IL-12 heterodimeric fc fusion proteins

By designing a heterodimeric Fc fusion protein, the problems of short half-life and toxicity of recombinant IL-12 in humans were solved, and the binding of IL-12 to the IL-12 receptor was optimized, resulting in more effective cell signaling and therapeutic effects.

CN113195523BActive Publication Date: 2025-11-04XENCOR INC
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Patent Information

Application Number
CN201980075579.9
Authority / Receiving Office
CN · China
Patent Type
Patents(China)
Current Assignee / Owner
Priority Date
2019-05-15
Filing Date
2019-10-03
Publication Date
2025-11-04
Estimated Expiration
2040-01-24

AI Technical Summary

Technical Problem

The use of recombinant IL-12 in humans is limited by its short half-life and toxicity issues, and the homodimer of the IL-12p40 subunit competitively binds to the IL-12 receptor, leading to antagonistic activity and affecting its cellular signaling function.

Method used

A heterodimeric Fc fusion protein was designed by substituting and modifying the amino acid subunits of IL-12p40 and IL-12p35 to form a heterodimer, which enhances its binding to the Fc domain, prolongs its half-life and reduces its toxicity, while optimizing its affinity for the IL-12 receptor.

Benefits of technology

It prolongs the half-life of IL-12, reduces toxicity, improves the binding efficiency with IL-12 receptor, enhances cell signaling function, and has better therapeutic effects.

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Abstract

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 treating cancer and can be used in combination with checkpoint blockade.
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Description

[0001] Cross-Reference to Related Applications

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 740,813, filed October 3, 2018, U.S. Provisional Application No. 62 / 810,038, filed February 25, 2019, and U.S. Provisional Application No. 62 / 848,512, filed May 15, 2019, all of which are hereby incorporated by reference in their entirety for all purposes.

[0003] SEQUENCE LISTING

[0004] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. This ASCII copy, created on November 21, 2019, is named 067461-5225-US_SL.txt and is 1,640,919 bytes in size. BACKGROUND

[0005] For T cells to mount an effective anti-tumor response, three things must happen. The T cell must first bind to an antigenic tumor peptide presented by MHC in the tumor environment. Second, a costimulatory molecule must bind to the T cell. And third, the T cell must be induced by cytokines such as IL-12 and IL-2 to produce a costimulatory cytokine such as IFNy that allows for differentiation and expansion. In the absence of cytokine induction, recognition of tumor peptide alone can lead to T cells becoming anergic, thereby leading to tolerance. Thus, a very promising approach in cancer immunotherapy is cytokine-based therapy. 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.

[0006] Recombinant IL-12 is a promising cytokine-based therapy because of its broad effects in activating the immune system. However, IL-12 has faced obstacles in human clinical trials to date due to toxicity. Like other cytokines, IL-12 has a short half-life, which necessitates frequent bolus injections.

[0007] In addition, IL-12 is composed of an alpha chain (p35 subunit; IL-12p35) and a beta chain (p40 subunit; IL-12p40) covalently linked to form a biologically active IL-12 heterodimer. IL-12 exerts its cell signaling function by binding to a dimeric IL-12 receptor complex composed of IL-12 receptor beta 1 (IL-12Rβ1) and IL-12 receptor beta 2 (IL-12Rβ2) on T cells and inducing IFNγ secretion. However, the IL-12p40 subunit can also exist in a homodimeric form, which reportedly antagonizes IL-12 activity by competing for binding to the IL-12 receptor.

[0008] The present application 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

[0009] In one aspect, the present application 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 Fc domain and the second Fc domain comprise a modification that promotes heterodimerization of the first Fc domain and the second Fc domain. In some embodiments, the present application provides a heterodimeric Fc fusion protein, wherein the IL-12p40 subunit has one or more amino acid substitutions selected from the group consisting of E59K, E59Q, D18N, 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.

[0010] In some embodiments, the present application provides a heterodimeric Fc fusion protein wherein the IL-12p40 subunit has an amino acid substitution selected from the group consisting 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, 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, 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 / C252S / K264E, E59K / K99E / N113Q / C252S / K264E, E59K / K99E / N200Q / 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 / 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.

[0011] In some embodiments, the present application provides a heterodimeric Fc fusion protein, wherein the IL-12p40 subunit has a polypeptide sequence 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)); 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 (S43K); xxxiii) SEQ ID NO: 379 (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: 326 (IL-12p40 (D34N / E59K)); 1) SEQ ID NO: 325 (IL-12p40 (E59K / K99E)); li) SEQ ID NO: 339 (IL-12p40 (D18K / E59K / K99E)); lii) SEQ ID NO: 343 (IL-12p40 (E59K / K99E / K264E)); liii) SEQ ID NO: 336 (IL-12p40 (E59K / K99Y)); liv) SEQ ID NO: 335 (IL-12p40 (E59Y / K99E));lv) SEQ ID NO: 338 (IL-12p40 (E45K / E59K / K99E)); lvi) SEQ ID NO: 340 (IL-12p40 (E59K / K99E / Q144E)); lvii) SEQ ID NO: 341 (IL-12p40 (E59K / K99E / Q144K)); lviii) SEQ ID NO: 342 (IL-12p40 (E59K / K99E / R159E)); Ix) SEQ ID NO: 343 (IL-12p40 (E59K / K99E / K264E)); lx) SEQ ID NO: 344 (IL-12p40 (D18K / E59K / K99E / K264E)); lxi) SEQ ID NO: 360 (IL-12p40 (C252S)); lxii) SEQ ID NO: 361 (IL-12p40 (D18K / E59K / K99E / C252S)); lxiii) SEQ ID NO: 362 (IL-12p40 (D18K / E59K / K99E / C252S / K264E)); lxiv) SEQ ID NO: 363 (IL-12p40 (E59K / K99Y / C252S)); lxv) SEQ ID NO: 364 (IL-12p40 (E59K / K99E / C252S / K264E)); lxvi) SEQ ID NO: 365 (IL-12p40 (E59K / K99E / C252S)); lxvii) SEQ ID NO: 254 (IL-12p40 (N103D / N113D)); lxviii) SEQ ID NO: 255 (IL-12p40 (N103D / N200D)); lxix) SEQ ID NO: 256 (IL-12p40 (N103D / N281D)); lxx) SEQ ID NO: 257 (IL-12p40 (N113D / N200D)); lxxi) SEQ ID NO: 258 (IL-12p40 (N113D / N281D)); lxxii) SEQ ID NO: 259 (IL-12p40 (N200D / N281D)); lxxiii) SEQ ID NO: 260 (IL-12p40 (N103D / N113D / N200D)); lxxiv) SEQ ID NO: 261 (IL-12p40 (N103D / N113D / N281D)); lxxv) SEQ ID NO: 262 (IL-12p40 (N103D / N200D / N281D));lxxvi) SEQ ID NO: 263 (IL-12p40 (N113D / N200D / N281D)); lxxvii) SEQ ID NO: 264 (IL-12p40 (N103Q)); lxxviii) SEQ ID NO: 265 (IL-12p40 (N113Q)); lxxix) SEQ ID NO: 266 (IL-12p40 (N200Q)); lxxx) SEQ ID NO: 267 (IL-12p40 (N281Q)); lxxxi) SEQ ID NO: 268 (IL-12p40 (N103Q / N113Q)); lxxxii) SEQ ID NO: 269 (IL-12p40 (N103Q / N200Q)); lxxxiii) SEQ ID NO: 270 (IL-12p40 (N103Q / N281Q)); lxxxiv) SEQ ID NO: 271 (IL-12p40 (N113Q / N200Q)); lxxxv) SEQ ID NO: 272 (IL-12p40 (N113Q / N281Q)); lxxxvi) SEQ ID NO: 273 (IL-12p40 (N200Q / N281Q)); lxxxvii) SEQ ID NO: 274 (IL-12p40 (N103Q / N113Q / N200Q)); lxxxviii) SEQ ID NO: 275 (IL-12p40 (N103Q / N113Q / N281Q)); lxxxix) SEQ ID NO: 276 (IL-12p40 (N103Q / N200Q / N281Q)); xc) SEQ ID NO: 277 (IL-12p40 (N113Q / N200Q / N281Q)); xci) SEQ ID NO: 278 (IL-12p40 (N103Q / N113Q / N200Q / N281Q)); xcii) SEQ ID NO: 327 (IL-12p40 (D34N / E59K / K99E)); xciii) SEQ ID NO: 328 (IL-12p40 (D34K / E59K / K99E)); xciv) SEQ ID NO: 329 (IL-12p40 (E32Q / D34N / E59K / K99E)); xcv) SEQ ID NO: 331 (IL-12p40 (E32K / D34N / E59K / K99E)); xcvi) SEQ ID NO: 337 (IL-12p40 (E59Y / K99Y));xcvii) SEQ ID NO: 366 (IL-12p40 (E59K / K99E / N103Q / C252S / K264E)); xcviii) SEQ ID NO: 367 (IL-12p40 (E59K / K99E / N113Q / C252S / K264E)); xcix) SEQ ID NO: 368 (IL-12p40 (E59K / K99E / N200Q / C252S / K264E)); c) SEQ ID NO: 369 (IL-12p40 (E59K / K99E / N281Q / C252S / K264E)); ci) SEQ ID NO: 370 (IL-12p40 (E59K / K99E / N103Q / N113Q / C252S / K264E)); cii) SEQ ID NO: 371 (IL-12p40 (E59K / K99E / N103Q / N200Q / C252S / K264E)); ciii) SEQ ID NO: 372 (IL-12p40 (E59K / K99E / N103Q / N281Q / C252S / K264E)); civ) SEQ ID NO: 373 (IL-12p40 (E59K / K99E / N113Q / N200Q / C252S / K264E)); cv) SEQ ID NO: 374 (IL-12p40 (E59K / K99E / N113Q / N281Q / C252S / K264E)); cvi) SEQ ID NO: 375 (IL-12p40 (E59K / K99E / N200Q / N281Q / C252S / K264E)); cvii) SEQ ID NO: 376 (IL-12p40 (E59K / K99E / N103Q / N113Q / N200Q / C252S / K264E)); cviii) SEQ ID NO: 377 (IL-12p40 (E59K / K99E / N103Q / N200Q / N281Q / C252S / K264E)); and cx) SEQ ID NO: 378 (IL-12p40 (E59K / K99E / N113Q / N200Q / N281Q / C252S / K264E)).

