IL-12 heterodimeric FC-fusion protein

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Heterodimeric Fc fusion proteins with modified IL-12 subunits address the toxicity and stability issues of recombinant IL-12, improving cancer immunotherapy by enhancing immune activation and extending half-life.

JP7801418B2Active Publication Date: 2026-01-16XENCOR INC

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Patent Information

Application Number: JP2024229412
Authority / Receiving Office: JP · JP
Patent Type: Patents
Current Assignee / Owner
Priority Date: 2019-05-15
Filing Date: 2024-12-25
Publication Date: 2026-01-16
Estimated Expiration: 2039-10-03

Application Information

Patent Timeline

15 May 2019

Priority

25 Dec 2024

Application

JP2024229412

16 Jan 2026

Publication

JP7801418B2

IPC: C07K19/00; C07K14/54; C07K16/28; C12N15/62; C12N15/63; C12N1/15; C12N1/19; C12N1/21; C12N5/10; C12P21/02

CPC: A61K38/00; C07K14/5434; C07K2319/30; A61K38/208; C07K1/18; A61P35/00; C07K19/00; A61K47/6813

Inventors: バーネット,マシュー; デスジャルレイス,ジョン,アール.

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AI Technical Summary

Technical Problem

Recombinant IL-12 faces challenges in human clinical trials due to toxicity and short half-life, necessitating frequent injections, and its p40 subunit can form antagonistic homodimers that interfere with its activity.

Method used

Development of heterodimeric Fc fusion proteins comprising variant IL-12p40 and IL-12p35 subunits covalently linked to Fc domains with specific amino acid modifications to enhance stability and reduce toxicity, promoting heterodimerization and altering receptor affinity.

Benefits of technology

The modified IL-12 heterodimeric Fc fusion proteins offer improved stability and reduced toxicity, potentially enhancing cytokine-based cancer immunotherapy by extending half-life and optimizing immune activation.

✦ Generated by Eureka AI based on patent content.

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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

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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 occur. First, T cells must engage antigenic tumor peptides presented by MHC in the tumor environment. Second, costimulatory molecules must bind to T cells. Third, T cells must be induced by cytokines such as IL-12 and IL-2 to produce costimulatory cytokines such as IFNγ, which enable differentiation and proliferation. Recognition of tumor peptides alone in the absence of cytokine induction renders T cells anergic, thereby leading to resistance. Therefore, cytokine-based therapy is a very promising approach in cancer immunotherapy. 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. Like other cytokines, the short half-life of IL-12 necessitates frequent bolus injections.

[0004] Additionally, 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, 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: 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 provides a method for the preparation of IL-12p40 subunits comprising the steps of: 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 , 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

[0010] The present invention provides heterodimeric Fc fusion proteins 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 producing 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-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) 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)), lxxviii) SEQ ID NO:XXX(IL-12p40(N113Q)), lxxix) SEQ ID NO:XXX(IL-12p40(N200Q)), lxxx) SEQ ID NO:XXX(IL-12p40(N281Q)), lx 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 the 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 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).

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

[0011] In some embodiments, the invention provides heterodimeric Fc-fusion proteins wherein the 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 the 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 provides a method for the preparation of variant IL-12p35 subunits comprising administering to a mammalian subject the method of the present invention a method for the preparation of variant IL-12p35 subunits comprising administering to a mammalian subject the method of the present invention a method for the preparation of variant IL-12p35 subunits comprising administering to a mammalian subject the method of the present invention 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 the first and 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 producing a medicament comprising administering to said first and / or said second Fc domain but with 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 heterodimeric Fc fusion proteins, wherein the first and second Fc domains further comprise the amino acid substitutions M428L / N424S.

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

[0021] a) i) SEQ ID NO: 47 (XENP27201 strand 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) SEQ ID NO: 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 chain 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:XXX (XENP33005 strand 2), 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, XENP33009, XENP33010, and XENP33011.

[0024] In some embodiments, the present invention provides heterodimeric Fc-fusion proteins, 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 present 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 present invention provides one or more nucleic acids encoding heterodimeric Fc-fusion proteins.

[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 methods for making a heterodimeric Fc-fusion protein, the methods comprising culturing a host cell under conditions such that the heterodimeric Fc-fusion protein is produced.

[0030] In some embodiments, the invention provides methods for purifying a heterodimeric Fc-fusion protein, the method comprising: a) providing a composition comprising the 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 IL-12p40 subunits, wherein 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, 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, Heterodimeric Fc fusion proteins are provided, 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 provides IL-12p35 subunits comprising any 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, L75, N76, E79, N85, L89, F96, M97, L124, M125, Q130, Q135, N and A196, wherein the amino acid residues are 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.

[0033] In another aspect, the present invention provides a first fusion protein comprising: a) a first protein domain and a first Fc domain, wherein the first protein domain is a first Fc domain; and b) a second fusion protein comprising a second protein domain and a second Fc domain, wherein the second protein domain is 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 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 and T366S / L368A / Y407V:T366W (optionally including the bridging disulfide, T366S / L368A / Y407V / Y349C:T366W / S354C), according to EU numbering.

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

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

[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 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).

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

[0040] In some embodiments, the present invention relates to a method for treating IL-12p40 deficiency, wherein the IL-12p40 subunit is a variant IL-12p40 subunit and / or the IL-12p35 subunit is a variant IL-12p40 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 the 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 the 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 IL-12p40 subunits, wherein 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, 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, Heterodimeric Fc fusion proteins are provided, 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 provides IL-12p40 subunits comprising: 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.

