-12p40 subunits comprising administering to the patient a IL-12p40 subunit comprising administering to the patient a IL-12p40 subunit comprising administering to the patient a IL-12p40 subunit comprising administering to the patient a IL-12p40 subunit comprising administering to the patient a IL-12p40 subunit comprising administering to the patient a IL-12p40 subunit comprising administering to the patient a IL-12p40 subunit comprising administering to the patient a IL-12p40 subunit S43K / E49Q, E59Q / K163E, E59Q / K99E, E59Q / K258E, E59Q / K260E, E59K / K99E , D18K / E59K / K99E, E59K / K99E / K264E, E59K / K99Y, E59Y / K99Y, E59Y / K99E, E 45K / E59K / K99E, E59K / K99E / Q144E, E59K / K99E / Q144K, E59K / K99E / R159E, E59K / K99E / K264E, D18K / E59K / K99E / K264E, DI8K / E59K / K99E / C252S, D18K / E59K / K99E / C252S / K264E, E59K / K99Y / C252S, E59K / K99E / C252S / K264E, E5 9K / K99E / C252S, N103D / N113D, N103D / N200D, N103D / N281D, N113D / N200D, N 113D / N281D, N200D / N281D, N103D / N113D / N200D, N103D / N113D / N281D, N10 3D / N200D / N281D, N113D / N200D / N281D, N103Q / N113Q, N103Q / N200Q, N103Q / N281Q, N113Q / N200Q, N113Q / N281Q, N200Q / N281Q, N103Q / N113Q / N200Q, N1 03Q / N113Q / N281Q, N103Q / N200Q / N281Q, N113Q / N200Q / N281Q, N103Q / N113Q / N200Q / N281Q, E59K / K99E / N103Q / C252S / K264E, E59K / K99E / N113Q / C252S / K264E, E59K / K99E / N200Q / C252S / K264E, E59K / K99E / N281Q / C252S / K264E,E59K / K99E / N103Q / N113Q / C252S / K264E, E59K / K99E / N103Q / N200Q / C252S / K264E, E59K / K99E / N103Q / N281Q / C252S / K264E, E59K / K99E / N113Q / N200Q / C252S / K264E, E59K / K99E / N113Q / N281Q / C252S / K264E, E59K / K99E / N200Q / N281Q / C252S / K264E, E Heterodimeric complexes having amino acid substitutions selected from the group consisting of 59K / K99E / N103Q / N113Q / N200Q / C252S / K264E, E59K / K99E / N103Q / N200Q / N281Q / C252S / K264E, E59K / K99E / N113Q / N200Q / N281Q / C252S / K264E, and E59K / K99E / N103Q / N113Q / N200Q / N281Q / C252S / K264E.

[0159] In some embodiments, the present invention relates to a method for the preparation of IL-12p40 subunits comprising the steps of: i) SEQ ID NO:57 (IL-12p40(N103D)); ii) SEQ ID NO:58 (IL-12p40(N113D)); iii) SEQ ID NO:59 (IL-12p40(N200D)); iv) SEQ ID NO:60 (IL-12p40(N281D)), v) SEQ ID NO:61 (IL-12p40(N103D / N113D / N200D / N281D)), vi) SEQ ID NO:62 (IL-12p40(Q42E)), vii) SEQ ID NO:63 (IL-12p40(E45Q)), viii) SEQ ID NO:64 (IL-12p40(Q56E)), ix) SEQ ID NO:65 (IL-12p40(E59Q)), x) SEQ ID NO:66 (IL-12p40(D62N)), xi) SEQ ID NO:67 (IL-12p40(Q42E / E45Q)), xii) SEQ ID NO:68 (IL-12p 40(E45Q / Q56E)), xiii) SEQ ID NO:69 (IL-12p40(Q42E / E59Q)), xiv) SEQ ID NO:70 (IL-12p40(Q56E / E59Q)), xv) SEQ ID NO:71 (IL-12p40(Q42E / E45Q / Q56E)), xvi) SEQ ID NO:72 (IL-12p40(E45Q / Q56E / E59Q)), xvii) SEQ ID NO:73 (IL-12p40(D161N)), xviii) SEQ ID NO:74 (IL-12p40(E73Q)), xix) SEQ ID NO:75 (IL-12p40(Q144E)), xx) SEQ ID NO:76 (IL-12p40(E262Q)), xxi) SEQ ID NO: 77 (IL-12p40(E100Q)), xxii) SEQ ID NO: 78 (IL-12p40(D18N)), xxiii) SEQ ID NO: 79 (IL-12p40(E33Q)), xxiv) SEQ ID NO: 80 (IL-12p40(Q229E)), xxv) SEQ ID NO: 81 (IL-12p40(E235Q)), xxvi) SEQ ID NO: 82 (IL-12p40(Q256N)), xxvii) SEQ ID NO: 83 (IL-12p40(E299Q)), xxviii) SEQ ID NO: 84 (IL-12p40(D87N)) , xxix) IL-12p40(E32Q), xxx) IL-12p40(D34N), xxxi) IL-12p40(S43E), xxxii) IL-12p40(S43K), xxxiii) Sequence number XXX(IL-12p40(E59K / K99E / N103Q / N11) 3Q / N200Q / N281Q / C252S / K264E)), xxxiv) SEQ ID NO: 205 (IL-12p40(E59K)), xxxv) IL-12p40(K99E), xxxvi) IL-12p40(K163E), xxxvii) IL-12p40(E187Q),xxxviii) IL-12p40(K258E), xxxix) IL-12p40(K260E), xl) SEQ ID NO: 206 (IL-12p40(E32Q / E59Q)), xli) SEQ ID NO: 207 (IL-12p40(D34N / E59Q)), xlii) SEQ ID NO: 208 (IL-12p40(E59Q / E187Q)), xliii) SEQ ID NO: 209 (IL-12p40(S43E / E59Q)), xliv) SEQ ID NO: 210 (IL-12p40(S43K / E49Q)), xlv) SEQ ID NO: 211 (IL-12p40(E59Q / K 163E), xlvi) SEQ ID NO:212 (IL-12p40(E59Q / K99E)), xlvii) SEQ ID NO:213 (IL-12p40(E59Q / K258E)), xlviii) SEQ ID NO:214 (IL-12p40(E59Q / K260E)), xlix) SEQ ID NO:XXX (IL-12p40(D34N / E59K)), l) SEQ ID NO:XXX (IL-12p40(E59K / K99E)), li) SEQ ID NO:XXX (IL-12p40(D18K / E59K / K99E)), lii) SEQ ID NO:XXX (IL-12p40(E59K / ) SEQ ID NO: XXX(IL-12p40(E59K / K99Y)), liv) SEQ ID NO: XXX(IL-12p40(E59Y / K99E)), lv) SEQ ID NO: XXX(IL-12p40(E45K / E59K / K99E)), lvi) SEQ ID NO: XXX(IL-12p40(E59K / K99E / Q144E)), lvii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / Q144K)), lviii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / R159E)), lix) SEQ ID NO: XXX(IL-12p40(E59K / K99Y)), liv) SEQ ID NO: XXX(IL-12p40(E59Y / K99E)), lv) SEQ ID NO: XXX(IL-12p40(E45K / E59K / K99E)), lvi) SEQ ID NO: XXX(IL-12p40(E59K / K99E / Q144E)), lvii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / Q144K)), lviii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / R159E)), Sequence number XXX(IL-12p40(E59K / K99E / K264E)), lx) Sequence number XXX(IL-12p40(D18K / E59K / K99E / K264E)), lxi) Sequence number XXX(IL-12p40(C252S)), lxii) Sequence number XXX(IL-12p40(DI8K / E59K / K99E / C252S)), lxiii) Sequence number XXX(IL-12p40(D18K / E59K / K99E / C252S / K264E)), lxiv) Sequence number XXX(IL-12p40(E59K / K99Y / C252S)),lxv) SEQ ID NO:XXX (IL-12p40(E59K / K99E / C252S / K2, 64E), lxvi) SEQ ID NO:XXX(IL-12p40(E59K / K99E / C252S)), lxvii) SEQ ID NO:XXX(IL-12p40(N103D / N113D)), lxviii) SEQ ID NO:XXX(IL-12p40(N103D / N200D)), lxix) SEQ ID NO:XXX(IL-12p40(N103D / N281D)), lxx) SEQ ID NO:XXX(IL-12p40(N113D / N200D)), lxxi) SEQ ID NO:XXX(IL-12p40(N113D / N281D)), lxxii) SEQ ID NO:XXX (IL-12p40(N200D / N281D)), lxxiii) SEQ ID NO:XXX(IL-12p50(N103D / N113D / N200D)), lxxiv) SEQ ID NO:XXX(IL-12p40(N103D / N113D / N281D)), lxxv) SEQ ID NO:XXX(IL-12p40(N103D / N200D / N281D)), lxxvi) SEQ ID NO:XXX(IL-12p40(N113D / N200D / N281D)), lxxvii) SEQ ID NO:XXX(IL-12p40(N103Q)), lxxviii) SEQ ID NO:X XX(IL-12p40(N113Q)), lxxix) SEQ ID NO:XXX(IL-12p40(N200Q)), lxxx) SEQ ID NO:XXX(IL-12p40(N281Q)), lxxxi) SEQ ID NO:XXX(IL-12p40(N103Q / N113Q)), lxxxii) SEQ ID NO:XXX(IL-12p40(N103Q / N200Q)), lxxxiii) SEQ ID NO:XXX(IL-12p40(N103Q / N281Q)), lxxxiv) SEQ ID NO:XXX(IL-12p40(N113Q / N200Q)), lxxxv) SEQ ID NO:XXX No. XXX(IL-12p40(N113Q / N281Q)), lxxxvi) No. XXX(IL-12p40(N200Q / N281Q)), lxxxvii) No. XXX(IL-12p40(N103Q / N113Q / N200Q)), lxxxviii) No. XXX(IL-12p40(N103Q / N113Q / N281Q)), lxxxix) No. XXX(IL-12p40(N103Q / N200Q / N281Q)), xc) No. XXX(IL-12p40(N113Q / N200Q / N281Q)),xci) SEQ ID NO: XXX(IL-12p40(N103Q / N113Q / N200Q / N281Q)), xcii) SEQ ID NO: XXX(IL-12p40(D34N / E59K / K99E)), xciii) SEQ ID NO: XXX(IL-12p40(D34K / E59K / K99E)), xciv) SEQ ID NO: XXX(IL-12p40(E32Q / D34N / E59K / K99E)), xcv) SEQ ID NO: XXX(IL-12p40(E32K / D34N / E59K / K99E)), xcvi) SEQ ID NO: XXX(IL-12 p40(E59Y / K99Y)), xcvii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N103Q / C252S / K264E)), xcviii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N113Q / C252S / K264E)), xcix) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N200Q / C252S / K264E)), c) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N281Q / C252S / K264E)), ci) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N103Q / N113Q / C252S / K264E)), cii) SEQ ID NO:XXX(IL-12p40(E59K / K99E / N103Q / N200Q / C252S / K264E)), ciii) SEQ ID NO:XXX(IL-12p40(E59K / K99E / N103Q / N281Q / C252S / K264E)), civ) SEQ ID NO:XXX(IL-12p40(E59K / K99E / N113Q / N200Q / C252S / K264E)), cv) SEQ ID NO:XXX (IL-12p40(E59K / K99E / N113Q / N281Q / C252S / K264E)), cvi) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N200Q / N281Q / C252S / K264E)), cvii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N103Q / N113Q / N200Q / C252S / K264E)), cviii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N103Q / N200Q / N281Q / C252S / K264E)),and cix) SEQ ID NO:XXX (IL-12p40(E59K / K99E / N113Q / N200Q / N281Q / C252S / K264E)).

[0160] In some embodiments, the present invention relates to a method for the preparation of IL-12p35 subunits comprising administering to the patient a IL-12p35 subunit comprising administering to the patient a IL-12p35 subunit comprising administering to the patient a IL-12p35 subunit comprising administering to the patient a IL-12p35 subunit comprising administering to the patient a IL-12p35 subunit comprising administering to the patient a IL-12p35 subunit 5, N136, E143, Q146, N151, E153, K158, E162, E163, D165, I171, R181, I182, R183, V185, T186, D188, R189, V190, S192, Y193, N195, and A196.

[0161] In some embodiments, the invention provides a heterodimeric complex, wherein the IL-12p35 subunit has one or more amino acid substitutions selected from the group consisting of N21D, Q35D, E38Q, D55Q, D55K, N71D, N71Q, L75A, N76D, E79Q, N85D, N85Q, L89A, F96A, M97A, L124A, M125A, Q130E, Q135E, N136D, E143Q, Q146E, N151D, N151K, E153K, E153Q, K158E, E162Q, E163Q, D165N, I171A, N195D, and N195Q.

[0162] In some embodiments, the invention provides a heterodimeric complex, wherein the IL-12p35 subunit has an amino acid substitution selected from the group consisting of N71D / N85D / N195D, N151D / E153Q, N151D / D165N, Q130E / N151D, N151D / K158E, E79Q / N151D, D55Q / N151D, N136D / N151D, N21D / N151D, E143Q / N151D, N71Q / N85Q, N71Q / N195Q, N85Q / N195Q, N71Q / N85Q / N195Q, N71D / N85D, N71D / N195D, and N85D / N195D.

[0163] In some embodiments, the present invention relates to a method for the preparation of IL-12p35 subunits comprising administering to a patient a therapeutically effective amount of the IL-12p35 subunit, the method comprising administering to a patient a therapeutically effective amount of the IL-12p35 subunit, the method comprising administering to a patient a therapeutically effective amount of the IL-12p35 subunit, the method comprising administering to a patient a therapeutically effective amount of the IL-12p35 subunit, (N151D)), viii) SEQ ID NO: 120 (IL-12p35(Q135E)), ix) SEQ ID NO: 121 (IL-12p35(Q35D)), x) SEQ ID NO: 122 (IL-12p35(Q146E)), xi) SEQ ID NO: 123 (IL-12p35(N76D)), xii) SEQ ID NO: 124 (IL-12p35(E162Q)), xiii) SEQ ID NO: 125 (IL-12p35(E163Q)), xiv) IL-12p35(N21D), xv) SEQ ID NO: XXX (IL-12p35(D55Q)), xvi) I xvii) IL-12p35(Q130E), xviii) IL-12p35(N136D), xix) IL-12p35(E143Q), xx) SEQ ID NO: 227 (IL-12p35(N151K)), xxi) SEQ ID NO: 226 (IL-12p35(E153K)), xxii) IL-12p35(K158E), xxiii) IL-12p35(D165N), xxiv) SEQ ID NO: 225 (IL-12p35(N151D / E153Q)), xxv) SEQ ID NO: 228 (IL-12 p35(N151D / D165N)), xxvi) SEQ ID NO: 229 (IL-12p35(Q130E / N151D)), xxvii) SEQ ID NO: 230 (IL-12p35(N151D / K158E)), xxviii) SEQ ID NO: 231 (IL-12p35(E79Q / N151D)), xxix) SEQ ID NO: 232 (IL-12p35(D55Q / N151D)), xxx) SEQ ID NO: 233 (IL-12p35(N136D / N151D)), xxxi) SEQ ID NO: 234 (IL-12p35(N21D / N151D )), xxxii) SEQ ID NO:235 (IL-12p35(E143Q / N151D)), xxxiii) SEQ ID NO:XXX (IL-12p35(F96A)), xxxiv) SEQ ID NO:XXX (IL-12p35(M97A)), xxxv) SEQ ID NO:XXX (IL-12p35(L89A)), xxxvi) SEQ ID NO:XXX (IL-12p35(L124A)), xxxvii) SEQ ID NO:X XX(IL-12p35(M125A)), xxxviii) SEQ ID NO:XXX(IL-12p35(L75A)), xxxiv) SEQ ID NO:XXX(IL-12p35(I171A)), xxxv) SEQ ID NO:XXX(IL-12p35(N71Q)), xxxvi) SEQ ID NO:XXX(IL-12p35(N85Q)), xxxvii) SEQ ID NO:XXX(IL-12p35(N195Q)), xxxviii) SEQ ID NO: XXX(IL-12p35(N71Q / N85Q)), xxxix) SEQ ID NO: XXX(IL-12p35(N71Q / N195Q)), xl) SEQ ID NO: XXX(IL-12p35(N85Q / N195Q), xli) SEQ ID NO: XXX(IL-12p35(N71Q / N85Q / N195Q)), xlii) SEQ ID NO: XXX(IL-12p35(N71D / N8 xlii) SEQ ID NO:XXX(IL-12p35(N71D / N195D), xliv) SEQ ID NO:XXX(IL-12p35(N85D / N195D)), xlv) SEQ ID NO:XXX(IL-12p35(D55Q)), and xlvi) SEQ ID NO:XXX(IL-12p35(D55K)).

[0164] In some embodiments, the present invention provides a composition comprising a heterodimeric complex for use in treating cancer in a subject.

[0165] In some embodiments, the invention provides one or more nucleic acids encoding the heterodimeric complex.

[0166] In some embodiments, the invention provides a host cell comprising the one or more nucleic acids encoding the heterodimeric complex.

[0167] In some embodiments, the invention provides a method of making a heterodimeric complex, the method comprising culturing a host cell under conditions whereby the heterodimeric complex is produced.

[0168] In some embodiments, the invention provides a method for purifying a heterodimeric complex, the method comprising: a) providing a composition comprising a heterodimeric complex; b) loading the composition onto an ion exchange column; and c) collecting fractions containing the heterodimeric complex.

[0169] In another aspect, the present invention provides variant IL-12p40 subunits having altered affinity for IL-12 receptor subunit beta-1 (IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2), and / or the IL-12 receptor complex.

[0170] In some embodiments, the present invention provides a variant IL-12p40 subunit, the variant IL-12p40 subunit being selected from the group consisting of E3, D7, E12, D14, W15, P17, D18, A19, P20, G21, E22, M23, D29, E32, E33, D34, L40, D41, Q42, S43, E45, L47, T54, I55, Q 56, K58, E59, F60, G61, D62, Q65, Y66, E73, K84, E86, D87, G88, I89, W90, D93, D97, K99, E100, K102 , N103, K104, F106, E110, N113, Y114, D129, D142, Q144, E156, R159, D161, N162, K163, D166, D170 , Q172, D174, A176, C177, P178, A179, A180, E181, S183, P185, E187, N200, S204, F206, R208, D209, D214, N218, Q220, N226, Q229, E231, E235, T242, P243, S245, Y246, F247, S248, Q256, C252, K258, K260, E262, K264, D265, D270, N281, Q289, D290, R291, Y292, Y293, and E299. In some embodiments, the present invention provides variant IL-12p40 subunits, the variant IL-12p40 subunits being D18N, D18K, E32Q, E33Q, D34N, D34K, Q42E, S43E, S43K, E45Q, Q56E, E59Q, E59K, D62N, E73Q, D87N, K99E, K99Y, E100Q, N103 D, N103Q, N113D, N113Q, Q144E, D161N, R159E, K163E, E187Q, N200D, N200Q, N218Q, Q229E, E235Q, C252S, Q256N, K258E, K260E, E262Q, K264E, N281D, N281Q, and E299Q.

[0171] In some embodiments, the present invention provides variant IL-12p40 subunits, the variant IL-12p40 subunits being N103D / N113D / N200D / N281D, Q42E / E45Q, E45Q / Q56E, Q42E / E59Q, Q56E / E59Q, Q42E / E45Q / Q56E, E45Q / Q56E / E59Q, E32Q / E59Q, D34N / E59K, D34N / E59K / K99E, D34K / E59K / K99E, E32Q / D34N / E59K / K99E, E32K / D34N / E59K / K99E, D ... 4N / E59Q, E59Q / E187Q, S43E / E59Q, S43K / E49Q, E59Q / K163E, E59Q / K99E, E5 9Q / K258E, E59Q / K260E, E59K / K99E, D18K / E59K / K99E, E59K / K99E / K264E, E5 9K / K99Y, E59Y / K99Y, E59Y / K99E, E45K / E59K / K99E, E59K / K99E / Q144E, E59 K / K99E / Q144K, E59K / K99E / R159E, E59K / K99E / K264E, D18K / E59K / K99E / K26 4E, DI8K / E59K / K99E / C252S, D18K / E59K / K99E / C252S / K264E, E59K / K99Y / C 252S, E59K / K99E / C252S / K264E, E59K / K99E / C252S, N103D / N113D, N103D / N2 00D, N103D / N281D, N113D / N200D, N113D / N281D, N200D / N281D, N103D / N113 D / N200D, N103D / N113D / N281D, N103D / N200D / N281D, N113D / N200D / N281D, N 103Q / N113Q, N103Q / N200Q, N103Q / N281Q, N113Q / N200Q, N113Q / N281Q, N20 0Q / N281Q, N103Q / N113Q / N200Q, N103Q / N113Q / N281Q, N103Q / N200Q / N281Q, N113Q / N200Q / N281Q, N103Q / N113Q / N200Q / N281Q, E59K / K99E / N103Q / C252S / K264E, E59K / K99E / N113Q / C252S / K264E, E59K / K99E / N200Q / C252S / K264E,E59K / K99E / N281Q / C252S / K264E, E59K / K99E / N103Q / N113Q / C252S / K264E, E59K / K99E / N103Q / N 200Q / C252S / K264E, E59K / K99E / N103Q / N281Q / C252S / K264E, E59K / K99E / N113Q / N200Q / C252S / K 264E, E59K / K99E / N113Q / N281Q / C252S / K264E, E59K / K99E / N200Q / N281Q / C252S / K264E, E59K / K9 9E / N103Q / N113Q / N200Q / C252S / K264E, E59K / K99E / N103Q / N200Q / N281Q / C252S / K264E, E59K / K, and E59K / K99E / N103Q / N113Q / N200Q / N281Q / C252S / K264E.

[0172] In some embodiments, the present invention provides variant IL-12p40 subunits, the variant IL-12p40 subunits being selected from the group consisting of i) SEQ ID NO:57 (IL-12p40(N103D)), ii) SEQ ID NO:58 (IL-12p40(N113D)), iii) SEQ ID NO:59 (IL-12p40(N200D)), iv) SEQ ID NO:60 (IL-12p40(N281D)), v) SEQ ID NO:61 (IL-12p40(N103D / N113D / N200D / N281D)), vi) SEQ ID NO:62 (IL-12p40(Q42E)), v) SEQ ID NO:63 (IL-12p40(Q42E)), ii) SEQ ID NO:63 (IL-12p40(E45Q)), viii) SEQ ID NO:64 (IL-12p40(Q56E)), ix) SEQ ID NO:65 (IL-12p40(E59Q)), x) SEQ ID NO:66 (IL-12p40(D62N)), xi) SEQ ID NO:67 (IL-12p40(Q42E / E45Q)), xii) SEQ ID NO:68 (IL-12p40(E45Q / Q56E)), xiii) SEQ ID NO:69 (IL-12p40(Q42E / E59Q)), xiv) SEQ ID NO:70 (IL-12p40(Q56E / E59Q)), xv) SEQ ID NO:71 ( IL-12p40(Q42E / E45Q / Q56E)), xvi) SEQ ID NO: 72 (IL-12p40(E45Q / Q56E / E59Q)), xvii) SEQ ID NO: 73 (IL-12p40(D161N)), xviii) SEQ ID NO: 74 (IL-12p40(E73Q)), xix) SEQ ID NO: 75 (IL-12p40(Q144E)), xx) SEQ ID NO: 76 (IL-12p40(E262Q)), xxi) SEQ ID NO: 77 (IL-12p40(E100Q)), xxii) SEQ ID NO: 78 (IL-12p40(D18N)), xxiii) SEQ ID NO: 7 9 (IL-12p40(E33Q)), xxiv) SEQ ID NO: 80 (IL-12p40(Q229E)), xxv) SEQ ID NO: 81 (IL-12p40(E235Q)), xxvi) SEQ ID NO: 82 (IL-12p40(Q256N)), xxvii) SEQ ID NO: 83 (IL-12p40(E299Q)), xxviii) SEQ ID NO: 84 (IL-12p40(D87N)), xxix) IL-12p40(E32Q), xxx) IL-12p40(D34N), xxxi) IL-12p40(S43E), xxxii) IL-12p40(S43K),xxxiii) SEQ ID NO: XXX (IL-12p40(E59K / K99E / N103Q / N113Q / N200Q / N281Q / C252S / K264E)), xxxiv) SEQ ID NO: 205 (IL-12p40(E59K)), xxxv) IL-12p40(K99E), xxxvi) IL-12p40(K163E), xxxvii) IL-12p40(E187Q), xxxviii) IL-12p40(K258E), xxxix) IL-12p40(K260E), xl) SEQ ID NO:206 (IL-12p40(E32Q / E59Q)), xli) SEQ ID NO:207 (IL-12p40(D34N / E59Q)), xlii) SEQ ID NO:208 (IL-12p40(E59Q / E187Q)), xliii) SEQ ID NO:209 (IL-12p40(S43E / E59Q)), xliv) SEQ ID NO:210 (IL-12p40(S43K / E49Q)), xlv) SEQ ID NO:211 (IL-12p40(E59Q / K163E)), xlvi) SEQ ID NO:212 (IL-1 2p40(E59Q / K99E)), xlvii) SEQ ID NO:213 (IL-12p40(E59Q / K258E)), xlviii) SEQ ID NO:214 (IL-12p40(E59Q / K260E)), xlix) SEQ ID NO:XXX (IL-12p40(D34N / E59K)), l) SEQ ID NO:XXX (IL-12p40(E59K / K99E)), li) SEQ ID NO:XXX (IL-12p40(D18K / E59K / K99E)), lii) SEQ ID NO:XXX (IL-12p40(E59K / K99E / K264E)), liii) SEQ ID NO: XXX(IL-12p40(E59K / K99Y)), liv) SEQ ID NO: XXX(IL-12p40(E59Y / K99E)), lv) SEQ ID NO: XXX(IL-12p40(E45K / E59K / K99E)), lvi) SEQ ID NO: XXX(IL-12p40(E59K / K99E / Q144E)), lvii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / Q144K)), lviii) SEQ ID NO: XXX(IL-12, p40(E59K / K99E / R159E)), lx) SEQ ID NO:XXX(IL-12p40(E59K / K99E / K264E)), lx) SEQ ID NO:XXX(IL-12p40(D18K / E59K / K99E / K264E)), lxi) SEQ ID NO:XXX(IL-12p40(C252S)), lxii) SEQ ID NO:XXX(IL-12p40(DI8K / E59K / K99E / C252S)), lxiii) SEQ ID NO:XXX(IL-12p40(D18K / E59K / K99E / C252S / K264E)), lxiv) SEQ ID NO:XXX(IL- lxv) SEQ ID NO: XXX(IL-12p40(E59K / K99Y / C252S / K264E)), lxvi) SEQ ID NO: XXX(IL-12p40(E59K / K99E / C252S)), lxvii) SEQ ID NO: XXX(IL-12p40(N103D / N113D)), lxviii) SEQ ID NO: XXX(IL-12p40(N103D / N200D)), lxix) SEQ ID NO: XXX(IL-12p40(N103D / N281D)), lxx) SEQ ID NO: XXX(IL-12p40(N113D / N200D)), lxxi) SEQ ID NO: XXX(IL-12p40(N113D / N281D)), lxxii) SEQ ID NO: XXX(IL-12p40(N200D / N281D)), lxxiii) SEQ ID NO: XXX(IL-12p50(N103D / N113D / N200D)), lxxiv) SEQ ID NO: XXX(IL-12p40(N103D / N113D / N281D)), lxxv) SEQ ID NO: XXX(IL-12p40(N103D / N200D / N281D)), lxxvi) SEQ ID NO: XXX(IL-12p40(N113D / N200D / N 281D), lxxvii) SEQ ID NO: XXX(IL-12p40(N103Q)), lxxviii) SEQ ID NO: XXX(IL-12p40(N113Q)), lxxix) SEQ ID NO: XXX(IL-12p40(N200Q)), lxxx) SEQ ID NO: XXX(IL-12p40(N281Q)), lxxxi) SEQ ID NO: XXX(IL-12p40(N103Q / N113Q)), lxxxii) SEQ ID NO: XXX(IL-12p40(N103Q / N200Q)), lxxxiii) SEQ ID NO: XXX(IL-12p40(N103Q / N281Q)),lxxxiv) SEQ ID NO: XXX(IL-12p40(N113Q / N200Q)), lxxxv) SEQ ID NO: XXX(IL-12p40(N113Q / N281Q)), lxxxvi) SEQ ID NO: XXX(IL-12p40(N200Q / N281Q)), lxxxvii) SEQ ID NO: XXX(IL-12p40(N103Q / N113Q / N200Q)), lxxxviii) SEQ ID NO: XXX(IL-12p40(N103Q / N113Q / N281Q)), lxxxix) SEQ ID NO: XXX(IL-12p40(N103Q / N200Q / N2 81Q), xc) SEQ ID NO: XXX(IL-12p40(N113Q / N200Q / N281Q)), xci) SEQ ID NO: XXX(IL-12p40(N103Q / N113Q / N200Q / N281Q)), xcii) SEQ ID NO: XXX(IL-12p40(D34N / E59K / K99E)), xciii) SEQ ID NO: XXX(IL-12p40(D34K / E59K / K99E)), xciv) SEQ ID NO: XXX(IL-12p40(E32Q / D34N / E59K / K99E)), xcv) SEQ ID NO: XXX(IL-12p40(E32K / D 34N / E59K / K99E), xcvi) SEQ ID NO: XXX(IL-12p40(E59Y / K99Y)), xcvii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N103Q / C252S / K264E)), xcviii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N113Q / C252S / K264E)), xcix) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N200Q / C252S / K264E)), c) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N281Q / C252S / K264E)), ci) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N103Q / N113Q / C252S / K264E)), cii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N103Q / N200Q / C252S / K264E)), ciii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N103Q / N281Q / C252S / K264E)), civ) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N113Q / N200Q / C252S / K264E)),cv) SEQ ID NO:XXX (IL-12p40(E59K / K99E / N113Q / N281Q / C, and cix) SEQ ID NO:XXX(IL-12p40(E59K / K99E / N113Q / N200Q / N281Q / C252S / K264E)).

[0173] In some embodiments, the invention provides compositions comprising a variant IL-12p40 subunit.

[0174] In some embodiments, the invention provides nucleic acids encoding variant IL-12p40 subunits.

[0175] In some embodiments, the invention provides a host cell comprising the nucleic acid encoding a variant IL-12p40 subunit.

[0176] In some embodiments, the invention provides a method of making a variant IL-12p40 subunit, the method comprising culturing a host cell of claim H8 under conditions whereby the variant IL-12p40 subunit is produced.

[0177] In another aspect, the present invention provides variant IL-12p35 subunits having altered affinity for IL-12 receptor subunit beta-1 (IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2), and / or the IL-12 receptor complex.

[0178] In some embodiments, the present invention provides a variant IL-12p35 subunit, the variant IL-12p35 subunit being selected from the group consisting of Q20, N21, Q35, E38, S44, E45, E46, H49, K54, D55, T59, V60, E61, C63, L64, P65, E67, L68, N71, S73, C74, L75, N76, E79, N85, L89, F96, and having one or more amino acid modifications at amino acid residues selected from the group consisting of M97, L124, M125, Q130, Q135, N136, E143, Q146, N151, E153, K158, E162, E163, D165, I171, R181, I182, R183, V185, T186, D188, R189, V190, S192, Y193, N195, and A196.

[0179] In some embodiments, the invention provides a variant IL-12p35 subunit having one or more amino acid substitutions selected from the group consisting of N21D, Q35D, E38Q, D55Q, D55K, N71D, N71Q, L75A, N76D, E79Q, N85D, N85Q, L89A, F96A, M97A, L124A, M125A, Q130E, Q135E, N136D, E143Q, Q146E, N151D, N151K, E153K, E153Q, K158E, E162Q, E163Q, D165N, I171A, N195D, and N195Q.