[0012] In some embodiments, the present application provides a heterodimeric Fc fusion protein, 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).

[0013] In some embodiments, the present application provides a heterodimeric Fc fusion protein, wherein the IL-12p35 subunit is a variant IL-12p35 subunit.

[0014] In some embodiments, the present application provides a heterodimeric Fc fusion protein, wherein the IL-12p40 subunit is a variant IL-12p40 subunit having altered affinity for IL-12 receptor subunit beta-1 (IL-12Rpi), IL-12 receptor subunit beta-2 (IL-12Rpi), and / or IL-12 receptor complex; and / or the IL-12p35 subunit is a variant IL-12p35 subunit having altered affinity for IL-12 receptor subunit beta-1 (IL-12Rpi), IL-12 receptor subunit beta-2 (IL-12Rpi), and / or IL-12 receptor complex.

[0015] In some embodiments, the present application provides a heterodimeric Fc fusion protein, 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.

[0016] In some embodiments, the present application provides a heterodimeric Fc fusion protein wherein the variant IL-12 p35 subunit has an amino acid substitution selected from the group consisting of N71D / N85D / N195D, N151D / E153Q, N151D / D165N, Q130E / N151D, N151D / K158E, E79Q / N151D, D55Q / N151D, N136D / N151D, N21D / N151D, E143Q / N151D, N71Q / N85Q, N71Q / N195Q, N85Q / N195Q, N71Q / N85Q / N195Q, N71D / N85D, N71D / N195D, and N85D / N195D.

[0017] In some embodiments, the present application provides a heterodimeric Fc fusion protein wherein the variant IL-12p35 subunit has a polypeptide sequence 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: 333 (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: 345 (IL-12p35(F96A)); xxxiv) SEQ ID NO: 346 (IL-12p35(M97A)); xxxv) SEQ ID NO: 347 (IL-12p35(L89A)); xxxvi) SEQ ID NO: 348 (IL-12p35(L124A)); xxxvii) SEQ ID NO: 349 (IL-12p35(M125A)); xxxviii) SEQ ID NO: 350 (IL-12p35(L75A)); xxxiv) SEQ ID NO: 351 (IL-12p35(I171A)); xxxv) SEQ ID NO: 279 (IL-12p35(N71Q)); xxxvi) SEQ ID NO: 280 (IL-12p35(N85Q)); xxxvii) SEQ ID NO: 281 (IL-12p35(N195Q)); xxxviii) SEQ ID NO: 282 (IL-12p35(N71Q / N85Q)); xxxix) SEQ ID NO: 283 (IL-12p35(N71Q / N195Q)); xl) SEQ ID NO: 284 (IL-12p35(N85Q / N195Q); xli) SEQ ID NO: 285 (IL-12p35(N71Q / N85Q / N195Q)); xlii) SEQ ID NO: 286 (IL-12p35(N71D / N85D)); xliii) SEQ ID NO: 287 (IL-12p35(N71D / N195D); xliv) SEQ ID NO: 288 (IL-12p35(N85D / N195D)); xlv) SEQ ID NO: 333 (IL-12p35(D55Q)) and xlvi) SEQ ID NO: 334 (IL-12p35(D55K)).

[0018] In some embodiments, the present application provides a heterodimeric Fc fusion protein, wherein the modification that promotes heterodimerization of the first Fc domain and the second Fc domain is 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.

[0019] In some embodiments, the present application provides a heterodimeric Fc fusion protein, wherein the variant IL-12p40 subunit domain is connected to the first Fc domain using a first domain linker and / or the IL-12p35 subunit domain is connected to the second Fc domain using a second domain linker.

[0020] In some embodiments, the present application provides a heterodimeric Fc fusion protein, wherein the first Fc domain and / or the second Fc domain has an additional set of amino acid substitutions comprising Q295E / N384D / Q418E / N421D, according to EU numbering.

[0021] In some embodiments, the present application provides a heterodimeric Fc fusion protein, wherein the first Fc domain and / or the 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.

[0022] In some embodiments, the present application provides a heterodimeric Fc fusion protein, wherein the first Fc domain and / or the second Fc domain further comprises the amino acid substitutions M428L / N424S.

[0023] In some embodiments, the present application provides a heterodimeric Fc fusion protein, wherein the heterodimeric Fc fusion protein comprises:

[0024] a) has a polypeptide sequence selected from the group consisting of: i) SEQ ID NO: 47 (XENP27201 chain 1), ii) SEQ ID NO: 85 (XenD24752), iii) SEQ ID NO: 86 (XenD24753), iv) SEQ ID NO: 87 (XenD24754), v) SEQ ID NO: 88 (XenD24755), vi) SEQ ID NO: 89 (XenD24756), vii) SEQ ID NO: 90 (XenD24757), viii) SEQ ID NO: 91 (XenD24758), ix) SEQ ID NO: 92 (XenD24759), x) SEQ ID NO: 93 (XenD24760), xi) SEQ ID NO: 94 (XenD24761), xii) SEQ ID NO: 95 (XenD24762), xiii) SEQ ID NO: 96 (XenD24763), xiv) SEQ ID NO: 97 (XenD24764), xv) SEQ ID NO: 98 (XenD24765), xvi) SEQ ID NO: 99 (XenD24766), xvii) SEQ ID NO: 100 (XenD24767), xviii) SEQ ID NO: 101 (XenD24768), xix) SEQ ID NO: 102 (XenD24769), xx) SEQ ID NO: 103 (XenD24770), xxi) SEQ ID NO: 104 (XenD24771), xxii) SEQ ID NO: 105 (XenD24772), xxiii) 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), xxxiii) SEQ ID NO: 218 (XenD25925), xxxiv) SEQ IDNO: 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: 291 (XenD26411), xli) SEQ ID NO: 292 (XenD26412), xlii) SEQ ID NO: 293 (XenD26413), xliii) SEQ ID NO: 294 (XenD26414), xliv) SEQ ID NO: 295 (XenD26415), xlv) SEQ ID NO: 296 (XenD26416), xlvi) SEQ ID NO: 297 (XenD26417), xlvii) SEQ ID NO: 298 (XenD26418), xlviii) SEQ ID NO: 301 (XenD27070), xlix) SEQ ID NO: 302 (XenD27071), I) SEQ ID NO: 303 (XenD27072), li) SEQ ID NO: 304 (XenD27073), lii) SEQ ID NO: 305 (XenD27074), liii) SEQ ID NO: 306 (XenD27075), liv) SEQ ID NO: 307 (XenD27076), lv) SEQ ID NO: 308 (XenD27077), lvi) SEQ ID NO: 309 (XenD27078), lvii) SEQ ID NO: 317 (XenD28173), lviii) SEQ ID NO: 318 (XenD24876), lix) SEQ ID NO: 320 (XenD27162), lx) SEQ ID NO: 321 (XenD27163), lxi) SEQ ID NO: 323 (XenD27164), lxii) SEQ ID NO: 324 (XenD27165) lxiii) SEQ ID NO: 357 (XENP31582 chain 1), lxiv) SEQ ID NO: 358 (XENP31583 chain 1), lxv) SEQ ID NO: 359 (XENP31584 chain 1), lxvi) SEQ ID NO: 380 (XENP32187 chain 1), lxvii) SEQ IDlx) SEQ ID NO: 381 (XENP32188 Chain 1), lxvi) SEQ ID NO: 382 (XENP32189 Chain 1), lxvii) SEQ ID NO: 425 (XENP32190 Chain 1), lxviii) SEQ ID NO: 384 (XENP32191 Chain 1), lxix) SEQ ID NO: 385 (XENP32991 Chain 1), lxx) SEQ ID NO: 386 (XENP32992 Chain 1), lxxi) SEQ ID NO: 387 (XENP32993 Chain 1), lxxii) SEQ ID NO: 388 (XENP32994 Chain 1), lxxiii) SEQ ID NO: 389 (XENP32995 Chain 1), lxxiv) SEQ ID NO: 390 (XENP32996 Chain 1), lxxv) SEQ ID NO: 391 (XENP32997 Chain 1), lxxvi) SEQ ID NO: 392 (XENP32998 Chain 1), lxxvi) SEQ ID NO: 393 (XENP32999 Chain 1), lxxvii) SEQ ID NO: 394 (XENP33000 Chain 1), lxxviii) SEQ ID NO: 395 (XENP33001 Chain 1), lxxx) SEQ ID NO: 396 (XENP33002 Chain 1), lxxxi) SEQ ID NO: 397 (XENP33003 Chain 1), lxxxii) SEQ ID NO: 398 (XENP33004 Chain 1), lxxxiii) SEQ ID NO: 426 (XENP33005 Chain 1), lxxxiv) SEQ ID NO: 427 (XENP33006 Chain 1), lxxxv) SEQ ID NO: 428 (XENP33007 Chain 1), lxxxvi) SEQ ID NO: 429 (XENP33008 Chain 1), lxxxvii) SEQ ID NO: 429 (XENP33008 Chain 1), lxxxviii) SEQ ID NO: 430 (XENP33009 Chain 1), lxxxix) SEQ ID NO: 431 (XENP33010 Chain 1), and xc) SEQ ID NO: 383 (XENP33011 Chain 1); and

[0025] b) has a polypeptide sequence selected from the group consisting of: 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: 299 (XenD26427), xxvii) SEQ ID NO: 300 (XenD26428), xxviii) SEQ ID NO: 311 (XenD27089), xxix) SEQ ID NO: 312 (XenD27090), xxx) SEQ ID NO: 313 (XenD27091), xxxi) SEQ ID NO: 314 (XenD27092), xxxii) SEQ ID NO: 315 (XenD27093), xxxiii) SEQ IDNO: 316 (XenD27094), xxxix) SEQ ID NO: 319 (XenD24877), xl) SEQ ID NO: 322 (XenD27166), xli) SEQ ID NO: 421 (XENP31582 Chain 2), xlii) SEQ ID NO: 422 (XENP31583 Chain 2), xliii) SEQ ID NO: 423 (XENP31584 Chain 2), xliv) SEQ ID NO: 402 (XENP32187 Chain 2), xlv) SEQ ID NO: 403 (XENP32188 Chain 2), xlvi) SEQ ID NO: 404 (XENP32189 Chain 2), xlvii) SEQ ID NO: 405 (XENP32190 Chain 2), xlviii) SEQ ID NO: 406 (XENP32191 Chain 2), xlix) SEQ ID NO: 407 (XENP32991 Chain 2), I) SEQ ID NO: 408 (XENP32992 Chain 2), li) SEQ ID NO: 409 (XENP32993 Chain 2), lii) SEQ ID NO: 410 (XENP32994 Chain 2), liii) SEQ ID NO: 411 (XENP32995 Chain 2), liv) SEQ ID NO: 412 (XENP32996 Chain 2), lv) SEQ ID NO: 413 (XENP32997 Chain 2), lvi) SEQ ID NO: 414 (XENP32998 Chain 2), lvii) SEQ ID NO: 415 (XENP32999 Chain 2), lviii) SEQ ID NO: 416 (XENP33000 Chain 2), lix) SEQ ID NO: 417 (XENP33001 Chain 2), Ix) SEQ ID NO: 418 (XENP33002 Chain 2), lxi) SEQ ID NO: 419 (XENP33003 Chain 2), lxii) SEQ ID NO: 420 (XENP33004 Chain 2), lxiii) SEQ ID NO: 399 (XENP33005 Chain 2), lxiv) SEQ ID NO: 400 (XENP33006 Chain 2), lxv) SEQ ID NO: 401 (XENP33007 Chain 2), lxvi) SEQ ID NO: 289 (XENP33008 Chain 2), lxvii) SEQ ID NO: 289 (XENP33008 Chain 2), lxviii) SEQ ID NO: 290 (XENP33009 Chain 2), lxix) SEQ ID NO: 291 (XENP33010 Chain 2), lxx) SEQ ID NO: 292 (XENP33011 Chain 2), lxxi) SEQ ID NO: 293 (XENP33012 Chain 2), lxxii) SEQ ID NO: 294 (XENP33013 Chain 2), lxxiii) SEQ ID NO: 295 (XENP33014 Chain 2), lxxiv) SEQ ID NO: 296 (XENP33015 Chain 2), lxxv) SEQ ID NO: 297 (XENP33016 Chain 2), lxxvi) SEQ ID NO: 298 (XENP33017 Chain 2), lxxvii) SEQ ID NO: 299 (XENP33018 Chain 2), lxxviii) SEQ ID NO: 300 (XENP33019 Chain 2), lxxix) SEQ ID NO: 301 (XENP33020 Chain 2), lxxx) SEQ ID NO: 302 (XENP33021 Chain 2), lxxxi) SEQ ID NO: 303 (XENP33022 Chain 2), lxxxii) SEQ ID NO: 304 (XENP33023 Chain 2), lxxxiii) SEQ ID NO: 305 (XENP33024 Chain 2), lxxxiv) SEQ ID NO: 306 (XENP33025 Chain 2), lxxxv) SEQ ID NO: 307 (XENP33026 Chain 2), lxxxvi) SEQ ID NO: 308 (XENP33027 Chain 2), lxxxvii) SEQ ID NO: 309 (XENP33028 Chain 2), lxxxviii) SEQ ID NO: 310 (XENP33029 Chain 2), lxxxixID NO: 352 (XENP33010 chain 2) and lxx) SEQ ID NO: 354 (XENP33011 chain 2).