[0044] In some embodiments, the present invention relates to IL-12p40 subunits 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 / 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 Heterodimeric Fc fusion proteins are provided 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 IL-12p40 subunits 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 / 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 producing 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(E45E)), 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 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 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 / K264 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 provides IL-12p35 subunits comprising any 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, L75, N76, E79, N85, L89, F96, M97, L124, M125, Q130, Q135, N and A196, wherein the amino acid residues are 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.

[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 producing a IL-12p35 subunit 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: 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)), 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 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)).

[0050] In some embodiments, the present 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 present 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 present invention provides one or more nucleic acids encoding heterodimeric Fc-fusion proteins.

[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 methods for making a heterodimeric Fc-fusion protein, the methods comprising culturing a host cell under conditions such that the heterodimeric Fc-fusion protein is produced.

[0056] In some embodiments, the invention provides methods for purifying a heterodimeric Fc-fusion protein, the method comprising: a) providing a composition comprising the 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, and 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 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 and T366S / L368A / Y407V:T366W (optionally including the bridging disulfide, T366S / L368A / Y407V / Y349C:T366W / S354C), according to EU numbering.

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

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

[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 producing a human IL-12p40 subunit, the method comprising the steps of: (1) providing a human IL-12 subunit beta (IL-12p40) precursor sequence; (2) providing a human IL-12 subunit beta (IL-12p40) mature form sequence; and (3) providing a human IL-12 subunit beta (IL-12p40) mature form sequence. and / or wherein 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).

[0063] In some embodiments, the invention provides heterodimeric Fc fusion proteins, wherein the 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 the 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 the 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 IL-12p40 subunits, wherein 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, 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, Heterodimeric Fc fusion proteins are provided, 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 provides IL-12p40 subunits comprising: 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.

[0068] In some embodiments, the present invention provides a method for the preparation of IL-12p40 subunits comprising the steps of: 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 , 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

[0010] The present invention provides heterodimeric Fc fusion proteins 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 producing IL-12p40 subunits comprising administering to a mammalian subject the method of the present invention, the method comprising administering to a mammalian subject the method of the present invention, the method comprising administering to a mammalian subject the method of the present invention, the method comprising administering to a mammalian subject the method of the present invention 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 / N220Q / 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-1 cix) 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)).

[0070] In some embodiments, the present invention relates to IL-12p35 subunits comprising any 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 producing a IL-12p35 subunit comprising administering to a patient in need thereof a IL-12p35 sequence comprising the IL-12p35 subunit of the present invention, the IL-12p35 subunit comprising the IL-12p35 subunit of the present invention being: 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: 119 120 (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 heterodimeric Fc fusion proteins 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 present invention provides a heterodimeric Fc-fusion protein, wherein the heterodimeric Fc-fusion protein is XENP27203.

[0076] In some embodiments, the present 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 present invention provides one or more nucleic acids encoding heterodimeric Fc-fusion proteins.

[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 methods for making a heterodimeric Fc-fusion protein, the methods comprising culturing a host cell under conditions such that the heterodimeric Fc-fusion protein is produced.

[0081] In some embodiments, the invention provides methods for purifying a heterodimeric Fc-fusion protein, the method comprising: a) providing a composition comprising the 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 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, 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 and T366S / L368A / Y407V:T366W (optionally including a bridging disulfide, T366S / L368A / Y407V / Y349C:T366W / S354C).

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

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

[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 heterodimeric Fc fusion proteins, wherein the 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 provides a method for treating IL-12p40 deficiency, comprising administering to a subject an IL-12p40 subunit ... The present invention provides heterodimeric Fc fusion proteins that are variant IL-12p35 subunits with altered affinity for the IL-12 receptor complex.

[0091] In some embodiments, the present invention relates to IL-12p40 subunits, wherein 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, 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, Heterodimeric Fc fusion proteins are provided, 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 provides IL-12p40 subunits comprising: 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.

[0093] In some embodiments, the present invention relates to IL-12p40 subunits, 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 and E59K / K99E / N103Q / N113Q / N200Q / N281Q / C252S / K264E.

[0094] In some embodiments, the present invention relates to a method for producing 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(E45E)), 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 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 cviii) SEQ ID NO:XXX(IL-12p40(E59K / K99E / N103Q / 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)).

[0095] In some embodiments, the present invention provides IL-12p35 subunits comprising any 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, L75, N76, E79, N85, L89, F96, M97, L124, M125, Q130, Q135, N and A196, wherein the amino acid residues are 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.

[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 producing IL-12p35 subunits, wherein the IL-12p35 subunit is 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- 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 heterodimeric Fc fusion proteins 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 present 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 present invention provides one or more nucleic acids encoding heterodimeric Fc-fusion proteins.

[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 methods for making a heterodimeric Fc-fusion protein, the methods comprising culturing a host cell under conditions such that the heterodimeric Fc-fusion protein is produced.

[0105] In some embodiments, the invention provides methods for purifying a heterodimeric Fc-fusion protein, the method comprising: a) providing a composition comprising the 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 present 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 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 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 and T366S / L368A / Y407V:T366W (optionally including the bridging disulfide, T366S / L368A / Y407V / Y349C:T366W / S354C), according to EU numbering.