[0180] In some embodiments, the present invention provides variant IL-12p35 subunits, the variant IL-12p35 subunits being N71D / N85D / N195D, N151D / E153Q, N151D / D165N, Q130E / N151D, N151D / K158E, E79Q / N151D, D55Q / N151D, N136D / N151D, N21D / N151D, E143Q / N151D, N71Q / N85Q, N71Q / N195Q, N85Q / N195Q, N71Q / N85Q / N195Q, N71D / N85D, N71D / N195D, and N85D / N195D.

[0181] In some embodiments, the invention provides a variant IL-12p35 subunit, the variant IL-12p35 subunit having amino acid substitutions N71D / N85D / N195D.

[0182] In some embodiments, the present invention provides a variant IL-12p35 subunit, the variant IL-12p35 subunit being selected from the group consisting of i) SEQ ID NO: 113 (IL-12p35(N71D)), ii) SEQ ID NO: 114 (IL-12p35(N85D)), iii) SEQ ID NO: 115 (IL-12p35(N195D)), iv) SEQ ID NO: 116 (IL-12p35(N71D / N85D / N195D)), v) SEQ ID NO: 117 (IL-12p35(E153Q)), vi) SEQ ID NO: 118 (IL-12p35(E38Q)), vii) SEQ ID NO: 1 19 (IL-12p35(N151D)), viii) SEQ ID NO: 120 (IL-12p35(Q135E)), ix) SEQ ID NO: 121 (IL-12p35(Q35D)), x) SEQ ID NO: 122 (IL-12p35(Q146E)), xi) SEQ ID NO: 123 (IL-12p35(N76D)), xii) SEQ ID NO: 124 (IL-12p35(E162Q)), xiii) SEQ ID NO: 125 (IL-12p35(E163Q)), xiv) IL-12p35(N21D), xv) SEQ ID NO: XXX (IL-12p35(D55Q)), xvi) IL-12p3 5(E79Q), xvii) IL-12p35(Q130E), xviii) IL-12p35(N136D), xix) IL-12p35(E143Q), xx) SEQ ID NO: 227 (IL-12p35(N151K)), xxi) SEQ ID NO: 226 (IL-12p35(E153K)), xxii) IL-12p35(K158E), xxiii) IL-12p35(D165N), xxiv) SEQ ID NO: 225 (IL-12p35(N151D / E153Q)), xxv) SEQ ID NO: 228 (IL-12p35(N151D / D165N)), xxvi) SEQ ID NO:229 (IL-12p35(Q130E / N151D)), xxvii) SEQ ID NO:230 (IL-12p35(N151D / K158E)), xxviii) SEQ ID NO:231 (IL-12p35(E79Q / N151D)), xxix) SEQ ID NO:232 (IL-12p35(D55Q / N151D)), xxx) SEQ ID NO:233 (IL-12p35(N136D / N151D)), xxxi) SEQ ID NO:234 ​​(IL-12p35(N21D / N151D)), xxxii) SEQ ID NO:235 (IL-12p35(E143Q / N151D)),xxxiii) SEQ ID NO:XXX(IL-12p35(F96A)), xxxiv) SEQ ID NO:XXX(IL-12p35(M97A)), xxxv) SEQ ID NO:XXX(IL-12p35(L89A)), xxxvi) SEQ ID NO:XXX(IL-12p35(L124A)), xxxvii) SEQ ID NO:XXX(IL-12p35(M125A)), xxxviii) SEQ ID NO:XXX(IL-12p35(L75A)), xxxiv) SEQ ID NO:XXX(IL-12p35(I171A)), xxxv) SEQ ID NO:XXX(IL-12p35(N71Q)), xxxvi) SEQ ID NO:XXX(IL-12p35(N85Q)), xxxvii) SEQ ID NO:XXX(IL-12p35(N195Q ... XXX(IL-12p35(N71Q / N85Q)), xxxix) SEQ ID NO:XXX(IL-12p35(N71Q / N195Q)), xl) SEQ ID NO:XXX(IL-12p35(N85Q / N195Q), xli) SEQ ID NO:XXX(IL-12p35(N71Q / N85Q / N195Q)), xlii) SEQ ID NO:XXX(IL-12p35(N71D / N85D)), xliii) SEQ ID NO:XXX(IL-12p35(N71D / N195D), xliv) SEQ ID NO:XXX(IL-12p35(N85D / N195D)), xlv) SEQ ID NO:XXX(IL-12p35(D55Q)), and xlvi) SEQ ID NO:XXX(IL-12p35(D55K)).

[0183] In some embodiments, the invention provides compositions comprising a variant IL-12p35 subunit.

[0184] In some embodiments, the invention provides nucleic acids encoding variant IL-12p35 subunits.

[0185] In some embodiments, the invention provides a host cell comprising the nucleic acid encoding a variant IL-12p35 subunit.

[0186] In some embodiments, the invention provides a variant IL-12p35 subunit, the method comprising culturing a host cell under conditions whereby the variant IL-12p35 subunit is produced.

[0187] In another aspect, the invention provides a fusion protein comprising: a) a first fusion protein comprising an IL-12p40 subunit domain covalently linked to a first Fc domain; and b) a second fusion protein comprising an IL-12p35 subunit domain covalently linked to a second Fc domain, said first and second Fc domains having the following structure according to EU numbering: L368D / K370S and S364K; L368D / K370S and S364K / E357L; L368D / The present invention provides heterodimeric proteins, which are a set of amino acid substitutions selected from the group consisting of: K370S and S364K / E357Q; T411E / K360E / Q362E and D401K; L368E / K370S and S364K; K370S and S364K / E357Q; T366S / L368A / Y407V and T366W; and T366S / L368A / Y407V / Y349C and T366W / S354C;

[0188] In some embodiments, the invention provides a heterodimeric protein, in which the IL-12p40 subunit domain is linked to the N-terminus of the first Fc domain and the IL-12p35 subunit domain is linked to the N-terminus of the second Fc domain.

[0189] In some embodiments, the invention provides a heterodimeric protein, in which the IL-12p40 subunit domain is linked to the C-terminus of the first Fc domain and the IL-12p35 subunit domain is linked to the C-terminus of the second Fc domain.

[0190] In some embodiments, the invention provides a heterodimeric protein, wherein the IL-12p40 subunit domain is linked to the first Fc domain using a domain linker and the IL-12p35 subunit domain is linked to the second Fc domain using a domain linker.

[0191] In some embodiments, the invention provides a heterodimeric protein, wherein said first and second Fc domains comprise an IgG1 hinge-CH2-CH3.

[0192] In some embodiments, the invention provides a heterodimeric protein, wherein the first fusion protein comprises, from N-terminus to C-terminus, IL-12p40 subunit domain-domain linker-hinge-CH2-CH3 and the second fusion protein comprises, from N-terminus to C-terminus, IL-12p35 subunit domain-domain linker-hinge-CH2-CH3.

[0193] In some embodiments, the invention relates to a first fusion protein comprising, from N-terminus to C-terminus, a hinge-CH2-CH3-domain linker-IL-12p40 subunit domain. and said second fusion protein comprises, from N-terminus to C-terminus, hinge-CH2-CH3-domain linker-IL-12p35 subunit domain.

[0194] In some embodiments, the present invention relates to a method for the preparation of IL-12p40 subunits comprising administering to a patient a therapeutically effective amount of the IL-12p40 subunit, the method comprising administering to a patient a therapeutically effective amount of the IL-12p40 subunit, the therapeutically effective amount of the IL-12p40 subunit being: i) SEQ ID NO:57 (IL-12p40(N103D)); ii) SEQ ID NO:58 (IL-12p40(N113D)); iii) SEQ ID NO:59 (IL-12p40(N200D)); iv) SEQ ID NO:60 (IL-12p40(N281D)); v) SEQ ID NO:61 (IL-12p40(N103D / N113D / N200D / N281D)); vi) SEQ ID NO:62 (IL-12p40(Q42E)); vii) SEQ ID NO:63 (IL-12p40(E45 Q)), viii) SEQ ID NO:64 (IL-12p40(Q56E)), ix) SEQ ID NO:65 (IL-12p40(E59Q)), x) SEQ ID NO:66 (IL-12p40(D62N)), xi) SEQ ID NO:67 (IL-12p40(Q42E / E45Q)), xii) SEQ ID NO:68 (IL-12p40(E45Q / Q56E)), xiii) SEQ ID NO:69 (IL-12p40(Q42E / E59Q)), xiv) SEQ ID NO:70 (IL-12p40(Q56E / E59Q)), xv) SEQ ID NO:71 (IL-12p40(Q42E / E 45Q / Q56E)), xvi) SEQ ID NO: 72 (IL-12p40(E45Q / Q56E / E59Q)), xvii) SEQ ID NO: 73 (IL-12p40(D161N)), xviii) SEQ ID NO: 74 (IL-12p40(E73Q)), xix) SEQ ID NO: 75 (IL-12p40(Q144E)), xx) SEQ ID NO: 76 (IL-12p40(E262Q)), xxi) SEQ ID NO: 77 (IL-12p40(E100Q)), xxii) SEQ ID NO: 78 (IL-12p40(D18N)), xxiii) SEQ ID NO: 79 (IL-12p 40(E33Q)), xxiv) SEQ ID NO: 80 (IL-12p40(Q229E)), xxv) SEQ ID NO: 81 (IL-12p40(E235Q)), xxvi) SEQ ID NO: 82 (IL-12p40(Q256N)), xxvii) SEQ ID NO: 83 (IL-12p40(E299Q)), xxviii) SEQ ID NO: 84 (IL-12p40(D87N)), xxix) IL-12p40(E32Q), xxx) IL-12p40(D34N), xxxi) IL-12p40(S43E), xxxii) IL-12p40(S43K),xxxiii) SEQ ID NO: XXX (IL-12p40(E59K / K99E / N103Q / N113Q / N200Q / N281Q / C252S / K264E)), xxxiv) SEQ ID NO: 205 (IL-12p40(E59K)), xxxv) IL-12p40(K99E), xxxvi) IL-12p40(K163E), xxxvii) IL-12p40(E187Q), xxxviii) IL-12p40(K258E), xxxix) IL-12p40(K260E), xl) SEQ ID NO: 206 ( IL-12p40(E32Q / E59Q)), xli) SEQ ID NO: 207 (IL-12p40(D34N / E59Q)), xlii) SEQ ID NO: 208 (IL-12p40(E59Q / E187Q)), xliii) SEQ ID NO: 209 (IL-12p40(S43E / E59Q)), xliv) SEQ ID NO: 210 (IL-12p40(S43K / E49Q)), xlv) SEQ ID NO: 211 (IL-12p40(E59Q / K163E)), xlvi) SEQ ID NO: 212 (IL-12p40(E59Q / K99E) ), xlvii) SEQ ID NO: 213 (IL-12p40(E59Q / K258E)), xlviii) SEQ ID NO: 214 (IL-12p40(E59Q / K260E)), xlix) SEQ ID NO: XXX (IL-12p40(D34N / E59K)), l) SEQ ID NO: XXX (IL-12p40(E59K / K99E)), li) SEQ ID NO: XXX (IL-12p40(D18K / E59K / K99E)), lii) SEQ ID NO: XXX (IL-12p40(E59K / K99E / K264E)), liii) SEQ ID NO: No. XXX(IL-12p40(E59K / K99Y)), liv) SEQ ID NO: XXX(IL-12p40(E59Y / K99E)), lv) SEQ ID NO: XXX(IL-12p40(E45K / E59K / K99E)), lvi) SEQ ID NO: XXX(IL-12p40(E59K / K99E / Q144E)), lvii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / Q144K)), lviii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / R159E)), lix) SEQ ID NO: No. XXX(IL-12p40(E59K / K99E / K264E)), lx) SEQ ID NO: XXX(IL-12p40(D18K / E59K / K99E / K264E)), lxi) SEQ ID NO: XXX(IL-12p40(C252S)), lxii) SEQ ID NO: XXX(IL-12p40(DI8K / E59K / K99E / C252S)), lxiii) SEQ ID NO: XXX(IL-12p40(D18K / E59K / K99E / C252S / K264E)), lxiv) SEQ ID NO: XXX(IL-12p40(E59K / K99Y / C252S)), lxv) SEQ ID NO: No. XXX(IL-12p40(E59K / K99E / C252S / K264E)), lxvi) SEQ ID NO: XXX(IL-12p40(E59K / K99E / C252S)), lxvii) SEQ ID NO: XXX(IL-12p40(N103D / N113D)), lxviii) SEQ ID NO: XXX(IL-12p40(N103D / N200D)), lxix) SEQ ID NO: XXX(IL-12p40(N103D / N281D)), lxx) SEQ ID NO: XXX(IL-12p40(N113D / N200D)), lxxi) SEQ ID NO: XXX(IL-12p40(N11 lxxii) SEQ ID NO: XXX(IL-12p40(N200D / N281D)), lxxiii) SEQ ID NO: XXX(IL-12p50(N103D / N113D / N200D)), lxxiv) SEQ ID NO: XXX(IL-12p40(N103D / N113D / N281D)), lxxv) SEQ ID NO: XXX(IL-12p40(N103D / N200D / N281D)), lxxvi) SEQ ID NO: XXX(IL-12p40(N113D / N200D / N281D)), lxxvii) SEQ ID NO: XXX(IL-12p40(N103Q )), lxxviii) SEQ ID NO: XXX(IL-12p40(N113Q)), lxxix) SEQ ID NO: XXX(IL-12p40(N200Q)), lxxx) SEQ ID NO: XXX(IL-12p40(N281Q)), lxxxi) SEQ ID NO: XXX(IL-12p40(N103Q / N113Q)), lxxxii) SEQ ID NO: XXX(IL-12p40(N103Q / N200Q)), lxxxiii) SEQ ID NO: XXX(IL-12p40(N103Q / N281Q)), lxxxiv) SEQ ID NO: XXX(IL-12p40(N113Q / N200Q)),lxxxv) SEQ ID NO:XXX(IL-12p40(N113Q / N281Q)), lxxxvi) SEQ ID NO:XXX(IL-12p40(N200Q / N281Q)), lxxxvii) SEQ ID NO:XXX(IL-12p40(N103Q / N113Q / N200Q)), lxxxviii) SEQ ID NO:XXX(IL-12p40(N103Q / N113Q / N281Q)), lxxxix) SEQ ID NO:XXX(IL-12p40(N103Q / N200Q / N281Q)), xc) SEQ ID NO:XXX(IL-12p40(N113Q / N200Q / N281Q )), xci) SEQ ID NO: XXX(IL-12p40(N103Q / N113Q / N200Q / N281Q)), xcii) SEQ ID NO: XXX(IL-12p40(D34N / E59K / K99E)), xciii) SEQ ID NO: XXX(IL-12p40(D34K / E59K / K99E)), xciv) SEQ ID NO: XXX(IL-12p40(E32Q / D34N / E59K / K99E)), xcv) SEQ ID NO: XXX(IL-12p40(E32K / D34N / E59K / K99E)), xcvi) SEQ ID NO: XXX(IL-12p40(E59Y / K99Y )), xcvii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N103Q / C252S / K264E)), xcviii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N113Q / C252S / K264E)), xcix) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N200Q / C252S / K264E)), c) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N281Q / C252S / K264E)), ci) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N103Q / N113Q / C252S / K264E)), cii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N103Q / N200Q / C252S / K264E)), ciii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N103Q / N281Q / C252S / K264E)), civ) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N113Q / N200Q / C252S / K264E)), cv) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N113Q / N281Q / C252S / K264E)),cvi) SEQ ID NO: XXX (IL-1, 2p40(E59K / K99E / N200Q / N281Q / C252S / K264E)), cvii) SEQ ID NO:XXX(IL-12p40(E59K / K99E / N103Q / N113Q / N200Q / C252S / K264E)), cviii) SEQ ID NO:XXX(IL-12p40(E59K / K99E / N103Q / N200Q / N281Q / C252S / K264E)), and cix) SEQ ID NO:XXX(IL-12p40(E59K / K99E / N113Q / N200Q / N281Q / C252S / K264E)).

[0195] In some embodiments, the present invention relates to a method for the preparation of a IL-12p35 subunit comprising administering to a patient a sequence identical to that of SEQ ID NO:113 (IL-12p35(N71D)), ii) SEQ ID NO:114 (IL-12p35(N85D)), iii) SEQ ID NO:115 (IL-12p35(N195D)), iv) SEQ ID NO:116 (IL-12p35(N71D / N85D / N195D)), v) SEQ ID NO:117 (IL-12p35(E153Q)), vi) SEQ ID NO:118 (IL-12p35(E38Q)), vii) SEQ ID NO:119 (IL-12p35(N151D)), viii) SEQ ID NO:120 (IL-12p35(N151D)), or viii) SEQ ID NO: 120 (IL-12p35(Q135E)), ix) SEQ ID NO: 121 (IL-12p35(Q35D)), x) SEQ ID NO: 122 (IL-12p35(Q146E)), xi) SEQ ID NO: 123 (IL-12p35(N76D)), xii) SEQ ID NO: 124 (IL-12p35(E162Q)), xiii) SEQ ID NO: 125 (IL-12p35(E163Q)), xiv) IL-12p35(N21D), xv) SEQ ID NO: XXX (IL-12p35(D55Q)), xvi) IL-12p35(E79Q), xvii) IL-12p35(Q130E), x viii) IL-12p35(N136D), xix) IL-12p35(E143Q), xx) SEQ ID NO: 227 (IL-12p35(N151K)), xxi) SEQ ID NO: 226 (IL-12p35(E153K)), xxii) IL-12p35(K158E)), xxiii) IL-12p35(D165N), xxiv) SEQ ID NO: 225 (IL-12p35(N151D / E153Q)), xxv) SEQ ID NO: 228 (IL-12p35(N151D / D165N)), xxvi) SEQ ID NO: 229 (IL-12p35(Q130E / N151D)) , xxvii) SEQ ID NO: 230 (IL-12p35(N151D / K158E)), xxviii) SEQ ID NO: 231 (IL-12p35(E79Q / N151D)), xxix) SEQ ID NO: 232 (IL-12p35(D55Q / N151D)), xxx) SEQ ID NO: 233 (IL-12p35(N136D / N151D)), xxxi) SEQ ID NO: 234 (IL-12p35(N21D / N151D)), xxxii) SEQ ID NO: 235 (IL-12p35(E143Q / N151D)), xxxiii) SEQ ID NO: XXX (IL-12p35(F96A)),xxxiv) SEQ ID NO:XXX(IL-12p35(M97A)), xxxv) SEQ ID NO:XXX(IL-12p35(L89A)), xxxvi) SEQ ID NO:XXX(IL-12p35(L124A)), xxxvii) SEQ ID NO:XXX(IL-12p35(M125A)), xxxviii) SEQ ID NO:XXX(IL-12p35(L75A)), xxxiv) SEQ ID NO:XXX(IL-12p35(I171A)), xxxv) SEQ ID NO:XXX(IL-12p35(N71Q)), xxxvi) SEQ ID NO:XXX(IL-12p35(N85Q)), xxxvii) SEQ ID NO:XXX(IL-12p35(N195Q)), xxxviii) SEQ ID NO:XXX(IL-12p35(N71Q / N85 The present invention provides a heterodimeric protein having a sequence selected from the group consisting of SEQ ID NO:XXX(IL-12p35(N71Q / N195Q)), xxxix) SEQ ID NO:XXX(IL-12p35(N71Q / N195Q)), lx) SEQ ID NO:XXX(IL-12p35(N85Q / N195Q), lxi) SEQ ID NO:XXX(IL-12p35(N71Q / N85Q / N195Q)), lxii) SEQ ID NO:XXX(IL-12p35(N71D / N85D)), lxiii) SEQ ID NO:XXX(IL-12p35(N71D / N195D), lxiv) SEQ ID NO:XXX(IL-12p35(N85D / N195D)), lxv) SEQ ID NO:XXX(IL-12p35(D55Q)), and lxvi) SEQ ID NO:XXX(IL-12p35(D55K)).

[0196] In another aspect, the invention comprises a heterodimeric protein comprising: a) a first Fc domain; and b) a fusion protein comprising i) a second Fc domain, ii) an IL-12p40 subunit domain, and iii) an IL-12p35 subunit domain, wherein the first and second Fc domains are designated as follows according to EU numbering: L368D / K370S and S364K; L368D / K370S and S364K / E357 L; L368D / K370S and S364K / E357Q; T411E / K360E / Q362E and D401K; L368E / K370S and S364K; K370S and S364K / E357Q; T366S / L368A / Y407V and T366W; and T366S / L368A / Y407V / Y349C and T366W / S354C;

[0197] In some embodiments, the invention provides a heterodimeric protein, wherein the fusion protein comprises, from N-terminus to C-terminus, IL-12p40 subunit domain-domain linker-IL-12p35 subunit domain-domain linker-hinge-CH2-CH3.

[0198] In some embodiments, the invention provides a heterodimeric protein, wherein the fusion protein comprises, from N-terminus to C-terminus, IL-12p35 subunit domain-domain linker-IL-12p40 subunit domain-domain linker-hinge-CH2-CH3.

[0199] In some embodiments, the invention provides a heterodimeric protein, wherein the fusion protein comprises, from N-terminus to C-terminus, hinge-CH2-CH3-domain linker IL-12p35 subunit domain-domain linker-IL-12p40 subunit domain.

[0200] In some embodiments, the invention provides a heterodimeric protein, wherein the fusion protein comprises, from N-terminus to C-terminus, hinge-CH2-CH3-domain linker IL-12p40 subunit domain-domain linker-IL-12p35 subunit domain.

[0201] In some embodiments, the present invention relates to a method for the preparation of IL-12p40 subunits comprising the steps of: i) SEQ ID NO:57 (IL-12p40(N103D)); ii) SEQ ID NO:58 (IL-12p40(N113D)); iii) SEQ ID NO:59 (IL-12p40(N200D)); iv) SEQ ID NO:60 (IL-12p40(N281D)); v) SEQ ID NO:61 (IL-12p40(N103D / N113D / N200D / N281D)); ) SEQ ID NO:62 (IL-12p40(Q42E)), vii) SEQ ID NO:63 (IL-12p40(E45Q)), viii) SEQ ID NO:64 (IL-12p40(Q56E)), ix) SEQ ID NO:65 (IL-12p40(E59Q)), x) SEQ ID NO:66 (IL-12p40(D62N)), xi) SEQ ID NO:67 (IL-12p40(Q42E / E45Q)), xii) SEQ ID NO:68 (IL-12p40(E45Q / Q56E )), xiii) SEQ ID NO: 69 (IL-12p40(Q42E / E59Q)), xiv) SEQ ID NO: 70 (IL-12p40(Q56E / E59Q)), xv) SEQ ID NO: 71 (IL-12p40(Q42E / E45Q / Q56E)), xvi) SEQ ID NO: 72 (IL-12p40(E45Q / Q56E / E59Q)), xvii) SEQ ID NO: 73 (IL-12p40(D161N)), xviii) SEQ ID NO: 74 (IL-12p4 0(E73Q)), xix) SEQ ID NO:75 (IL-12p40(Q144E)), xx) SEQ ID NO:76 (IL-12p40(E262Q)), xxi) SEQ ID NO:77 (IL-12p40(E100Q)), xxii) SEQ ID NO:78 (IL-12p40(D18N)), xxiii) SEQ ID NO:79 (IL-12p40(E33Q)), xxiv) SEQ ID NO:80 (IL-12p40(Q229E)), xxv) SEQ ID NO:81 (IL-12p40(I 82 (IL-12p40(Q256N)), xxvii) SEQ ID NO: 83 (IL-12p40(E299Q)), xxviii) SEQ ID NO: 84 (IL-12p40(D87N)), xxix) IL-12p40(E32Q), xxx) IL-12p40(D34N), xxxi) IL-12p40(S43E), xxxii) IL-12p40(S43K), xxxiii) SEQ ID NO: XXX (IL-12p40(E59K / K99E / N103Q / N113Q / N200Q / N281Q / C252S / K264E), xxxiv) SEQ ID NO: 205 (IL-12p40(E59K)), xxxv) IL-12p40(K99E), xxxvi) IL-12p40(K163E), xxxvii) IL-12p40(E187Q), xxxviii) IL-12p40(K258E), xxxix) IL-12p40(K260E), xl) SEQ ID NO: 206 (IL-12p40(E32Q / E59Q)), xli) SEQ ID NO: 207 (IL-12p40(D34N / E59Q)), xlii) SEQ ID NO: 208 (IL-12p40(E59Q / E187Q) ), xliii) SEQ ID NO: 209 (IL-12p40(S43E / E59Q)), xliv) SEQ ID NO: 210 (IL-12p40(S43K / E49Q)), xlv) SEQ ID NO: 211 (IL-12p40(E59Q / K163E)), xlvi) SEQ ID NO: 212 (IL-12p40(E59Q / K99E)), xlvii) SEQ ID NO: 213 (IL-12p40(E59Q / K258E)), xlviii) SEQ ID NO: 214 (IL-12p40(E59Q / K260E)), xlix) SEQ ID NO: XXX (IL-12p40(D34N / E59K)), l) SEQ ID NO: XXX(IL-12p40(E59K / K99E)), li) SEQ ID NO: XXX(IL-12p40(D18K / E59K / K99E)), lii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / K264E)), liii) SEQ ID NO: XXX(IL-12p40(E59K / K99Y)), liv) SEQ ID NO: XXX(IL-12p40(E59Y / K99E)), lv) SEQ ID NO: XXX(IL-12p40(E45K / E59K / K99E)), lvi) SEQ ID NO: XXX(IL-12p40(E59K / K99E / Q144E)),lvii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / Q144K)), lviii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / R159E)), lix) SEQ ID NO: XXX(IL-12p40(E59K / K99E / K264E)), lx) SEQ ID NO: XXX(IL-12p40(D18K / E59K / K99E / K264E)), lxi) SEQ ID NO: XXX(IL-12p40(C252S)), lxii) SEQ ID NO: XXX(IL-12p40(DI8K / E59K / K99E / C252S)), lxiii ) SEQ ID NO: XXX(IL-12p40(D18K / E59K / K99E / C252S / K264E)), lxiv) SEQ ID NO: XXX(IL-12p40(E59K / K99Y / C252S)), lxv) SEQ ID NO: XXX(IL-12p40(E59K / K99E / C252S / K264E)), lxvi) SEQ ID NO: XXX(IL-12p40(E59K / K99E / C252S)), lxvii) SEQ ID NO: XXX(IL-12p40(N103D / N113D)), lxviii) SEQ ID NO: XXX(IL-12p40(N103D / N20 0D)), lxix) SEQ ID NO: XXX(IL-12p40(N103D / N281D)), lxx) SEQ ID NO: XXX(IL-12p40(N113D / N200D)), lxxi) SEQ ID NO: XXX(IL-12p40(N113D / N281D)), lxxii) SEQ ID NO: XXX(IL-12p40(N200D / N281D)), lxxiii) SEQ ID NO: XXX(IL-12p50(N103D / N113D / N200D)), lxxiv) SEQ ID NO: XXX(IL-12p40(N103D / N113D / N281D)), lxxv) SEQ ID NO: Sequence number XXX(IL-12p40(N103D / N200D / N281D)), lxxvi) Sequence number XXX(IL-12p40(N113D / N200D / N281D)), lxxvii) Sequence number XXX(IL-12p40(N103Q)), lxxviii) Sequence number XXX(IL-12p40(N113Q)), lxxix) Sequence number XXX(IL-12p40(N200Q)), lxxx) Sequence number XXX(IL-12p40(N281Q)), lxxxi) Sequence number XXX(IL-12p40(N103Q / N113Q)),lxxxii) SEQ ID NO: XXX (IL-12p40 (N103Q / N200Q)), lx, xxiii) SEQ ID NO:XXX(IL-12p40(N103Q / N281Q)), lxxxiv) SEQ ID NO:XXX(IL-12p40(N113Q / N200Q)), lxxxv) SEQ ID NO:XXX(IL-12p40(N113Q / N281Q)), lxxxvi) SEQ ID NO:XXX(IL-12p40(N200Q / N281Q)), lxxxvii) SEQ ID NO:XXX(IL-12p40(N103Q / N113Q / N200Q)), lxxxviii) SEQ ID NO:XXX(IL-12p40(N103Q / N113Q / N281 Q)), lxxxix) SEQ ID NO: XXX(IL-12p40(N103Q / N200Q / N281Q)), xc) SEQ ID NO: XXX(IL-12p40(N113Q / N200Q / N281Q)), xci) SEQ ID NO: XXX(IL-12p40(N103Q / N113Q / N200Q / N281Q)), xcii) SEQ ID NO: XXX(IL-12p40(D34N / E59K / K99E)), xciii) SEQ ID NO: XXX(IL-12p40(D34K / E59K / K99E)), xciv) SEQ ID NO: XXX(IL-12p40(E3 2Q / D34N / E59K / K99E)), xcv) SEQ ID NO: XXX(IL-12p40(E32K / D34N / E59K / K99E)), xcvi) SEQ ID NO: XXX(IL-12p40(E59Y / K99Y)), xcvii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N103Q / C252S / K264E)), xcviii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N113Q / C252S / K264E)), xcix) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N2 00Q / C252S / K264E)), c) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N281Q / C252S / K264E)), ci) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N103Q / N113Q / C252S / K264E)), cii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N103Q / N200Q / C252S / K264E)), ciii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N103Q / N281Q / C252S / K264E)),civ) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N113Q / N200Q / C252S / K264E)), cv) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N113Q / N281Q / C252S / K264E)), cvi) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N200Q / N281Q / C252S / K264E)), cvii) SEQ ID NO: XXX(IL-12p40(E59K / K99 cviii) SEQ ID NO: XXX (IL-12p40(E59K / K99E / N103Q / N200Q / N281Q / C252S / K264E)), and cix) SEQ ID NO: XXX (IL-12p40(E59K / K99E / N113Q / N200Q / N281Q / C252S / K264E)).

[0202] In some embodiments, the present invention relates to a method for the preparation of a IL-12p35 subunit comprising administering to a patient a IL-12p35 sequence comprising the steps of: i) SEQ ID NO:113 (IL-12p35(N71D)), ii) SEQ ID NO:114 (IL-12p35(N85D)), iii) SEQ ID NO:115 (IL-12p35(N195D)), iv) SEQ ID NO:116 (IL-12p35(N71D / N85D / N195D)), v) SEQ ID NO:117 (IL-12p35(E153Q)), vi) SEQ ID NO:118 (IL-12p35(E38Q)), vii) SEQ ID NO:119 (IL-12p35(N151D)), viii) SEQ ID NO:120 (IL-12p35(Q135E)), ix) SEQ ID NO:121 (IL-12p35(Q35D)), x) SEQ ID NO: No. 122 (IL-12p35(Q146E)), xi) SEQ ID NO: 123 (IL-12p35(N76D)), xii) SEQ ID NO: 124 (IL-12p35(E162Q)), xiii) SEQ ID NO: 125 (IL-12p35(E163Q)), xiv) SEQ ID NO: XXX (IL-12p35(D5 5Q), xvi) IL-12p35(E79Q), xvii) IL-12p35(Q130E), xviii) IL-12p35(N136D), xix) IL-12p35(E143Q), xx) SEQ ID NO: 227 (IL-12p35(N151K)), xxi) SEQ ID NO: 226 (IL-12p35(E153K)), xxii) I IL-12p35(K158E), xxiii) IL-12p35(D165N), xxiv) SEQ ID NO: 225 (IL-12p35(N151D / E153Q)), xxv) SEQ ID NO: 228 (IL-12p35(N151D / D165N)), xxvi) SEQ ID NO: 229 (IL-12p35(Q130E / N151D)), xxvii) SEQ ID NO: 230 (IL-12p35(N151D / K158E)), xxviii) SEQ ID NO: 231 (IL-12p35(E79Q / N151D)), xxix) SEQ ID NO: 232 (IL-12p35(D55Q / N15 1D)), xxx) SEQ ID NO:233 (IL-12p35(N136D / N151D)), xxxi) SEQ ID NO:234 ​​(IL-12p35(N21D / N151D)), xxxii) SEQ ID NO:235 (IL-12p35(E143Q / N151D)), xxxiii) SEQ ID NO:XXX(IL-12p35(F96A)), xxxiv) SEQ ID NO:XXX(IL-12p35(M97A)), xxxv) SEQ ID NO:XXX(IL-12p35(L89A)), xxxvi) SEQ ID NO:XXX(IL-12p35(L124A)), xxxvii) SEQ ID NO:XXX( IL-12p35(M125A)), xxxviii) SEQ ID NO:XXX(IL-12p35(L75A)), xxxiv) SEQ ID NO:XXX(IL-12p35(I171A)), xxxv) SEQ ID NO:XXX(IL-12p35(N71Q)), xxxvi) SEQ ID NO:XXX(IL-12p35(N85Q)), xxxvii) SEQ ID NO:XXX(IL-12p35(N195Q)), xxxviii) SEQ ID NO:XXX(IL-12p35(N71Q / N85Q)), xxxix) SEQ ID NO:XXX(IL-12p35(N71Q / N195Q)), lx) SEQ ID NO:XXX(IL-12p35(N71Q / N195Q)), lxiv) SEQ ID NO:XXX(IL-12p35(N85D / N195D)); lxv) SEQ ID NO:XXX(IL-12p35(D55Q)); and lxvi) SEQ ID NO:XXX(IL-12p35(D55K)).The present invention provides a heterodimeric protein.