[0026] In some embodiments, the present application provides a heterodimeric Fc fusion protein, wherein the heterodimeric Fc fusion protein is 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, XENP28838, XENP28839, XENP28840, XENP28841, XENP28842, XENP28843, XENP28844, XENP28845, XENP28846, XENP28847, XENP28848, XENP28849, XENP28850, XENP28851, XENP28852, XENP29949, XENP29950, XENP29951, XENP29952, XENP30597, XENP30598, XENP30599, XENP30600, XENP30601, XENP30602, XENP30603, XENP30604, XENP30605, XENP30606, XENP30307, XENP30308, XENP30609, XENP31250, XENP31251, XENP31252, XENP31253, XENP31254, XENP31255, XENP31256, XENP31257, XENP31258, XENP31259, XENP31260, XENP31261, XENP31262, XENP31263, XENP31264, XENP31265, XENP31286, XENP31142, XENP31143, XENP31144, XENP31145, XENP31146, XENP31582, XENP31583, XENP31584, XENP32186, XENP32187, XENP32188, XENP32189, XENP32190, XENP32191, XENP32991, XENP32992, XENP32993, XENP32994, XENP32995, XENP32996, XENP32997, XENP32998, XENP32999, XENP33000, XENP33001, XENP33002,XENP33003, XENP33004, XENP33005, XENP33006, XENP33007, XENP33008, XENP33008, XENP33009, XENP33010, and XENP33011.

[0027] In some embodiments, the present application provides a heterodimeric Fc fusion protein, wherein the heterodimeric Fc fusion protein is selected from the group consisting of: XENP31251, XENP31254, XENP31258, XENP32186, XENP32187, XENP32188, XENP32189, XENP32190, and XENP32191.

[0028] In some embodiments, the present application provides a heterodimeric Fc fusion protein further comprising one or more modifications to the IL-12 p40 subunit selected from the group consisting of: N103Q, N113Q, N200Q, and N281Q.

[0029] In some embodiments, the present application provides a composition comprising a heterodimeric Fc fusion protein for use in treating cancer in a subject.

[0030] In some embodiments, the present application provides one or more nucleic acids encoding a heterodimeric Fc fusion protein.

[0031] In some embodiments, the present application provides a host cell comprising the one or more nucleic acids encoding a heterodimeric Fc fusion protein.

[0032] In some embodiments, the present application provides a method of making a heterodimeric Fc fusion protein comprising culturing a host cell under conditions wherein the heterodimeric Fc fusion protein is produced.

[0033] In some embodiments, the present application provides a method of purifying a heterodimeric Fc fusion protein 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.

[0034] In some embodiments, the present application provides a heterodimeric Fc fusion protein wherein the IL-12 p40 subunit has one or more amino acid modifications at an amino acid residue 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, 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.

[0035] In some embodiments, the present application provides a heterodimeric Fc fusion protein wherein the IL-12 p35 subunit has one or more amino acid modifications at an amino acid residue 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, Q135, N136, E143, Q146, N151, E153, K158, E162, E163, D165, I171, R181, I182, R183, V185, T186, D188, R189, V190, S192, Y193, N195, and A196.

[0036] In another aspect, the application provides a heterodimeric Fc fusion protein comprising: a) a first fusion protein comprising a first protein domain and a first Fc domain, wherein the first protein domain is covalently connected to the C-terminus of the 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 connected to the C-terminus of the Fc domain; wherein the first Fc domain and the second Fc domain comprise modifications that promote heterodimerization of the first Fc domain and the second Fc domain, and wherein the first protein domain comprises an IL-12 p40 subunit and the second protein domain comprises an IL-12 p35 subunit.

[0037] In some embodiments, the application provides a heterodimeric Fc fusion protein, wherein the modifications that promote heterodimerization of the first Fc domain and the second Fc domain 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 comprising a bridging disulfide bond T366S / L368A / Y407V / Y349C:T366W / S354C) according to EU numbering.

[0038] In some embodiments, the application provides a heterodimeric Fc fusion protein, wherein the first protein domain is connected to the first Fc domain using a first domain linker and / or the second protein domain is connected to the second Fc domain using a second domain linker.

[0039] In some embodiments, the application provides a heterodimeric Fc fusion protein, wherein the first Fc domain and / or the second Fc domain has an additional set of amino acid substitutions comprising Q295E / N384D / Q418E / N421D according to EU numbering.

[0040] In some embodiments, the present application provides a heterodimeric Fc fusion protein, wherein the first Fc domain and / or the second Fc domain has an additional group 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.

[0041] In some embodiments, the present application provides a heterodimeric Fc fusion protein, wherein the variant 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).

[0042] In some embodiments, the present application provides a heterodimeric Fc fusion protein, wherein the first Fc domain and / or the second Fc domain further comprises the amino acid substitutions M428L / N424S.

[0043] In some embodiments, the present application provides a heterodimeric Fc fusion protein, wherein the IL-12p40 subunit is a variant IL-12p40 subunit and / or the IL-12p35 subunit is a variant IL-12p35 subunit.

[0044] In some embodiments, the present application provides a heterodimeric Fc fusion protein, wherein the IL-12p40 subunit is a variant IL-12p40 subunit having altered affinity for IL-12 receptor subunit beta-1 (IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2), and / or IL-12 receptor complex; and / or the IL-12p35 subunit is a variant IL-12p35 subunit having altered affinity for IL-12 receptor subunit beta-1 (IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2), and / or IL-12 receptor complex.

[0045] In some embodiments, the present application provides a heterodimeric Fc fusion protein, wherein the IL-12 p40 subunit has one or more amino acid modifications at an amino acid residue 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, 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.

[0046] In some embodiments, the present application provides a heterodimeric Fc fusion protein, wherein the IL-12 p40 subunit has one or more amino acid substitutions selected from the group consisting of D18N, D18K, E32Q, E33Q, D34N, D34K, Q42E, S43E, S43K, E45Q, Q56E, E59Q, E59K, D62N, E73Q, D87N, K99E, K99Y, E100Q, N103D, N103Q, N113D, N113Q, Q144E, D161N, R159E, K163E, E187Q, N200D, N200Q, N218Q, Q229E, E235Q, C252S, Q256N, K258E, K260E, E262Q, K264E, N281D, N281Q, and E299Q.

[0047] In some embodiments, the present application provides a heterodimeric Fc fusion protein wherein the IL-12p40 subunit has an amino acid substitution selected from the group consisting 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, 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, 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 / C252S / K264E, E59K / K99E / N113Q / C252S / K264E, E59K / K99E / N200Q / 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 / 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.

[0048] In some embodiments, the present application provides a heterodimeric Fc fusion protein wherein the IL-12p40 subunit has an amino acid substitution selected from the group consisting 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, 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, 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 / C252S / K264E, E59K / K99E / N113Q / C252S / K264E, E59K / K99E / N200Q / 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 / 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.