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

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

[0110] In some embodiments, the invention provides a method for producing a medicament for the treatment of ... Heterodimeric Fc fusion proteins having an additional set are provided.

[0111] 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 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).

[0112] In some embodiments, the invention provides heterodimeric Fc fusion proteins, wherein the 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 the 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 the 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 IL-12p40 subunits, wherein 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, 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, Heterodimeric Fc fusion proteins are provided, 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 IL-12p40 subunits, wherein the IL-12p40 subunit is 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, N20 ... 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 provides a method for the preparation of IL-12p40 subunits comprising the steps of: 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 , 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

[0010] The present invention provides heterodimeric Fc fusion proteins 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 producing 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-12p4 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)), x 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) 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: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 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 / N103Q / 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 provides IL-12p35 subunits comprising: 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 producing a IL-12p35 subunit comprising administering to a patient in need thereof a IL-12p35 sequence comprising the IL-12p35 subunit of the present invention, the IL-12p35 subunit comprising the IL-12p35 subunit of the present invention being: 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: 119 120 (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 xli) 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 present invention provides one or more nucleic acids encoding heterodimeric Fc-fusion proteins.

[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 methods for making a heterodimeric Fc-fusion protein, the methods comprising culturing a host cell under conditions such that the heterodimeric Fc-fusion protein is produced.

[0127] In some embodiments, the invention provides methods for purifying a heterodimeric Fc-fusion protein, the method comprising: a) providing a composition comprising the 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 present 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 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 modifications that promote heterodimerization of the first and second Fc domains are 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 heterodimeric Fc fusion proteins, which are a set of amino acid substitutions selected from:

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

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

[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 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).

[0134] In some embodiments, the invention provides heterodimeric Fc fusion proteins, wherein the 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 the 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 the 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 IL-12p40 subunits, wherein 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, L4 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 provides IL-12p40 subunits comprising: 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.

[0139] In some embodiments, the present invention provides a method for the preparation of IL-12p40 subunits comprising the steps of: 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 , 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 producing 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(E45E)), 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 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 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(D18K / E59K / K99E / C252S / K264E)), lxvi) SEQ ID NO: XXX (IL-12p40(E59K / K99E / C252S / K264E)), 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 and cix) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N103Q / N200Q / N281Q / C252S / K264E)).

[0141] In some embodiments, the present invention provides IL-12p35 subunits comprising any 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, L75, N76, E79, N85, L89, F96, M97, L124, M125, Q130, Q135, N and A196, wherein the amino acid residues are 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.

[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 producing a IL-12p35 subunit 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: 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)), 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 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)).

[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 present invention provides one or more nucleic acids encoding heterodimeric Fc-fusion proteins.

[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 methods for making a heterodimeric Fc-fusion protein, the methods comprising culturing a host cell under conditions such that the heterodimeric Fc-fusion protein is produced.

[0149] In some embodiments, the present 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 fractions 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 heterodimeric complexes wherein the IL-12p40 subunit is covalently linked to the IL-12p35 subunit using a domain linker.

[0152] In some embodiments, the invention provides heterodimeric complexes 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 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 the 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.

[0156] In some embodiments, the present invention relates to IL-12p40 subunits, wherein the IL-12p40 subunit is 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, D209, D214, N218, Q220, N226, Q229, E231, E235, T242, P243, S245, Y and Y292, Y293, and E299.

[0157] In some embodiments, the present invention relates to IL-12p40 subunits, wherein the IL-12p40 subunit is D18N, D18K, E32Q, E33Q, D34N, D34K, Q42E, S43E, S43K, E45Q, Q56E, E59Q, E59K, D62N, E73Q, D87N, K99E, K99Y, E100Q, N103D, N103Q, N113D, N113Q, Q1 and E299Q, wherein the heterodimeric complex has 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 provides a method for the preparation of IL-12p40 subunits comprising the steps of: 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 , 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 are provided 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 producing 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 / IL-12p40(E45K / E59K / K99E)), 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(E45K / E59K / K99E)), 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 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-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 IL-12p35 subunits, wherein the IL-12p35 subunit is 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, M97, L124, M125, Q130, Q13 and A196, wherein the amino acid residues are selected from the group consisting of: 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 heterodimeric complexes 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 heterodimeric complexes, 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.

[0163] In some embodiments, the present invention relates to a method for producing IL-12p35 subunits 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) 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 host cells comprising one or more of the nucleic acids encoding the heterodimeric complex.

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

[0168] In some embodiments, the present 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 variant IL-12p40 subunits, the variant IL-12p40 subunits 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, 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 having amino acid substitutions selected from the group consisting of 99E / N113Q / N200Q / N281Q / C252S / K264E, 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 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- 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 / 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 present invention provides compositions comprising a variant IL-12p40 subunit.

[0174] In some embodiments, the present 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 such that 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 comprising any 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, L75, N76, E79, N85, L89, F96, and 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 present 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 present invention provides a variant IL-12p35 subunit, wherein the variant IL-12p35 subunit has amino acid substitutions N71D / N85D / N195D.

[0182] In some embodiments, the present invention provides variant IL-12p35 subunits, the variant IL-12p35 subunits 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)), xxxviii) SEQ ID NO:XXX 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 present invention provides compositions comprising a variant IL-12p35 subunit.