[0203] In another aspect, the invention provides a homodimeric Fc fusion protein comprising a first monomer and a second monomer, each comprising, from N-terminus to C-terminus, an IL-12p40 subunit domain-optional first domain linker-IL-12p35 subunit domain-optional second domain linker-Fc domain.

[0204] In some embodiments, the invention provides homodimeric Fc fusion proteins, wherein the modifications that promote homodimerization of the Fc domain are a set of amino acid substitutions selected from the group consisting of: L368D / K370S; S364K; S364K / E357L; S364K / E357Q; T411E / K360E / Q362E; D401K; T366S / L368A / Y407V; T366W; T366S / L368A / Y407V / Y349C; and T366W / S354C, according to EU numbering.

[0205] In some embodiments, the invention provides a homodimeric Fc fusion protein, wherein said first domain linker and said second domain linker have the same amino acid sequence.

[0206] In some embodiments, the invention provides homodimeric Fc fusion proteins, wherein the Fc domain has an additional set of amino acid substitutions comprising: Q295E / N384D / Q418E / N421D, according to EU numbering.

[0207] In some embodiments, the present invention provides a method for the preparation of a ribosome-specific Fc domain comprising the steps of: G236R / L328R, E233P / L234V / L235A / G236_ / S239K, E Homodimeric Fc fusion proteins are provided having an additional set of amino acid substitutions selected from the group consisting of 233P / L234V / L235A / G236_ / S239K / A327G, E233P / L234V / L235A / G236_ / S267K / A327G, E233P / L234V / L235A / G236_, and E233P / L234V / L235A / G236_ / S267K.

[0208] In some embodiments, the invention provides homodimeric Fc fusion proteins, wherein the IL-12p40 subunit has a polypeptide sequence selected from the group consisting of SEQ ID NO:XX (human IL-12 subunit beta (IL-12p40) precursor sequence) and SEQ ID NO:XX (human IL-12 subunit beta (IL-12p40) mature form sequence), and the IL-12p35 subunit has a polypeptide sequence selected from the group consisting of SEQ ID NO:XX (human IL-12 subunit alpha (IL-12p35) precursor sequence) and SEQ ID NO:XX (human IL-12 subunit alpha (IL-12p35) mature form sequence).

[0209] In some embodiments, the invention provides a homodimeric Fc fusion protein, wherein each of the Fc domains further comprises the amino acid substitutions M428L / N424S.

[0210] In some embodiments, the invention provides homodimeric Fc fusion proteins, wherein said IL-12p40 subunit is a variant IL-12p40 subunit and / or said IL-12p35 subunit is a variant IL-12p35 subunit.

[0211] In some embodiments, the invention provides homodimeric Fc fusion proteins wherein said IL-12p40 subunit is IL-12 receptor subunit beta-1 (IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2), and / or a variant IL-12p40 subunit with altered affinity for the IL-12 receptor complex, and / or said IL-12p35 subunit is IL-12 receptor subunit beta-1 (IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2), and / or a variant IL-12p35 subunit with altered affinity for the IL-12 receptor complex.

[0212] In some embodiments, the IL-12p40 subunit is selected from the group consisting of E3, D7, E12, D14, W15, P17, D18, A19, P20, G21, E22, M23, D29, E32, E33, D34, L40, D41, Q42, S43, E45, L47, T54, I55, Q56, K58, E59, F60, G 61, D62, Q65, Y66, E73, K84, E86, D87, G88, I89, W90, D93, D97, K99, E100, K102, N103, K104, F106, E110, N113, Y114, D129, D142, Q144, E156, R159, D161, N162, K163, D166, D 170, Q172, D174, A176, C177, P178, A179, A180, E181, S183, P185, E187, N200, S204, F 206, R208, D209, D214, N218, Q220, N226, Q229, E231, E235, T242, P243, S245, Y246, F2 47, S248, C252S, , Q256, K158, K260, E262, K264, D265, D270, N281, Q289, D290, R291, Y292, Y293, and E299.

[0213] In some embodiments, the present invention relates to an IL-12p40 subunit comprising the amino acid sequence of D18N, D18K, E32Q, E33Q, D34N, D34K, Q42E, S43E, S43K, , E45Q, Q56E, E59Q, E59K, D62N, E73Q, D87N, K99E, K99Y, E100Q, N103D, N103Q, N113D, N113Q, Q144E, D161N, R159E, K163E, E187Q, N200D, N200Q, N218Q, Q229E, E235Q, C252S, Q256N, K258E, K260E, E262Q, K264E, N281D, N281Q, and E299Q.

[0214] In some embodiments, the present invention relates to a method for the preparation of IL-12p40 subunits comprising administering to the patient a IL-12p40 subunit comprising administering to the patient a IL-12p40 subunit comprising administering to the patient a IL-12p40 subunit comprising administering to the patient a IL-12p40 subunit comprising administering to the patient a IL-12p40 subunit comprising administering to the patient a IL-12p40 subunit comprising administering to the patient a IL-12p40 subunit comprising administering to the patient a IL-12p40 subunit S43K / E49Q, E59Q / K163E, E59Q / K99E, E59Q / K258E, E59Q / K260E, E59K / K99E , D18K / E59K / K99E, E59K / K99E / K264E, E59K / K99Y, E59Y / K99Y, E59Y / K99E, E 45K / E59K / K99E, E59K / K99E / Q144E, E59K / K99E / Q144K, E59K / K99E / R159E, E59K / K99E / K264E, D18K / E59K / K99E / K264E, DI8K / E59K / K99E / C252S, D18K / E59K / K99E / C252S / K264E, E59K / K99Y / C252S, E59K / K99E / C252S / K264E, E5 9K / K99E / C252S, N103D / N113D, N103D / N200D, N103D / N281D, N113D / N200D, N 113D / N281D, N200D / N281D, N103D / N113D / N200D, N103D / N113D / N281D, N10 3D / N200D / N281D, N113D / N200D / N281D, N103Q / N113Q, N103Q / N200Q, N103Q / N281Q, N113Q / N200Q, N113Q / N281Q, N200Q / N281Q, N103Q / N113Q / N200Q, N1 03Q / N113Q / N281Q, N103Q / N200Q / N281Q, N113Q / N200Q / N281Q, N103Q / N113Q / N200Q / N281Q, E59K / K99E / N103Q / C252S / K264E, E59K / K99E / N113Q / C252S / K264E, E59K / K99E / N200Q / C252S / K264E, E59K / K99E / N281Q / C252S / K264E,E59K / K99E / N103Q / N113Q / C252S / K264E, E59K / K99E / N103Q / N200Q / C252S / K264E, E59K / K99E / N103Q / N281Q / C252S / K264E, E 59K / K99E / N113Q / N200Q / C252S / K264E, E59K / K99E / N113Q / N281Q / C252S / K264E, E59K / K99E / N200Q / N281Q / C252S / K264E, E59 The present invention provides homodimeric Fc fusion proteins having amino acid substitutions selected from the group consisting of K / K99E / N103Q / N113Q / N200Q / C252S / K264E, E59K / K99E / N103Q / N200Q / N281Q / C252S / K264E, E59K / K99E / N113Q / N200Q / N281Q / C252S / K264E, and E59K / K99E / N103Q / N113Q / N200Q / N281Q / C252S / K264E.

[0215] In some embodiments, the present invention relates to a method for the preparation of a IL-12p40 subunit comprising administering to a patient a IL-12p40 subunit comprising administering to a patient a IL-12p40 subunit comprising administering to a patient a IL-12p40 subunit comprising administering to a patient a IL-12p40 subunit comprising administering to a patient a IL-12p40 subunit comprising administering to a patient a IL-12p40 subunit , viii) SEQ ID NO: 64 (IL-12p40(Q56E)), ix) SEQ ID NO: 65 (IL-12p40(E59Q)), x) SEQ ID NO: 66 (IL-12p40(D62N)), xi) SEQ ID NO: 67 (IL-12p40(Q42E / E45Q)), xii) SEQ ID NO: 68 (IL-12p40(E45Q / Q56E)), xiii) SEQ ID NO: 69 (IL-12p40(Q42E / E59Q)), xiv) SEQ ID NO: 70 (IL-12p40(Q56E / E59Q)), xv) SEQ ID NO: 71 (IL-12p40(Q42E / E45Q / Q56E)), x vi) SEQ ID NO: 72 (IL-12p40(E45Q / Q56E / E59Q)), xvii) SEQ ID NO: 73 (IL-12p40(D161N)), xviii) SEQ ID NO: 74 (IL-12p40(E73Q)), xix) SEQ ID NO: 75 (IL-12p40(Q144E)), xx) SEQ ID NO: 76 (IL-12p40(E262Q)), xxi) SEQ ID NO: 77 (IL-12p40(E100Q)), xxii) SEQ ID NO: 78 (IL-12p40(D18N)), xxiii) SEQ ID NO: 79 (IL-12p40(E33Q)), xxiv) SEQ ID NO: 80 ( IL-12p40(Q229E)), xxv) SEQ ID NO: 81 (IL-12p40(E235Q)), xxvi) SEQ ID NO: 82 (IL-12p40(Q256N)), xxvii) SEQ ID NO: 83 (IL-12p40(E299Q)), xxviii) SEQ ID NO: 84 (IL-12p40(D87N)), xxix) IL-12p40(E32Q), xxx) IL-12p40(D34N), xxxi) IL-12p40(S43E), xxxii) IL-12p40(S43K), xxxiii) SEQ ID NO: XXX (IL-12p40(E59K / K99 E / N103Q / N113Q / N200Q / N281Q / C252S / K264E), xxxiv) SEQ ID NO: 205 (IL-12p40(E59K)), xxxv) IL-12p40(K99E), xxxvi) IL-12p40(K163E), xxxvii) IL-12p40(E187Q), xxxviii) IL-12p40(K258E), xxxix) IL-12p40(K260E), xl) SEQ ID NO: 206 (IL-12p40(E32Q / E59Q)), xli) SEQ ID NO: 207 (IL-12p40(D34N / E59Q)),xlii) SEQ ID NO: 208 (IL-12p40(E59Q / E187Q)), xliii) SEQ ID NO: 209 (IL-12p40(S43E / E59Q)), xliv) SEQ ID NO: 210 (IL-12p40(S43K / E49Q)), xlv) SEQ ID NO: 211 (IL-12p40(E59Q / K163E)), xlvi) SEQ ID NO: 212 (IL-12p40(E59Q / K99E)), xlvii) SEQ ID NO: 213 (IL-12p40(E59Q / K258E)), xlviii) SEQ ID NO: 214 (IL-12p40(E59Q / K260E)), xlix) SEQ ID NO:XXX(IL-12p40(D34N / E59K)), l) SEQ ID NO:XXX(IL-12p40(E59K / K99E)), li) SEQ ID NO:XXX(IL-12p40(D18K / E59K / K99E)), lii) SEQ ID NO:XXX(IL-12p40(E59K / K99E / K264E)), liii) SEQ ID NO:XXX(IL-12p40(E59K / K99Y)), liv) SEQ ID NO:XXX(IL-12p40(E59Y / K99E)), lv) SEQ ID NO:XXX(IL-12p40(E45K / E59K / K99E)), lvi) SEQ ID NO: XXX(IL-12p40(E59K / K99E / Q144E)), lvii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / Q144K)), lviii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / R159E)), lix) SEQ ID NO: XXX(IL-12p40(E59K / K99E / K264E)), lx) SEQ ID NO: XXX(IL-12p40(D18K / E59K / K99E / K264E)), lxi) SEQ ID NO: XXX(IL-12p40(C252S)), lxii) SEQ ID NO: XXX(IL-12p40(I L-12p40(DI8K / E59K / K99E / C252S)), lxiii) SEQ ID NO: XXX(IL-12p40(D18K / E59K / K99E / C252S / K264E)), lxiv) SEQ ID NO: XXX(IL-12p40(E59K / K99Y / C252S)), lxv) SEQ ID NO: XXX(IL-12p40(E59K / K99E / C252S / K264E)), lxvi) SEQ ID NO: XXX(IL-12p40(E59K / K99E / C252S)), lxvii) SEQ ID NO: XXX(IL-12p40(N103D / N113D)),lxviii) SEQ ID NO: XXX (IL-12p40(N103D / N200D)); , lxix) SEQ ID NO:XXX(IL-12p40(N103D / N281D)), lxx) SEQ ID NO:XXX(IL-12p40(N113D / N200D)), lxxi) SEQ ID NO:XXX(IL-12p40(N113D / N281D)), lxxii) SEQ ID NO:XXX(IL-12p40(N200D / N281D)), lxxiii) SEQ ID NO:XXX(IL-12p50(N103D / N113D / N200D)), lxxiv) SEQ ID NO:XXX(IL-12p40(N103D / N113D / N281D)), lxxv) SEQ ID NO:X XX(IL-12p40(N103D / N200D / N281D)), lxxvi) SEQ ID NO:XXX(IL-12p40(N113D / N200D / N281D)), lxxvii) SEQ ID NO:XXX(IL-12p40(N103Q)), lxxviii) SEQ ID NO:XXX(IL-12p40(N113Q)), lxxix) SEQ ID NO:XXX(IL-12p40(N200Q)), lxxx) SEQ ID NO:XXX(IL-12p40(N281Q)), lxxxi) SEQ ID NO:XXX(IL-12p40(N103Q / N113Q)), lxxxi i) SEQ ID NO:XXX(IL-12p40(N103Q / N200Q)), lxxxiii) SEQ ID NO:XXX(IL-12p40(N103Q / N281Q)), lxxxiv) SEQ ID NO:XXX(IL-12p40(N113Q / N200Q)), lxxxv) SEQ ID NO:XXX(IL-12p40(N113Q / N281Q)), lxxxvi) SEQ ID NO:XXX(IL-12p40(N200Q / N281Q)), lxxxvii) SEQ ID NO:XXX(IL-12p40(N103Q / N113Q / N200Q)), lxxxviii) SEQ ID NO:XXX(IL-12p40(N103Q / N113Q / N200Q)), XXX(IL-12p40(N103Q / N113Q / N281Q)), lxxxix) SEQ ID NO: XXX(IL-12p40(N103Q / N200Q / N281Q)), xc) SEQ ID NO: XXX(IL-12p40(N113Q / N200Q / N281Q)), xci) SEQ ID NO: XXX(IL-12p40(N103Q / N113Q / N200Q / N281Q)), xcii) SEQ ID NO: XXX(IL-12p40(D34N / E59K / K99E)), xciii) SEQ ID NO: XXX(IL-12p40(D34K / E59K / K99E)),xciv) SEQ ID NO: XXX (IL-12p40(E32Q / D34N / E59K / K99E)), xcv) SEQ ID NO: XXX (IL-12p40(E32K / D34N / E59K / K99E)), xcvi) SEQ ID NO: XXX (IL-12p40(E59Y / K99Y)), xcvii) SEQ ID NO: XXX (IL-12p40(E59K / K99E / N103Q / C252S / K264E)), xcviii) SEQ ID NO: XXX (IL-12p40(E59K / K99E / N113Q / C252S / K264E)) , xcix) SEQ ID NO:XXX(IL-12p40(E59K / K99E / N200Q / C252S / K264E)), c) SEQ ID NO:XXX(IL-12p40(E59K / K99E / N281Q / C252S / K264E)), ci) SEQ ID NO:XXX(IL-12p40(E59K / K99E / N103Q / N113Q / C252S / K264E)), cii) SEQ ID NO:XXX(IL-12p40(E59K / K99E / N103Q / N200Q / C252S / K264E)), ciii) SEQ ID NO:XXX (IL-12p40(E59K / K99E / N103Q / N281Q / C252S / K264E)), civ) SEQ ID NO:XXX(IL-12p40(E59K / K99E / N113Q / N200Q / C252S / K264E)), cv) SEQ ID NO:XXX(IL-12p40(E59K / K99E / N113Q / N281Q / C252S / K264E)), cvi) SEQ ID NO:XXX(IL-12p40(E59K / K99E / N200Q / N281Q / C252S / K264E)), cvii) SEQ ID NO:XX X(IL-12p40(E59K / K99E / N103Q / N113Q / N200Q / C252S / K264E)), cviii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N103Q / N200Q / N281Q / C252S / K264E)), and cix) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N113Q / N200Q / N281Q / C252S / K264E)).

[0216] In some embodiments, the present invention relates to an IL-12p35 subunit comprising one or more of the following amino acids: Q20, N21, Q35, E38, S44, E45, E46, H49, K54, D55, T59, The present invention provides homodimeric Fc fusion proteins having one or more amino acid modifications at an amino acid residue selected from the group consisting of V60, E61, C63, L64, P65, E67, L68, N71, S73, C74, L75, N76, E79, N85, L89, F96, M97, L124, M125, Q130, Q135, N136, E143, Q146, N151, E153, K158, E162, E163, D165, I171, R181, I182, R183, V185, T186, D188, R189, V190, S192, Y193, N195, and A196.

[0217] In some embodiments, the invention provides homodimeric Fc fusion proteins, wherein the IL-12p35 subunit has one or more amino acid substitutions selected from the group consisting of N21D, Q35D, E38Q, D55Q, D55K, N71D, N71Q, L75A, N76D, E79Q, N85D, N85Q, L89A, F96A, M97A, L124A, M125A, Q130E, Q135E, N136D, E143Q, Q146E, N151D, N151K, E153K, E153Q, K158E, E162Q, E163Q, D165N, I171A, N195D, and N195Q.

[0218] In some embodiments, the invention provides homodimeric Fc fusion proteins, wherein the IL-12p35 subunit has amino acid substitutions selected from the group consisting of N71D / N85D / N195D, N151D / E153Q, N151D / D165N, Q130E / N151D, N151D / K158E, E79Q / N151D, D55Q / N151D, N136D / N151D, N21D / N151D, E143Q / N151D, N71Q / N85Q, N71Q / N195Q, N85Q / N195Q, N71Q / N85Q / N195Q, N71D / N85D, N71D / N195D, and N85D / N195D.

[0219] In some embodiments, the present invention relates to a method for the preparation of a IL-12p35 subunit comprising administering to a patient a IL-12p35 sequence comprising the steps of: i) SEQ ID NO:113 (IL-12p35(N71D)), ii) SEQ ID NO:114 (IL-12p35(N85D)), iii) SEQ ID NO:115 (IL-12p35(N195D)), iv) SEQ ID NO:116 (IL-12p35(N71D / N85D / N195D)), v) SEQ ID NO:117 (IL-12p35(E153Q)), vi) SEQ ID NO:118 (IL-12p35(E38Q)), vii) SEQ ID NO:119 (IL-12p35(N151D)), viii) SEQ ID NO:120 (IL-12p35(N151D)), viiii) SEQ ID NO:121 (IL-12p35(N151D)), 0 (IL-12p35(Q135E)), ix) SEQ ID NO: 121 (IL-12p35(Q35D)), x) SEQ ID NO: 122 (IL-12p35(Q146E)), xi) SEQ ID NO: 123 (IL-12p35(N76D)), xii) SEQ ID NO: 124 (IL-12p35(E162Q)), xiii) SEQ ID NO: 125 (IL-12p35(E163Q)), xiv) IL-12p35(N21D), xv) SEQ ID NO: XXX (IL-12p35(D55Q)), xvi) IL-12p35(E79Q), xvii) IL-12p35(Q130E), xviii )IL-12p35(N136D), xix)IL-12p35(E143Q), xx)SEQ ID NO:227(IL-12p35(N151K)), xxi)SEQ ID NO:226(IL-12p35(E153K)), xxii)IL-12p35(K158E), xxiii)IL-12p35(D165N), xxiv)SEQ ID NO:225(IL-12p35(N151D / E153Q)), xxv)SEQ ID NO:228(IL-12p35(N151D / D165N)), xxvi)SEQ ID NO:229(IL-12p35(Q130E / N151D)), xxvii) SEQ ID NO:230 (IL-12p35(N151D / K158E)), xxviii) SEQ ID NO:231 (IL-12p35(E79Q / N151D)), xxix) SEQ ID NO:232 (IL-12p35(D55Q / N151D)), xxx) SEQ ID NO:233 (IL-12p35(N136D / N151D)), xxxi) SEQ ID NO:234 ​​(IL-12p35(N21D / N151D)), xxxii) SEQ ID NO:235 (IL-12p35(E143Q / N151D)), xxxiii) SEQ ID NO:XXX (IL-12p35(F96A)), xxxiv) SEQ ID NO: No. XXX(IL-12p35(M97A)), xxxv) SEQ ID NO: XXX(IL-12p35(L89A)), xxxvi) SEQ ID NO: XXX(IL-12p35(L124A)), xxxvii) SEQ ID NO: XXX(IL-12p35(M125A)), xxxviii) SEQ ID NO: XXX(IL-12p35(L75A)), xxxiv) SEQ ID NO: XXX(IL-12p35(I171A)), xxxv) SEQ ID NO: XXX(IL-12p35(N71Q)), xxxvi) SEQ ID NO: XXX(IL-12p35(N85Q)), xxxvii) SEQ ID NO: XXX(IL-12p35(N195Q)), xxxviii) SEQ ID NO: XXX(IL-12p35(N71Q / N85Q)), xxxix xli) SEQ ID NO:XXX(IL-12p35(N71Q / N195Q)), xl) SEQ ID NO:XXX(IL-12p35(N85Q / N195Q), xli) SEQ ID NO:XXX(IL-12p35(N71Q / N85Q / N195Q)), xlii) SEQ ID NO:XXX(IL-12p35(N71D / N85D)), xliii) SEQ ID NO:XXX(IL-12p35(N71D / N195D), xliv) SEQ ID NO:XXX(IL-12p35(N85D / N195D)), xlv) SEQ ID NO:XXX(IL-12p35(D55Q)), and xlvi) SEQ ID NO:XXX(IL-12p35(D55K)).

[0220] In some embodiments, a homodimeric Fc fusion protein is provided, wherein said first and second monomers are XENP31289.

[0221] In some embodiments, a homodimeric Fc fusion protein is provided, wherein said first and second monomers are XENP31291.

[0222] In some embodiments, the present invention provides a homodimeric Fc-fusion protein composition comprising a homodimeric Fc-fusion protein for use in treating cancer in a subject.

[0223] In some embodiments, the invention provides one or more nucleic acids encoding a homodimeric Fc fusion protein.

[0224] In some embodiments, the invention provides host cells comprising one or more of the nucleic acids encoding the homodimeric Fc fusion proteins.

[0225] In some embodiments, the invention provides a method of making a homodimeric Fc-fusion protein, the method comprising culturing a host cell under conditions whereby the homodimeric Fc-fusion protein is produced.

[0226] In some embodiments, the invention provides a method for purifying a homodimeric Fc-fusion protein, the method comprising: a) providing a composition comprising a homodimeric Fc-fusion protein; b) loading the composition onto an ion exchange column; and c) collecting fractions containing the homodimeric Fc-fusion protein.

[0227] In another aspect, the invention provides a homodimeric Fc fusion protein comprising a first monomer and a second monomer, each comprising, from N-terminus to C-terminus, IL-12p35 subunit domain-optional first domain linker-IL-12p40 subunit domain-optional second domain linker-Fc domain.

[0228] In some embodiments, the invention provides a method for the preparation of Fc domains comprising the steps of: L368D / K370S; S364K; S364K / E357L; S364K / E357Q; T411E / K360E / Q362E; D401K; T The present invention provides homodimeric Fc fusion proteins, which have a set of amino acid substitutions selected from the group consisting of: 366S / L368A / Y407V; T366W; T366S / L368A / Y407V / Y349C; and T366W / S354C.

[0229] In some embodiments, the invention provides a homodimeric Fc fusion protein, wherein said first domain linker and said second domain linker have the same amino acid sequence.

[0230] In some embodiments, the invention provides homodimeric Fc fusion proteins, wherein the Fc domain has an additional set of amino acid substitutions comprising: Q295E / N384D / Q418E / N421D, according to EU numbering.

[0231] In some embodiments, the invention provides homodimeric Fc fusion proteins, wherein the Fc domain has an additional set of amino acid substitutions selected from the group consisting of G236R / L328R, E233P / L234V / L235A / G236_ / S239K, E233P / L234V / L235A / G236_ / S239K / A327G, E233P / L234V / L235A / G236_ / S267K / A327G, E233P / L234V / L235A / G236_, and E233P / L234V / L235A / G236_ / S267K, according to EU numbering.

[0232] In some embodiments, the invention provides homodimeric Fc fusion proteins, wherein the IL-12p40 subunit has a polypeptide sequence selected from the group consisting of SEQ ID NO:3 (human IL-12 subunit beta (IL-12p40) precursor sequence) and SEQ ID NO:4 (human IL-12 subunit beta (IL-12p40) mature form sequence), and the IL-12p35 subunit has a polypeptide sequence selected from the group consisting of SEQ ID NO:1 (human IL-12 subunit alpha (IL-12p35) precursor sequence) and SEQ ID NO:2 (human IL-12 subunit alpha (IL-12p35) mature form sequence).

[0233] In some embodiments, the invention provides a homodimeric Fc fusion protein, wherein each of the Fc domains further comprises the amino acid substitutions M428L / N424S.

[0234] In some embodiments, the invention provides homodimeric Fc fusion proteins, wherein said IL-12p40 subunit is a variant IL-12p40 subunit and / or said IL-12p35 subunit is a variant IL-12p35 subunit.

[0235] In some embodiments, the invention provides homodimeric Fc fusion proteins wherein said IL-12p40 subunit is IL-12 receptor subunit beta-1 (IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2), and / or a variant IL-12p40 subunit with altered affinity for the IL-12 receptor complex, and / or said IL-12p35 subunit is IL-12 receptor subunit beta-1 (IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2), and / or a variant IL-12p35 subunit with altered affinity for the IL-12 receptor complex.

[0236] In some embodiments, the IL-12p40 subunit is selected from the group consisting of E3, D7, E12, D14, W15, P17, D18, A19, P20, G21, E22, M23, D29, E32, E33, D34, L40, D41, Q42, S43, E45, L47, T54, I55, Q56, K58, E59, F60, G61, D62, Q65, Y66, E73, , K84, E86, D87, G88, I89, W90, D93, D97, K99, E100, K102, N103, K104, F106, E110, N113, Y114, D129, D142, Q144, E156, R159 , D161, N162, K163, D166, D170, Q172, D174, A176, C177, P178, A179, A180, E181, S183, P185, E187, N200, S204, F206, R208, D 209, D214, N218, Q220, N226, Q229, E231, E235, T242, P243, S245, Y246, F247, S248, C252S, , Q256, K158, K260, E262, K264, D265, D270, N281, Q289, D290, R291, Y292, Y293, and E299.

[0237] In some embodiments, the present invention relates to an IL-12p40 subunit comprising D18N, D18K, E32Q, E33Q, D34N, D34K, Q42E, S43E, S43K, E45Q, Q56E, E59Q, E59K, D62N, E73Q, D87N, K99E, K99Y, E100Q, N103D, N103Q, N113D, N113Q, Q144 The present invention provides homodimeric Fc fusion proteins having one or more amino acid substitutions selected from the group consisting of E, D161N, R159E, K163E, E187Q, N200D, N200Q, N218Q, Q229E, E235Q, C252S, Q256N, K258E, K260E, E262Q, K264E, N281D, N281Q, and E299Q.