[0049] In some embodiments, the present application provides a heterodimeric Fc fusion protein, wherein the IL-12p40 subunit has a polypeptide sequence 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)); 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 (S43K); xxxiii) SEQ ID NO: 379 (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: 326 (IL-12p40 (D34N / E59K)); 1) SEQ ID NO: 325 (IL-12p40 (E59K / K99E)); li) SEQ ID NO: 339 (IL-12p40 (D18K / E59K / K99E)); lii) SEQ ID NO: 343 (IL-12p40 (E59K / K99E / K264E)); liii) SEQ ID NO: 336 (IL-12p40 (E59K / K99Y)); liv) SEQ ID NO: 335 (IL-12p40 (E59Y / K99E));lv) SEQ ID NO: 338 (IL-12p40 (E45K / E59K / K99E)); lvi) SEQ ID NO: 340 (IL-12p40 (E59K / K99E / Q144E)); lvii) SEQ ID NO: 341 (IL-12p40 (E59K / K99E / Q144K)); lviii) SEQ ID NO: 342 (IL-12p40 (E59K / K99E / R159E)); Ix) SEQ ID NO: 343 (IL-12p40 (E59K / K99E / K264E)); lx) SEQ ID NO: 344 (IL-12p40 (D18K / E59K / K99E / K264E)); lxi) SEQ ID NO: 360 (IL-12p40 (C252S)); lxii) SEQ ID NO: 361 (IL-12p40 (D18K / E59K / K99E / C252S)); lxiii) SEQ ID NO: 362 (IL-12p40 (D18K / E59K / K99E / C252S / K264E)); lxiv) SEQ ID NO: 363 (IL-12p40 (E59K / K99Y / C252S)); lxv) SEQ ID NO: 364 (IL-12p40 (E59K / K99E / C252S / K264E)); lxvi) SEQ ID NO: 365 (IL-12p40 (E59K / K99E / C252S)); lxvii) SEQ ID NO: 254 (IL-12p40 (N103D / N113D)); lxviii) SEQ ID NO: 255 (IL-12p40 (N103D / N200D)); lxix) SEQ ID NO: 256 (IL-12p40 (N103D / N281D)); lxx) SEQ ID NO: 257 (IL-12p40 (N113D / N200D)); lxxi) SEQ ID NO: 258 (IL-12p40 (N113D / N281D)); lxxii) SEQ ID NO: 259 (IL-12p40 (N200D / N281D)); lxxiii) SEQ ID NO: 260 (IL-12p40 (N103D / N113D / N200D)); lxxiv) SEQ ID NO: 261 (IL-12p40 (N103D / N113D / N281D)); lxxv) SEQ ID NO: 262 (IL-12p40 (N103D / N200D / N281D));lxxvi) SEQ ID NO: 263 (IL-12p40 (N113D / N200D / N281D)); lxxvii) SEQ ID NO: 264 (IL-12p40 (N103Q)); lxxviii) SEQ ID NO: 265 (IL-12p40 (N113Q)); lxxix) SEQ ID NO: 266 (IL-12p40 (N200Q)); lxxx) SEQ ID NO: 267 (IL-12p40 (N281Q)); lxxxi) SEQ ID NO: 268 (IL-12p40 (N103Q / N113Q)); lxxxii) SEQ ID NO: 269 (IL-12p40 (N103Q / N200Q)); lxxxiii) SEQ ID NO: 270 (IL-12p40 (N103Q / N281Q)); lxxxiv) SEQ ID NO: 271 (IL-12p40 (N113Q / N200Q)); lxxxv) SEQ ID NO: 272 (IL-12p40 (N113Q / N281Q)); lxxxvi) SEQ ID NO: 273 (IL-12p40 (N200Q / N281Q)); lxxxvii) SEQ ID NO: 274 (IL-12p40 (N103Q / N113Q / N200Q)); lxxxviii) SEQ ID NO: 275 (IL-12p40 (N103Q / N113Q / N281Q)); lxxxix) SEQ ID NO: 276 (IL-12p40 (N103Q / N200Q / N281Q)); xc) SEQ ID NO: 277 (IL-12p40 (N113Q / N200Q / N281Q)); xci) SEQ ID NO: 278 (IL-12p40 (N103Q / N113Q / N200Q / N281Q)); xcii) SEQ ID NO: 327 (IL-12p40 (D34N / E59K / K99E)); xciii) SEQ ID NO: 328 (IL-12p40 (D34K / E59K / K99E)); xciv) SEQ ID NO: 329 (IL-12p40 (E32Q / D34N / E59K / K99E)); xcv) SEQ ID NO: 331 (IL-12p40 (E32K / D34N / E59K / K99E)); xcvi) SEQ ID NO: 337 (IL-12p40 (E59Y / K99Y));xcvii) SEQ ID NO: 366 (IL-12p40 (E59K / K99E / N103Q / C252S / K264E)); xcviii) SEQ ID NO: 367 (IL-12p40 (E59K / K99E / N113Q / C252S / K264E)); xcix) SEQ ID NO: 368 (IL-12p40 (E59K / K99E / N200Q / C252S / K264E)); c) SEQ ID NO: 369 (IL-12p40 (E59K / K99E / N281Q / C252S / K264E)); ci) SEQ ID NO: 370 (IL-12p40 (E59K / K99E / N103Q / N113Q / C252S / K264E)); cii) SEQ ID NO: 371 (IL-12p40 (E59K / K99E / N103Q / N200Q / C252S / K264E)); ciii) SEQ ID NO: 372 (IL-12p40 (E59K / K99E / N103Q / N281Q / C252S / K264E)); civ) SEQ ID NO: 373 (IL-12p40 (E59K / K99E / N113Q / N200Q / C252S / K264E)); cv) SEQ ID NO: 374 (IL-12p40 (E59K / K99E / N113Q / N281Q / C252S / K264E)); cvi) SEQ ID NO: 375 (IL-12p40 (E59K / K99E / N200Q / N281Q / C252S / K264E)); cvii) SEQ ID NO: 376 (IL-12p40 (E59K / K99E / N103Q / N113Q / N200Q / C252S / K264E));cviii) SEQ ID NO: 377 (IL-12p40 (E59K / K99E / N103Q / N200Q / N281Q / C252S / K264E)) and cix) SEQ ID NO: 378 (IL-12p40 (E59K / K99E / N113Q / N200Q / N281Q / C252S / K264E)). In some embodiments, the present application provides a heterodimeric Fc fusion protein in which the IL-12 p35 subunit has one or more amino acid modifications as amino acid residues 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, Q135, N136, E143, Q146, N151, E153, K158, E162, E163, D165, I171, R181, I182, R183, V185, T186, D188, R189, V190, S192, Y193, N195, and A196.

[0050] In some embodiments, the present application provides a heterodimeric Fc fusion protein in which the IL-12 p35 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.

[0051] In some embodiments, the present application provides a heterodimeric Fc fusion protein wherein the IL-12 p35 subunit has an amino acid substitution selected from the group consisting of N71D / N85D / N195D, N151D / E153Q, N151D / D165N, Q130E / N151D, N151D / K158E, E79Q / N151D, D55Q / N151D, N136D / N151D, N21D / N151D, E143Q / N151D, N71Q / N85Q, N71Q / N195Q, N85Q / N195Q, N71Q / N85Q / N195Q, N71D / N85D, N71D / N195D, and N85D / N195D.

[0052] In some embodiments, the present application provides a heterodimeric Fc fusion protein wherein the IL-12 p35 subunit has a polypeptide sequence 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: 333 (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: 345 (IL-12p35(F96A)); xxxiv) SEQ ID NO: 346 (IL-12p35(M97A)); xxxv) SEQ ID NO: 347 (IL-12p35(L89A)); xxxvi) SEQ ID NO: 348 (IL-12p35(L124A)); xxxvii) SEQ ID NO: 349 (IL-12p35(M125A)); xxxviii) SEQ ID NO: 350 (IL-12p35(L75A)); xxxiv) SEQ ID NO: 351 (IL-12p35(I171A)); xxxv) SEQ ID NO: 279 (IL-12p35(N71Q)); xxxvi) SEQ ID NO: 280 (IL-12p35(N85Q)); xxxvii) SEQ ID NO: 281 (IL-12p35(N195Q)); xxxviii) SEQ ID NO: 282 (IL-12p35(N71Q / N85Q)); xxxix) SEQ ID NO: 283 (IL-12p35(N71Q / N195Q)); xl) SEQ ID NO: 284 (IL-12p35(N85Q / N195Q); xli) SEQ ID NO: 285 (IL-12p35(N71Q / N85Q / N195Q)); xlii) SEQ ID NO: 286 (IL-12p35(N71D / N85D)); xliii) SEQ ID NO: 287 (IL-12p35(N71D / N195D); xliv) SEQ ID NO: 288 (IL-12p35(N85D / N195D)); xlv) SEQ ID NO: 333 (IL-12p35(D55Q)) and xlvi) SEQ ID NO: 334 (IL-12p35(D55K)).

[0053] In some embodiments, the present application provides a heterodimeric Fc fusion protein, wherein the heterodimeric Fc fusion protein comprises: a) the first fusion protein having the polypeptide sequence of SEQ ID NO: 49 (XENP27202 Chain 1), and b) the second fusion protein having the polypeptide sequence of SEQ ID NO: 50 (XENP27202 Chain 2).

[0054] In some embodiments, the present application provides a heterodimeric Fc fusion protein, wherein the heterodimeric Fc fusion protein is XENP27202.

[0055] In some embodiments, the present application provides a composition comprising a heterodimeric Fc fusion protein for use in treating cancer in a subject.

[0056] In some embodiments, the present application provides one or more nucleic acids encoding a heterodimeric Fc fusion protein.

[0057] In some embodiments, the present application provides a host cell comprising the one or more nucleic acids encoding a heterodimeric Fc fusion protein.

[0058] In some embodiments, the present application provides a method of making a heterodimeric Fc fusion protein, the method comprising culturing a host cell under conditions wherein the heterodimeric Fc fusion protein is produced.

[0059] In some embodiments, the present application provides a method of 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.