[0184] In some embodiments, the present 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 in which 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, wherein the first and second Fc domains have the following structure according to EU numbering: L368D / K370S and S364K; L368D / K370S and S364K / E357L; L368D / Heterodimeric proteins are provided, wherein the set of amino acid substitutions is 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 heterodimeric proteins 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 heterodimeric proteins 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 heterodimeric proteins, 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 heterodimeric proteins, wherein the first and second Fc domains comprise an IgG1 hinge-CH2-CH3.

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

[0193] In some embodiments, the invention provides a method for producing a fusion protein comprising, from N-terminus to C-terminus, a hinge-CH2-CH3-domain linker-IL-12p40 subunit domain. and the 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 producing 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(E45E)), 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 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, 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 producing a IL-12p35 subunit comprising administering to a patient in need thereof a IL-12p35 sequence comprising the IL-12p35 subunit of the present invention, the IL-12p35 subunit comprising the IL-12p35 subunit of the present invention being: 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: 119 120 (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 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 have the following amino acid sequences 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- 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 producing 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(N103D / N113D / N200D / N281D)), vi) SEQ ID NO: 63 (IL-12p40(N103D / N113D / N200D / N281D)), vi) SEQ ID NO: 64 (IL-12p40(N103D / N113D / N200D / N281D)), vi) SEQ ID NO: 65 (IL-12p40(N103D / N113D / N200D / N281D)), vi) SEQ ID NO: 66 (IL-12p40(N103D / N113D / N200D / N281D)), vi) SEQ ID NO: 67 (IL-12p40(N103D / N113D / N200D / N281D)), vi) SEQ ID NO: 68 (IL-12p40(N103D / N113D / N200D / N281D)), vi) SEQ ID NO: 69 (IL-12p40(N103 ) 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 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(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:XXX 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 c) SEQ ID NO: XXX (IL-12p40(E59K / K99E / N281Q / C252S / K264E)), cii) SEQ ID NO: XXX (IL-12p40(E59K / K99E / N103Q / N113Q / 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)), 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 provides a method for producing a IL-12p35 subunit comprising administering to a patient in need thereof ... 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(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 having a sequence selected from the group consisting of 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));Heterodimeric proteins are provided.

[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-an optional first domain linker-an IL-12p35 subunit domain-an optional second domain linker-an 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 homodimeric Fc-fusion proteins, wherein the first domain linker and the 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, according to EU numbering, Q295E / N384D / Q418E / N421D.

[0207] In some embodiments, the present invention provides a method for the production of a mAb-based 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 homodimeric Fc fusion proteins, 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 the 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 the 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 IL-12p40 subunits comprising the amino acids 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 provides a method for the preparation of IL-12p40 subunits comprising the steps of: 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 , 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 Homodimeric 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.

[0215] In some embodiments, the present invention relates to a method for producing 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-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 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 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 IL-12p35 subunits comprising the amino acids Q20, N21, Q35, E38, S44, E45, E46, H49, K54, D55, T59, Homodimeric Fc fusion proteins are provided, comprising 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 producing IL-12p35 subunits 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 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(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 the first and second monomers are XENP31289.

[0221] In some embodiments, a homodimeric Fc-fusion protein is provided, wherein the 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 present 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 methods for making a homodimeric Fc-fusion protein, the methods comprising culturing a host cell under conditions such that the homodimeric Fc-fusion protein is produced.

[0226] In some embodiments, the invention provides methods for purifying a homodimeric Fc-fusion protein, the method comprising: a) providing a composition comprising the 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, an IL-12p35 subunit domain-an optional first domain linker-an IL-12p40 subunit domain-an optional second domain linker-an Fc domain.

[0228] In some embodiments, the present invention provides a method for enhancing homodimerization of the Fc domain, wherein the modifications that promote homodimerization of the Fc domain are, according to EU numbering, L368D / K370S; S364K; S364K / E357L; S364K / E357Q; T411E / K360E / Q362E; D401K; T Homodimeric Fc fusion proteins are provided in which the set of amino acid substitutions is selected from the group consisting of: 366S / L368A / Y407V; T366W; T366S / L368A / Y407V / Y349C; and T366W / S354C.

[0229] In some embodiments, the invention provides homodimeric Fc-fusion proteins, wherein the first domain linker and the 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, according to EU numbering, Q295E / N384D / Q418E / N421D.

[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 homodimeric Fc fusion proteins, 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 the 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 the 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 and E299, wherein the amino acid residues are selected from the group consisting of: 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 IL-12p40 subunits, wherein the IL-12p40 subunit is 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 provides a method for the preparation of IL-12p40 subunits comprising the steps of: 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 , 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 provides a method for the production of IL-12p40 subunits 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 Sequence number 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 / K264E)), 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 cviii) SEQ ID NO:XXX(IL-12p40(E59K / K99E / N103Q / 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 provides IL-12p35 subunits comprising any 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, L75, N76, E79, N85, L89, F96, M97, L124, M125, Q130, Q135, N and A196, wherein the amino acid residues are 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 producing a IL-12p35 subunit comprising administering to a patient in need thereof a IL-12p35 sequence comprising the IL-12p35 subunit 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(I 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: 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 the first and second monomers are XENP31289.

[0244] In some embodiments, a homodimeric Fc-fusion protein is provided, wherein the 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 present 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 methods for making a homodimeric Fc-fusion protein, the methods comprising culturing a host cell under conditions such that the homodimeric Fc-fusion protein is produced.