[0238] In some embodiments, the present invention relates to a method for the preparation of IL-12p40 subunits comprising administering to the patient a IL-12p40 subunit comprising administering to the patient a IL-12p40 subunit comprising administering to the patient a IL-12p40 subunit comprising administering to the patient a IL-12p40 subunit comprising administering to the patient a IL-12p40 subunit comprising administering to the patient a IL-12p40 subunit comprising administering to the patient a IL-12p40 subunit comprising administering to the patient a IL-12p40 subunit S43K / E49Q, E59Q / K163E, E59Q / K99E, E59Q / K258E, E59Q / K260E, E59K / K99E , D18K / E59K / K99E, E59K / K99E / K264E, E59K / K99Y, E59Y / K99Y, E59Y / K99E, E 45K / E59K / K99E, E59K / K99E / Q144E, E59K / K99E / Q144K, E59K / K99E / R159E, E59K / K99E / K264E, D18K / E59K / K99E / K264E, DI8K / E59K / K99E / C252S, D18K / E59K / K99E / C252S / K264E, E59K / K99Y / C252S, E59K / K99E / C252S / K264E, E5 9K / K99E / C252S, N103D / N113D, N103D / N200D, N103D / N281D, N113D / N200D, N 113D / N281D, N200D / N281D, N103D / N113D / N200D, N103D / N113D / N281D, N10 3D / N200D / N281D, N113D / N200D / N281D, N103Q / N113Q, N103Q / N200Q, N103Q / N281Q, N113Q / N200Q, N113Q / N281Q, N200Q / N281Q, N103Q / N113Q / N200Q, N1 03Q / N113Q / N281Q, N103Q / N200Q / N281Q, N113Q / N200Q / N281Q, N103Q / N113Q / N200Q / N281Q, E59K / K99E / N103Q / C252S / K264E, E59K / K99E / N113Q / C252S / K264E, E59K / K99E / N200Q / C252S / K264E, E59K / K99E / N281Q / C252S / K264E,E59K / K99E / N103Q / N113Q / C252S / K264E, E59K / K99E / N103Q / N200Q / C252S / K264E, E59K / K99E / N1 03Q / N281Q / C252S / K264E, E59K / K99E / N113Q / N200Q / C252S / K264E, E59K / K99E / N113Q / N281Q / C, and E59K / K99E / N103Q / N113Q / N200Q / N281Q / C252S / K264E. In some embodiments, the present invention relates to an IL-12p40 subunit comprising: i) SEQ ID NO:57 (IL-12p40(N103D)), ii) SEQ ID NO:58 (IL-12p40(N113D)), iii) SEQ ID NO:59 (IL-12p40(N200D)), iv) SEQ ID NO:60 (IL-12p40(N281D)), v) SEQ ID NO:61 (IL-12p40(N103D / N113D / N200D / N281D)), vi) SEQ ID NO:62 (IL-12p40(Q42E)), vii) SEQ ID NO:63 (IL-12p40(E45Q)), viii) SEQ ID NO:64 (IL-12p40(Q56E)), ix) SEQ ID NO:65 (IL -12p40(E59Q)), x) SEQ ID NO: 66 (IL-12p40(D62N)), xi) SEQ ID NO: 67 (IL-12p40(Q42E / E45Q)), xii) SEQ ID NO: 68 (IL-12p40(E45Q / Q56E)), xiii) SEQ ID NO: 69 (IL-12p40(Q42E / E59Q)), xiv) SEQ ID NO: 70 (IL-12p40(Q56E / E59Q)), xv) SEQ ID NO: 71 (IL-12p40(Q42E / E45Q / Q56E)), xvi) SEQ ID NO: 72 (IL-12p40(E45Q / Q56E / E59Q)), xvii) SEQ ID NO:73 (IL-12p40(D161N)), xviii) SEQ ID NO:74 (IL-12p40(E73Q)), xix) SEQ ID NO:75 (IL-12p40(Q144E)), xx) SEQ ID NO:76 (IL-12p40(E262Q)), xxi) SEQ ID NO:77 (IL-12p40(E100Q)), xxii) SEQ ID NO:78 (IL-12p40(D18N)), xxiii) SEQ ID NO:79 (IL-12p40(E33Q)), xxiv) SEQ ID NO:80 (IL-12p40(Q229E)), xxv) SEQ ID NO:81 (IL-12p40(E23 5Q), xxvi) SEQ ID NO: 82 (IL-12p40(Q256N)), xxvii) SEQ ID NO: 83 (IL-12p40(E299Q)), xxviii) SEQ ID NO: 84 (IL-12p40(D87N)), xxix) IL-12p40(E32Q), xxx) IL-12p40(D34N), xxxi) IL-12p40(S43E), xxxii) IL-12p40(S43K), xxxiii) SEQ ID NO: XXX (IL-12p40(E59K / K99E / N103Q / N113Q / N200Q / N281Q / C252S / K264E)),xxxiv) SEQ ID NO: 205 (IL-12p40(E59K)), xxxv) IL-12p40(K99E), xxxvi) IL-12p40(K163E), xxxvii) IL-12p40(E187Q), xxxviii) IL-12p40(K258E), xxxix) IL-12p40(K260E), xl) SEQ ID NO: 206 (IL-12p40(E32Q / E59Q)), xl i) SEQ ID NO: 207 (IL-12p40(D34N / E59Q)), xlii) SEQ ID NO: 208 (IL-12p40(E59Q / E187Q)), xliii) SEQ ID NO: 209 (IL-12p40(S43E / E59Q)), xliv) SEQ ID NO: 210 (IL-12p40(S43K / E49Q)), xlv) SEQ ID NO: 211 (IL-12p40(E59Q / K163E)), xlvi) SEQ ID NO: 212 (IL-12p40(E59Q / K163E)), Seq. ID No. 212 (IL-12p40(E59Q / K99E)), xlvii) SEQ ID No. 213 (IL-12p40(E59Q / K258E)), xlviii) SEQ ID No. 214 (IL-12p40(E59Q / K260E)), xlix) SEQ ID No. XXX (IL-12p40(D34N / E59K)), l) SEQ ID No. XXX (IL-12p40(E59K / K99E)), li) SEQ ID No. XX X(IL-12p40(D18K / E59K / K99E)), lii) SEQ ID NO:XXX(IL-12p40(E59K / K99E / K264E)), liii) SEQ ID NO:XXX(IL-12p40(E59K / K99Y)), liv) SEQ ID NO:XXX(IL-12p40(E59Y / K99E)), lv) SEQ ID NO:XXX(IL-12p40(E45K / E59K / K99E)), lvi, ) SEQ ID NO: XXX(IL-12p40(E59K / K99E / Q144E)), lvii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / Q144K)), lviii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / R159E)), lix) SEQ ID NO: XXX(IL-12p40(E59K / K99E / K264E)), lx) SEQ ID NO: XXX(IL-12p40(D18K / E59K / K99E / K264E)), lxi) SEQ ID NO: XXX(IL-12p40(C252S)), lxii) SEQ ID NO: XXX( IL-12p40(DI8K / E59K / K99E / C252S)), lxiii) SEQ ID NO: XXX(IL-12p40(D18K / E59K / K99E / C252S / K264E)), lxiv) SEQ ID NO: XXX(IL-12p40(E59K / K99Y / C252S)), lxv) SEQ ID NO: XXX(IL-12p40(E59K / K99E / C252S / K264E)), lxvi) SEQ ID NO: XXX(IL-12p40(E59K / K99E / C252S)), lxvii) SEQ ID NO: XXX(IL-12p40(N103D / N113 D)), lxviii) SEQ ID NO:XXX(IL-12p40(N103D / N200D)), lxix) SEQ ID NO:XXX(IL-12p40(N103D / N281D)), lxx) SEQ ID NO:XXX(IL-12p40(N113D / N200D)), lxxi) SEQ ID NO:XXX(IL-12p40(N113D / N281D)), lxxii) SEQ ID NO:XXX(IL-12p40(N200D / N281D)), lxxiii) SEQ ID NO:XXX(IL-12p50(N103D / N113D / N200D)), lxxiv) SEQ ID NO:XX X(IL-12p40(N103D / N113D / N281D)), lxxv) SEQ ID NO:XXX(IL-12p40(N103D / N200D / N281D)), lxxvi) SEQ ID NO:XXX(IL-12p40(N113D / N200D / N281D)), lxxvii) SEQ ID NO:XXX(IL-12p40(N103Q)), lxxviii) SEQ ID NO:XXX(IL-12p40(N113Q)), lxxix) SEQ ID NO:XXX(IL-12p40(N200Q)), lxxx) SEQ ID NO:XXX(IL-12p40(N281Q)),lxxxi) SEQ ID NO:XXX(IL-12p40(N103Q / N113Q)), lxxxii) SEQ ID NO:XXX(IL-12p40(N103Q / N200Q)), lxxxiii) SEQ ID NO:XXX(IL-12p40(N103Q / N281Q)), lxxxiv) SEQ ID NO:XXX(IL-12p40(N113Q / N200Q)), lxxxv) SEQ ID NO:XXX(IL-12p40(N113Q / N281Q)), lxxxvi) SEQ ID NO:XXX(IL-12p40(N200Q / N281Q)), lxxxvii) SEQ ID NO:XXX (IL-12p40(N103Q / N113Q / N200Q)), lxxxviii) SEQ ID NO:XXX(IL-12p40(N103Q / N113Q / N281Q)), lxxxix) SEQ ID NO:XXX(IL-12p40(N103Q / N200Q / N281Q)), xc) SEQ ID NO:XXX(IL-12p40(N113Q / N200Q / N281Q)), xci) SEQ ID NO:XXX(IL-12p40(N103Q / N113Q / N200Q / N281Q)), xcii) SEQ ID NO:XXX(IL-12p40(D34N / E59K / K99E )), xciii) SEQ ID NO: XXX(IL-12p40(D34K / E59K / K99E)), xciv) SEQ ID NO: XXX(IL-12p40(E32Q / D34N / E59K / K99E)), xcv) SEQ ID NO: XXX(IL-12p40(E32K / D34N / E59K / K99E)), xcvi) SEQ ID NO: XXX(IL-12p40(E59Y / K99Y)), xcvii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N103Q / C252S / K264E)), xcviii) SEQ ID NO: XXX(IL-12p40(E 59K / K99E / N113Q / C252S / K264E)), xcix) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N200Q / C252S / K264E)), c) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N281Q / C252S / K264E)), ci) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N103Q / N113Q / C252S / K264E)), cii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N103Q / N200Q / C252S / K264E)),ciii) SEQ ID NO: XXX (IL-12p40 (E59K / K99E / N103Q / N281Q / C252S / K264E)); civ) sequence; Sequence number XXX(IL-12p40(E59K / K99E / N113Q / N200Q / C252S / K264E)), cv) Sequence number XXX(IL-12p40(E59K / K99E / N113Q / N281Q / C252S / K264E)), cvi) Sequence number XXX(IL-12p40(E59K / K99E / N200Q / N281Q / C252S / K264E)), cvii) Sequence number XXX(IL-12p40(E59K / K99E / N103Q 10) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N113Q / N200Q / N281Q / C252S / K264E)), cviii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N103Q / N200Q / N281Q / C252S / K264E)), and cix) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N113Q / N200Q / N281Q / C252S / K264E)).

[0239] In some embodiments, the present invention relates to an IL-12 p35 subunit comprising one or more of the following: Q20, N21, Q35, E38, S44, E45, E46, H49, K54, D55, T59, V60, E61, C63, L64, P65, E67, L68, N71, S73, C74, L75, N76, E79, N85, L89, F96, M97, L124, M125, Q130, Q135, N The present invention provides homodimeric Fc fusion proteins having one or more amino acid modifications at amino acid residues selected from the group consisting of 136, E143, Q146, N151, E153, K158, E162, E163, D165, I171, R181, I182, R183, V185, T186, D188, R189, V190, S192, Y193, N195, and A196.

[0240] In some embodiments, the invention provides homodimeric Fc fusion proteins, wherein the IL-12p35 subunit has one or more amino acid substitutions selected from the group consisting of N21D, Q35D, E38Q, D55Q, D55K, N71D, N71Q, L75A, N76D, E79Q, N85D, N85Q, L89A, F96A, M97A, L124A, M125A, Q130E, Q135E, N136D, E143Q, Q146E, N151D, N151K, E153K, E153Q, K158E, E162Q, E163Q, D165N, I171A, N195D, and N195Q.

[0241] In some embodiments, the invention provides homodimeric Fc fusion proteins, wherein the IL-12p35 subunit has amino acid substitutions selected from the group consisting of N71D / N85D / N195D, N151D / E153Q, N151D / D165N, Q130E / N151D, N151D / K158E, E79Q / N151D, D55Q / N151D, N136D / N151D, N21D / N151D, E143Q / N151D, N71Q / N85Q, N71Q / N195Q, N85Q / N195Q, N71Q / N85Q / N195Q, N71D / N85D, N71D / N195D, and N85D / N195D.

[0242] In some embodiments, the present invention relates to a method for the preparation of a IL-12p35 subunit comprising administering to a patient a IL-12p35 sequence comprising the steps of: i) SEQ ID NO:113 (IL-12p35(N71D)), ii) SEQ ID NO:114 (IL-12p35(N85D)), iii) SEQ ID NO:115 (IL-12p35(N195D)), iv) SEQ ID NO:116 (IL-12p35(N71D / N85D / N195D)), v) SEQ ID NO:117 (IL-12p35(E153Q)), vi) SEQ ID NO:118 (IL-12p35(E38Q)), vii) SEQ ID NO:11 9 (IL-12p35(N151D)), viii) SEQ ID NO: 120 (IL-12p35(Q135E)), ix) SEQ ID NO: 121 (IL-12p35(Q35D)), x) SEQ ID NO: 122 (IL-12p35(Q146E)), xi) SEQ ID NO: 123 (IL-12p35(N76D)), xii) SEQ ID NO: 124 (IL-12p35(E162Q)), xiii) SEQ ID NO: 125 (IL-12p35(E163Q)), xiv) IL-12p35(N21D), xv) SEQ ID NO: XXX (IL-12p35(N151D)), L-12p35(D55Q)), xvi) IL-12p35(E79Q), xvii) IL-12p35(Q130E), xviii) IL-12p35(N136D), xix) IL-12p35(E143Q), xx) SEQ ID NO: 227 (IL-12p35(N151K)), xxi) SEQ ID NO: 226 (IL-12p35(E153K)), xxii) IL-12p35(K158E), xxiii) IL-12p35(D165N), xxiv) SEQ ID NO: 225 (IL-12p35(N151D / E153Q)), xxv) SEQ ID NO: 2 28 (IL-12p35(N151D / D165N)), xxvi) SEQ ID NO: 229 (IL-12p35(Q130E / N151D)), xxvii) SEQ ID NO: 230 (IL-12p35(N151D / K158E)), xxviii) SEQ ID NO: 231 (IL-12p35(E79Q / N151D)), xxix) SEQ ID NO: 232 (IL-12p35(D55Q / N151D)), xxx) SEQ ID NO: 233 (IL-12p35(N136D / N151D)), xxxi) SEQ ID NO: 234 (IL-12p35(N21D / N151D)), xx xii) SEQ ID NO:235 (IL-12p35(E143Q / N151D)), xxxiii) SEQ ID NO:XXX (IL-12p35(F96A)), xxxiv) SEQ ID NO:XXX (IL-12p35(M97A)), xxxv) SEQ ID NO:XXX (IL-12p35(L89A)), xxxvi) SEQ ID NO:XXX (IL-12p35(L124A)), xxxvii) SEQ ID NO:XXX (IL-12p35(M125A)), xxxviii) SEQ ID NO:XXX (IL-12p35(L75A)), xxxiv) SEQ ID NO:XXX (IL-12p35( I171A), xxxv) SEQ ID NO:XXX(IL-12p35(N71Q)), xxxvi) SEQ ID NO:XXX(IL-12p35(N85Q)), xxxvii) SEQ ID NO:XXX(IL-12p35(N195Q)), xxxviii) SEQ ID NO:XXX(IL-12p35(N71Q / N85Q)), xxxix) SEQ ID NO:XXX(IL-12p35(N71Q / N195Q)), xl) SEQ ID NO:XXX(IL-12p35(N85Q / N195Q), xli) SEQ ID NO:XXX(IL-12p35(N71Q / N85Q / N195Q)),xlii) SEQ ID NO:XXX(IL-12p35(N71D / N85D)), xliii) SEQ ID NO:XXX(IL-12p35(N71D / N195D), xliv) SEQ ID NO:XXX(IL-12p35(N85D / N195D)), xlv) SEQ ID NO:XXX(IL-12p35(D55Q)), and xlvi) SEQ ID NO:XXX(IL-12p35(D55K)).

[0243] In some embodiments, a homodimeric Fc fusion protein is provided, wherein said first and second monomers are XENP31289.

[0244] In some embodiments, a homodimeric Fc fusion protein is provided, wherein said first and second monomers are XENP31291.

[0245] In some embodiments, the present invention provides a homodimeric Fc-fusion protein composition comprising a homodimeric Fc-fusion protein for use in treating cancer in a subject.

[0246] In some embodiments, the invention provides one or more nucleic acids encoding a homodimeric Fc fusion protein.

[0247] In some embodiments, the invention provides host cells comprising one or more of the nucleic acids encoding the homodimeric Fc fusion proteins.

[0248] In some embodiments, the invention provides a method of making a homodimeric Fc-fusion protein, the method comprising culturing a host cell under conditions whereby the homodimeric Fc-fusion protein is produced.

[0249] In some embodiments, the invention provides a method for purifying a homodimeric Fc-fusion protein, the method comprising: a) providing a composition comprising a homodimeric Fc-fusion protein; b) loading the composition onto an ion exchange column; and c) collecting fractions containing the homodimeric Fc-fusion protein.

[0250] In another aspect, the invention provides a homodimeric Fc fusion protein comprising a first monomer and a second monomer, each comprising, from N-terminus to C-terminus, Fc domain-optional first domain linker-IL-12p40 subunit domain-optional second domain linker-IL-12p35 subunit domain.

[0251] In some embodiments, the invention provides homodimeric Fc fusion proteins, wherein the modifications that promote homodimerization of the Fc domain are a set of amino acid substitutions selected from the group consisting of: L368D / K370S; S364K; S364K / E357L; S364K / E357Q; T411E / K360E / Q362E; D401K; T366S / L368A / Y407V; T366W; T366S / L368A / Y407V / Y349C; and T366W / S354C, according to EU numbering.

[0252] In some embodiments, the invention provides a homodimeric Fc fusion protein, wherein said first domain linker and said second domain linker have the same amino acid sequence.

[0253] In some embodiments, the invention provides homodimeric Fc fusion proteins, wherein the Fc domain has an additional set of amino acid substitutions comprising: Q295E / N384D / Q418E / N421D, according to EU numbering.

[0254] In some embodiments, the invention provides homodimeric Fc fusion proteins, wherein the Fc domain has an additional set of amino acid substitutions selected from the group consisting of G236R / L328R, E233P / L234V / L235A / G236_ / S239K, E233P / L234V / L235A / G236_ / S239K / A327G, E233P / L234V / L235A / G236_ / S267K / A327G, E233P / L234V / L235A / G236_, and E233P / L234V / L235A / G236_ / S267K, according to EU numbering.

[0255] In some embodiments, the invention provides homodimeric Fc fusion proteins, wherein the IL-12p40 subunit has a polypeptide sequence selected from the group consisting of SEQ ID NO:3 (human IL-12 subunit beta (IL-12p40) precursor sequence) and SEQ ID NO:4 (human IL-12 subunit beta (IL-12p40) mature form sequence), and the IL-12p35 subunit has a polypeptide sequence selected from the group consisting of SEQ ID NO:1 (human IL-12 subunit alpha (IL-12p35) precursor sequence) and SEQ ID NO:2 (human IL-12 subunit alpha (IL-12p35) mature form sequence).

[0256] In some embodiments, the invention provides a homodimeric Fc fusion protein, wherein each of the Fc domains further comprises the amino acid substitutions M428L / N424S.

[0257] In some embodiments, the invention provides homodimeric Fc fusion proteins, wherein said IL-12p40 subunit is a variant IL-12p40 subunit and / or said IL-12p35 subunit is a variant IL-12p35 subunit.

[0258] In some embodiments, the invention provides homodimeric Fc fusion proteins wherein said IL-12p40 subunit is IL-12 receptor subunit beta-1 (IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2), and / or a variant IL-12p40 subunit with altered affinity for the IL-12 receptor complex, and / or said IL-12p35 subunit is IL-12 receptor subunit beta-1 (IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2), and / or a variant IL-12p35 subunit with altered affinity for the IL-12 receptor complex.

[0259] In some embodiments, the IL-12p40 subunit is selected from the group consisting of E3, D7, E12, D14, W15, P17, D18, A19, P20, G21, E22, M23, D29, E32, E33, D34, L40, D41, Q42, S43, E45, L47, T54, I55, Q56, K58, E59, F60, G 61, D62, Q65, Y66, E73, K84, E86, D87, G88, I89, W90, D93, D97, K99, E100, K102, N103, K104, F106, E110, N113, Y114, D129, D142, Q144, E156, R159, D161, N162, K163, D166, D 170, Q172, D174, A176, C177, P178, A179, A180, E181, S183, P185, E187, N200, S204, F 206, R208, D209, D214, N218, Q220, N226, Q229, E231, E235, T242, P243, S245, Y246, F2 47, S248, C252S, , Q256, K158, K260, E262, K264, D265, D270, N281, Q289, D290, R291, Y292, Y293, and E299.

[0260] In some embodiments, the present invention relates to an IL-12p40 subunit comprising D18N, D18K, E32Q, E33Q, D34N, D34K, Q42E, S43E, S43K, E45Q, Q56E, E59Q, E59K, D62N, E73Q, D87N, K99E, K99Y, E100Q, N103D, N103Q, N113D, N113Q, Q144 The present invention provides homodimeric Fc fusion proteins having one or more amino acid substitutions selected from the group consisting of E, D161N, R159E, K163E, E187Q, N200D, N200Q, N218Q, Q229E, E235Q, C252S, Q256N, K258E, K260E, E262Q, K264E, N281D, N281Q, and E299Q.

[0261] In some embodiments, the present invention relates to an IL-12p40 subunit comprising N103D / N113D / N200D / N281D, Q42E / E45Q, E45Q / Q56E, Q42E / E59Q, Q56E / E59Q, Q42E / E45Q / Q56E, E45Q / Q56E / E59Q, E32Q / E59Q, D34N / E59K, D34N / E59K / K99E, D34K / E59K / K99E, E32Q / D34N / E59K / K99E, E32K / D34N / E59K / K99E, D34N / E59Q, E59Q / E187Q, S43E / E59Q, S43K / E49Q, E59Q / K163E, E59Q / K99E, E59Q / K258E, E59Q / K2 60E, E59K / K99E, D18K / E59K / K99E, E59K / K99E / K264E, E59K / K99Y, E59Y / K99Y, E59Y / K99E, E45K / E59K / K99E, E59K / K99E / Q144E, E59K / K99E / Q144K, E59K / K99E / R159E, E59K / K99E / K2 64E, D18K / E59K / K99E / K264E, DI8K / E59K / K99E / C252S, D18K / E59K / K99E / C252S / K264E, E5 9K / K99Y / C252S, E59K / K99E / C252S / K264E, E59K / K99E / C252S, N103D / N113D, N103D / N200D , N103D / N281D, N113D / N200D, N113D / N281D, N200D / N281D, N103D / N113D / N200D, N103D / N113D / N281D, N103D / N200D / N281D , N113D / N200D / N281D, N103Q / N113Q, N103Q / N200Q, N103Q / N281Q, N113Q / N200Q, N113Q / N281Q, N200Q / N281Q, N103Q / N113Q / N200Q, N103Q / N113Q / N281Q, N103Q / N200Q / N281Q, N113Q / N200Q / N281Q, N103Q / N113Q / N200Q / N281Q, E59K / K99E / N103Q / C 252S / K264E, E59K / K99E / N113Q / C252S / K264E, E59K / K99E / N200Q / C252S / K264E, E59K / K99E / N281Q / C252S / K264E, E59K / K9 9E / N103Q / N113Q / C252S / K264E, E59K / K99E / N103Q / N200Q / C252S / K264E, E59K / K99E / N103Q / N281Q / C252S / K264E, E59K / K9 9E / N113Q / N200Q / C252S / K264E, E59K / K99E / N113Q / N281Q / C252S / K264E, E59K / K99E / N200Q / N281Q / C252S / K264E, E59K / K9 Homodimeric Fc fusion proteins are provided having amino acid substitutions selected from the group consisting of 9E / N103Q / N113Q / N200Q / C252S / K264E, E59K / K99E / N103Q / N200Q / N281Q / C252S / K264E, E59K / K99E / N113Q / N200Q / N281Q / C252S / K264E, and E59K / K99E / N103Q / N113Q / N200Q / N281Q / C252S / K264E.

[0262] In some embodiments, the present invention relates to a method for the preparation of IL-12p40 subunits comprising administering to a patient a therapeutically effective amount of the IL-12p40 subunit, the method comprising administering to a patient a therapeutically effective amount of the IL-12p40 subunit, the therapeutically effective amount of the IL-12p40 subunit being: i) SEQ ID NO:57 (IL-12p40(N103D)); ii) SEQ ID NO:58 (IL-12p40(N113D)); iii) SEQ ID NO:59 (IL-12p40(N200D)); iv) SEQ ID NO:60 (IL-12p40(N281D)); v) SEQ ID NO:61 (IL-12p40(N103D / N113D / N200D / N281D)); vi) SEQ ID NO:62 (IL-12p40(Q42E)); vii) SEQ ID NO:63 (IL-12p40(E45 Q)), viii) SEQ ID NO:64 (IL-12p40(Q56E)), ix) SEQ ID NO:65 (IL-12p40(E59Q)), x) SEQ ID NO:66 (IL-12p40(D62N)), xi) SEQ ID NO:67 (IL-12p40(Q42E / E45Q)), xii) SEQ ID NO:68 (IL-12p40(E45Q / Q56E)), xiii) SEQ ID NO:69 (IL-12p40(Q42E / E59Q)), xiv) SEQ ID NO:70 (IL-12p40(Q56E / E59Q)), xv) SEQ ID NO:71 (IL-12p40(Q42E / E 45Q / Q56E)), xvi) SEQ ID NO: 72 (IL-12p40(E45Q / Q56E / E59Q)), xvii) SEQ ID NO: 73 (IL-12p40(D161N)), xviii) SEQ ID NO: 74 (IL-12p40(E73Q)), xix) SEQ ID NO: 75 (IL-12p40(Q144E)), xx) SEQ ID NO: 76 (IL-12p40(E262Q)), xxi) SEQ ID NO: 77 (IL-12p40(E100Q)), xxii) SEQ ID NO: 78 (IL-12p40(D18N)), xxiii) SEQ ID NO: 79 (IL-12p 40(E33Q)), xxiv) SEQ ID NO: 80 (IL-12p40(Q229E)), xxv) SEQ ID NO: 81 (IL-12p40(E235Q)), xxvi) SEQ ID NO: 82 (IL-12p40(Q256N)), xxvii) SEQ ID NO: 83 (IL-12p40(E299Q)), xxviii) SEQ ID NO: 84 (IL-12p40(D87N)), xxix) IL-12p40(E32Q), xxx) IL-12p40(D34N), xxxi) IL-12p40(S43E), xxxii) IL-12p40(S43K),xxxiii) SEQ ID NO: XXX (IL-12p40(E59K / K99E / N103Q / N113Q / N200Q / N281Q / C252S / K264E)), xxxiv) SEQ ID NO: 205 (IL-12p40(E59K)), xxxv) IL-12p40(K99E), xxxvi) IL-12p40(K163E), xxxvii) IL-12p40(E187Q), xxxviii), IL-12p40(K258E), xxxix) IL-12p40(K260E), xl) SEQ ID NO: 206 (IL-12p40(E32Q / E59Q)), xli) SEQ ID NO: 207 (IL-12p40(D34N / E59Q)), xlii) SEQ ID NO: 208 (IL-12p40(E59Q / E187Q)), xliii) SEQ ID NO: 209 (IL-12p40(S43E / E59Q)), xliv) SEQ ID NO: 210 (IL-12p40(S43K / E49Q)), xlv) SEQ ID NO: 211 (IL-12p40(E59Q / K163E)) , xlvi) SEQ ID NO: 212 (IL-12p40(E59Q / K99E)), xlvii) SEQ ID NO: 213 (IL-12p40(E59Q / K258E)), xlviii) SEQ ID NO: 214 (IL-12p40(E59Q / K260E)), xlix) SEQ ID NO: XXX (IL-12p40(D34N / E59K)), l) SEQ ID NO: XXX (IL-12p40(E59K / K99E)), li) SEQ ID NO: XXX (IL-12p40(D18K / E59K / K99E)), lii) SEQ ID NO: XXX (IL-12p40(E59K / K99E / K264E)), liii) SEQ ID NO: XXX(IL-12p40(E59K / K99Y)), liv) SEQ ID NO: XXX(IL-12p40(E59Y / K99E)), lv) SEQ ID NO: XXX(IL-12p40(E45K / E59K / K99E)), lvi) SEQ ID NO: XXX(IL-12p40(E59K / K99E / Q144E)), lvii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / Q144K)), lviii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / R159E)), lix) SEQ ID NO: No. XXX(IL-12p40(E59K / K99E / K264E)), lx) No. XXX(IL-12p40(D18K / E59K / K99E / K264E)), lxi) No. XXX(IL-12p40(C252S)), lxii) No. XXX(IL-12p40(DI8K / E59K / K99E / C252S)), lxiii) No. XXX(IL-12p40(D18K / E59K / K99E / C252S / K264E)), lxiv) No. XXX(IL-12p40(E59K / K99Y / C252S)),lxv) SEQ ID NO:XXX(IL-12p40(E59K / K99E / C252S / K264E)), lxvi) SEQ ID NO:XXX(IL-12p40(E59K / K99E / C252S)), lxvii) SEQ ID NO:XXX(IL-12p40(N103D / N113D)), lxviii) SEQ ID NO:XXX(IL-12p40(N103D / N200D)), lxix) SEQ ID NO:XXX(IL-12p40(N103D / N281D)), lxx) SEQ ID NO:XXX(IL-12p40(N113D / N200D)), lxxi) SEQ ID NO:X XX(IL-12p40(N113D / N281D)), lxxii) SEQ ID NO:XXX(IL-12p40(N200D / N281D)), lxxiii) SEQ ID NO:XXX(IL-12p50(N103D / N113D / N200D)), lxxiv) SEQ ID NO:XXX(IL-12p40(N103D / N113D / N281D)), lxxv) SEQ ID NO:XXX(IL-12p40(N103D / N200D / N281D)), lxxvi) SEQ ID NO:XXX(IL-12p40(N113D / N200D / N281D)), lxxvii ) SEQ ID NO:XXX(IL-12p40(N103Q)), lxxviii) SEQ ID NO:XXX(IL-12p40(N113Q)), lxxix) SEQ ID NO:XXX(IL-12p40(N200Q)), lxxx) SEQ ID NO:XXX(IL-12p40(N281Q)), lxxxi) SEQ ID NO:XXX(IL-12p40(N103Q / N113Q)), lxxxii) SEQ ID NO:XXX(IL-12p40(N103Q / N200Q)), lxxxiii) SEQ ID NO:XXX(IL-12p40(N103Q / N281Q)), lxxxiv) SEQ ID NO:XXX(IL-12p40(N113Q / N200Q)), lxxxv) SEQ ID NO:XXX(IL-12p40(N113Q / N281Q)), lxxxvi) SEQ ID NO:XXX(IL-12p40(N200Q / N281Q)), lxxxvii) SEQ ID NO:XXX(IL-12p40(N103Q / N113Q / N200Q)), lxxxviii) SEQ ID NO:XXX(IL-12p40(N103Q / N113Q / N281Q)), lxxxix) SEQ ID NO:XXX(IL-12p40(N103Q / N200Q / N281Q)),xc) SEQ ID NO: XXX (IL-12p40(N113Q / N200Q / N281Q)), xci) SEQ ID NO: XXX (IL-12p40(N103Q / N113Q / N2, 00Q / N281Q), xcii) SEQ ID NO: XXX(IL-12p40(D34N / E59K / K99E)), xciii) SEQ ID NO: XXX(IL-12p40(D34K / E59K / K99E)), xciv) SEQ ID NO: XXX(IL-12p40(E32Q / D34N / E59K / K99E)), xcv) SEQ ID NO: XXX(IL-12p40(E32K / D34N / E59K / K99E)), xcvi) SEQ ID NO: XXX(IL-12p40(E59Y / K99Y)), xcvii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N103Q / C252S / K264E)), xcviii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N113Q / C252S / K264E)), xcix) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N200Q / C252S / K264E)), c) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N281Q / C252S / K264E)), ci) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N103Q / N113Q / C252S / K264E)), cii) SEQ ID NO: XXX(IL-12p40(E59 K / K99E / N103Q / N200Q / C252S / K264E)), ciii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N103Q / N281Q / C252S / K264E)), civ) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N113Q / N200Q / C252S / K264E)), cv) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N113Q / N281Q / C252S / K264E)), cvi) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N200Q / N281Q / C25 2S / K264E)), cvii) SEQ ID NO:XXX(IL-12p40(E59K / K99E / N103Q / N113Q / N200Q / C252S / K264E)), cviii) SEQ ID NO:XXX(IL-12p40(E59K / K99E / N103Q / N200Q / N281Q / C252S / K264E)), and cix) SEQ ID NO:XXX(IL-12p40(E59K / K99E / N113Q / N200Q / N281Q / C252S / K264E)).

[0263] In some embodiments, the present invention relates to an IL-12 p35 subunit comprising one or more of the following: Q20, N21, Q35, E38, S44, E45, E46, H49, K54, D55, T59, V60, E61, C63, L64, P65, E67, L68, N71, S73, C74, L75, N76, E79, N85, L89, F96, M97, L124, M125, Q130, Q135, N The present invention provides homodimeric Fc fusion proteins having one or more amino acid modifications at amino acid residues selected from the group consisting of 136, E143, Q146, N151, E153, K158, E162, E163, D165, I171, R181, I182, R183, V185, T186, D188, R189, V190, S192, Y193, N195, and A196.

[0264] In some embodiments, the invention provides homodimeric Fc fusion proteins, wherein the IL-12p35 subunit has one or more amino acid substitutions selected from the group consisting of N21D, Q35D, E38Q, D55Q, D55K, N71D, N71Q, L75A, N76D, E79Q, N85D, N85Q, L89A, F96A, M97A, L124A, M125A, Q130E, Q135E, N136D, E143Q, Q146E, N151D, N151K, E153K, E153Q, K158E, E162Q, E163Q, D165N, I171A, N195D, and N195Q.