[0060] In another aspect, the present application 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 wherein 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 Fc domain and the second Fc domain comprise modifications that promote heterodimerization of the first Fc domain and the second Fc domain, and wherein the first protein domain comprises an IL-12 p40 subunit and the second protein domain comprises an IL-12 p35 subunit.

[0061] In some embodiments, the present application provides a heterodimeric Fc fusion protein wherein the modification that promotes heterodimerization of the first Fc domain and the second Fc domain is 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 comprising a bridging disulfide T366S / L368A / Y407V / Y349C:T366W / S354C) according to EU numbering.

[0062] In some embodiments, the present application provides a heterodimeric Fc fusion protein wherein the first protein domain is connected to the second protein domain using a first domain linker and / or the second protein domain is connected to the first Fc domain using a second domain linker.

[0063] In some embodiments, the present application provides a heterodimeric Fc fusion protein wherein the first Fc domain and / or the second Fc domain has an additional set of amino acid substitutions comprising Q295E / N384D / Q418E / N421D according to EU numbering.

[0064] In some embodiments, the present application provides a heterodimeric Fc fusion protein wherein the first Fc domain and / or the 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.

[0065] In some embodiments, the present application provides a heterodimeric Fc fusion protein, wherein the variant IL-12 p40 subunit has a polypeptide sequence selected from the group consisting of SEQ ID NO: 3 (human IL-12 subunit beta (IL-12 p40) precursor sequence) and SEQ ID NO: 4 (human IL-12 subunit beta (IL-12 p40) mature form sequence), and / or the IL-12 p35 subunit has a polypeptide sequence selected from the group consisting of SEQ ID NO: 1 (human IL-12 subunit alpha (IL-12 p35) precursor sequence) and SEQ ID NO: 2 (human IL-12 subunit alpha (IL-12 p35) mature form sequence).

[0066] In some embodiments, the present application provides a heterodimeric Fc fusion protein, wherein the first Fc domain and / or the second Fc domain further comprises the amino acid substitutions M428L / N424S.

[0067] In some embodiments, the present application provides a heterodimeric Fc fusion protein, wherein the IL-12 p40 subunit is a variant IL-12 p40 subunit and / or the IL-12 p35 subunit is a variant IL-12 p35 subunit.

[0068] In some embodiments, the present application provides a heterodimeric Fc fusion protein, wherein the IL-12 p40 subunit is a variant IL-12 p40 subunit having altered affinity for IL-12 receptor subunit beta-1 (IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2), and / or IL-12 receptor complex; and / or the IL-12 p35 subunit is a variant IL-12 p35 subunit having altered affinity for IL-12 receptor subunit beta-1 (IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2), and / or IL-12 receptor complex.

[0069] In some embodiments, the present application provides a heterodimeric Fc fusion protein, wherein the IL-12 p40 subunit has one or more amino acid modifications at an amino acid residue 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, D209, D214, N218, Q220, N226, Q229, E231, E235, T242, P243, S245, Y246, F247, S248, Q256, K158, C252, K260, E262, K264, D265, D270, N281, Q289, D290, R291, Y292, Y293, and E299.

[0070] In some embodiments, the present application provides a heterodimeric Fc fusion protein, wherein the IL-12 p40 subunit has one or more amino acid substitutions selected from the group consisting of D18N, D18K, E32Q, E33Q, D34N, D34K, Q42E, S43E, S43K, E45Q, Q56E, E59Q, E59K, D62N, E73Q, D87N, K99E, K99Y, E100Q, N103D, N103Q, N113D, N113Q, Q144E, D161N, R159E, K163E, E187Q, N200D, N200Q, N218Q, Q229E, E235Q, C252S, Q256N, K258E, K260E, E262Q, K264E, N281D, N281Q, and E299Q.

[0071] In some embodiments, the present application provides a heterodimeric Fc fusion protein wherein the IL-12p40 subunit has an amino acid substitution selected from the group consisting 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, 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, 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 / C252S / K264E, E59K / K99E / N113Q / C252S / K264E, E59K / K99E / N200Q / 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 / 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.

[0072] In some embodiments, the present application provides a heterodimeric Fc fusion protein, wherein the IL-12p40 subunit has a polypeptide sequence 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)); 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 (S43K); xxxiii) SEQ ID NO: 379 (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: 326 (IL-12p40 (D34N / E59K)); 1) SEQ ID NO: 325 (IL-12p40 (E59K / K99E)); li) SEQ ID NO: 339 (IL-12p40 (D18K / E59K / K99E)); lii) SEQ ID NO: 343 (IL-12p40 (E59K / K99E / K264E)); liii) SEQ ID NO: 336 (IL-12p40 (E59K / K99Y)); liv) SEQ ID NO: 335 (IL-12p40 (E59Y / K99E));lv) SEQ ID NO: 338 (IL-12p40 (E45K / E59K / K99E)); lvi) SEQ ID NO: 340 (IL-12p40 (E59K / K99E / Q144E)); lvii) SEQ ID NO: 341 (IL-12p40 (E59K / K99E / Q144K)); lviii) SEQ ID NO: 342 (IL-12p40 (E59K / K99E / R159E)); Ix) SEQ ID NO: 343 (IL-12p40 (E59K / K99E / K264E)); lx) SEQ ID NO: 344 (IL-12p40 (D18K / E59K / K99E / K264E)); lxi) SEQ ID NO: 360 (IL-12p40 (C252S)); lxii) SEQ ID NO: 361 (IL-12p40 (D18K / E59K / K99E / C252S)); lxiii) SEQ ID NO: 362 (IL-12p40 (D18K / E59K / K99E / C252S / K264E)); lxiv) SEQ ID NO: 363 (IL-12p40 (E59K / K99Y / C252S)); lxv) SEQ ID NO: 364 (IL-12p40 (E59K / K99E / C252S / K264E)); lxvi) SEQ ID NO: 365 (IL-12p40 (E59K / K99E / C252S)); lxvii) SEQ ID NO: 254 (IL-12p40 (N103D / N113D)); lxviii) SEQ ID NO: 255 (IL-12p40 (N103D / N200D)); lxix) SEQ ID NO: 256 (IL-12p40 (N103D / N281D)); lxx) SEQ ID NO: 257 (IL-12p40 (N113D / N200D)); lxxi) SEQ ID NO: 258 (IL-12p40 (N113D / N281D)); lxxii) SEQ ID NO: 259 (IL-12p40 (N200D / N281D)); lxxiii) SEQ ID NO: 260 (IL-12p40 (N103D / N113D / N200D)); lxxiv) SEQ ID NO: 261 (IL-12p40 (N103D / N113D / N281D)); lxxv) SEQ ID NO: 262 (IL-12p40 (N103D / N200D / N281D));lxxvi) SEQ ID NO: 263 (IL-12p40 (N113D / N200D / N281D)); lxxvii) SEQ ID NO: 264 (IL-12p40 (N103Q)); lxxviii) SEQ ID NO: 265 (IL-12p40 (N113Q)); lxxix) SEQ ID NO: 266 (IL-12p40 (N200Q)); lxxx) SEQ ID NO: 267 (IL-12p40 (N281Q)); lxxxi) SEQ ID NO: 268 (IL-12p40 (N103Q / N113Q)); lxxxii) SEQ ID NO: 269 (IL-12p40 (N103Q / N200Q)); lxxxiii) SEQ ID NO: 270 (IL-12p40 (N103Q / N281Q)); lxxxiv) SEQ ID NO: 271 (IL-12p40 (N113Q / N200Q)); lxxxv) SEQ ID NO: 272 (IL-12p40 (N113Q / N281Q)); lxxxvi) SEQ ID NO: 273 (IL-12p40 (N200Q / N281Q)); lxxxvii) SEQ ID NO: 274 (IL-12p40 (N103Q / N113Q / N200Q)); lxxxviii) SEQ ID NO: 275 (IL-12p40 (N103Q / N113Q / N281Q)); lxxxix) SEQ ID NO: 276 (IL-12p40 (N103Q / N200Q / N281Q)); xc) SEQ ID NO: 277 (IL-12p40 (N113Q / N200Q / N281Q)); xci) SEQ ID NO: 278 (IL-12p40 (N103Q / N113Q / N200Q / N281Q)); xcii) SEQ ID NO: 327 (IL-12p40 (D34N / E59K / K99E)); xciii) SEQ ID NO: 328 (IL-12p40 (D34K / E59K / K99E)); xciv) SEQ ID NO: 329 (IL-12p40 (E32Q / D34N / E59K / K99E)); xcv) SEQ ID NO: 331 (IL-12p40 (E32K / D34N / E59K / K99E)); xcvi) SEQ ID NO: 337 (IL-12p40 (E59Y / K99Y));xcvii) SEQ ID NO: 366 (IL-12p40 (E59K / K99E / N103Q / C252S / K264E)); xcviii) SEQ ID NO: 367 (IL-12p40 (E59K / K99E / N113Q / C252S / K264E)); xcix) SEQ ID NO: 368 (IL-12p40 (E59K / K99E / N200Q / C252S / K264E)); c) SEQ ID NO: 369 (IL-12p40 (E59K / K99E / N281Q / C252S / K264E)); ci) SEQ ID NO: 370 (IL-12p40 (E59K / K99E / N103Q / N113Q / C252S / K264E)); cii) SEQ ID NO: 371 (IL-12p40 (E59K / K99E / N103Q / N200Q / C252S / K264E)); ciii) SEQ ID NO: 372 (IL-12p40 (E59K / K99E / N103Q / N281Q / C252S / K264E)); civ) SEQ ID NO: 373 (IL-12p40 (E59K / K99E / N113Q / N200Q / C252S / K264E)); cv) SEQ ID NO: 374 (IL-12p40 (E59K / K99E / N113Q / N281Q / C252S / K264E)); cvi) SEQ ID NO: 375 (IL-12p40 (E59K / K99E / N200Q / N281Q / C252S / K264E)); cvii) SEQ ID NO: 376 (IL-12p40 (E59K / K99E / N103Q / N113Q / N200Q / C252S / K264E)); cviii) SEQ ID NO: 377 (IL-12p40 (E59K / K99E / N103Q / N200Q / N281Q / C252S / K264E)); and cx) SEQ ID NO: 378 (IL-12p40 (E59K / K99E / N113Q / N200Q / N281Q / C252S / K264E)).