[0249] In some embodiments, the invention provides methods for purifying a homodimeric Fc-fusion protein, the method comprising: a) providing a composition comprising the 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 homodimeric Fc-fusion proteins, wherein the first domain linker and the 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, according to EU numbering, Q295E / N384D / Q418E / N421D.

[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 homodimeric Fc fusion proteins, 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 the 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 the 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 IL-12p40 subunits, wherein the IL-12p40 subunit is 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 IL-12p40 subunits 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 producing 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(E45E)), 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 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) 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: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 and cix) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N113Q / N200Q / N281Q / C252S / K264E)).

[0263] In some embodiments, the present invention provides IL-12p35 subunits comprising any 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, L75, N76, E79, N85, L89, F96, M97, L124, M125, Q130, Q135, N and A196, wherein the amino acid residues are 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 present invention provides that the IL-12p35 subunit is 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 producing a IL-12p35 subunit comprising administering to a patient in need thereof a IL-12p35 sequence comprising the IL-12p35 subunit of the present invention, the IL-12p35 subunit comprising the IL-12p35 subunit of the present invention being: 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: 119 120 (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 the first and second monomers are XENP31289.

[0268] In some embodiments, a homodimeric Fc-fusion protein is provided, wherein the 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 present 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 methods for making a homodimeric Fc-fusion protein, the methods comprising culturing a host cell under conditions such that the homodimeric Fc-fusion protein is produced.

[0273] In some embodiments, the invention provides methods for purifying a homodimeric Fc-fusion protein, the method comprising: a) providing a composition comprising the 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 homodimeric Fc-fusion proteins, wherein the first domain linker and the 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, according to EU numbering, Q295E / N384D / Q418E / N421D.

[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 provides a method for producing IL-12p40 subunits 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 homodimeric Fc fusion proteins, 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 the 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 the 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 IL-12p40 subunits, wherein the IL-12p40 subunit is 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 provides an IL-12p40 subunit comprising N103D / 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 and E59K / K99E / N103Q / N200Q / N281Q / C252S / K264E.

[0286] In some embodiments, the present invention relates to a method for producing 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(I IL-12p40(E45Q / Q56E)), 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 No. XXX(IL-12p40(N113Q / N200Q)), lxxxv) Sequence No. XXX(IL-12p40(N113Q / N281Q)), lxxxvi) Sequence No. XXX(IL-12p40(N200Q / N281Q)), lxxxvii) Sequence No. XXX(IL-12p40(N103Q / N113Q / N200Q)), lxxxviii) Sequence No. XXX(IL-12p40(N103Q / N113Q / N281Q)), lxxxix) Sequence No. XXX(IL-12p40(N103Q / N200Q / N281Q)), xc) Sequence 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: cvii) SEQ ID NO:XXX(IL-12p40(E59K / K99E / 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)).

[0287] In some embodiments, the present invention provides IL-12p35 subunits comprising any 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, L75, N76, E79, N85, L89, F96, M97, L124, M125, Q130, Q135, N and A196, wherein the amino acid residues are 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 producing a IL-12p35 subunit comprising administering to a patient in need thereof a IL-12p35 sequence comprising the IL-12p35 subunit of the present invention, the IL-12p35 subunit comprising the IL-12p35 subunit of the present invention being: 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: 119 120 (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, xli) 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 the first and second monomers are XENP31289.

[0292] In some embodiments, a homodimeric Fc-fusion protein is provided, wherein the 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 present 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 methods for making a homodimeric Fc-fusion protein, the methods comprising culturing a host cell under conditions such that the homodimeric Fc-fusion protein is produced.

[0297] In some embodiments, the invention provides methods for purifying a homodimeric Fc-fusion protein, the method comprising: a) providing a composition comprising the 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 homodimeric Fc-fusion proteins, wherein the first domain linker and the second domain linker have the same amino acid sequence.

[0301] In some embodiments, the present invention provides a method for the production of Fc domains comprising administering to a mammalian subject the present invention, wherein the Fc domain is Q29 according to EU numbering. Homodimeric Fc fusion proteins are provided with 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 homodimeric Fc fusion proteins, 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 the 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 the 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 α, β, β- and β-glucan.

[0308] In some embodiments, the present invention relates to IL-12p40 subunits, wherein the IL-12p40 subunit is 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 provides a method for the preparation of IL-12p40 subunits comprising the steps of: 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 , 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 Homodimeric 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. In some embodiments, the present invention provides a method for the production of IL-12p40 subunits 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(Q256N)), 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-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:XXX (IL-12p40(N113D / N200D)), 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 / xcvi) SEQ ID NO: XXX (IL-12p40(E59K / K99E / N103Q / C252S / K264E)), 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)), or 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 provides IL-12p35 subunits comprising any 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, L75, N76, E79, N85, L89, F96, M97, L124, M125, Q130, Q135, N and A196, wherein the amino acid residues are 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 producing IL-12p35 subunits 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-12p 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 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-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 homodimeric Fc-fusion proteins 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 the first and second monomers are XENP31289.

[0315] In some embodiments, a homodimeric Fc-fusion protein is provided, wherein the 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 present 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 methods for making a homodimeric Fc-fusion protein, the methods comprising culturing a host cell under conditions such that the homodimeric Fc-fusion protein is produced.

[0320] In some embodiments, the invention provides methods for purifying a homodimeric Fc-fusion protein, the method comprising: a) providing a composition comprising the 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 homodimeric Fc-fusion proteins, wherein the first domain linker and the 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, according to EU numbering, Q295E / N384D / Q418E / N421D.