[0265] In some embodiments, the invention relates to a method for the preparation of IL-12p35 subunits comprising administering to the patient a IL-12p35 subunit selected from the group consisting of N71D / N85D / N195D, N151D / E153Q, N151D / D165N, Q130E / N151D, N151D / K158E, E79Q / N151D, D55Q / N151D, N136D / N151D, N21D / N151D, E143Q / N151D, N71Q / N85Q, N71Q / N195Q, N85Q / N195Q, N71Q / N85Q / N195Q, N71D / N85D, N71D / N195D, and N85D / N195D. The present invention provides homodimeric Fc fusion proteins having an amino acid substitution selected from the group consisting of:

[0266] In some embodiments, the present invention relates to a method for the preparation of a IL-12p35 subunit comprising administering to a patient a sequence identical to that of SEQ ID NO:113 (IL-12p35(N71D)), ii) SEQ ID NO:114 (IL-12p35(N85D)), iii) SEQ ID NO:115 (IL-12p35(N195D)), iv) SEQ ID NO:116 (IL-12p35(N71D / N85D / N195D)), v) SEQ ID NO:117 (IL-12p35(E153Q)), vi) SEQ ID NO:118 (IL-12p35(E38Q)), vii) SEQ ID NO:119 (IL-12p35(N151D)), viii) SEQ ID NO:120 (IL-12p35(N151D)), or viii) SEQ ID NO: 120 (IL-12p35(Q135E)), ix) SEQ ID NO: 121 (IL-12p35(Q35D)), x) SEQ ID NO: 122 (IL-12p35(Q146E)), xi) SEQ ID NO: 123 (IL-12p35(N76D)), xii) SEQ ID NO: 124 (IL-12p35(E162Q)), xiii) SEQ ID NO: 125 (IL-12p35(E163Q)), xiv) IL-12p35(N21D), xv) SEQ ID NO: XXX (IL-12p35(D55Q)), xvi) IL-12p35(E79Q), xvii) IL-12p35(Q130E), x viii) IL-12p35(N136D), xix) IL-12p35(E143Q), xx) SEQ ID NO: 227 (IL-12p35(N151K)), xxi) SEQ ID NO: 226 (IL-12p35(E153K)), xxii) IL-12p35(K158E), xxiii) IL-12p35(D165N), xxiv) SEQ ID NO: 225 (IL-12p35(N151D / E153Q)), xxv) SEQ ID NO: 228 (IL-12p35(N151D / D165N)), xxvi) SEQ ID NO: 229 (IL-12p35(Q130E / N151D)) , xxvii) SEQ ID NO: 230 (IL-12p35(N151D / K158E)), xxviii) SEQ ID NO: 231 (IL-12p35(E79Q / N151D)), xxix) SEQ ID NO: 232 (IL-12p35(D55Q / N151D)), xxx) SEQ ID NO: 233 (IL-12p35(N136D / N151D)), xxxi) SEQ ID NO: 234 (IL-12p35(N21D / N151D)), xxxii) SEQ ID NO: 235 (IL-12p35(E143Q / N151D)), xxxiii) SEQ ID NO: XXX (IL-12p35(F96A)),xxxiv) SEQ ID NO:XXX(IL-12p35(M97A)), xxxv) SEQ ID NO:XXX(IL-12p35(L89A)), xxxvi) SEQ ID NO:XXX(IL-12p35(L124A)), xxxvii) SEQ ID NO:XXX(IL-12p35(M125A)), xxxviii) SEQ ID NO:XXX(IL-12p35(L75A)), xxxiv) SEQ ID NO:XXX(IL-12p35(I171A)), xxxv) SEQ ID NO:XXX(IL-12p35(N71Q)), xxxvi) SEQ ID NO:XXX(IL-12p35(N85Q)), xxxvii) SEQ ID NO:XXX(IL-12p35(N195Q)), xxxviii) SEQ ID NO:XXX(IL-12p35(N71Q / N85Q)), xxix) SEQ ID NO:XXX(IL-12p35(N71Q / N195Q)), xl) SEQ ID NO:XXX(IL-12p35(N85Q / N195Q), xli) SEQ ID NO:XXX(IL-12p35(N71Q / N85Q / N195Q)), xlii) SEQ ID NO:XXX(IL-12p35(N71D / N85D)), xliii) SEQ ID NO:XXX(IL-12p35(N71D / N195D), xliv) SEQ ID NO:XXX(IL-12p35(N85D / N195D)), xlv) SEQ ID NO:XXX(IL-12p35(D55Q)), and xlvi) SEQ ID NO:XXX(IL-12p35(D55K)).

[0267] In some embodiments, a homodimeric Fc fusion protein is provided, wherein said first and second monomers are XENP31289.

[0268] In some embodiments, a homodimeric Fc fusion protein is provided, wherein said first and second monomers are XENP31291.

[0269] In some embodiments, the present invention provides a homodimeric Fc-fusion protein composition comprising a homodimeric Fc-fusion protein for use in treating cancer in a subject.

[0270] In some embodiments, the invention provides one or more nucleic acids encoding a homodimeric Fc fusion protein.

[0271] In some embodiments, the invention provides host cells comprising one or more of the nucleic acids encoding the homodimeric Fc fusion proteins.

[0272] In some embodiments, the invention provides a method of making a homodimeric Fc-fusion protein, the method comprising culturing a host cell under conditions whereby the homodimeric Fc-fusion protein is produced.

[0273] In some embodiments, the invention provides a method for purifying a homodimeric Fc-fusion protein, the method comprising: a) providing a composition comprising a homodimeric Fc-fusion protein; b) loading the composition onto an ion exchange column; and c) collecting fractions containing the homodimeric Fc-fusion protein.

[0274] In another aspect, the invention provides a homodimeric Fc fusion protein comprising a first monomer and a second monomer, each comprising, from N-terminus to C-terminus, Fc domain-optional first domain linker-IL-12p35 subunit domain-optional second domain linker-IL-12p40 subunit domain.

[0275] In some embodiments, the invention provides homodimeric Fc fusion proteins, wherein the modifications that promote homodimerization of the Fc domain are a set of amino acid substitutions selected from the group consisting of: L368D / K370S; S364K; S364K / E357L; S364K / E357Q; T411E / K360E / Q362E; D401K; T366S / L368A / Y407V; T366W; T366S / L368A / Y407V / Y349C; and T366W / S354C, according to EU numbering.

[0276] In some embodiments, the invention provides a homodimeric Fc fusion protein, wherein said first domain linker and said second domain linker have the same amino acid sequence.

[0277] In some embodiments, the invention provides homodimeric Fc fusion proteins, wherein the Fc domain has an additional set of amino acid substitutions comprising: Q295E / N384D / Q418E / N421D, according to EU numbering.

[0278] In some embodiments, the invention provides homodimeric Fc fusion proteins, wherein the Fc domain has an additional set of amino acid substitutions selected from the group consisting of G236R / L328R, E233P / L234V / L235A / G236_ / S239K, E233P / L234V / L235A / G236_ / S239K / A327G, E233P / L234V / L235A / G236_ / S267K / A327G, E233P / L234V / L235A / G236_, and E233P / L234V / L235A / G236_ / S267K, according to EU numbering.

[0279] In some embodiments, the present invention relates to a method for producing an IL-12p40 subunit comprising the steps of: SEQ ID NO:3 (human IL-12 subunit beta (IL-12p40) precursor sequence); and SEQ ID NO: and SEQ ID NO:4 (human IL-12 subunit beta (IL-12p40) mature form sequence), and the IL-12p35 subunit has a polypeptide sequence selected from the group consisting of SEQ ID NO:1 (human IL-12 subunit alpha (IL-12p35) precursor sequence) and SEQ ID NO:2 (human IL-12 subunit alpha (IL-12p35) mature form sequence).

[0280] In some embodiments, the invention provides a homodimeric Fc fusion protein, wherein each of the Fc domains further comprises the amino acid substitutions M428L / N424S.

[0281] In some embodiments, the invention provides homodimeric Fc fusion proteins, wherein said IL-12p40 subunit is a variant IL-12p40 subunit and / or said IL-12p35 subunit is a variant IL-12p35 subunit.

[0282] In some embodiments, the invention provides homodimeric Fc fusion proteins wherein said IL-12p40 subunit is IL-12 receptor subunit beta-1 (IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2), and / or a variant IL-12p40 subunit with altered affinity for the IL-12 receptor complex, and / or said IL-12p35 subunit is IL-12 receptor subunit beta-1 (IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2), and / or a variant IL-12p35 subunit with altered affinity for the IL-12 receptor complex.

[0283] In some embodiments, the IL-12p40 subunit is selected from the group consisting of E3, D7, E12, D14, W15, P17, D18, A19, P20, G21, E22, M23, D29, E32, E33, D34, L40, D41, Q42, S43, E45, L47, T54, I55, Q56, K58, E59, F60, G 61, D62, Q65, Y66, E73, K84, E86, D87, G88, I89, W90, D93, D97, K99, E100, K102, N103, K104, F106, E110, N113, Y114, D129, D142, Q144, E156, R159, D161, N162, K163, D166, D 170, Q172, D174, A176, C177, P178, A179, A180, E181, S183, P185, E187, N200, S204, F 206, R208, D209, D214, N218, Q220, N226, Q229, E231, E235, T242, P243, S245, Y246, F2 47, S248, C252S, , Q256, K158, K260, E262, K264, D265, D270, N281, Q289, D290, R291, Y292, Y293, and E299.

[0284] In some embodiments, the present invention relates to an IL-12p40 subunit comprising D18N, D18K, E32Q, E33Q, D34N, D34K, Q42E, S43E, S43K, E45Q, Q56E, E59Q, E59K, D62N, E73Q, D87N, K99E, K99Y, E100Q, N103D, N103Q, N113D, N113Q, Q144 The present invention provides homodimeric Fc fusion proteins having one or more amino acid substitutions selected from the group consisting of E, D161N, R159E, K163E, E187Q, N200D, N200Q, N218Q, Q229E, E235Q, C252S, Q256N, K258E, K260E, E262Q, K264E, N281D, N281Q, and E299Q.

[0285] In some embodiments, the present invention relates to an IL-12p40 subunit having the structure:<h2 style=";text-align:left;direction:ltr">D / N113D / N200D / N281D、Q42E / E45Q、E45Q / Q56E、Q42E / E59Q、Q56E / E59Q、Q4 2E / E45Q / Q56E、E45Q / Q56E / E59Q、E32Q / E59Q、D34N / E59K、D34N / E59K / K99E 、D34K / E59K / K99E、E32Q / D34N / E59K / K99E、E32K / D34N / E59K / K99E、D34N / E 59Q、E59Q / E187Q、S43E / E59Q、S43K / E49Q、E59Q / K163E、E59Q / K99E、E59Q / K 258E、E59Q / K260E、E59K / K99E、D18K / E59K / K99E、E59K / K99E / K264E、E59K / K99Y、E59Y / K99Y、E59Y / K99E、E45K / E59K / K99E、E59K / K99E / Q144E、E59K / K 99E / Q144K, E59K / K99E / R159E, E59K / K99E / K264E, D18K / E59K / K99E / K264E, DI8K / E59K / K99E / C252S, D18K / E59K / K99E / C252S / K264E, E59K / K99Y / C252 S、E59K / K99E / C252S / K264E、E59K / K99E / C252S、N103D / N113D、N103D / N200 D、N103D / N281D、N113D / N200D、N113D / N281D、N200D / N281D、N103D / N113D / N200D、N103D / N113D / N281D、N103D / N200D / N281D、N113D / N200D / N281D、N1 03Q / N113Q、N103Q / N200Q、N103Q / N281Q、N113Q / N200Q、N113Q / N281Q、N200Q / N281Q、N103Q / N113Q / N200Q、N103Q / N113Q / N281Q、N103Q / N200Q / N281Q、N 113Q / N200Q / N281Q、N103Q / N113Q / N200Q / N281Q、E59K / K99E / N103Q / C252S / K264E、E59K / K99E / N113Q / C252S / K264E、E59K / K99E / N200Q / C252S / K264E、E59K / K99E / N281Q / C252S / K264E、E59K / K99E / N103Q / N113Q / C252S / K264E、E59K / K99E / N103Q / N200Q / C252S / K264E, E59K / K99E / N103Q / N281Q / C252S / K264E, E59K / K99E / N113Q / N200Q / C252S / K264E, E59K / K99E / N113Q / N281Q / C252S / K264E, E59K / K99E / N200Q / N281Q / C252S / K264E, E59K / K99E / N103Q / N113 The present invention provides homodimeric Fc fusion proteins having amino acid substitutions selected from the group consisting of E59K / K99E / N103Q / N200Q / N281Q / C252S / K264E, E59K / K99E / N113Q / N200Q / N281Q / C252S / K264E, E59K / K99E / N103Q / N113Q / N200Q / N281Q / C252S / K264E, and E59K / K99E / N103Q / N113Q / N200Q / N281Q / C252S / K264E.

[0286] In some embodiments, the present invention relates to a method for the preparation of IL-12p40 subunits comprising administering to a patient a therapeutically effective amount of the IL-12p40 subunit, the method comprising administering to a patient a therapeutically effective amount of the IL-12p40 subunit, the method comprising administering to a patient a therapeutically effective amount of the IL-12p40 subunit, the method comprising administering to a patient a therapeutically effective amount of the IL-12p40 subunit, L-12p40(E59Q)), x) SEQ ID NO: 66 (IL-12p40(D62N)), xi) SEQ ID NO: 67 (IL-12p40(Q42E / E45Q)), xii) SEQ ID NO: 68 (IL-12p40(E45Q / Q56E)), xiii) SEQ ID NO: 69 (IL-12p40(Q42E / E59Q)), xiv) SEQ ID NO: 70 (IL-12p40(Q56E / E59Q)), xv) SEQ ID NO: 71 (IL-12p40(Q42E / E45Q / Q56E)), xvi) SEQ ID NO: 72 (IL-12p40(E45Q / Q56E / E59Q)), xvii) SEQ ID NO: 73 (IL-12p40(D161N)), xviii) SEQ ID NO: 74 (IL-12p40(E73Q)), xix) SEQ ID NO: 75 (IL-12p40(Q144E)), xx) SEQ ID NO: 76 (IL-12p40(E262Q)), xxi) SEQ ID NO: 77 (IL-12p40(E100Q)), xxii) SEQ ID NO: 78 (IL-12p40(D18N)), xxiii) SEQ ID NO: 79 (IL-12p40(E33Q)), xxiv) SEQ ID NO: 80 (IL-12p40(Q229E)), xxv) SEQ ID NO: 81 (IL-12p40(E235Q)), xxvi) SEQ ID NO: 82 (IL-12p40(Q256N) ), xxvii) SEQ ID NO: 83 (IL-12p40(E299Q)), xxviii) SEQ ID NO: 84 (IL-12p40(D87N)), xxix) IL-12p40(E32Q), xxx) IL-12p40(D34N), xxxi) IL-12p40(S43E), xxxii) IL-12p40(S43K), xxxiii) SEQ ID NO: XXX (IL-12p40(E59K / K99E / N103Q / N113Q / N200Q / N281Q / C252S / K264E)), xxxiv) SEQ ID NO: 205 (IL-12p40(E59K)) , xxxv) IL-12p40(K99E), xxxvi) IL-12p40(K163E), xxxvii) IL-12p40(E187Q), xxxviii) IL-12p40(K258E), xxxix) IL-12p40(K260E), xl) SEQ ID NO: 206 (IL-12p40(E32Q / E59Q)), xli) SEQ ID NO: 207 (IL-12p40(D34N / E59Q)), xlii) SEQ ID NO: 208 (IL-12p40(E59Q / E187Q)), xliii) SEQ ID NO: 209 (IL-12p40(S43E / E59 Q), xliv) SEQ ID NO: 210 (IL-12p40(S43K / E49Q)), xlv) SEQ ID NO: 211 (IL-12p40(E59Q / K163E)), xlvi) SEQ ID NO: 212 (IL-12p40(E59Q / K99E)), xlvii) SEQ ID NO: 213 (IL-12p40(E59Q / K258E)), xlviii) SEQ ID NO: 214 (IL-12p40(E59Q / K260E)), xlix) SEQ ID NO: XXX (IL-12p40(D34N / E59K)), l) SEQ ID NO: XXX (IL-12p40(E59K / K99E)),li) SEQ ID NO: XXX(IL-12p40(D18K / E59K / K99E)), lii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / K264E)), liii) SEQ ID NO: XXX(IL-12p40(E59K / K99Y)), liv) SEQ ID NO: XXX(IL-12p40(E59Y / K99E)), lv) SEQ ID NO: XXX(IL-12p40(E45K / E59K / K99E)), lvi) SEQ ID NO: XXX(IL-12p40(E59K / K99E / Q144E)), lvii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / Q144K)), lviii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / R159E)), lix) SEQ ID NO: XXX(IL-12p40(E59K / K99E / K264E)), lx) SEQ ID NO: XXX(IL-12p40(D18K / E59K / K99E / K264E)), lxi) SEQ ID NO: XXX(IL-12p40(C252S)), lxii) SEQ ID NO: XXX(IL-12p40(DI8K / E59K / K99E / C252S)), lxiii) SEQ ID NO: XXX(IL-12p40(D18K / E59K / K 99E / C252S / K264E)), lxiv) SEQ ID NO: XXX(IL-12p40(E59K / K99Y / C252S)), lxv) SEQ ID NO: XXX(IL-12p40(E59K / K99E / C252S / K264E)), lxvi) SEQ ID NO: XXX(IL-12p40(E59K / K99E / C252S)), lxvii) SEQ ID NO: XXX(IL-12p40(N103D / N113D)), lxviii) SEQ ID NO: XXX(IL-12p40(N103D / N200D)), lxix) SEQ ID NO: XXX(IL-12p40(N103D / N281D)), lxx) SEQ ID NO: XXX(IL-12p40(N113D / N200D)), lxxi) SEQ ID NO: XXX(IL-12p40(N113D / N281D)), lxxii) SEQ ID NO: XXX(IL-12p40(N200D / N281D)), lxxiii) SEQ ID NO: XXX(IL-12p50(N103D / N113D / N200D)), lxxiv) SEQ ID NO: XXX(IL-12p40(N103D / N113D / N281D)), lxxv) SEQ ID NO: XXX(IL-12p40(N103D / N200D / N281D)),lxxvi) SEQ ID NO: XXX (IL-12p40 (N113D / N200D / N281D)), lxx, vii) SEQ ID NO:XXX(IL-12p40(N103Q)), lxxviii) SEQ ID NO:XXX(IL-12p40(N113Q)), lxxix) SEQ ID NO:XXX(IL-12p40(N200Q)), lxxx) SEQ ID NO:XXX(IL-12p40(N281Q)), lxxxi) SEQ ID NO:XXX(IL-12p40(N103Q / N113Q)), lxxxii) SEQ ID NO:XXX(IL-12p40(N103Q / N200Q)), lxxxiii) SEQ ID NO:XXX(IL-12p40(N103Q / N281Q)), lxxxiv) SEQ ID NO:XXX(IL-12p40(N103Q / N281Q)), Sequence number XXX(IL-12p40(N113Q / N200Q)), lxxxv) Sequence number XXX(IL-12p40(N113Q / N281Q)), lxxxvi) Sequence number XXX(IL-12p40(N200Q / N281Q)), lxxxvii) Sequence number XXX(IL-12p40(N103Q / N113Q / N200Q)), lxxxviii) Sequence number XXX(IL-12p40(N103Q / N113Q / N281Q)), lxxxix) Sequence number XXX(IL-12p40(N103Q / N200Q / N281Q)), xc) Sequence No. XXX(IL-12p40(N113Q / N200Q / N281Q)), xci) No. XXX(IL-12p40(N103Q / N113Q / N200Q / N281Q)), xcii) No. XXX(IL-12p40(D34N / E59K / K99E)), xciii) No. XXX(IL-12p40(D34K / E59K / K99E)), xciv) No. XXX(IL-12p40(E32Q / D34N / E59K / K99E)), xcv) No. XXX(IL-12p40(E32K / D34N / E59K / K99E )), xcvi) SEQ ID NO: XXX(IL-12p40(E59Y / K99Y)), xcvii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N103Q / C252S / K264E)), xcviii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N113Q / C252S / K264E)), xcix) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N200Q / C252S / K264E)), c) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N281Q / C252S / K264E)),ci) SEQ ID NO: XXX (IL-12p40(E59K / K99E / N103Q / N113Q / C252S / K264E)), cii) SEQ ID NO: XXX (IL-12p40(E59K / K99E / N103Q / N200Q / C252S / K264E)), ciii) SEQ ID NO: XXX (IL-12p40(E59K / K99E / N103Q / N281Q / C252S / K264E)), civ) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N113Q / N200Q / C252S / K264E)), cv) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N113Q / N281Q / C252S / K264E)), cvi) SEQ ID NO: No. XXX(IL-12p40(E59K / K99E / N200Q / N281Q / C252S / K264E)), cvii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N103Q / N113Q / N200Q / C252S / K264E)), cviii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N103Q / N200Q / N281Q / C252S / K264E)), and cix) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N113Q / N200Q / N281Q / C252S / K264E)).

[0287] In some embodiments, the present invention relates to an IL-12 p35 subunit comprising one or more of the following: Q20, N21, Q35, E38, S44, E45, E46, H49, K54, D55, T59, V60, E61, C63, L64, P65, E67, L68, N71, S73, C74, L75, N76, E79, N85, L89, F96, M97, L124, M125, Q130, Q135, N The present invention provides homodimeric Fc fusion proteins having one or more amino acid modifications at amino acid residues selected from the group consisting of 136, E143, Q146, N151, E153, K158, E162, E163, D165, I171, R181, I182, R183, V185, T186, D188, R189, V190, S192, Y193, N195, and A196.

[0288] In some embodiments, the invention provides homodimeric Fc fusion proteins, wherein the IL-12p35 subunit has one or more amino acid substitutions selected from the group consisting of N21D, Q35D, E38Q, D55Q, D55K, N71D, N71Q, L75A, N76D, E79Q, N85D, N85Q, L89A, F96A, M97A, L124A, M125A, Q130E, Q135E, N136D, E143Q, Q146E, N151D, N151K, E153K, E153Q, K158E, E162Q, E163Q, D165N, I171A, N195D, and N195Q.

[0289] In some embodiments, the invention provides homodimeric Fc fusion proteins, wherein the IL-12p35 subunit has amino acid substitutions selected from the group consisting of N71D / N85D / N195D, N151D / E153Q, N151D / D165N, Q130E / N151D, N151D / K158E, E79Q / N151D, D55Q / N151D, N136D / N151D, N21D / N151D, E143Q / N151D, N71Q / N85Q, N71Q / N195Q, N85Q / N195Q, N71Q / N85Q / N195Q, N71D / N85D, N71D / N195D, and N85D / N195D.

[0290] In some embodiments, the present invention relates to a method for the preparation of a IL-12p35 subunit comprising administering to a patient a sequence identical to that of SEQ ID NO:113 (IL-12p35(N71D)), ii) SEQ ID NO:114 (IL-12p35(N85D)), iii) SEQ ID NO:115 (IL-12p35(N195D)), iv) SEQ ID NO:116 (IL-12p35(N71D / N85D / N195D)), v) SEQ ID NO:117 (IL-12p35(E153Q)), vi) SEQ ID NO:118 (IL-12p35(E38Q)), vii) SEQ ID NO:119 (IL-12p35(N151D)), viii) SEQ ID NO:120 (IL-12p35(N151D)), or viii) SEQ ID NO: 120 (IL-12p35(Q135E)), ix) SEQ ID NO: 121 (IL-12p35(Q35D)), x) SEQ ID NO: 122 (IL-12p35(Q146E)), xi) SEQ ID NO: 123 (IL-12p35(N76D)), xii) SEQ ID NO: 124 (IL-12p35(E162Q)), xiii) SEQ ID NO: 125 (IL-12p35(E163Q)), xiv) IL-12p35(N21D), xv) SEQ ID NO: XXX (IL-12p35(D55Q)), xvi) IL-12p35(E79Q), xvii) IL-12p35(Q130E), x viii) IL-12p35(N136D), xix) IL-12p35(E143Q), xx) SEQ ID NO: 227 (IL-12p35(N151K)), xxi) SEQ ID NO: 226 (IL-12p35(E153K)), xxii) IL-12p35(K158E), xxiii) IL-12p35(D165N), xxiv) SEQ ID NO: 225 (IL-12p35(N151D / E153Q)), xxv) SEQ ID NO: 228 (IL-12p35(N151D / D165N)), xxvi) SEQ ID NO: 229 (IL-12p35(Q130E / N151D)) , xxvii) SEQ ID NO: 230 (IL-12p35(N151D / K158E)), xxviii) SEQ ID NO: 231 (IL-12p35(E79Q / N151D)), xxix) SEQ ID NO: 232 (IL-12p35(D55Q / N151D)), xxx) SEQ ID NO: 233 (IL-12p35(N136D / N151D)), xxxi) SEQ ID NO: 234 (IL-12p35(N21D / N151D)), xxxii) SEQ ID NO: 235 (IL-12p35(E143Q / N151D)), xxxiii) SEQ ID NO: XXX (IL-12p35(F96A)),xxxiv) SEQ ID NO:XXX(IL-12p35(M97A)), xxxv) SEQ ID NO:XXX(IL-12p35(L89A)), xxxvi) SEQ ID NO:XXX(IL-12p35(L124A)), xxxvii) SEQ ID NO:XXX(IL-12p35(M125A)), xxxviii) SEQ ID NO:XXX(IL-12p35(L75A)), xxxiv) SEQ ID NO:XXX(IL-12p35(I171A)), xxxv) SEQ ID NO:XXX(IL-12p35(N71Q)), xxxvi) SEQ ID NO:XXX(IL-12p35(N85Q)), xxxvii) SEQ ID NO:XXX(IL-12p35(N195Q)), xxxviii) SEQ ID NO:XXX(IL-12p35(N71Q / N85Q)), xxxix) SEQ ID NO:XXX(IL, The present invention provides homodimeric Fc fusion proteins having a polypeptide sequence selected from the group consisting of SEQ ID NO: XXX(IL-12p35(N71Q / N195Q)), xl) SEQ ID NO: XXX(IL-12p35(N85Q / N195Q), xli) SEQ ID NO: XXX(IL-12p35(N71Q / N85Q / N195Q)), xlii) SEQ ID NO: XXX(IL-12p35(N71D / N85D)), xliii) SEQ ID NO: XXX(IL-12p35(N71D / N195D), xliv) SEQ ID NO: XXX(IL-12p35(N85D / N195D)), xlv) SEQ ID NO: XXX(IL-12p35(D55Q)), and xlvi) SEQ ID NO: XXX(IL-12p35(D55K)).

[0291] In some embodiments, a homodimeric Fc fusion protein is provided, wherein said first and second monomers are XENP31289.

[0292] In some embodiments, a homodimeric Fc fusion protein is provided, wherein said first and second monomers are XENP31291.

[0293] In some embodiments, the present invention provides a homodimeric Fc-fusion protein composition comprising a homodimeric Fc-fusion protein for use in treating cancer in a subject.

[0294] In some embodiments, the invention provides one or more nucleic acids encoding a homodimeric Fc fusion protein.

[0295] In some embodiments, the invention provides host cells comprising one or more of the nucleic acids encoding the homodimeric Fc fusion proteins.

[0296] In some embodiments, the invention provides a method of making a homodimeric Fc-fusion protein, the method comprising culturing a host cell under conditions whereby the homodimeric Fc-fusion protein is produced.

[0297] In some embodiments, the invention provides a method for purifying a homodimeric Fc-fusion protein, the method comprising: a) providing a composition comprising a homodimeric Fc-fusion protein; b) loading the composition onto an ion exchange column; and c) collecting fractions containing the homodimeric Fc-fusion protein.

[0298] In another aspect, the invention provides a homodimeric Fc fusion protein comprising a first monomer and a second monomer, each comprising, from N-terminus to C-terminus, IL-12p40 subunit domain-first domain linker-IL-12p35 subunit domain-second domain linker-Fc domain.

[0299] In some embodiments, the invention provides homodimeric Fc fusion proteins, wherein the modifications that promote homodimerization of the Fc domain are a set of amino acid substitutions selected from the group consisting of: L368D / K370S; S364K; S364K / E357L; S364K / E357Q; T411E / K360E / Q362E; D401K; T366S / L368A / Y407V; T366W; T366S / L368A / Y407V / Y349C; and T366W / S354C, according to EU numbering.

[0300] In some embodiments, the invention provides a homodimeric Fc fusion protein, wherein said first domain linker and said second domain linker have the same amino acid sequence.

[0301] In some embodiments, the present invention relates to a method for the preparation of a ribosome-specific Fc domain comprising administering to a mammalian subject the ribosome-specific Fc domain of the present invention, Homodimeric Fc fusion proteins are provided that have an additional set of amino acid substitutions, including 5E / N384D / Q418E / N421D.

[0302] In some embodiments, the invention provides homodimeric Fc fusion proteins, wherein the Fc domain has an additional set of amino acid substitutions selected from the group consisting of G236R / L328R, E233P / L234V / L235A / G236_ / S239K, E233P / L234V / L235A / G236_ / S239K / A327G, E233P / L234V / L235A / G236_ / S267K / A327G, E233P / L234V / L235A / G236_, and E233P / L234V / L235A / G236_ / S267K, according to EU numbering.

[0303] In some embodiments, the invention provides homodimeric Fc fusion proteins, wherein the IL-12p40 subunit has a polypeptide sequence selected from the group consisting of SEQ ID NO:3 (human IL-12 subunit beta (IL-12p40) precursor sequence) and SEQ ID NO:4 (human IL-12 subunit beta (IL-12p40) mature form sequence), and the IL-12p35 subunit has a polypeptide sequence selected from the group consisting of SEQ ID NO:1 (human IL-12 subunit alpha (IL-12p35) precursor sequence) and SEQ ID NO:2 (human IL-12 subunit alpha (IL-12p35) mature form sequence).

[0304] In some embodiments, the invention provides a homodimeric Fc fusion protein, wherein each of the Fc domains further comprises the amino acid substitutions M428L / N424S.

[0305] In some embodiments, the invention provides homodimeric Fc fusion proteins, wherein said IL-12p40 subunit is a variant IL-12p40 subunit and / or said IL-12p35 subunit is a variant IL-12p35 subunit.

[0306] In some embodiments, the invention provides homodimeric Fc fusion proteins wherein said IL-12p40 subunit is IL-12 receptor subunit beta-1 (IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2), and / or a variant IL-12p40 subunit with altered affinity for the IL-12 receptor complex, and / or said IL-12p35 subunit is IL-12 receptor subunit beta-1 (IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2), and / or a variant IL-12p35 subunit with altered affinity for the IL-12 receptor complex.

[0307] In some embodiments, the IL-12p40 subunit is selected from the group consisting of E3, D7, E12, D14, W15, P17, D18, A19, P20, G21, E22, M23, D29, E32, E33, D34, L40, D41, Q42, S43, E45, L47, T54, I55, Q56, K58, E59, F60, G61, D62, Q65, Y66, E73, K84, E86, D87, G88, I89, W90, D93, D97, K99, E100, K102, N103, K104, F106, E110, N113, Y114, D129, D142, Q144, E156, R15 9, D161, N162, K163, D166, D170, Q172, D174, A176, C177, P178, A179, A180, E18 1, S183, P185, E187, N200, S204, F206, R208, D209, D214, N218, Q220, N226, Q22 9, E231, E235, T242, P243, S245, Y246, F247, S248, C252S, , Q256, K158, K260, E262, K264, D265, D270, N281, Q289, D290, R291, Y292, Y293, and E299. The present invention provides homodimeric Fc fusion proteins having one or more amino acid modifications at amino acid residues that are selected from the group consisting of ribozyme, ribozyme, ribozyme-binding domain ... and ribozyme-binding domain.

[0308] In some embodiments, the present invention relates to an IL-12p40 subunit comprising D18N, D18K, E32Q, E33Q, D34N, D34K, Q42E, S43E, S43K, E45Q, Q56E, E59Q, E59K, D62N, E73Q, D87N, K99E, K99Y, E100Q, N103D, N103Q, N113D, N113Q, Q144 The present invention provides homodimeric Fc fusion proteins having one or more amino acid substitutions selected from the group consisting of E, D161N, R159E, K163E, E187Q, N200D, N200Q, N218Q, Q229E, E235Q, C252S, Q256N, K258E, K260E, E262Q, K264E, N281D, N281Q, and E299Q.