[0073] In some embodiments, the present application provides a heterodimeric Fc fusion protein, wherein the IL-12 p35 subunit has one or more amino acid modifications 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, Q135, N136, E143, Q146, N151, E153, K158, E162, E163, D165, I171, R181, I182, R183, V185, T186, D188, R189, V190, S192, Y193, N195, and A196 as amino acid residues.

[0074] In some embodiments, the present application provides a heterodimeric Fc fusion protein, wherein the IL-12 p35 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.

[0075] In some embodiments, the present application provides a heterodimeric Fc fusion protein, wherein the IL-12 p35 subunit has one or more 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.

[0076] In some embodiments, the present application provides a heterodimeric Fc fusion protein wherein the IL-12 p35 subunit has a polypeptide sequence 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: 333 (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: 345 (IL-12p35(F96A)); xxxiv) SEQ ID NO: 346 (IL-12p35(M97A)); xxxv) SEQ ID NO: 347 (IL-12p35(L89A)); xxxvi) SEQ ID NO: 348 (IL-12p35(L124A)); xxxvii) SEQ ID NO: 349 (IL-12p35(M125A)); xxxviii) SEQ ID NO: 350 (IL-12p35(L75A)); xxxiv) SEQ ID NO: 351 (IL-12p35(I171A)); xxxv) SEQ ID NO: 279 (IL-12p35(N71Q)); xxxvi) SEQ ID NO: 280 (IL-12p35(N85Q)); xxxvii) SEQ ID NO: 281 (IL-12p35(N195Q)); xxxviii) SEQ ID NO: 282 (IL-12p35(N71Q / N85Q)); xxxix) SEQ ID NO: 283 (IL-12p35(N71Q / N195Q)); xl) SEQ ID NO: 284 (IL-12p35(N85Q / N195Q); xli) SEQ ID NO: 285 (IL-12p35(N71Q / N85Q / N195Q)); xlii) SEQ ID NO: 286 (IL-12p35(N71D / N85D)); xliii) SEQ ID NO: 287 (IL-12p35(N71D / N195D); xliv) SEQ ID NO: 288 (IL-12p35(N85D / N195D)); xlv) SEQ ID NO: 333 (IL-12p35(D55Q)) and xlvi) SEQ ID NO: 334 (IL-12p35(D55K)).

[0077] In some embodiments, the present application provides a heterodimeric Fc fusion protein, wherein the heterodimeric Fc fusion protein comprises: 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).

[0078] In some embodiments, the present application provides a heterodimeric Fc fusion protein, wherein the heterodimeric Fc fusion protein is XENP27203.

[0079] In some embodiments, the present application provides a heterodimeric Fc fusion protein, wherein the heterodimeric Fc fusion protein is XENP31290.

[0080] In some embodiments, the present application provides a composition comprising a heterodimeric Fc fusion protein for use in treating cancer in a subject.

[0081] In some embodiments, the present application provides one or more nucleic acids encoding a heterodimeric Fc fusion protein.

[0082] In some embodiments, the present application provides a host cell comprising the one or more nucleic acids encoding a heterodimeric Fc fusion protein.

[0083] In some embodiments, the present application provides a method of making a heterodimeric Fc fusion protein, the method comprising culturing a host cell under conditions wherein the heterodimeric Fc fusion protein is produced.

[0084] In some embodiments, the present application provides a method of 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.

[0085] In another aspect, the application 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 attached to the second protein domain, and wherein the second protein domain is covalently attached to the N-terminus of the first Fc domain; and b) a second Fc domain; wherein the first Fc domain and the second Fc domain comprise modifications that promote heterodimerization of the first Fc domain and the second Fc domain, and wherein the first protein domain comprises an IL-12 p35 subunit and the second protein domain comprises an IL-12 p40 subunit.

[0086] In some embodiments, the application provides a heterodimeric Fc fusion protein, wherein the modifications that promote heterodimerization of the first Fc domain and the second Fc domain 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 comprising a bridging disulfide T366S / L368A / Y407V / Y349C:T366W / S354C) according to EU numbering.

[0087] In some embodiments, the application provides a heterodimeric Fc fusion protein, wherein the first protein domain is connected to the second protein domain using a first domain linker and / or the second protein domain is connected to the first Fc domain using a second domain linker.

[0088] In some embodiments, the application provides a heterodimeric Fc fusion protein, wherein the first Fc domain and / or the second Fc domain has an additional set of amino acid substitutions comprising Q295E / N384D / Q418E / N421D according to EU numbering.

[0089] In some embodiments, the present application provides a heterodimeric Fc fusion protein, wherein the first Fc domain and / or the 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.

[0090] In some embodiments, the present application provides a heterodimeric Fc fusion protein, wherein the variant 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).

[0091] In some embodiments, the present application provides a heterodimeric Fc fusion protein, wherein the first Fc domain and / or the second Fc domain further comprises the amino acid substitutions M428L / N424S.

[0092] In some embodiments, the present application provides a heterodimeric Fc fusion protein, wherein the IL-12p40 subunit is a variant IL-12p40 subunit and / or the IL-12p35 subunit is a variant IL-12p35 subunit.

[0093] In some embodiments, the present application provides a heterodimeric Fc fusion protein, wherein the IL-12p40 subunit is a variant IL-12p40 subunit having altered affinity for IL-12 receptor subunit beta-1 (IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2), and / or IL-12 receptor complex; and / or the IL-12p35 subunit is a variant IL-12p35 subunit having altered affinity for IL-12 receptor subunit beta-1 (IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2), and / or IL-12 receptor complex.

[0094] In some embodiments, the present application provides a heterodimeric Fc fusion protein, wherein the IL-12 p40 subunit has one or more amino acid modifications at an amino acid residue 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, 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.

[0095] In some embodiments, the present application provides a heterodimeric Fc fusion protein, wherein the IL-12 p40 subunit has one or more amino acid substitutions selected from the group consisting of D18N, D18K, E32Q, E33Q, D34N, D34K, Q42E, S43E, S43K, E45Q, Q56E, E59Q, E59K, D62N, E73Q, D87N, K99E, K99Y, E100Q, N103D, N103Q, N113D, N113Q, Q144E, D161N, R159E, K163E, E187Q, N200D, N200Q, N218Q, Q229E, E235Q, C252S, Q256N, K258E, K260E, E262Q, K264E, N281D, N281Q, and E299Q.