[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 homodimeric Fc fusion proteins, 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 the 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 the 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 IL-12p40 subunits, wherein the IL-12p40 subunit is 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 IL-12p40 subunits 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 / 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 Homodimeric 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. In some embodiments, the present invention provides a method for the production of IL-12p40 subunits 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, K / 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 / 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(E32K / D34N / E59K / K99E)), 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 / 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 and cix) SEQ ID NO: XXX(IL-12p40(E59K / K99E / N113Q / N200Q / N281Q / C252S / K264E)).

[0333] In some embodiments, the present invention provides IL-12p35 subunits comprising any 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, L75, N76, E79, N85, L89, F96, M97, L124, M125, Q130, Q135, N and A196, wherein the amino acid residues are 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 provides a method for producing a IL-12p35 subunit comprising administering to a mammalian subject the IL-12p35 subunit of which the IL-12p35 subunit is 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: 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 the first and second monomers are XENP31289.

[0338] In some embodiments, a homodimeric Fc-fusion protein is provided, wherein the 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 present 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 methods for making a homodimeric Fc-fusion protein, the methods comprising culturing a host cell under conditions such that the homodimeric Fc-fusion protein is produced.

[0343] In some embodiments, the invention provides methods for purifying a homodimeric Fc-fusion protein, the method comprising: a) providing a composition comprising the 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 homodimeric Fc-fusion proteins, wherein the first domain linker and the 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, according to EU numbering, Q295E / N384D / Q418E / N421D.

[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 homodimeric Fc fusion proteins, wherein each of the Fc domains further comprises the amino acid substitutions M428L / N424S.

[0351] In some embodiments, the present invention provides a method for the preparation of IL-12p40 subunits comprising administering to a mammalian subject the method ... 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 the 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 the 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 IL-12p40 subunits, wherein the IL-12p40 subunit is 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 IL-12p40 subunits 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 / 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 Homodimeric Fc fusion proteins are provided 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 provides a method for the production of IL-12p40 subunits 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- 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 provides IL-12p35 subunits comprising any 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, L75, N76, E79, N85, L89, F96, M97, L124, M125, Q130, Q135, N and A196, wherein the amino acid residues are 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 IL-12p35 subunits comprising the amino acids N71D / N85D / N195D, N151D / E153Q, N151D / D165N, Q130E / N151D, N151D / K158E, E79Q / N151D, D55Q / N151 and N85D / N195D.

[0359] In some embodiments, the present invention relates to a method for producing a IL-12p35 subunit comprising administering to a patient in need thereof a IL-12p35 sequence comprising the IL-12p35 subunit of the present invention, the IL-12p35 subunit comprising the IL-12p35 subunit of the present invention being: 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: 119 120 (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 the first and second monomers are XENP31289.

[0361] In some embodiments, a homodimeric Fc-fusion protein is provided, wherein the 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 present 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 methods for making a homodimeric Fc-fusion protein, the methods comprising culturing a host cell under conditions such that the homodimeric Fc-fusion protein is produced.

[0366] In some embodiments, the invention provides methods for purifying a homodimeric Fc-fusion protein, the method comprising: a) providing a composition comprising the 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 homodimeric Fc-fusion proteins, wherein the first domain linker and the 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, according to EU numbering, Q295E / N384D / Q418E / N421D.

[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 homodimeric Fc fusion proteins, 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 the 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 the 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 IL-12p40 subunits, wherein the IL-12p40 subunit is 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 provides a method for the preparation of IL-12p40 subunits comprising the steps of: 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 , 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 Homodimeric 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. In some embodiments, the present invention provides a method for the production of IL-12p40 subunits 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) 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 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- cix) 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)).

[0379] In some embodiments, the present invention relates to IL-12p35 subunits comprising any 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 monomeric 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 producing a IL-12p35 subunit comprising administering to a patient in need thereof a IL-12p35 sequence comprising the IL-12p35 subunit of the present invention, the IL-12p35 subunit comprising the IL-12p35 subunit of the present invention being: 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: 119 120 (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 the first and second monomers are XENP31289.

[0384] In some embodiments, a homodimeric Fc-fusion protein is provided, wherein the 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 present 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 methods for making a homodimeric Fc-fusion protein, the methods comprising culturing a host cell under conditions such that the homodimeric Fc-fusion protein is produced.

[0389] In some embodiments, the invention provides methods for purifying a homodimeric Fc-fusion protein, the method comprising: a) providing a composition comprising the 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 methods 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 present 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 methods 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 prior to treatment.

[0398] In some embodiments, the invention provides methods 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 prior to 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 methods 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 T cell proliferation in a patient in need of such treatment. The present invention provides a method of inducing T cell proliferation using 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 present invention provides an IL-12p40 subunit. In another aspect, the present invention provides an IL-12p35 subunit. In a further aspect, the present invention provides a heterodimeric complex comprising a) an IL-12p40 subunit and b) an IL-12p35 subunit. [Brief explanation of the drawings]