[0309] In some embodiments, the present invention relates to a method for the preparation of IL-12p40 subunits comprising administering to the patient a IL-12p40 subunit comprising administering to the patient a IL-12p40 subunit comprising administering to the patient a IL-12p40 subunit comprising administering to the patient a IL-12p40 subunit comprising administering to the patient a IL-12p40 subunit comprising administering to the patient a IL-12p40 subunit comprising administering to the patient a IL-12p40 subunit comprising administering to the patient a IL-12p40 subunit S43K / E49Q, E59Q / K163E, E59Q / K99E, E59Q / K258E, E59Q / K260E, E59K / K99E , D18K / E59K / K99E, E59K / K99E / K264E, E59K / K99Y, E59Y / K99Y, E59Y / K99E, E 45K / E59K / K99E, E59K / K99E / Q144E, E59K / K99E / Q144K, E59K / K99E / R159E, E59K / K99E / K264E, D18K / E59K / K99E / K264E, DI8K / E59K / K99E / C252S, D18K / E59K / K99E / C252S / K264E, E59K / K99Y / C252S, E59K / K99E / C252S / K264E, E5 9K / K99E / C252S, N103D / N113D, N103D / N200D, N103D / N281D, N113D / N200D, N 113D / N281D, N200D / N281D, N103D / N113D / N200D, N103D / N113D / N281D, N10 3D / N200D / N281D, N113D / N200D / N281D, N103Q / N113Q, N103Q / N200Q, N103Q / N281Q, N113Q / N200Q, N113Q / N281Q, N200Q / N281Q, N103Q / N113Q / N200Q, N1 03Q / N113Q / N281Q, N103Q / N200Q / N281Q, N113Q / N200Q / N281Q, N103Q / N113Q / N200Q / N281Q, E59K / K99E / N103Q / C252S / K264E, E59K / K99E / N113Q / C252S / K264E, E59K / K99E / N200Q / C252S / K264E, E59K / K99E / N281Q / C252S / K264E,E59K / K99E / N103Q / N113Q / C252S / K264E, E59K / K99E / N103Q / N200Q / C252S / K264E, E59K / K99E / N103Q / N281Q / C252S / K264E, E 59K / K99E / N113Q / N200Q / C252S / K264E, E59K / K99E / N113Q / N281Q / C252S / K264E, E59K / K99E / N200Q / N281Q / C252S / K264E, E59 The present invention provides homodimeric Fc fusion proteins having amino acid substitutions selected from the group consisting of K / K99E / N103Q / N113Q / N200Q / C252S / K264E, E59K / K99E / N103Q / N200Q / N281Q / C252S / K264E, E59K / K99E / N113Q / N200Q / N281Q / C252S / K264E, and E59K / K99E / N103Q / N113Q / N200Q / N281Q / C252S / K264E. In some embodiments, the present invention relates to an IL-12p40 subunit comprising: i) SEQ ID NO: 57 (IL-12p40(N103D)), ii) SEQ ID NO: 58 (IL-1 2p40(N113D)), iii) SEQ ID NO:59 (IL-12p40(N200D)), iv) SEQ ID NO:60 (IL-12p40(N281D)), v) SEQ ID NO:61 (IL-12p40(N103D / N113D / N200D / N281D)), vi) SEQ ID NO:62 (IL-12p40(Q42E)), vii) SEQ ID NO:63 (IL-12p40(E45Q)), viii) SEQ ID NO:64 (IL-12p40(Q56E)), ix) SEQ ID NO:65 (IL-12p40(E59Q)), x) SEQ ID NO:66 (IL-12p40(D62N)), xi) SEQ ID NO: 67 (IL-12p40(Q42E / E45Q)), xii) SEQ ID NO: 68 (IL-12p40(E45Q / Q56E)), xiii) SEQ ID NO: 69 (IL-12p40(Q42E / E59Q)), xiv) SEQ ID NO: 70 (IL-12p40(Q56E / E59Q)), xv) SEQ ID NO: 71 (IL-12p40(Q42E / E45Q / Q56E)), xvi) SEQ ID NO: 72 (IL-12p40(E45Q / Q56E / E59Q)), xvii) SEQ ID NO: 73 (IL-12p40(D161N)), xviii) SEQ ID NO: 74 (IL-12p40 (E73Q)), xix) SEQ ID NO: 75 (IL-12p40(Q144E)), xx) SEQ ID NO: 76 (IL-12p40(E262Q)), xxi) SEQ ID NO: 77 (IL-12p40(E100Q)), xxii) SEQ ID NO: 78 (IL-12p40(D18N)), xxiii) SEQ ID NO: 79 (IL-12p40(E33Q)), xxiv) SEQ ID NO: 80 (IL-12p40(Q229E)), xxv) SEQ ID NO: 81 (IL-12p40(E235Q)), xxvi) SEQ ID NO: 82 (IL-12p40(Q256N)), xxvii) SEQ ID NO: 83 (IL-12p40(I L-12p40(E299Q)), xxviii) SEQ ID NO: 84 (IL-12p40(D87N)), xxix) IL-12p40(E32Q), xxx) IL-12p40(D34N), xxxi) IL-12p40(S43E), xxxii) IL-12p40(S43K), xxxiii) SEQ ID NO: XXX (IL-12p40(E59K / K99E / N103Q / N113Q / N200Q / N281Q / C252S / K264E)), xxxiv) SEQ ID NO: 205 (IL-12p40(E59K)), xxxv) IL-12p40(K99E),xxxvi) IL-12p40(K163E), xxxvii) IL-12p40(E187Q), xxxviii) IL-12p40(K258E), xxxix) IL-12p40(K260E), xl) SEQ ID NO: 206 (IL-12p40(E32Q / E59Q)), xli) SEQ ID NO: 207 (IL-12p40(D34N / E59Q)), xlii) SEQ ID NO: 208 (IL-12p40(E59Q / E187Q)), xliii) SEQ ID NO: 209 (IL-12p40(S43E / E59Q)), xliv) SEQ ID NO: 210 (IL-1 2p40(S43K / E49Q)), xlv) SEQ ID NO:211 (IL-12p40(E59Q / K163E)), xlvi) SEQ ID NO:212 (IL-12p40(E59Q / K99E)), xlvii) SEQ ID NO:213 (IL-12p40(E59Q / K258E)), xlviii) SEQ ID NO:214 (IL-12p40(E59Q / K260E)), xlix) SEQ ID NO:XXX (IL-12p40(D34N / E59K)), l) SEQ ID NO:XXX (IL-12p40(E59K / K99E)), li) SEQ ID NO:XXX (IL-12p40(D 18K / E59K / K99E), lii) SEQ ID NO:XXX(IL-12p40(E59K / K99E / K264E)), liii) SEQ ID NO:XXX(IL-12p40(E59K / K99Y)), liv) SEQ ID NO:XXX(IL-12p40(E59Y / K99E)), lv) SEQ ID NO:XXX(IL-12p40(E45K / E59K / K99E)), lvi) SEQ ID NO:XXX(IL-12p40(E59K / K99E / Q144E)), lvii) SEQ ID NO:XXX(IL-12p40(E59K / K99E / Q144K)), lviii ) SEQ ID NO: XXX(IL-12p40(E59K / K99E / R159E)), lx) SEQ ID NO: XXX(IL-12p40(E59K / K99E / K264E)), lx) SEQ ID NO: XXX(IL-12p40(D18K / E59K / K99E / K264E)), lxi) SEQ ID NO: XXX(IL-12p40(C252S)), lxii) SEQ ID NO: XXX(IL-12p40(DI8K / E59K / K99E / C252S)), lxiii) SEQ ID NO: XXX(IL-12p40(D18K / E59K / K99E / C252S / K264E)),lxiv) SEQ ID NO:XXX (IL-12p40(E59K / K99Y / C25, 2S)), lxv) SEQ ID NO: XXX(IL-12p40(E59K / K99E / C252S / K264E)), lxvi) SEQ ID NO: XXX(IL-12p40(E59K / K99E / C252S)), lxvii) SEQ ID NO: XXX(IL-12p40(N103D / N113D)), lxviii) SEQ ID NO: XXX(IL-12p40(N103D / N200D)), lxix) SEQ ID NO: XXX(IL-12p40(N103D / N281D)), lxx) SEQ ID NO: XXX(IL-12p40(N113D / N200D)), lxxi) SEQ ID NO: Sequence number XXX(IL-12p40(N113D / N281D)), lxxii) Sequence number XXX(IL-12p40(N200D / N281D)), lxxiii) Sequence number XXX(IL-12p50(N103D / N113D / N200D)), lxxiv) Sequence number XXX(IL-12p40(N103D / N113D / N281D)), lxxv) Sequence number XXX(IL-12p40(N103D / N200D / N281D)), lxxvi) Sequence number XXX(IL-12p40(N113D / N200D / N281D)), lxx vii) SEQ ID NO:XXX(IL-12p40(N103Q)), lxxviii) SEQ ID NO:XXX(IL-12p40(N113Q)), lxxix) SEQ ID NO:XXX(IL-12p40(N200Q)), lxxx) SEQ ID NO:XXX(IL-12p40(N281Q)), lxxxi) SEQ ID NO:XXX(IL-12p40(N103Q / N113Q)), lxxxii) SEQ ID NO:XXX(IL-12p40(N103Q / N200Q)), lxxxiii) SEQ ID NO:XXX(IL-12p40(N103Q / N281Q)), lxxxi v) SEQ ID NO:XXX(IL-12p40(N113Q / N200Q)), lxxxv) SEQ ID NO:XXX(IL-12p40(N113Q / N281Q)), lxxxvi) SEQ ID NO:XXX(IL-12p40(N200Q / N281Q)), lxxxvii) SEQ ID NO:XXX(IL-12p40(N103Q / N113Q / N200Q)), lxxxviii) SEQ ID NO:XXX(IL-12p40(N103Q / N113Q / N281Q)), lxxxix) SEQ ID NO:XXX(IL-12p40(N103Q / N200Q / N281Q)),xc) SEQ ID NO: XXX(IL-12p40(N113Q / N200Q / N281Q)), xci) SEQ ID NO: XXX(IL-12p40(N103Q / N113Q / N200Q / N281Q)), xcii) SEQ ID NO: XXX(IL-12p40(D34N / E59K / K99E)), xciii) SEQ ID NO: XXX(IL-12p40(D34K / E59K / K99E)), xciv) SEQ ID NO: XXX(IL-12p40(E32Q / D34N / E59K / K99E)), xcv) SEQ ID NO: XXX(IL-12p40(E32K / D34N / E59K / K99E)), xcvi) SEQ ID NO: XXX(IL-12p40(E59Y / K99Y)), xcvii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N103Q / C252S / K264E)), xcviii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N113Q / C252S / K264E)), xcix) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N200Q / C252S / K264E)), c) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N281Q / C 252S / K264E)), ci) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N103Q / N113Q / C252S / K264E)), cii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N103Q / N200Q / C252S / K264E)), ciii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N103Q / N281Q / C252S / K264E)), civ) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N113Q / N200Q / C252S / K264E)), cv) SEQ ID NO: XXX (IL-12p40(E59K / K99E / N113Q / N281Q / C252S / K264E)), cvi) SEQ ID NO: XXX (IL-12p40(E59K / K99E / N200Q / N281Q / C252S / K264E)), cvii) SEQ ID NO: XXX (IL-12p40(E59K / K99E / N103Q / N113Q / N200Q / C252S / K264E)), cviii) SEQ ID NO: XXX (IL-12p40(E59K / K99E / N103Q / N200Q / N281Q / C252S / K264E)),and cix) SEQ ID NO: XXX (IL-12p40(E59K / K99E / N113Q / N200Q / N281Q / C252S / K264E)), The present invention provides a homodimeric Fc fusion protein having a polypeptide sequence selected from the group consisting of:

[0310] In some embodiments, the present invention relates to an IL-12 p35 subunit comprising one or more of the following: Q20, N21, Q35, E38, S44, E45, E46, H49, K54, D55, T59, V60, E61, C63, L64, P65, E67, L68, N71, S73, C74, L75, N76, E79, N85, L89, F96, M97, L124, M125, Q130, Q135, N The present invention provides homodimeric Fc fusion proteins having one or more amino acid modifications at amino acid residues selected from the group consisting of 136, E143, Q146, N151, E153, K158, E162, E163, D165, I171, R181, I182, R183, V185, T186, D188, R189, V190, S192, Y193, N195, and A196.

[0311] In some embodiments, the invention provides homodimeric Fc fusion proteins, wherein the IL-12p35 subunit has one or more amino acid substitutions selected from the group consisting of N21D, Q35D, E38Q, D55Q, D55K, N71D, N71Q, L75A, N76D, E79Q, N85D, N85Q, L89A, F96A, M97A, L124A, M125A, Q130E, Q135E, N136D, E143Q, Q146E, N151D, N151K, E153K, E153Q, K158E, E162Q, E163Q, D165N, I171A, N195D, and N195Q.

[0312] In some embodiments, the invention provides homodimeric Fc fusion proteins, wherein the IL-12p35 subunit has amino acid substitutions selected from the group consisting of N71D / N85D / N195D, N151D / E153Q, N151D / D165N, Q130E / N151D, N151D / K158E, E79Q / N151D, D55Q / N151D, N136D / N151D, N21D / N151D, E143Q / N151D, N71Q / N85Q, N71Q / N195Q, N85Q / N195Q, N71Q / N85Q / N195Q, N71D / N85D, N71D / N195D, and N85D / N195D.

[0313] In some embodiments, the present invention relates to a method for the preparation of IL-12p35 subunits comprising administering to a patient a therapeutically effective amount of the IL-12p35 subunit, the method comprising administering to a patient a therapeutically effective amount of the IL-12p35 subunit, the method comprising administering to a patient a therapeutically effective amount of the IL-12p35 subunit, the method comprising administering to a patient a therapeutically effective amount of the IL-12p35 subunit, 35(E38Q)), vii) SEQ ID NO:119 (IL-12p35(N151D)), viii) SEQ ID NO:120 (IL-12p35(Q135E)), ix) SEQ ID NO:121 (IL-12p35(Q35D)), x) SEQ ID NO:122 (IL-12p35(Q146E)), xi) SEQ ID NO:123 (IL-12p35(N76D)), xii) SEQ ID NO:124 (IL-12p35(E162Q)), xiii) SEQ ID NO:125 (IL-12p35 (E163Q)), xiv) IL-12p35(N21D), xv) SEQ ID NO:XXX (IL-12p35(D55Q)), xvi) IL-12p35(E79Q), xvii) IL-12p35(Q130E), xviii) IL-12p35(N136D), xix) IL-12p35(E143Q), xx) SEQ ID NO:227 (IL-12p35(N151K)), xxi) SEQ ID NO:226 (IL-12p35(E153K)), xxii) I 225 (IL-12p35(N151D / E153Q)), xxv) SEQ ID NO:228 (IL-12p35(N151D / D165N)), xxvi) SEQ ID NO:229 (IL-12p35(Q130E / N151D)), xxvii) SEQ ID NO:230 (IL-12p35(N151D / K158E)), xxviii) SEQ ID NO:231 (IL-1 2p35(E79Q / N151D)), xxix) SEQ ID NO:232 (IL-12p35(D55Q / N151D)), xxx) SEQ ID NO:233 (IL-12p35(N136D / N151D)), xxxi) SEQ ID NO:234 ​​(IL-12p35(N21D / N151D)), xxxii) SEQ ID NO:235 (IL-12p35(E143Q / N151D)), xxxiii) SEQ ID NO:XXX (IL-12p35(F96A)), xxxiv) SEQ ID NO:XXX (IL-12p35(M97A)), xxxv) SEQ ID NO:XXX(IL-12p35(L89A)), xxxvi) SEQ ID NO:XXX(IL-12p35(L124A)), xxxvii) SEQ ID NO:XXX(IL-12p35(M125A)), xxxviii) SEQ ID NO:XXX(IL-12p35(L75A)), xxxiv) SEQ ID NO:XXX(IL-12p35(I171A)), xxxv) SEQ ID NO:XXX(IL-12p35(N71Q)), xxxv i) SEQ ID NO:XXX(IL-12p35(N85Q)), xxxvii) SEQ ID NO:XXX(IL-12p35(N195Q)), xxxviii) SEQ ID NO:XXX(IL-12p35(N71Q / N85Q)), xxxix) SEQ ID NO:XXX(IL-12p35(N71Q / N195Q)), xl) SEQ ID NO:XXX(IL-12p35(N85Q / N195Q), xli) SEQ ID NO:XXX(IL-12p35(N71Q / N85Q / N195Q)), xli The present invention provides a homodimeric Fc fusion protein having a polypeptide sequence selected from the group consisting of: i) SEQ ID NO:XXX(IL-12p35(N71D / N85D)); xliii) SEQ ID NO:XXX(IL-12p35(N71D / N195D); xliv) SEQ ID NO:XXX(IL-12p35(N85D / N195D)); xlv) SEQ ID NO:XXX(IL-12p35(D55Q)); and xlvi) SEQ ID NO:XXX(IL-12p35(D55K)).

[0314] In some embodiments, a homodimeric Fc fusion protein is provided, wherein said first and second monomers are XENP31289.

[0315] In some embodiments, a homodimeric Fc fusion protein is provided, wherein said first and second monomers are XENP31291.

[0316] In some embodiments, the present invention provides a homodimeric Fc-fusion protein composition comprising a homodimeric Fc-fusion protein for use in treating cancer in a subject.

[0317] In some embodiments, the invention provides one or more nucleic acids encoding a homodimeric Fc fusion protein.

[0318] In some embodiments, the invention provides host cells comprising one or more of the nucleic acids encoding the homodimeric Fc fusion proteins.

[0319] In some embodiments, the invention provides a method of making a homodimeric Fc-fusion protein, the method comprising culturing a host cell under conditions whereby the homodimeric Fc-fusion protein is produced.

[0320] In some embodiments, the invention provides a method for purifying a homodimeric Fc-fusion protein, the method comprising: a) providing a composition comprising a homodimeric Fc-fusion protein; b) loading the composition onto an ion exchange column; and c) collecting fractions containing the homodimeric Fc-fusion protein.

[0321] In another aspect, the invention provides a homodimeric Fc fusion protein comprising a first monomer and a second monomer, each comprising, from N-terminus to C-terminus, IL-12p35 subunit domain-first domain linker-IL-12p40 subunit domain-second domain linker-Fc domain.

[0322] In some embodiments, the invention provides homodimeric Fc fusion proteins, wherein the modifications that promote homodimerization of the Fc domain are a set of amino acid substitutions selected from the group consisting of: L368D / K370S; S364K; S364K / E357L; S364K / E357Q; T411E / K360E / Q362E; D401K; T366S / L368A / Y407V; T366W; T366S / L368A / Y407V / Y349C; and T366W / S354C, according to EU numbering.

[0323] In some embodiments, the invention provides a homodimeric Fc fusion protein, wherein said first domain linker and said second domain linker have the same amino acid sequence.

[0324] In some embodiments, the invention provides homodimeric Fc fusion proteins, wherein the Fc domain has an additional set of amino acid substitutions comprising: Q295E / N384D / Q418E / N421D, according to EU numbering.

[0325] In some embodiments, the invention provides homodimeric Fc fusion proteins, wherein the Fc domain has an additional set of amino acid substitutions selected from the group consisting of G236R / L328R, E233P / L234V / L235A / G236_ / S239K, E233P / L234V / L235A / G236_ / S239K / A327G, E233P / L234V / L235A / G236_ / S267K / A327G, E233P / L234V / L235A / G236_, and E233P / L234V / L235A / G236_ / S267K, according to EU numbering.

[0326] In some embodiments, the invention provides homodimeric Fc fusion proteins, wherein the IL-12p40 subunit has a polypeptide sequence selected from the group consisting of SEQ ID NO:3 (human IL-12 subunit beta (IL-12p40) precursor sequence) and SEQ ID NO:4 (human IL-12 subunit beta (IL-12p40) mature form sequence), and the IL-12p35 subunit has a polypeptide sequence selected from the group consisting of SEQ ID NO:1 (human IL-12 subunit alpha (IL-12p35) precursor sequence) and SEQ ID NO:2 (human IL-12 subunit alpha (IL-12p35) mature form sequence).

[0327] In some embodiments, the invention provides a homodimeric Fc fusion protein, wherein each of the Fc domains further comprises the amino acid substitutions M428L / N424S.

[0328] In some embodiments, the invention provides homodimeric Fc fusion proteins, wherein said IL-12p40 subunit is a variant IL-12p40 subunit and / or said IL-12p35 subunit is a variant IL-12p35 subunit.

[0329] In some embodiments, the invention provides homodimeric Fc fusion proteins wherein said IL-12p40 subunit is IL-12 receptor subunit beta-1 (IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2), and / or a variant IL-12p40 subunit with altered affinity for the IL-12 receptor complex, and / or said IL-12p35 subunit is IL-12 receptor subunit beta-1 (IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2), and / or a variant IL-12p35 subunit with altered affinity for the IL-12 receptor complex.

[0330] In some embodiments, the IL-12p40 subunit is selected from the group consisting of E3, D7, E12, D14, W15, P17, D18, A19, P20, G21, E22, M23, D29, E32, E33, D34, L40, D41, Q42, S43, E45, L47, T54, I55, Q56, K58, E59, F60, G 61, D62, Q65, Y66, E73, K84, E86, D87, G88, I89, W90, D93, D97, K99, E100, K102, N103, K104, F106, E110, N113, Y114, D129, D142, Q144, E156, R159, D161, N162, K163, D166, D 170, Q172, D174, A176, C177, P178, A179, A180, E181, S183, P185, E187, N200, S204, F 206, R208, D209, D214, N218, Q220, N226, Q229, E231, E235, T242, P243, S245, Y246, F2 47, S248, C252S, , Q256, K158, K260, E262, K264, D265, D270, N281, Q289, D290, R291, Y292, Y293, and E299.

[0331] In some embodiments, the present invention relates to an IL-12p40 subunit comprising D18N, D18K, E32Q, E33Q, D34N, D34K, Q42E, S43E, S43K, E45Q, Q56E, E59Q, E59K, D62N, E73Q, D87N, K99E, K99Y, E100Q, N103D, N103Q, N113D, N113Q, Q144 The present invention provides homodimeric Fc fusion proteins having one or more amino acid substitutions selected from the group consisting of E, D161N, R159E, K163E, E187Q, N200D, N200Q, N218Q, Q229E, E235Q, C252S, Q256N, K258E, K260E, E262Q, K264E, N281D, N281Q, and E299Q.

[0332] In some embodiments, the present invention relates to a method for the preparation of IL-12p40 subunits comprising administering to the patient a sequence similar to that described herein, comprising administering to the patient a sequence similar to that described herein, and / or administering to the patient a sequence similar to that described herein, , S43K / E49Q, E59Q / K163E, E59Q / K99E, E59Q / K258E, E59Q / K260E, E59K / K99 E, D18K / E59K / K99E, E59K / K99E / K264E, E59K / K99Y, E59Y / K99Y, E59Y / K99E, E45K / E59K / K99E, E59K / K99E / Q144E, E59K / K99E / Q144K, E59K / K99E / R159E , E59K / K99E / K264E, D18K / E59K / K99E / K264E, DI8K / E59K / K99E / C252S, D18K / E59K / K99E / C252S / K264E, E59K / K99Y / C252S, E59K / K99E / C252S / K264E, E 59K / K99E / C252S, N103D / N113D, N103D / N200D, N103D / N281D, N113D / N200D, N113D / N281D, N200D / N281D, N103D / N113D / N200D, N103D / N113D / N281D, N1 03D / N200D / N281D, N113D / N200D / N281D, N103Q / N113Q, N103Q / N200Q, N103Q / N281Q, N113Q / N200Q, N113Q / N281Q, N200Q / N281Q, N103Q / N113Q / N200Q, N 103Q / N113Q / N281Q, N103Q / N200Q / N281Q, N113Q / N200Q / N281Q, N103Q / N113 Q / N200Q / N281Q, E59K / K99E / N103Q / C252S / K264E, E59K / K99E / N113Q / C252 S / K264E, E59K / K99E / N200Q / C252S / K264E, E59K / K99E / N281Q / C252S / K264E , E59K / K99E / N103Q / N113Q / C252S / K264E, E59K / K99E / N103Q / N200Q / C252S / K264E, E59K / K99E / N103Q / N281Q / C252S / K264E, E 59K / K99E / N113Q / N200Q / C252S / K264E, E59K / K99E / N113Q / N281Q / C252S / K264E, E59K / K99E / N200Q / N281Q / C252S / K264E, E59 Provided are homodimeric Fc fusion proteins having amino acid substitutions selected from the group consisting of K / K99E / N103Q / N113Q / N200Q / C252S / K264E, E59K / K99E / N103Q / N200Q / N281Q / C252S / K264E, E59K / K99E / N113Q / N200Q / N281Q / C252S / K264E, and E59K / K99E / N103Q / N113Q / N200Q / N281Q / C252S / K264E. In some embodiments, the present invention relates to an IL-12p40 subunit comprising: i) SEQ ID NO:57 (IL-12p40(N103D)), ii) SEQ ID NO:58 (IL-12p40(N113D)), iii) SEQ ID NO:59 (IL-12p40(N200D)), iv) SEQ ID NO:60 (IL-12p40(N281D)), v) SEQ ID NO:61 (IL-12p40(N103D / N113D / N200D / N281D)), vi) SEQ ID NO:62 (IL-12p40(Q42E)), vii) SEQ ID NO:63 (IL-12p40(E45Q)), viii) SEQ ID NO:64 (IL-12p40(Q56E)), ix) SEQ ID NO:65 (IL -12p40(E59Q)), x) SEQ ID NO: 66 (IL-12p40(D62N)), xi) SEQ ID NO: 67 (IL-12p40(Q42E / E45Q)), xii) SEQ ID NO: 68 (IL-12p40(E45Q / Q56E)), xiii) SEQ ID NO: 69 (IL-12p40(Q42E / E59Q)), xiv) SEQ ID NO: 70 (IL-12p40(Q56E / E59Q)), xv) SEQ ID NO: 71 (IL-12p40(Q42E / E45Q / Q56E)), xvi) SEQ ID NO: 72 (IL-12p40(E45Q / Q56E / E59Q)), xvii) SEQ ID NO:73 (IL-12p40(D161N)), xviii) SEQ ID NO:74 (IL-12p40(E73Q)), xix) SEQ ID NO:75 (IL-12p40(Q144E)), xx) SEQ ID NO:76 (IL-12p40(E262Q)), xxi) SEQ ID NO:77 (IL-12p40(E100Q)), xxii) SEQ ID NO:78 (IL-12p40(D18N)), xxiii) SEQ ID NO:79 (IL-12p40(E33Q)), xxiv) SEQ ID NO:80 (IL-12p40(Q229E)), xxv) SEQ ID NO:81 (IL-12p40(E23 5Q), xxvi) SEQ ID NO: 82 (IL-12p40(Q256N)), xxvii) SEQ ID NO: 83 (IL-12p40(E299Q)), xxviii) SEQ ID NO: 84 (IL-12p40(D87N)), xxix) IL-12p40(E32Q), xxx) IL-12p40(D34N), xxxi) IL-12p40(S43E), xxxii) IL-12p40(S43K), xxxiii) SEQ ID NO: XXX (IL-12p40(E59K / K99E / N103Q / N113Q / N200Q / N281Q / C252S / K264E)),xxxiv) SEQ ID NO: 205 (IL-12p40(E59K)), xxxv) IL-12p40(K99E), xxxvi) IL-12p40(K163E), xxxvii) IL-12p40(E187Q), xxxviii) IL-12p40(K258E), xxxix) IL-12p40(K260E), xl) SEQ ID NO: 206 (IL-12p40(E32Q / E59Q)), xli) SEQ ID NO: 207 (IL-12p40(D34N / E59Q)), xlii) SEQ ID NO: 208 (IL-12p40(E59Q / E187Q)), xliii) SEQ ID NO: 209 (IL-12p40(S43E / E 59Q), xliv) SEQ ID NO:210 (IL-12p40(S43K / E49Q)), xlv) SEQ ID NO:211 (IL-12p40(E59Q / K163E)), xlvi) SEQ ID NO:212 (IL-12p40(E59Q / K99E)), xlvii) SEQ ID NO:213 (IL-12p40(E59Q / K258E)), xlviii) SEQ ID NO:214 (IL-12p40(E59Q / K260E)), xlix) SEQ ID NO:XXX (IL-12p40(D34N / E59K)), l) SEQ ID NO:XXX (IL-12p40(E59K / K99E)), li) SEQ ID NO:XXX (IL-12p40(D18, IL-12p40(E59K / K99E / K264E)), liii) SEQ ID NO: XXX(IL-12p40(E59K / K99Y)), liv) SEQ ID NO: XXX(IL-12p40(E59Y / K99E)), lv) SEQ ID NO: XXX(IL-12p40(E45K / E59K / K99E)), lvi) SEQ ID NO: XXX(IL-12p40(E59K / K99E / Q144E)), lvii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / Q144K)), lviii) SEQ ID NO: XXX( IL-12p40(E59K / K99E / R159E)), lix) SEQ ID NO: XXX(IL-12p40(E59K / K99E / K264E)), lx) SEQ ID NO: XXX(IL-12p40(D18K / E59K / K99E / K264E)), lxi) SEQ ID NO: XXX(IL-12p40(C252S)), lxii) SEQ ID NO: XXX(IL-12p40(DI8K / E59K / K99E / C252S)), lxiii) SEQ ID NO: XXX(IL-12p40(D18K / E59K / K99E / C252S / K264E)), lxiv) SEQ ID NO: XXX(IL -12p40(E59K / K99Y / C252S)), lxv) SEQ ID NO: XXX(IL-12p40(E59K / K99E / C252S / K264E)), lxvi) SEQ ID NO: XXX(IL-12p40(E59K / K99E / C252S)), lxvii) SEQ ID NO: XXX(IL-12p40(N103D / N113D)), lxviii) SEQ ID NO: XXX(IL-12p40(N103D / N200D)), lxix) SEQ ID NO: XXX(IL-12p40(N103D / N281D)), lxx) SEQ ID NO: XXX(IL-12p40(N113D / N2 00D), lxxi) SEQ ID NO: XXX(IL-12p40(N113D / N281D)), lxxii) SEQ ID NO: XXX(IL-12p40(N200D / N281D)), lxxiii) SEQ ID NO: XXX(IL-12p50(N103D / N113D / N200D)), lxxiv) SEQ ID NO: XXX(IL-12p40(N103D / N113D / N281D)), lxxv) SEQ ID NO: XXX(IL-12p40(N103D / N200D / N281D)), lxxvi) SEQ ID NO: XXX(IL-12p40(N113D / N200D / N281D)),lxxvii) SEQ ID NO:XXX(IL-12p40(N103Q)), lxxviii) SEQ ID NO:XXX(IL-12p40(N113Q)), lxxix) SEQ ID NO:XXX(IL-12p40(N200Q)), lxxx) SEQ ID NO:XXX(IL-12p40(N281Q)), lxxxi) SEQ ID NO:XXX(IL-12p40(N103Q / N113Q)), lxxxii) SEQ ID NO:XXX(IL-12p40(N103Q / N200Q)), lxxxiii) SEQ ID NO:XXX(IL-12p40(N103Q / N281Q)), lxxxiv) SEQ ID NO: No. XXX(IL-12p40(N113Q / N200Q)), lxxxv) SEQ ID NO: XXX(IL-12p40(N113Q / N281Q)), lxxxvi) SEQ ID NO: XXX(IL-12p40(N200Q / N281Q)), lxxxvii) SEQ ID NO: XXX(IL-12p40(N103Q / N113Q / N200Q)), lxxxviii) SEQ ID NO: XXX(IL-12p40(N103Q / N113Q / N281Q)), lxxxix) SEQ ID NO: XXX(IL-12p40(N103Q / N200Q / N281Q)), xc) SEQ ID NO: XXX(IL-12p40(N103Q / N200Q / N281Q)), IL-12p40(N113Q / N200Q / N281Q)), xci) SEQ ID NO: XXX(IL-12p40(N103Q / N113Q / N200Q / N281Q)), xcii) SEQ ID NO: XXX(IL-12p40(D34N / E59K / K99E)), xciii) SEQ ID NO: XXX(IL-12p40(D34K / E59K / K99E)), xciv) SEQ ID NO: XXX(IL-12p40(E32Q / D34N / E59K / K99E)), xcv) SEQ ID NO: XXX(IL-12p40(E32K / D34N / E59K / K99E)), xcvi) SEQ ID NO: XXX(IL-12p40(E59Y / K99Y)), xcvii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N103Q / C252S / K264E)), xcviii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N113Q / C252S / K264E)), xcix) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N200Q / C252S / K264E)), c) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N281Q / C252S / K264E)), ci) SEQ ID NO: XXX(IL-12, p40(E59K / K99E / N103Q / N113Q / C252S / K264E)), cii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N103Q / N200Q / C252S / K264E)), ciii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N103Q / N281Q / civ) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N113Q / N200Q / C252S / K264E)), cv) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N113Q / N281Q / C252S / K264E)), cvi) SEQ ID NO: XXX(IL The present invention provides homodimeric Fc fusion proteins having a polypeptide sequence selected from the group consisting of SEQ ID NO: XXX (IL-12p40(E59K / K99E / N200Q / N281Q / C252S / K264E)), cvii) SEQ ID NO: XXX (IL-12p40(E59K / K99E / N103Q / N113Q / N200Q / C252S / K264E)), cviii) SEQ ID NO: XXX (IL-12p40(E59K / K99E / N103Q / N200Q / N281Q / C252S / K264E)), and cix) SEQ ID NO: XXX (IL-12p40(E59K / K99E / N113Q / N200Q / N281Q / C252S / K264E)).