[0408] [Figure 1] 1A and 1B show the sequences of IL-12 and its receptor. [Figure 2] Figures 2A-2E show useful pairs of Fc heterodimerization variant sets (including skewed and pI variants). Variants without a corresponding "monomer 2" are pI variants that can be used alone for either monomer. [Figure 3] A list of isosteric variant antibody constant regions and their respective substitutions is provided below. pI_(-) indicates a low pI variant and pI_(+) indicates a high pI variant. These can be optionally and independently combined with other heterodimerization variants of the invention (and other variant species 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 be found in only one monomer. [Figure 5] Particularly useful embodiments of the "non-cytokine" components of the present invention are presented. [Figure 6]Several exemplary domain linkers are shown. In some embodiments, these linkers find use in linking the IL-12p35 subunit, the IL-12p40 subunit, or a single-chain IL-12 complex to the N-terminus of the Fc region. In some embodiments, these linkers find use in fusing the IL-12p35 subunit to the IL-12p40 subunit in a single-chain IL-12 complex. It is important to note that an scIL-12 complex can contain either IL-12p35 linked N-terminally to IL-12p40 or IL-12p40 linked N-terminally to IL-12p35. Also, in some cases described herein, the hinge portion of the Fc domain functions as a domain linker and can be similarly combined with any of these linkers. [Figure 7]7A-7E show the sequences of several useful IL-12-Fc fusion backbones based on human IgG1 that do not contain cytokine sequences (e.g., the IL-12 p35 subunit, the IL-12 p40 subunit, or the scIL-12 complex). Backbone 1 is based on human IgG1 (356E / 358M allotype) and includes the C220S, S364K / E357Q:L368D / K370S sucvariant on both chains, the Q295E / N384D / Q418E / N421D pI variant on one chain with the L368D / K370S sucvariant, and the E233P / L234V / L235A / G236del / S267K ablation variant on both chains. Backbone 2 is based on human IgG1 (356E / 358M allotype) and contains the C220S, S364K:L368D / K370S scubariants on both chains, the Q295E / N384D / Q418E / N421D pI variants on the chain with the L368D / K370S scubariant, and the E233P / L234V / L235A / G236del / S267K ablation variants on both chains. Backbone 3 is based on human IgG1 (356E / 358M allotype) and contains the C220S, S364K:L368E / K370S scuba variants on both chains, the Q295E / N384D / Q418E / N421D pI variants on the chain with the L368E / K370S scuba variant, and the E233P / L234V / L235A / G236del / S267K ablation variants 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 scuba variant, a Q295E / N384D / Q418E / N421D pI variant on the chain with the L368D / K370S scuba variant, 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 scubariants, Q295E / N384D / Q418E / N421D pI variants on the chain with the L368D / K370S scubariant, and E233P / L234V / L235A / G236del / S267K ablation variants on both chains, and N297A variants on both chains. Backbone 7 is identical to 6 except for the N297S mutation. 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 the S364K / E357Q:L368D / K370S scuba variant, the Q295E / N384D / Q418E / N421D pI variant on the chain with the L368D / K370S scuba variant, and the S228P (EU numbering, i.e., S241P in Kabat) variant on both chains, which ablate Fab arm exchange as known in the art. Backbone 9 is based on human IgG2 and contains the S364K / E357Q:L368D / K370S scuba variant, the 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 scubariant, the Q295E / N384D / Q418E / N421D pI variant on the chain with the L368D / K370S scubariant, 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 contains the C220S, S364K / E357Q:L368D / K370S scubariant on both chains, the P217R / P229R / N276K pI variant on one chain with the S364K / E357Q scubariant, and the E233P / L234V / L235A / G236del / S267K ablation variant on both chains. As will be appreciated by those of skill 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 schematically in Figure 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 can 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 sequence 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 several 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 can contain additional amino acid modifications (generally amino acid substitutions) in addition to the scuba variants, pI variants, and ablation variants included within the backbone of this figure. [Figure 8]Figures 8A-8F show exemplary formats for the IL-12-Fc fusion proteins of the invention. The N-terminal IL-12 heterodimer Fc fusion or "IL-12-hetero-Fc" (Figure 8A) format comprises an IL-12p40 subunit recombinantly fused to the N-terminus of one side of the heterodimeric Fc and an IL-12p35 subunit recombinantly fused to the N-terminus of the other side of the heterodimeric Fc. The IL-12p35 and IL-12p40 subunits may be linked to their respective Fc chains by a domain linker. The C-terminal IL-12 heterodimer Fc fusion or "hetero-Fc-IL-12" (Figure 8B) format comprises an IL-12p40 subunit recombinantly fused to the C-terminus of one side of the heterodimeric Fc and an IL-12p35 subunit recombinantly fused to the C-terminus of the other side of the heterodimeric Fc. The IL-12p35 and IL-12p40 subunits can be linked to their respective Fc chains by domain linkers. The N-terminal single-chain IL-12-Fc fusion or "scIL-12-Fc" (Figure 8C-D) format comprises a single-chain IL-12 complex (or "scIL-12 complex") recombinantly fused to the N-terminus of one side of a heterodimeric Fc (optionally via a domain linker), with the other side of the molecule being an "Fc-only" or "empty Fc" heterodimeric Fc. The C-terminal single-chain IL-12-Fc fusion or "Fc-scIL-12" (Figure 8E-F) format comprises a scIL-12 complex recombinantly fused to the C-terminus of one side of a heterodimeric Fc (optionally via a domain linker), with the other side of the molecule being an "Fc-only" or "empty Fc" heterodimeric Fc. The scIL-12 complex can contain either IL-12p35 linked N-terminally to IL-12p40 or IL-12p40 linked N-terminally to IL-12p35, typically but optionally with a domain linker. The order of the two subunits in the scIL-12 complex can be designated as follows: "scIL-12(p40 / p35)," where the IL-12p40 subunit is linked N-terminally to the IL-12p35 subunit, or "scIL-12(p35 / p40)," where IL-12p35 is linked N-terminally to the IL-12p40 subunit. [Figure 9]6 shows the sequence of an exemplary IL-12-Fc fusion protein of the XENP27201 "IL-12-hetero-Fc" format, containing wild-type IL-12p40 and wild-type IL-12p35 sequences. The linker is double underlined (although, as will be understood by those skilled in the art, the linker can be replaced by other linkers, some of which are shown in FIG. 6), and a slash ( / ) indicates the boundary(s) between the IL-12p35, IL-12p40, linker, and Fc regions. [Figure 10] 6 shows the sequence of an exemplary IL-12-Fc fusion protein in the "hetero-Fc-IL-12" format, XENP27202, containing wild-type IL-12p40 and wild-type IL-12p35 sequences. The linker is double underlined (although, as will be understood by those skilled in the art, the linker can be replaced by other linkers, some of which are shown in FIG. 6), and a slash ( / ) indicates the boundary(s) between the IL-12p35, IL-12p40, linker, and Fc regions. [Figure 11] 6 shows the sequences of exemplary IL-12-Fc fusion proteins in the "scIL-12-Fc" format, XENP27203 and XENP27204, which contain wild-type IL-12p40 and wild-type IL-12p35 sequences. The linker is double underlined (although, as will be understood by those skilled in the art, the linker can be replaced by other linkers, some of which are shown in FIG. 6), and a slash ( / ) indicates the boundary(s) between the IL-12p35, IL-12p40, linker, and Fc region. [Figure 12] Figures 12A-12B show A) chromatograms showing the purification part 2 of XENP27201 (protein A chromatography followed by anion exchange chromatography), and the purity and homogeneity of peak B isolated from the anion exchange separation shown in Figure 12A compared to peak A as determined by B) analytical size exclusion chromatography with multi-angle light scattering (aSEC-MALS) and C) analytical anion exchange chromatography (analytical AIEX). Figure 12B also shows the molecular weight of the protein species in the peak as determined by multi-angle light scattering. [Figure 13] Figures 13A-13C show A) chromatograms showing the purification part 2 of XENP27203 (protein A chromatography followed by anion exchange chromatography), and the purity and homogeneity of peak B isolated from the anion exchange separation shown in Figure 13A compared to peak A as determined by B) analytical size exclusion chromatography with multi-angle light scattering (SEC-MALS) and C) analytical anion exchange chromatography. Figure 13B also shows the molecular weight of the protein species in the peak as determined by multi-angle light scattering. [Figure 14] Figures A-14B show cartoon schematics of A) bivalent IL-12p40-Fc fusions and B) bivalent IL-12p35-Fc fusions. Each fusion contains either an IL-12p40 or IL-12p35 subunit recombinantly fused to the N-terminus of a homodimeric Fc. The subunits may have a domain linker between their respective C- and N-termini of the Fc region. [Figure 15] 14 shows the sequences of XENP27560, a bivalent IL-12p40-Fc fusion, and XENP27561, a bivalent IL-12p35-Fc fusion (cartoon schematic shown in FIG. 14), which contain wild-type IL-12p40 and wild-type IL-12p35 sequences. The linker is double underlined (although, as will be understood by those skilled in the art, the linker can be replaced by other linkers, some of which are shown in FIG. 6), and a slash ( / ) indicates the boundary(s) between the IL-12p35, IL-12p40, linker, and Fc regions. [Figure 16] Figures 16A-16D show STAT4 phosphorylation on A) CD4+CD45RA-CD25hi T cells, B) CD8+CD45RA-CD25hi T cells, C) γδ T cells, and D) NK cells following incubation of activated PBMCs with the indicated test articles. [Figure 17] Residues on IL-12p40 predicted to contribute to binding of IL-12p40 at the IL-12 receptor (based on the IL-12p40 mature form sequence) are shown. [Figure 18]Shown are aspartic acid, glutamic acid, asparagine, and glutamine residues on IL-12p40 (based on the IL-12p40 mature form sequence) identified as having an ASA score (water accessible surface area calculated using the 1.4 Å radius of the water molecule and the polyhedral representation of each atom) of at least 19 using the QuaSAR package in MOE. [Figure 19] Shown are residues on IL-12p40 (based on the IL-12p40 mature sequence) predicted to contact the IL-23 receptor (based on the crystal structure deposited in the PDB under accession number 5MZV) and the predicted contact type(s). "D" indicates a contact predicted based on proximity. "H" indicates a contact predicted based on potential hydrogen bonding. "I" indicates a contact predicted based on potential salt bridges. "A" indicates a contact predicted based on potential arene bonding. [Figure 20] 20A-20I show sequences for exemplary IL-12p40 variants d...

Claims

1. 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 having the amino acid substitutions M428L / N434S in the amino acid sequence set forth in SEQ ID NO: 404; Numbering follows EU numbering, Heterodimeric Fc fusion proteins.

2. a) a first nucleic acid encoding the first fusion protein of claim 1; and b) a second nucleic acid encoding the second fusion protein of 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 of 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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Cytokine fused to immunoglobulin fc heterodimer and pharmaceutical composition comprising same
WO2018030806A1

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Cytokine fused to immunoglobulin fc heterodimer and pharmaceutical composition comprising same