[0333] In some embodiments, the present invention relates to an IL-12 p35 subunit comprising one or more of the following: Q20, N21, Q35, E38, S44, E45, E46, H49, K54, D55, T59, V60, E61, C63, L64, P65, E67, L68, N71, S73, C74, L75, N76, E79, N85, L89, F96, M97, L124, M125, Q130, Q135, N The present invention provides homodimeric Fc fusion proteins having one or more amino acid modifications at amino acid residues selected from the group consisting of 136, E143, Q146, N151, E153, K158, E162, E163, D165, I171, R181, I182, R183, V185, T186, D188, R189, V190, S192, Y193, N195, and A196.

[0334] In some embodiments, the invention provides homodimeric Fc fusion proteins, wherein the IL-12p35 subunit has one or more amino acid substitutions selected from the group consisting of N21D, Q35D, E38Q, D55Q, D55K, N71D, N71Q, L75A, N76D, E79Q, N85D, N85Q, L89A, F96A, M97A, L124A, M125A, Q130E, Q135E, N136D, E143Q, Q146E, N151D, N151K, E153K, E153Q, K158E, E162Q, E163Q, D165N, I171A, N195D, and N195Q.

[0335] In some embodiments, the invention provides homodimeric Fc fusion proteins, wherein the IL-12p35 subunit has amino acid substitutions selected from the group consisting of N71D / N85D / N195D, N151D / E153Q, N151D / D165N, Q130E / N151D, N151D / K158E, E79Q / N151D, D55Q / N151D, N136D / N151D, N21D / N151D, E143Q / N151D, N71Q / N85Q, N71Q / N195Q, N85Q / N195Q, N71Q / N85Q / N195Q, N71D / N85D, N71D / N195D, and N85D / N195D.

[0336] In some embodiments, the present invention relates to a method for the preparation of a IL-12p35 subunit comprising administering to a patient a IL-12p35 polypeptide comprising the steps of: i) SEQ ID NO:113 (IL-12p35(N71D)), ii) SEQ ID NO:114 (IL-12p35(N85D)), iii) SEQ ID NO:115 (IL-12p35(N195D)), iv) SEQ ID NO:116 (IL-12p35(N71D / N85D / N195D)), v) SEQ ID NO:117 (IL-12p35(E153Q)), vi) SEQ ID NO:118 (IL-12p35(E38Q)), vii) SEQ ID NO:119 (IL-12p35(N151D)), viii) SEQ ID NO:120 (IL-12p35(Q135E)), ix) SEQ ID NO:121 ( IL-12p35(Q35D)), x) SEQ ID NO: 122 (IL-12p35(Q146E)), xi) SEQ ID NO: 123 (IL-12p35(N76D)), xii) SEQ ID NO: 124 (IL-12p35(E162Q)), xiii) SEQ ID NO: 125 (IL-12p35(E163Q)), xiv) IL-12p35(N21D), xv) SEQ ID NO: XXX (IL-12p35(D55Q)), xvi) IL-12p35(E79Q), xvii) IL-12p35(Q130E), xviii) IL-12p35(N136D), xix) (IL-12 p35(E143Q)), xx) SEQ ID NO: 227 (IL-12p35(N151K)), xxi) SEQ ID NO: 226 (IL-12p35(E153K)), xxii) IL-12p35(K158E), xxiii) IL-12p35(D165N), xxiv) SEQ ID NO: 225 (IL-12p35(N151D / E153Q)), xxv) SEQ ID NO: 228 (IL-12p35(N151D / D165N)), xxvi) SEQ ID NO: 229 (IL-12p35(Q130E / N151D)), xxvii) SEQ ID NO: 230 (IL-12p35(N151D / K158E), xxviii) SEQ ID NO: 231 (IL-12p35(E79Q / N151D)), xxix) SEQ ID NO: 232 (IL-12p35(D55Q / N151D)), xxx) SEQ ID NO: 233 (IL-12p35(N136D / N151D)), xxxi) SEQ ID NO: 234 (IL-12p35(N21D / N151D)), xxxii) SEQ ID NO: 235 (IL-12p35(E143Q / N151D)), xxxiii) SEQ ID NO: XXX (IL-12p35(F96A)), xxxiv) SEQ ID NO: XXX (IL-12p35(M97A) ), xxxv) SEQ ID NO:XXX(IL-12p35(L89A)), xxxvi) SEQ ID NO:XXX(IL-12p35(L124A)), xxxvii) SEQ ID NO:XXX(IL-12p35(M125A)), xxxviii) SEQ ID NO:XXX(IL-12p35(L75A)), xxxiv) SEQ ID NO:XXX(IL-12p35(I171A)), xxxv) SEQ ID NO:XXX(IL-12p35(N71Q)), xxxvi) SEQ ID NO:XXX(IL-12p35(N85Q)), xxxvii) SEQ ID NO:XXX(IL-12p35(N195Q)),xxxviii) SEQ ID NO:XXX(IL-12p35(N71Q / N85Q)), xxxix) SEQ ID NO:XXX(IL-12p35(N71Q / N195Q)), xl) SEQ ID NO:XXX(IL-12p35(N85Q / N195Q), xli) SEQ ID NO:XXX(IL-12p35(N71Q / N85Q / N195Q)), xlii) SEQ ID NO:XXX(IL-12p35(N71D / N85D) xlii) SEQ ID NO:XXX(IL-12p35(N71D / N195D), xliv) SEQ ID NO:XXX(IL-12p35(N85D / N195D)), xlv) SEQ ID NO:XXX(IL-12p35(D55Q)), and xlvi) SEQ ID NO:XXX(IL-12p35(D55K)).

[0337] In some embodiments, a homodimeric Fc fusion protein is provided, wherein said first and second monomers are XENP31289.

[0338] In some embodiments, a homodimeric Fc fusion protein is provided, wherein said first and second monomers are XENP31291.

[0339] In some embodiments, the present invention provides a homodimeric Fc-fusion protein composition comprising a homodimeric Fc-fusion protein for use in treating cancer in a subject.

[0340] In some embodiments, the invention provides one or more nucleic acids encoding a homodimeric Fc fusion protein.

[0341] In some embodiments, the invention provides host cells comprising one or more of the nucleic acids encoding the homodimeric Fc fusion proteins.

[0342] In some embodiments, the invention provides a method of making a homodimeric Fc-fusion protein, the method comprising culturing a host cell under conditions whereby the homodimeric Fc-fusion protein is produced.

[0343] In some embodiments, the invention provides a method for purifying a homodimeric Fc-fusion protein, the method comprising: a) providing a composition comprising a homodimeric Fc-fusion protein; b) loading the composition onto an ion exchange column; and c) collecting fractions containing the homodimeric Fc-fusion protein.

[0344] In another aspect, the invention provides a homodimeric Fc fusion protein comprising a first monomer and a second monomer, each comprising, from N-terminus to C-terminus, Fc domain-first domain linker-IL-12p40 subunit domain-second domain linker-IL-12p35 subunit domain.

[0345] In some embodiments, the invention provides homodimeric Fc fusion proteins, wherein the modifications that promote homodimerization of the Fc domain are a set of amino acid substitutions selected from the group consisting of: L368D / K370S; S364K; S364K / E357L; S364K / E357Q; T411E / K360E / Q362E; D401K; T366S / L368A / Y407V; T366W; T366S / L368A / Y407V / Y349C; and T366W / S354C, according to EU numbering.

[0346] In some embodiments, the invention provides a homodimeric Fc fusion protein, wherein said first domain linker and said second domain linker have the same amino acid sequence.

[0347] In some embodiments, the invention provides homodimeric Fc fusion proteins, wherein the Fc domain has an additional set of amino acid substitutions comprising: Q295E / N384D / Q418E / N421D, according to EU numbering.

[0348] In some embodiments, the invention provides homodimeric Fc fusion proteins, wherein the Fc domain has an additional set of amino acid substitutions selected from the group consisting of G236R / L328R, E233P / L234V / L235A / G236_ / S239K, E233P / L234V / L235A / G236_ / S239K / A327G, E233P / L234V / L235A / G236_ / S267K / A327G, E233P / L234V / L235A / G236_, and E233P / L234V / L235A / G236_ / S267K, according to EU numbering.

[0349] In some embodiments, the invention provides homodimeric Fc fusion proteins, wherein the IL-12p40 subunit has a polypeptide sequence selected from the group consisting of SEQ ID NO:3 (human IL-12 subunit beta (IL-12p40) precursor sequence) and SEQ ID NO:4 (human IL-12 subunit beta (IL-12p40) mature form sequence), and the IL-12p35 subunit has a polypeptide sequence selected from the group consisting of SEQ ID NO:1 (human IL-12 subunit alpha (IL-12p35) precursor sequence) and SEQ ID NO:2 (human IL-12 subunit alpha (IL-12p35) mature form sequence).

[0350] In some embodiments, the invention provides a homodimeric Fc fusion protein, wherein each of the Fc domains further comprises the amino acid substitutions M428L / N424S.

[0351] In some embodiments, the present invention relates to a method for the preparation of IL-12p40 subunits comprising administering to a patient a therapeutically effective amount of The present invention provides a homodimeric Fc fusion protein, wherein the IL-12p40 subunit is a variant IL-12p35 subunit.

[0352] In some embodiments, the invention provides homodimeric Fc fusion proteins wherein said IL-12p40 subunit is IL-12 receptor subunit beta-1 (IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2), and / or a variant IL-12p40 subunit with altered affinity for the IL-12 receptor complex, and / or said IL-12p35 subunit is IL-12 receptor subunit beta-1 (IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2), and / or a variant IL-12p35 subunit with altered affinity for the IL-12 receptor complex.

[0353] In some embodiments, the IL-12p40 subunit is selected from the group consisting of E3, D7, E12, D14, W15, P17, D18, A19, P20, G21, E22, M23, D29, E32, E33, D34, L40, D41, Q42, S43, E45, L47, T54, I55, Q56, K58, E59, F60, G 61, D62, Q65, Y66, E73, K84, E86, D87, G88, I89, W90, D93, D97, K99, E100, K102, N103, K104, F106, E110, N113, Y114, D129, D142, Q144, E156, R159, D161, N162, K163, D166, D 170, Q172, D174, A176, C177, P178, A179, A180, E181, S183, P185, E187, N200, S204, F 206, R208, D209, D214, N218, Q220, N226, Q229, E231, E235, T242, P243, S245, Y246, F2 47, S248, C252S, , Q256, K158, K260, E262, K264, D265, D270, N281, Q289, D290, R291, Y292, Y293, and E299.

[0354] In some embodiments, the present invention relates to an IL-12p40 subunit comprising D18N, D18K, E32Q, E33Q, D34N, D34K, Q42E, S43E, S43K, E45Q, Q56E, E59Q, E59K, D62N, E73Q, D87N, K99E, K99Y, E100Q, N103D, N103Q, N113D, N113Q, Q144 The present invention provides homodimeric Fc fusion proteins having one or more amino acid substitutions selected from the group consisting of E, D161N, R159E, K163E, E187Q, N200D, N200Q, N218Q, Q229E, E235Q, C252S, Q256N, K258E, K260E, E262Q, K264E, N281D, N281Q, and E299Q.

[0355] In some embodiments, the present invention relates to a method for the preparation of IL-12p40 subunits comprising administering to the patient a sequence similar to that described herein, comprising administering to the patient a sequence similar to that described herein, wherein the IL-12p40 subunit is N103D / N113D / N200D / N281D, Q42E / E45Q, E45Q / Q56E, Q42E / E59Q, Q56E / E59Q, Q42E / E45Q / Q56E, E45Q / Q56E / E59Q, E32Q / E59Q, D34N / E59K, D34N / E59K / K99E, D34K / E59K / K99E, E32Q / D34N / E59K / K99E, E32K / D34N / E59K / K99E, D34N / E59Q, E59Q / E187Q , S43E / E59Q, S43K / E49Q, E59Q / K163E, E59Q / K99E, E59Q / K258E, E59Q / K2 60E, E59K / K99E, D18K / E59K / K99E, E59K / K99E / K264E, E59K / K99Y, E59Y / K99Y, E59Y / K99E, E45K / E59K / K99E, E59K / K99E / Q144E, E59K / K99E / Q144 K, E59K / K99E / R159E, E59K / K99E / K264E, D18K / E59K / K99E / K264E, DI8K / E59K / K99E / C252S, D18K / E59K / K99E / C252S / K264E, E59K / K99Y / C252S , E59K / K99E / C252S / K264E, E59K / K99E / C252S, N103D / N113D, N103D / N2 00D, N103D / N281D, N113D / N200D, N113D / N281D, N200D / N281D, N103D / N113D / N200D, N103D / N113D / N281D, N103D / N200D / N281D, N113D / N200D / N281D, N103Q / N113Q, N103Q / N200Q, N103Q / N281Q, N113Q / N200Q, N11 3Q / N281Q, N200Q / N281Q, N103Q / N113Q / N200Q, N103Q / N113Q / N281Q, N1 03Q / N200Q / N281Q, N113Q / N200Q / N281Q, N103Q / N113Q / N200Q / N281Q, E59K / K99E / N103Q / C252S / K264E, E59K / K99E / N113Q / C252S / K264E, E59 K / K99E / N200Q / C252S / K264E, E59K / K99E / N281Q / C252S / K264E, E59K / K99E / N103Q / N113Q / C252S / K264E, E59K / K99E / N103Q / N200Q / C252S / K2 64E, E59K / K99E / N103Q / N281Q / C252S / K264E, E59K / K99E / N113Q / N200 Q / C252S / K264E, E59K / K99E / N113Q / N281Q / C252S / K264E, E59K / K99E / N The present invention provides homodimeric Fc fusion proteins having amino acid substitutions selected from the group consisting of 200Q / N281Q / C252S / K264E, E59K / K99E / N103Q / N113Q / N200Q / C252S / K264E, E59K / K99E / N103Q / N200Q / N281Q / C252S / K264E, E59K / K99E / N113Q / N200Q / N281Q / C252S / K264E, and E59K / K99E / N103Q / N113Q / N200Q / N281Q / C252S / K264E. In some embodiments, the present invention relates to an IL-12p40 subunit comprising: i) SEQ ID NO:57 (IL-12p40(N103D)), ii) SEQ ID NO:58 (IL-12p40(N113D)), iii) SEQ ID NO:59 (IL-12p40(N200D)), iv) SEQ ID NO:60 (IL-12p40(N281D)), v) SEQ ID NO:61 (IL-12p40(N103D / N113D / N200D / N281D)), vi) SEQ ID NO:62 (IL-12p40(Q42E)), vii) SEQ ID NO:63 (IL-12p40(E45Q)), viii) SEQ ID NO:64 (IL-12p40(Q56E)), ix) SEQ ID NO:65 (IL-12p4 0(E59Q)), x) SEQ ID NO:66 (IL-12p40(D62N)), xi) SEQ ID NO:67 (IL-12p40(Q42E / E45Q)), xii) SEQ ID NO:68 (IL-12p40(E45Q / Q56E)), xiii) SEQ ID NO:69 (IL-12p40(Q42E / E59Q)), xiv) SEQ ID NO:70 (IL-12p40(Q56E / E59Q)), xv) SEQ ID NO:71 (IL-12p40(Q42E / E45Q / Q56E)), xvi) SEQ ID NO:72 (IL-12p40(E45Q / Q56E / E59Q)), xvii) SEQ ID NO:73 (IL- IL-12p40(D161N)), xviii) SEQ ID NO: 74 (IL-12p40(E73Q)), xix) SEQ ID NO: 75 (IL-12p40(Q144E)), xx) SEQ ID NO: 76 (IL-12p40(E262Q)), xxi) SEQ ID NO: 77 (IL-12p40(E100Q)), xxii) SEQ ID NO: 78 (IL-12p40(D18N)), xxiii) SEQ ID NO: 79 (IL-12p40(E33Q)), xxiv) SEQ ID NO: 80 (IL-12p40(Q229E)), xxv) SEQ ID NO: 81 (IL-12p40(E235Q)), xxvi) SEQ ID NO: 8 2 (IL-12p40(Q256N)), xxvii) SEQ ID NO: 83 (IL-12p40(E299Q)), xxviii) SEQ ID NO: 84 (IL-12p40(D87N)), xxix) IL-12p40(E32Q), xxx) IL-12p40(D34N), xxxi) IL-12p40(S43E), xxxii) IL-12p40(S43K), xxxiii) SEQ ID NO: XXX (IL-12p40(E59K / K99E / N103Q / N113Q / N200Q / N281Q / C252S / K264E)), xxxiv) SEQ ID NO: 205 (IL-12.p40(E59K)), xxxv) IL-12p40(K99E), xxxvi) IL-12p40(K163E), xxxvii) IL-12p40(E187Q), xxxviii) IL-12p40(K258E), xxxix) IL-12p40(K260E), xl) SEQ ID NO: 206 (IL-12p40(E32Q / E59Q)), xli) SEQ ID NO: 207 (IL-12p40(D34N / E59Q)), xlii) SEQ ID NO: 208 (IL-12p40(E59Q / E187Q)), xliii) SEQ ID NO: 209 (IL -12p40(S43E / E59Q)), xliv) SEQ ID NO: 210 (IL-12p40(S43K / E49Q)), xlv) SEQ ID NO: 211 (IL-12p40(E59Q / K163E)), xlvi) SEQ ID NO: 212 (IL-12p40(E59Q / K99E)), xlvii) SEQ ID NO: 213 (IL-12p40(E59Q / K258E)), xlviii) SEQ ID NO: 214 (IL-12p40(E59Q / K260E)), xlix) SEQ ID NO: XXX (IL-12p40(D34N / E59K)), l) SEQ ID NO: XXX ( IL-12p40(E59K / K99E)), li) SEQ ID NO: XXX(IL-12p40(D18K / E59K / K99E)), lii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / K264E)), liii) SEQ ID NO: XXX(IL-12p40(E59K / K99Y)), liv) SEQ ID NO: XXX(IL-12p40(E59Y / K99E)), lv) SEQ ID NO: XXX(IL-12p40(E45K / E59K / K99E)), lvi) SEQ ID NO: XXX(IL-12p40(E59K / K99E / Q144E) ), lvii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / Q144K)), lviii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / R159E)), lix) SEQ ID NO: XXX(IL-12p40(E59K / K99E / K264E)), lx) SEQ ID NO: XXX(IL-12p40(D18K / E59K / K99E / K264E)), lxi) SEQ ID NO: XXX(IL-12p40(C252S)), lxii) SEQ ID NO: XXX(IL-12p40(DI8K / E59K / K99E / C252S)),lxiii) SEQ ID NO: XXX(IL-12p40(D18K / E59K / K99E / C252S / K264E)), lxiv) SEQ ID NO: XXX(IL-12p40(E59K / K99Y / C252S)), lxv) SEQ ID NO: XXX(IL-12p40(E59K / K99E / C252S / K264E)), lxvi) SEQ ID NO: XXX(IL-12p40(E59K / K99E / C252S)), lxvii) SEQ ID NO: XXX(IL-12p40(N103D / N113D)), lxviii) SEQ ID NO: XXX(IL-12p40(N10 lxix) SEQ ID NO: XXX(IL-12p40(N103D / N281D)), lxx) SEQ ID NO: XXX(IL-12p40(N113D / N200D)), lxxi) SEQ ID NO: XXX(IL-12p40(N113D / N281D)), lxxii) SEQ ID NO: XXX(IL-12p40(N200D / N281D)), lxxiii) SEQ ID NO: XXX(IL-12p50(N103D / N113D / N200D)), lxxiv) SEQ ID NO: XXX(IL-12p40(N103D / N113D / N281D)), lxxv) SEQ ID NO:XXX(IL-12p40(N103D / N200D / N281D)), lxxvi) SEQ ID NO:XXX(IL-12p40(N113D / N200D / N281D)), lxxvii) SEQ ID NO:XXX(IL-12p40(N103Q)), lxxviii) SEQ ID NO:XXX(IL-12p40(N113Q)), lxxix) SEQ ID NO:XXX(IL-12p40(N200Q)), lxxx) SEQ ID NO:XXX(IL-12p40(N281Q)), lxxxi) SEQ ID NO:XXX(IL-12p40(N103Q / N 113Q), lxxxii) SEQ ID NO: XXX(IL-12p40(N103Q / N200Q)), lxxxiii) SEQ ID NO: XXX(IL-12p40(N103Q / N281Q)), lxxxiv) SEQ ID NO: XXX(IL-12p40(N113Q / N200Q)), lxxxv) SEQ ID NO: XXX(IL-12p40(N113Q / N281Q)), lxxxvi) SEQ ID NO: XXX(IL-12p40(N200Q / N281Q)), lxxxvii) SEQ ID NO: XXX(IL-12p40(N103Q / N113Q / N200Q)),lxxxviii) SEQ ID NO: XXX (IL-12p40(N103Q / N113Q / N281Q)), lxxxix) SEQ ID NO: XXX (IL-12p40(N103Q / N200Q), / N281Q)), xc) SEQ ID NO: XXX(IL-12p40(N113Q / N200Q / N281Q)), xci) SEQ ID NO: XXX(IL-12p40(N103Q / N113Q / N200Q / N281Q)), xcii) SEQ ID NO: XXX(IL-12p40(D34N / E59K / K99E)), xciii) SEQ ID NO: XXX(IL-12p40(D34K / E59K / K99E)), xciv) SEQ ID NO: XXX(IL-12p40(E32Q / D34N / E59K / K99E)), xcv) SEQ ID NO: XXX(IL-12p40(E32 K / D34N / E59K / K99E)), xcvi) SEQ ID NO: XXX(IL-12p40(E59Y / K99Y)), xcvii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N103Q / C252S / K264E)), xcviii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N113Q / C252S / K264E)), xcix) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N200Q / C252S / K264E)), c) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N28 1Q / C252S / K264E)), ci) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N103Q / N113Q / C252S / K264E)), cii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N103Q / N200Q / C252S / K264E)), ciii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N103Q / N281Q / C252S / K264E)), civ) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N113Q / N200Q / C252S / K264E) ), cv) SEQ ID NO: XXX (IL-12p40(E59K / K99E / N113Q / N281Q / C252S / K264E)), cvi) SEQ ID NO: XXX (IL-12p40(E59K / K99E / N200Q / N281Q / C252S / K264E)), cvii) SEQ ID NO: XXX (IL-12p40(E59K / K99E / N103Q / N113Q / N200Q / C252S / K264E)), cviii) SEQ ID NO: XXX (IL-12p40(E59K / K99E / N103Q / N200Q / N281Q / C252S / K264E)),and cix) SEQ ID NO:XXX (IL-12p40(E59K / K99E / N113Q / N200Q / N281Q / C252S / K264E)).

[0356] In some embodiments, the present invention relates to an IL-12 p35 subunit comprising one or more of the following: Q20, N21, Q35, E38, S44, E45, E46, H49, K54, D55, T59, V60, E61, C63, L64, P65, E67, L68, N71, S73, C74, L75, N76, E79, N85, L89, F96, M97, L124, M125, Q130, Q135, N The present invention provides homodimeric Fc fusion proteins having one or more amino acid modifications at amino acid residues selected from the group consisting of 136, E143, Q146, N151, E153, K158, E162, E163, D165, I171, R181, I182, R183, V185, T186, D188, R189, V190, S192, Y193, N195, and A196.

[0357] In some embodiments, the invention provides homodimeric Fc fusion proteins, wherein the IL-12p35 subunit has one or more amino acid substitutions selected from the group consisting of N21D, Q35D, E38Q, D55Q, D55K, N71D, N71Q, L75A, N76D, E79Q, N85D, N85Q, L89A, F96A, M97A, L124A, M125A, Q130E, Q135E, N136D, E143Q, Q146E, N151D, N151K, E153K, E153Q, K158E, E162Q, E163Q, D165N, I171A, N195D, and N195Q.

[0358] In some embodiments, the present invention relates to an IL-12 p35 subunit comprising the amino acid sequence of N71D / N85D / N195D, N151D / E153Q, N151D / D165N, Q130E / N151D, N151D / K158E, E79Q / N151D, D55Q / N151 and N85D / N195D, N136D / N151D, N21D / N151D, E143Q / N151D, N71Q / N85Q, N71Q / N195Q, N85Q / N195Q, N71Q / N85Q / N195Q, N71D / N85D, N71D / N195D, and N85D / N195D.

[0359] In some embodiments, the present invention relates to a method for the preparation of a IL-12p35 subunit comprising administering to a patient a sequence identical to that of SEQ ID NO:113 (IL-12p35(N71D)), ii) SEQ ID NO:114 (IL-12p35(N85D)), iii) SEQ ID NO:115 (IL-12p35(N195D)), iv) SEQ ID NO:116 (IL-12p35(N71D / N85D / N195D)), v) SEQ ID NO:117 (IL-12p35(E153Q)), vi) SEQ ID NO:118 (IL-12p35(E38Q)), vii) SEQ ID NO:119 (IL-12p35(N151D)), viii) SEQ ID NO:120 (IL-12p35(N151D)), or viii) SEQ ID NO: 120 (IL-12p35(Q135E)), ix) SEQ ID NO: 121 (IL-12p35(Q35D)), x) SEQ ID NO: 122 (IL-12p35(Q146E)), xi) SEQ ID NO: 123 (IL-12p35(N76D)), xii) SEQ ID NO: 124 (IL-12p35(E162Q)), xiii) SEQ ID NO: 125 (IL-12p35(E163Q)), xiv) IL-12p35(N21D), xv) SEQ ID NO: XXX (IL-12p35(D55Q)), xvi) IL-12p35(E79Q), xvii) IL-12p35(Q130E), x viii) IL-12p35(N136D), xix) IL-12p35(E143Q), xx) SEQ ID NO: 227 (IL-12p35(N151K)), xxi) SEQ ID NO: 226 (IL-12p35(E153K)), xxii) IL-12p35(K158E), xxiii) IL-12p35(D165N), xxiv) SEQ ID NO: 225 (IL-12p35(N151D / E153Q)), xxv) SEQ ID NO: 228 (IL-12p35(N151D / D165N)), xxvi) SEQ ID NO: 229 (IL-12p35(Q130E / N151D)) , xxvii) SEQ ID NO: 230 (IL-12p35(N151D / K158E)), xxviii) SEQ ID NO: 231 (IL-12p35(E79Q / N151D)), xxix) SEQ ID NO: 232 (IL-12p35(D55Q / N151D)), xxx) SEQ ID NO: 233 (IL-12p35(N136D / N151D)), xxxi) SEQ ID NO: 234 (IL-12p35(N21D / N151D)), xxxii) SEQ ID NO: 235 (IL-12p35(E143Q / N151D)), xxxiii) SEQ ID NO: XXX (IL-12p35(F96A)),xxxiv) SEQ ID NO:XXX(IL-12p35(M97A)), xxxv) SEQ ID NO:XXX(IL-12p35(L89A)), xxxvi) SEQ ID NO:XXX(IL-12p35(L124A)), xxxvii) SEQ ID NO:XXX(IL-12p35(M125A)), xxxviii) SEQ ID NO:XXX(IL-12p35(L75A)), xxxiv) SEQ ID NO:XXX(IL-12p35(I171A)), xxxv) SEQ ID NO:XXX(IL-12p35(N71Q)), xxxvi) SEQ ID NO:XXX(IL-12p35(N85Q)), xxxvii) SEQ ID NO:XXX(IL-12p35(N195Q)), xxxviii) SEQ ID NO:XXX(IL-12p35(N71Q / N85Q)), xxix) SEQ ID NO:XXX(IL-12p35(N71Q / N195Q)), xl) SEQ ID NO:XXX(IL-12p35(N85Q / N195Q), xli) SEQ ID NO:XXX(IL-12p35(N71Q / N85Q / N195Q)), xlii) SEQ ID NO:XXX(IL-12p35(N71D / N85D)), xliii) SEQ ID NO:XXX(IL-12p35(N71D / N195D), xliv) SEQ ID NO:XXX(IL-12p35(N85D / N195D)), xlv) SEQ ID NO:XXX(IL-12p35(D55Q)), and xlvi) SEQ ID NO:XXX(IL-12p35(D55K)).

[0360] In some embodiments, a homodimeric Fc fusion protein is provided, wherein said first and second monomers are XENP31289.

[0361] In some embodiments, a homodimeric Fc fusion protein is provided, wherein said first and second monomers are XENP31291.

[0362] In some embodiments, the present invention provides a homodimeric Fc-fusion protein composition comprising a homodimeric Fc-fusion protein for use in treating cancer in a subject.

[0363] In some embodiments, the invention provides one or more nucleic acids encoding a homodimeric Fc fusion protein.

[0364] In some embodiments, the invention provides host cells comprising one or more of the nucleic acids encoding the homodimeric Fc fusion proteins.

[0365] In some embodiments, the invention provides a method of making a homodimeric Fc-fusion protein, the method comprising culturing a host cell under conditions whereby the homodimeric Fc-fusion protein is produced.

[0366] In some embodiments, the invention provides a method for purifying a homodimeric Fc-fusion protein, the method comprising: a) providing a composition comprising a homodimeric Fc-fusion protein; b) loading the composition onto an ion exchange column; and c) collecting fractions containing the homodimeric Fc-fusion protein.

[0367] In another aspect, the invention provides a homodimeric Fc fusion protein comprising a first monomer and a second monomer, each comprising, from N-terminus to C-terminus, Fc domain-first domain linker-IL-12p35 subunit domain-second domain linker-IL-12p40 subunit domain.

[0368] In some embodiments, the invention provides homodimeric Fc fusion proteins, wherein the modifications that promote homodimerization of the Fc domain are a set of amino acid substitutions selected from the group consisting of: L368D / K370S; S364K; S364K / E357L; S364K / E357Q; T411E / K360E / Q362E; D401K; T366S / L368A / Y407V; T366W; T366S / L368A / Y407V / Y349C; and T366W / S354C, according to EU numbering.

[0369] In some embodiments, the invention provides a homodimeric Fc fusion protein, wherein said first domain linker and said second domain linker have the same amino acid sequence.

[0370] In some embodiments, the invention provides homodimeric Fc fusion proteins, wherein the Fc domain has an additional set of amino acid substitutions comprising: Q295E / N384D / Q418E / N421D, according to EU numbering.

[0371] In some embodiments, the invention provides homodimeric Fc fusion proteins, wherein the Fc domain has an additional set of amino acid substitutions selected from the group consisting of G236R / L328R, E233P / L234V / L235A / G236_ / S239K, E233P / L234V / L235A / G236_ / S239K / A327G, E233P / L234V / L235A / G236_ / S267K / A327G, E233P / L234V / L235A / G236_, and E233P / L234V / L235A / G236_ / S267K, according to EU numbering.

[0372] In some embodiments, the invention provides homodimeric Fc fusion proteins, wherein the IL-12p40 subunit has a polypeptide sequence selected from the group consisting of SEQ ID NO:3 (human IL-12 subunit beta (IL-12p40) precursor sequence) and SEQ ID NO:4 (human IL-12 subunit beta (IL-12p40) mature form sequence), and the IL-12p35 subunit has a polypeptide sequence selected from the group consisting of SEQ ID NO:1 (human IL-12 subunit alpha (IL-12p35) precursor sequence) and SEQ ID NO:2 (human IL-12 subunit alpha (IL-12p35) mature form sequence).

[0373] In some embodiments, the invention provides a homodimeric Fc fusion protein, wherein each of the Fc domains further comprises the amino acid substitutions M428L / N424S.

[0374] In some embodiments, the invention provides homodimeric Fc fusion proteins, wherein said IL-12p40 subunit is a variant IL-12p40 subunit and / or said IL-12p35 subunit is a variant IL-12p35 subunit.

[0375] In some embodiments, the invention provides homodimeric Fc fusion proteins wherein said IL-12p40 subunit is IL-12 receptor subunit beta-1 (IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2), and / or a variant IL-12p40 subunit with altered affinity for the IL-12 receptor complex, and / or said IL-12p35 subunit is IL-12 receptor subunit beta-1 (IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2), and / or a variant IL-12p35 subunit with altered affinity for the IL-12 receptor complex.

[0376] In some embodiments, the IL-12p40 subunit is selected from the group consisting of E3, D7, E12, D14, W15, P17, D18, A19, P20, G21, E22, M23, D29, E32, E33, D34, L40, D41, Q42, S43, E45, L47, T54, I55, Q56, K58, E59, F60, G 61, D62, Q65, Y66, E73, K84, E86, D87, G88, I89, W90, D93, D97, K99, E100, K102, N103, K104, F106, E110, N113, Y114, D129, D142, Q144, E156, R159, D161, N162, K163, D166, D 170, Q172, D174, A176, C177, P178, A179, A180, E181, S183, P185, E187, N200, S204, F 206, R208, D209, D214, N218, Q220, N226, Q229, E231, E235, T242, P243, S245, Y246, F2 47, S248, C252S, , Q256, K158, K260, E262, K264, D265, D270, N281, Q289, D290, R291, Y292, Y293, and E299.

[0377] In some embodiments, the present invention relates to an IL-12p40 subunit comprising D18N, D18K, E32Q, E33Q, D34N, D34K, Q42E, S43E, S43K, E45Q, Q56E, E59Q, E59K, D62N, E73Q, D87N, K99E, K99Y, E100Q, N103D, N103Q, N113D, N113Q, Q144 The present invention provides homodimeric Fc fusion proteins having one or more amino acid substitutions selected from the group consisting of E, D161N, R159E, K163E, E187Q, N200D, N200Q, N218Q, Q229E, E235Q, C252S, Q256N, K258E, K260E, E262Q, K264E, N281D, N281Q, and E299Q.

[0378] In some embodiments, the present invention relates to a method for the preparation of IL-12p40 subunits comprising administering to the patient a IL-12p40 subunit comprising administering to the patient a IL-12p40 subunit comprising administering to the patient a IL-12p40 subunit comprising administering to the patient a IL-12p40 subunit comprising administering to the patient a IL-12p40 subunit comprising administering to the patient a IL-12p40 subunit comprising administering to the patient a IL-12p40 subunit comprising administering to the patient a IL-12p40 subunit S43K / E49Q, E59Q / K163E, E59Q / K99E, E59Q / K258E, E59Q / K260E, E59K / K99E , D18K / E59K / K99E, E59K / K99E / K264E, E59K / K99Y, E59Y / K99Y, E59Y / K99E, E 45K / E59K / K99E, E59K / K99E / Q144E, E59K / K99E / Q144K, E59K / K99E / R159E, E59K / K99E / K264E, D18K / E59K / K99E / K264E, DI8K / E59K / K99E / C252S, D18K / E59K / K99E / C252S / K264E, E59K / K99Y / C252S, E59K / K99E / C252S / K264E, E5 9K / K99E / C252S, N103D / N113D, N103D / N200D, N103D / N281D, N113D / N200D, N 113D / N281D, N200D / N281D, N103D / N113D / N200D, N103D / N113D / N281D, N10 3D / N200D / N281D, N113D / N200D / N281D, N103Q / N113Q, N103Q / N200Q, N103Q / N281Q, N113Q / N200Q, N113Q / N281Q, N200Q / N281Q, N103Q / N113Q / N200Q, N1 03Q / N113Q / N281Q, N103Q / N200Q / N281Q, N113Q / N200Q / N281Q, N103Q / N113Q / N200Q / N281Q, E59K / K99E / N103Q / C252S / K264E, E59K / K99E / N113Q / C252S / K264E, E59K / K99E / N200Q / C252S / K264E, E59K / K99E / N281Q / C252S / K264E,E59K / K99E / N103Q / N113Q / C252S / K264E, E59K / K99E / N103Q / N200Q / C252S / K264E, E59K / K99E / N103Q / N281Q / C252S / K264E, E 59K / K99E / N113Q / N200Q / C252S / K264E, E59K / K99E / N113Q / N281Q / C252S / K264E, E59K / K99E / N200Q / N281Q / C252S / K264E, E59 The present invention provides homodimeric Fc fusion proteins having amino acid substitutions selected from the group consisting of K / K99E / N103Q / N113Q / N200Q / C252S / K264E, E59K / K99E / N103Q / N200Q / N281Q / C252S / K264E, E59K / K99E / N113Q / N200Q / N281Q / C252S / K264E, and E59K / K99E / N103Q / N113Q / N200Q / N281Q / C252S / K264E. In some embodiments, the present invention relates to an IL-12p40 subunit comprising: i) SEQ ID NO:57 (IL-12p40(N103D)), ii) SEQ ID NO:58 (IL-12p40(N113D)), iii) SEQ ID NO:59 (IL-12p40(N200D)), iv) SEQ ID NO:60 (IL-12p40(N281D)), v) SEQ ID NO:61 (IL-12p40(N103D / N113D / N200D / N281D)), vi) SEQ ID NO:62 (IL-12p40(Q42E)), vii) SEQ ID NO:63 (IL-12p40(E45Q)), viii) SEQ ID NO:64 (IL-12p40(Q56E)), ix) SEQ ID NO: 65 (IL-12p40(E59Q)), x) SEQ ID NO:66 (IL-12p40(D62N)), xi) SEQ ID NO:67 (IL-12p40(Q42E / E45Q)), xii) SEQ ID NO:68 (IL-12p40(E45Q / Q56E)), xiii) SEQ ID NO:69 (IL-12p40(Q42E / E59Q)), xiv) SEQ ID NO:70 (IL-12p40(Q56E / E59Q)), xv) SEQ ID NO:71 (IL-12p40(Q42E / E45Q / Q56E)), xvi) SEQ ID NO:72 (IL-12p40(E45Q / Q56E / E5 9Q)), xvii) SEQ ID NO: 73 (IL-12p40(D161N)), xviii) SEQ ID NO: 74 (IL-12p40(E73Q)), xix) SEQ ID NO: 75 (IL-12p40(Q144E)), xx) SEQ ID NO: 76 (IL-12p40(E262Q)), xxi) SEQ ID NO: 77 (IL-12p40(E100Q)), xxii) SEQ ID NO: 78 (IL-12p40(D18N)), xxiii) SEQ ID NO: 79 (IL-12p40(E33Q)), xxiv) SEQ ID NO: 80 (IL-12p40(Q229E)), xxv) SEQ ID NO: 81 (IL- 12p40(E235Q)), xxvi) SEQ ID NO: 82 (IL-12p40(Q256N)), xxvii) SEQ ID NO: 83 (IL-12p40(E299Q)), xxviii) SEQ ID NO: 84 (IL-12p40(D87N)), xxix) IL-12p40(E32Q), xxx) IL-12p40(D34N), xxxi) IL-12p40(S43E), xxxii) IL-12p40(S43K), xxxiii) SEQ ID NO: XXX (IL-12p40(E59K / K99E / N103Q / N113Q / N200Q / N281Q / C252S / K264E), xxxiv) SEQ ID NO: 205 (IL-12p40(E59K)), xxxv) IL-12p40(K99E), xxxvi) IL-12p40(K163E), xxxvii) IL-12p40(E187Q), xxxviii) IL-12p40(K258E), xxxix) IL-12p40(K260E), xl) SEQ ID NO: 206 (IL-12p40(E32Q / E59Q)), xli) SEQ ID NO: 207 (IL-12p40(D34N / E59Q)), xlii) SEQ ID NO: 208 (IL-12p40(E59Q / E187Q )), xliii) SEQ ID NO: 209 (IL-12p40(S43E / E59Q)), xliv) SEQ ID NO: 210 (IL-12p40(S43K / E49Q)), xlv) SEQ ID NO: 211 (IL-12p40(E59Q / K163E)), xlvi) SEQ ID NO: 212 (IL-12p40(E59Q / K99E)), xlvii) SEQ ID NO: 213 (IL-12p40(E59Q / K258E)), xlviii) SEQ ID NO: 214 (IL-12p40(E59Q / K260E)), xlix) SEQ ID NO: XXX (IL-12p40(D34N / E59K)),l) SEQ ID NO:XXX(IL-12p40(E59K / K99E)), li) SEQ ID NO:XXX(IL-12p40(D18K / E59K / K99E)), lii) SEQ ID NO:XXX(IL-12p40(E59K / K99E / K264E)), liii) SEQ ID NO:XXX(IL-12p40(E59K / K99Y)), liv) SEQ ID NO:XXX(IL-12p40(E59Y / K99E)), lv) SEQ ID NO:XXX(IL-12p40(E45K / E59K / K99E)), lvi) SEQ ID NO:XXX(IL-12p40(E59K / K99E / Q144E)), lvii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / Q144K)), lviii) SEQ ID NO: XXX(IL-12p40(E59K / K99E / R159E)), lix) SEQ ID NO: XXX(IL-12p40(E59K / K99E / K264E)), lx) SEQ ID NO: XXX(IL-12p40(D18K / E59K / K99E / K264E)), lxi) SEQ ID NO: XXX(IL-12p40(C252S)), lxii) SEQ ID NO: XXX(IL-12p40(DI8K / E59K / K99E / C252S) ), lxiii) SEQ ID NO: XXX(IL-12p40(D18K / E59K / K99E / C252S / K264E)), lxiv) SEQ ID NO: XXX(IL-12p40(E59K / K99Y / C252S)), lxv) SEQ ID NO: XXX(IL-12p40(E59K / K99E / C252S / K264E)), lxvi) SEQ ID NO: XXX(IL-12p40(E59K / K99E / C252S)), lxvii) SEQ ID NO: XXX(IL-12p40(N103D / N113D)), lxviii) SEQ ID NO: XXX(IL-12p40(N1 03D / N200D)), lxix) SEQ ID NO: XXX(IL-12p40(N103D / N281D)), lxx) SEQ ID NO: XXX(IL-12p40(N113D / N200D)), lxxi) SEQ ID NO: XXX(IL-12p40(N113D / N281D)), lxxii) SEQ ID NO: XXX(IL-12p40(N200D / N281D)), lxxiii) SEQ ID NO: XXX(IL-12p50(N103D / N113D / N200D)), lxxiv) SEQ ID NO: XXX(IL-12p40(N103D / N113D / N281D)),lxxv) SEQ ID NO: XXX (IL-12p40 (N103D / N200D / N281D)), lx, xvi) SEQ ID NO:XXX(IL-12p40(N113D / N200D / N281D)), lxxvii) SEQ ID NO:XXX(IL-12p40(N103Q)), lxxviii) SEQ ID NO:XXX(IL-12p40(N113Q)), lxxix) SEQ ID NO:XXX(IL-12p40(N200Q)), lxxx) SEQ ID NO:XXX(IL-12p40(N281Q)), lxxxi) SEQ ID NO:XXX(IL-12p40(N103Q / N113Q)), lxxxii) SEQ ID NO:XXX(IL-12p40(N103Q / N200Q)), xxxiii) SEQ ID NO: XXX(IL-12p40(N103Q / N281Q)), lxxxiv) SEQ ID NO: XXX(IL-12p40(N113Q / N200Q)), lxxxv) SEQ ID NO: XXX(IL-12p40(N113Q / N281Q)), lxxxvi) SEQ ID NO: XXX(IL-12p40(N200Q / N281Q)), lxxxvii) SEQ ID NO: XXX(IL-12p40(N103Q / N113Q / N200Q)), lxxxviii) SEQ ID NO: XXX(IL-12p40(N103Q / N113Q / N281Q)), lx xxix) SEQ ID NO: XXX(IL-12p40(N103Q / N200Q / N281Q)), xc) SEQ ID NO: XXX(IL-12p40(N113Q / N200Q / N281Q)), xci) SEQ ID NO: XXX(IL-12p40(N103Q / N113Q / N200Q / N281Q)), xcii) SEQ ID NO: XXX(IL-12p40(D34N / E59K / K99E)), xciii) SEQ ID NO: XXX(IL-12p40(D34K / E59K / K99E)), xciv) SEQ ID NO: XXX(IL-12p40(E32Q / D34N / E59K / K99E)), xcv) SEQ ID NO: XXX (IL-12p40(E32K / D34N / E59K / K99E)), xcvi) SEQ ID NO: XXX (IL-12p40(E59Y / K99Y)), xcvii) SEQ ID NO: XXX (IL-12p40(E59K / K99E / N103Q / C252S / K264E)), xcviii) SEQ ID NO: XXX (IL-12p40(E59K / K99E / N113Q / C252S / K264E)), xcix) SEQ ID NO: XXX (IL-12p40(E59K / K99E / N200Q / C252S / K264E)),c) SEQ ID NO: XXX (IL-12p40(E59K / K99E / N281Q / C252S / K264E)), ci) SEQ ID NO: XXX (IL-12p40(E59K / K99E / N103Q / N113Q / C252S / K264E)), cii) SEQ ID NO: XXX (IL-12p40(E59K / K99E / N103Q / N200Q / C252S / K26 4E)), ciii) SEQ ID NO: XXX (IL-12p40(E59K / K99E / N103Q / N281Q / C252S / K264E)), civ) SEQ ID NO: XXX (IL-12p40(E59K / K99E / N113Q / N200Q / C252S / K264E)), cv) SEQ ID NO: XXX (IL-12p40(E59K / K99E / N113Q / N 281Q / C252S / K264E)), cvi) SEQ ID NO: XXX (IL-12p40(E59K / K99E / N200Q / N281Q / C252S / K264E)), cvii) SEQ ID NO: XXX (IL-12p40(E59K / K99E / N103Q / N113Q / N200Q / C252S / K264E)), cviii) SEQ ID NO: XXX (IL- and cix) SEQ ID NO:XXX (IL-12p40(E59K / K99E / N103Q / N200Q / N281Q / C252S / K264E)).

[0379] In some embodiments, the present invention relates to an IL-12 p35 subunit comprising one or more of the following: Q20, N21, Q35, E38, S44, E45, E46, H49, K54, D55, T59, V60, E61, C63, L64, P65, E67, L68, N71, S73, C74, L75, N76, E79, N85, L89, F96, M97, L124, M125, Q1 30, Q135, N136, E143, Q146, N151, E153, K158, E162, E163, D165, I171, R181, I182, R183, V185, T186, D188, R189, V190, S192, Y193, N195, and A196. A multimeric Fc fusion protein is provided.

[0380] In some embodiments, the invention provides homodimeric Fc fusion proteins, wherein the IL-12p35 subunit has one or more amino acid substitutions selected from the group consisting of N21D, Q35D, E38Q, D55Q, D55K, N71D, N71Q, L75A, N76D, E79Q, N85D, N85Q, L89A, F96A, M97A, L124A, M125A, Q130E, Q135E, N136D, E143Q, Q146E, N151D, N151K, E153K, E153Q, K158E, E162Q, E163Q, D165N, I171A, N195D, and N195Q.

[0381] In some embodiments, the invention provides homodimeric Fc fusion proteins, wherein the IL-12p35 subunit has amino acid substitutions selected from the group consisting of N71D / N85D / N195D, N151D / E153Q, N151D / D165N, Q130E / N151D, N151D / K158E, E79Q / N151D, D55Q / N151D, N136D / N151D, N21D / N151D, E143Q / N151D, N71Q / N85Q, N71Q / N195Q, N85Q / N195Q, N71Q / N85Q / N195Q, N71D / N85D, N71D / N195D, and N85D / N195D.

[0382] In some embodiments, the present invention relates to a method for the preparation of a IL-12p35 subunit comprising administering to a patient a sequence identical to that of SEQ ID NO:113 (IL-12p35(N71D)), ii) SEQ ID NO:114 (IL-12p35(N85D)), iii) SEQ ID NO:115 (IL-12p35(N195D)), iv) SEQ ID NO:116 (IL-12p35(N71D / N85D / N195D)), v) SEQ ID NO:117 (IL-12p35(E153Q)), vi) SEQ ID NO:118 (IL-12p35(E38Q)), vii) SEQ ID NO:119 (IL-12p35(N151D)), viii) SEQ ID NO:120 (IL-12p35(N151D)), or viii) SEQ ID NO: 120 (IL-12p35(Q135E)), ix) SEQ ID NO: 121 (IL-12p35(Q35D)), x) SEQ ID NO: 122 (IL-12p35(Q146E)), xi) SEQ ID NO: 123 (IL-12p35(N76D)), xii) SEQ ID NO: 124 (IL-12p35(E162Q)), xiii) SEQ ID NO: 125 (IL-12p35(E163Q)), xiv) IL-12p35(N21D), xv) SEQ ID NO: XXX (IL-12p35(D55Q)), xvi) IL-12p35(E79Q), xvii) IL-12p35(Q130E), x viii) IL-12p35(N136D), xix) IL-12p35(E143Q), xx) SEQ ID NO: 227 (IL-12p35(N151K)), xxi) SEQ ID NO: 226 (IL-12p35(E153K)), xxii) IL-12p35(K158E), xxiii) IL-12p35(D165N), xxiv) SEQ ID NO: 225 (IL-12p35(N151D / E153Q)), xxv) SEQ ID NO: 228 (IL-12p35(N151D / D165N)), xxvi) SEQ ID NO: 229 (IL-12p35(Q130E / N151D)) , xxvii) SEQ ID NO: 230 (IL-12p35(N151D / K158E)), xxviii) SEQ ID NO: 231 (IL-12p35(E79Q / N151D)), xxix) SEQ ID NO: 232 (IL-12p35(D55Q / N151D)), xxx) SEQ ID NO: 233 (IL-12p35(N136D / N151D)), xxxi) SEQ ID NO: 234 (IL-12p35(N21D / N151D)), xxxii) SEQ ID NO: 235 (IL-12p35(E143Q / N151D)), xxxiii) SEQ ID NO: XXX (IL-12p35(F96A)),xxxiv) SEQ ID NO:XXX(IL-12p35(M97A)), xxxv) SEQ ID NO:XXX(IL-12p35(L89A)), xxxvi) SEQ ID NO:XXX(IL-12p35(L124A)), xxxvii) SEQ ID NO:XXX(IL-12p35(M125A)), xxxviii) SEQ ID NO:XXX(IL-12p35(L75A)), xxxiv) SEQ ID NO:XXX(IL-12p35(I171A)), xxxv) SEQ ID NO:XXX(IL-12p35(N71Q)), xxxvi) SEQ ID NO:XXX(IL-12p35(N85Q)), xxxvi, i) SEQ ID NO:XXX(IL-12p35(N195Q)), xxxviii) SEQ ID NO:XXX(IL-12p35(N71Q / N85Q)), xxxix) SEQ ID NO:XXX(IL-12p35(N71Q / N195Q)), xl) SEQ ID NO:XXX(IL-12p35(N85Q / N195Q), xli) SEQ ID NO:XXX(IL-12p35(N71Q / N85Q / N195Q)), xlii) SEQ ID NO:XXX(IL-12 xlii) SEQ ID NO:XXX(IL-12p35(N71D / N85D)), xliv) SEQ ID NO:XXX(IL-12p35(N85D / N195D)), xlv) SEQ ID NO:XXX(IL-12p35(D55Q)), and xlvi) SEQ ID NO:XXX(IL-12p35(D55K)).

[0383] In some embodiments, a homodimeric Fc fusion protein is provided, wherein said first and second monomers are XENP31289.

[0384] In some embodiments, a homodimeric Fc fusion protein is provided, wherein said first and second monomers are XENP31291.

[0385] In some embodiments, the present invention provides a homodimeric Fc-fusion protein composition comprising a homodimeric Fc-fusion protein for use in treating cancer in a subject.

[0386] In some embodiments, the invention provides one or more nucleic acids encoding a homodimeric Fc fusion protein.

[0387] In some embodiments, the invention provides host cells comprising one or more of the nucleic acids encoding the homodimeric Fc fusion proteins.

[0388] In some embodiments, the invention provides a method of making a homodimeric Fc-fusion protein, the method comprising culturing a host cell under conditions whereby the homodimeric Fc-fusion protein is produced.

[0389] In some embodiments, the invention provides a method for purifying a homodimeric Fc-fusion protein, the method comprising: a) providing a composition comprising a homodimeric Fc-fusion protein; b) loading the composition onto an ion exchange column; and c) collecting fractions containing the homodimeric Fc-fusion protein.

[0390] In another aspect, the invention provides a method of treating cancer in a patient in need thereof comprising administering to said patient a therapeutically effective amount of a heterodimeric or homodimeric Fc-fusion protein according to any of the preceding paragraphs.

[0391] In some embodiments, the present invention provides a method of treating cancer in a patient in need thereof, further comprising administering a therapeutically effective amount of a checkpoint blockade antibody.

[0392] In some further embodiments, the invention provides methods of treating cancer in a patient in need thereof, wherein the checkpoint blockade antibody is selected from an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-TIM3 antibody, an anti-TIGIT antibody, an anti-LAG3 antibody, and an anti-CTLA-4 antibody.

[0393] In some further embodiments, the invention provides methods of treating cancer in a patient in need thereof, wherein the anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, , or pidilizumab.

[0394] In some further embodiments, the invention provides methods of treating cancer in a patient in need thereof, wherein the anti-PD-L1 antibody is atezolizumab, avelumab, or durvalumab.

[0395] In some further embodiments, the present invention provides methods of treating cancer in a patient in need thereof, wherein the patient exhibits an increase in lymphocytes following administration.

[0396] In some further embodiments, the invention provides methods of treating cancer in a patient in need thereof, wherein the patient exhibits an increase in peripheral CD8+ T cells following administration.

[0397] In some embodiments, the invention provides a method of treating cancer in a patient in need thereof, wherein an IL-12Fc fusion protein is administered prior to a checkpoint inhibitor to increase checkpoint expression pre-treatment.

[0398] In some embodiments, the invention provides a method of treating cancer in a patient in need thereof, wherein an IL-12Fc fusion protein is administered prior to a checkpoint inhibitor to increase checkpoint expression pre-treatment.

[0399] In another aspect, the invention provides a method of inducing T cell proliferation in a patient in need thereof, comprising administering to said patient a therapeutically effective amount of a heterodimeric or homodimeric Fc-fusion protein according to any of the preceding clauses.

[0400] In some further embodiments, the invention provides methods of inducing T cell proliferation in a patient in need thereof, further comprising administering a therapeutically effective amount of a checkpoint blockade antibody.

[0401] In some further embodiments, the invention provides methods of inducing T cell proliferation in a patient in need thereof, wherein the checkpoint blockade antibody is selected from an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-TIM3 antibody, an anti-TIGIT antibody, an anti-LAG3 antibody, and an anti-CTLA-4 antibody.

[0402] In some further embodiments, the invention provides methods of inducing T cell proliferation in a patient in need thereof, wherein the anti-PD-1 antibody is nivolumab, pembrolizumab, or pidilizumab.

[0403] In some other further embodiments, the invention provides methods of inducing T cell proliferation in a patient in need thereof, wherein the anti-PD-L1 antibody is atezolizumab, avelumab, or durvalumab.

[0404] In some further embodiments, the present invention provides methods of inducing T cell proliferation in a patient in need thereof, wherein the T cell proliferation is at least a two-fold increase in T cells.

[0405] In some embodiments, the invention provides a method of inducing T cell proliferation in a patient in need thereof, wherein an IL-12Fc fusion protein is administered prior to a checkpoint inhibitor to increase checkpoint expression pre-treatment.

[0406] In some embodiments, the present invention provides a method for the treatment of a patient in need of inducing T cell proliferation. The present invention provides a method of inducing T cell proliferation in a patient receiving an IL-12Fc fusion protein, wherein the IL-12Fc fusion protein is administered prior to a checkpoint inhibitor to increase checkpoint expression prior to treatment.

[0407] In one aspect, the invention provides an IL-12p40 subunit. In another aspect, the invention provides an IL-12p35 subunit. In a further aspect, the invention provides a heterodimeric complex comprising a) an IL-12p40 subunit and b) an IL-12p35 subunit. [Brief description of the drawings]

[0408] [Figure 1] 1A and 1B show the sequences of IL-12 and its receptor. [Diagram 2] 2A-2E show useful pairs of Fc heterodimerization variant sets (including skew and pI variants). Variants that do not have a corresponding "monomer 2" are pI variants that can be used alone for either monomer. [Diagram 3] A list of isosteric variant antibody constant regions and their respective substitutions is provided below. pI_(-) indicates low pI variants and pI_(+) indicates high pI variants. These can be optionally and independently combined with other heterodimerization variants of the invention (and other variant types outlined herein). [Figure 4] Useful ablation variants that ablate FcγR binding are shown (sometimes referred to as "knockout" or "KO" variants). Generally, ablation variants are found in both monomers, although in some cases they may only be found in one monomer. [Diagram 5] Particularly useful embodiments of the "non-cytokine" components of the present invention are presented. [Figure 6]Some exemplary domain linkers are shown. In some embodiments, these linkers find use in linking IL-12p35 subunits, IL-12p40 subunits, or single-chain IL-12 complexes to the N-terminus of the Fc region. In some embodiments, these linkers find use in fusing IL-12p35 subunits to IL-12p40 subunits in single-chain IL-12 complexes. It is important to note that scIL-12 complexes can include either IL-12p35 N-terminally linked to IL-12p40 or IL-12p40 N-terminally linked to IL-12p35. Also, in some cases described herein, the hinge portion of the Fc domain functions as a domain linker and can be combined with any of these linkers as well. [Figure 7]7A-7E show sequences of several useful IL-12-Fc fusion backbones based on human IgG1 that do not contain cytokine sequences (e.g., IL-12p35 subunit, IL-12p40 subunit, or scIL-12 complex). Backbone 1 is based on human IgG1 (356E / 358M allotype) and includes C220S on both chains, S364K / E357Q:L368D / K370S scubariant, Q295E / N384D / Q418E / N421D pI variant on one chain with L368D / K370S scubariant, and E233P / L234V / L235A / G236del / S267K ablation variant on both chains. Backbone 2 is based on human IgG1 (356E / 358M allotype) and contains C220S on both chains, an S364K:L368D / K370S scubariant, a Q295E / N384D / Q418E / N421D pI variant on the chain with the L368D / K370S scubariant, and an E233P / L234V / L235A / G236del / S267K ablation variant on both chains. Backbone 3 is based on human IgG1 (356E / 358M allotype) and contains C220S on both chains, an S364K:L368E / K370S scubariant, a Q295E / N384D / Q418E / N421D pI variant on the chain with the L368E / K370S scubariant, and an E233P / L234V / L235A / G236del / S267K ablation variant on both chains. Backbone 4 is based on human IgG1 (356E / 358M allotype) and contains C220S on both chains, a D401K:K360E / Q362E / T411E scubariant, a Q295E / N384D / Q418E / N421D pI variant on the chain with the K360E / Q362E / T411E scubariant, and E233P / L234V / L235A / G236del / S267K ablation variants on both chains.Backbone 5 is based on human IgG1 (356D / 358L allotype) and contains C220S on both chains, an S364K / E357Q:L368D / K370S scubariant, a Q295E / N384D / Q418E / N421D pI variant on the chain with the L368D / K370S scubariant, and an E233P / L234V / L235A / G236del / S267K ablation variant on both chains. Backbone 6 is based on human IgG1 (356E / 358M allotype) and contains C220S on both chains, S364K / E357Q:L368D / K370S scubariant, Q295E / N384D / Q418E / N421D pI variant on the chain with L368D / K370S scubariant, and E233P / L234V / L235A / G236del / S267K ablation variant on both chains, as well as N297A variant on both chains. Backbone 7 is identical to 6, except that the mutation is N297S. Alternative formats for backbones 6 and 7 can exclude the ablation variants E233P / L234V / L235A / G236del / S267K in both chains. Backbone 8 is based on human IgG4 and contains an S364K / E357Q:L368D / K370S scuba variant, a Q295E / N384D / Q418E / N421D pI variant on the chain with the L368D / K370S scuba variant, and an S228P (EU numbering, i.e., S241P in Kabat) variant on both chains that ablate Fab arm exchange as known in the art. Backbone 9 is based on human IgG2 and contains an S364K / E357Q:L368D / K370S scuba variant, a Q295E / N384D / Q418E / N421D pI variant on the chain with the L368D / K370S scuba variant. Backbone 10 is based on human IgG2 and contains the S364K / E357Q:L368D / K370S sq variant, the Q295E / N384D / Q418E / N421D pI variants on the chain with the L368D / K370S sq variant, and the S267K variant on both chains.Backbone 11 is identical to backbone 1 except that it contains the M428L / N434S Xtend mutations. Backbone 12 is based on human IgG1 (356E / 358M allotype) and contains C220S on both identical chains and E233P / L234V / L235A / G236del / S267K ablation variants on both identical chains. Backbone 13 is based on human IgG1 (356E / 358M allotype) and includes C220S on both chains, S364K / E357Q:L368D / K370S scubarients, P217R / P229R / N276K pI variants on one chain with S364K / E357Q scubarients, and E233P / L234V / L235A / G236del / S267K ablation variants on both chains. As will be appreciated by those skilled in the art and outlined below, these sequences can be used in any of the IL-12-Fc fusion formats outlined herein, including but not limited to the IL-12-heteroFc, heteroFc-IL-12, and scIL-12-Fc formats, as shown diagrammatically in FIG. 8. It should be noted that for hetero Fc-IL-12 fusions, the backbone may further comprise a deletion of K447. Additionally, any IL-12p35 and / or IL-12p40 variants may be incorporated into these Figure 7 backbones in any combination. Included within each of these backbones are sequences that are 90, 95, 98, and 99% identical (as defined herein) to the listed sequences, and / or contain from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions (compared to the "parent" in the figure, which already contains some amino acid modifications compared to the parent human IgG1 (or IgG2 or IgG4, depending on the backbone, as will be understood by those skilled in the art). That is, the listed backbones may contain additional amino acid modifications (generally amino acid substitutions) in addition to the scubariant, pI variants, and ablation variants included within the backbones of this figure. [Figure 8]8A-8F show exemplary formats for the IL-12-Fc fusion proteins of the invention. The N...

Claims

1. A heterodimeric Fc fusion protein comprising: a) a first fusion protein comprising an amino acid sequence having the amino acid substitutions M428L / N434S in the amino acid sequence set forth in SEQ ID NO: 382; b) a second fusion protein comprising an amino acid sequence as set forth in SEQ ID NO: 404 with the amino acid substitutions M428L / N434S; Numbering follows EU numbering, Heterodimeric Fc fusion proteins.

2. a) a first nucleic acid encoding a first fusion protein according to claim 1; b) a second nucleic acid encoding a second fusion protein according to claim 1; A set of nucleic acids encoding the heterodimeric Fc fusion protein of claim 1.

3. a) a first expression vector comprising the first nucleic acid according to claim 2; and b) a second expression vector comprising the second nucleic acid of claim 2; A set of expression vectors.

4. A host cell comprising the set of expression vectors described in claim 3.

5. A method for producing a heterodimeric Fc fusion protein, the method comprising culturing a host cell described in claim 4 under conditions in which the heterodimeric Fc fusion protein is expressed, and recovering the protein.

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