[0096] In some embodiments, the present application provides a heterodimeric Fc fusion protein wherein the IL-12p40 subunit has an amino acid substitution selected from the group consisting 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, 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, 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 / C252S / K264E, E59K / K99E / N113Q / C252S / K264E, E59K / K99E / N200Q / 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 / 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.

[0097] In some embodiments, the present application provides a heterodimeric Fc fusion protein, wherein the IL-12p40 subunit has a polypeptide sequence 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)); 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 (S43K); xxxiii) SEQ ID NO: 379 (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: 326 (IL-12p40 (D34N / E59K)); 1) SEQ ID NO: 325 (IL-12p40 (E59K / K99E)); li) SEQ ID NO: 339 (IL-12p40 (D18K / E59K / K99E)); lii) SEQ ID NO: 343 (IL-12p40 (E59K / K99E / K264E)); liii) SEQ ID NO: 336 (IL-12p40 (E59K / K99Y)); liv) SEQ ID NO: 335 (IL-12p40 (E59Y / K99E));lv) SEQ ID NO: 338 (IL-12p40 (E45K / E59K / K99E)); lvi) SEQ ID NO: 340 (IL-12p40 (E59K / K99E / Q144E)); lvii) SEQ ID NO: 341 (IL-12p40 (E59K / K99E / Q144K)); lviii) SEQ ID NO: 342 (IL-12p40 (E59K / K99E / R159E)); Ix) SEQ ID NO: 343 (IL-12p40 (E59K / K99E / K264E)); lx) SEQ ID NO: 344 (IL-12p40 (D18K / E59K / K99E / K264E)); lxi) SEQ ID NO: 360 (IL-12p40 (C252S)); lxii) SEQ ID NO: 361 (IL-12p40 (D18K / E59K / K99E / C252S)); lxiii) SEQ ID NO: 362 (IL-12p40 (D18K / E59K / K99E / C252S / K264E)); lxiv) SEQ ID NO: 363 (IL-12p40 (E59K / K99Y / C252S)); lxv) SEQ ID NO: 364 (IL-12p40 (E59K / K99E / C252S / K264E)); lxvi) SEQ ID NO: 365 (IL-12p40 (E59K / K99E / C252S)); lxvii) SEQ ID NO: 254 (IL-12p40 (N103D / N113D)); lxviii) SEQ ID NO: 255 (IL-12p40 (N103D / N200D)); lxix) SEQ ID NO: 256 (IL-12p40 (N103D / N281D)); lxx) SEQ ID NO: 257 (IL-12p40 (N113D / N200D)); lxxi) SEQ ID NO: 258 (IL-12p40 (N113D / N281D)); lxxii) SEQ ID NO: 259 (IL-12p40 (N200D / N281D)); lxxiii) SEQ ID NO: 260 (IL-12p40 (N103D / N113D / N200D)); lxxiv) SEQ ID NO: 261 (IL-12p40 (N103D / N113D / N281D)); lxxv) SEQ ID NO: 262 (IL-12p40 (N103D / N200D / N281D));lxxvi) SEQ ID NO: 263 (IL-12p40 (N113D / N200D / N281D)); lxxvii) SEQ ID NO: 264 (IL-12p40 (N103Q)); lxxviii) SEQ ID NO: 265 (IL-12p40 (N113Q)); lxxix) SEQ ID NO: 266 (IL-12p40 (N200Q)); lxxx) SEQ ID NO: 267 (IL-12p40 (N281Q)); lxxxi) SEQ ID NO: 268 (IL-12p40 (N103Q / N113Q)); lxxxii) SEQ ID NO: 269 (IL-12p40 (N103Q / N200Q)); lxxxiii) SEQ ID NO: 270 (IL-12p40 (N103Q / N281Q)); lxxxiv) SEQ ID NO: 271 (IL-12p40 (N113Q / N200Q)); lxxxv) SEQ ID NO: 272 (IL-12p40 (N113Q / N281Q)); lxxxvi) SEQ ID NO: 273 (IL-12p40 (N200Q / N281Q)); lxxxvii) SEQ ID NO: 274 (IL-12p40 (N103Q / N113Q / N200Q)); lxxxviii) SEQ ID NO: 275 (IL-12p40 (N103Q / N113Q / N281Q)); lxxxix) SEQ ID NO: 276 (IL-12p40 (N103Q / N200Q / N281Q)); xc) SEQ ID NO: 277 (IL-12p40 (N113Q / N200Q / N281Q)); xci) SEQ ID NO: 278 (IL-12p40 (N103Q / N113Q / N200Q / N281Q)); xcii) SEQ ID NO: 327 (IL-12p40 (D34N / E59K / K99E)); xciii) SEQ ID NO: 328 (IL-12p40 (D34K / E59K / K99E)); xciv) SEQ ID NO: 329 (IL-12p40 (E32Q / D34N / E59K / K99E)); xcv) SEQ ID NO: 331 (IL-12p40 (E32K / D34N / E59K / K99E)); xcvi) SEQ ID NO: 337 (IL-12p40 (E59Y / K99Y));xcvii) SEQ ID NO: 366 (IL-12p40 (E59K / K99E / N103Q / C252S / K264E)); xcviii) SEQ ID NO: 367 (IL-12p40 (E59K / K99E / N113Q / C252S / K264E)); xcix) SEQ ID NO: 368 (IL-12p40 (E59K / K99E / N200Q / C252S / K264E)); c) SEQ ID NO: 369 (IL-12p40 (E59K / K99E / N281Q / C252S / K264E)); ci) SEQ ID NO: 370 (IL-12p40 (E59K / K99E / N103Q / N113Q / C252S / K264E)); cii) SEQ ID NO: 371 (IL-12p40 (E59K / K99E / N103Q / N200Q / C252S / K264E)); ciii) SEQ ID NO: 372 (IL-12p40 (E59K / K99E / N103Q / N281Q / C252S / K264E)); civ) SEQ ID NO: 373 (IL-12p40 (E59K / K99E / N113Q / N200Q / C252S / K264E)); cv) SEQ ID NO: 374 (IL-12p40 (E59K / K99E / N113Q / N281Q / C252S / K264E)); cvi) SEQ ID NO: 375 (IL-12p40 (E59K / K99E / N200Q / N281Q / C252S / K264E)); cvii) SEQ ID NO: 376 (IL-12p40 (E59K / K99E / N103Q / N113Q / N200Q / C252S / K264E)); cviii) SEQ ID NO: 377 (IL-12p40 (E59K / K99E / N103Q / N200Q / N281Q / C252S / K264E)); and cx) SEQ ID NO: 378 (IL-12p40 (E59K / K99E / N113Q / N200Q / N281Q / C252S / K264E)).

[0098] In some embodiments, the present application provides a heterodimeric Fc fusion protein, wherein the IL-12 p35 subunit has one or more amino acid modifications 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, Q135, N136, E143, Q146, N151, E153, K158, E162, E163, D165, I171, R181, I182, R183, V185, T186, D188, R189, V190, S192, Y193, N195, and A196 as amino acid residues.

[0099] In some embodiments, the present application provides a heterodimeric Fc fusion protein, wherein the IL-12 p35 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.

[0100] In some embodiments, the present application provides a heterodimeric Fc fusion protein, wherein the IL-12 p35 subunit has one or more 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.

[0101] In some embodiments, the present application provides a heterodimeric Fc fusion protein wherein the IL-12 p35 subunit has a polypeptide sequence 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: 333 (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: 345 (IL-12p35(F96A)); xxxiv) SEQ ID NO: 346 (IL-12p35(M97A)); xxxv) SEQ ID NO: 347 (IL-12p35(L89A)); xxxvi) SEQ ID NO: 348 (IL-12p35(L124A)); xxxvii) SEQ ID NO: 349 (IL-12p35(M125A)); xxxviii) SEQ ID NO: 350 (IL-12p35(L75A)); xxxiv) SEQ ID NO: 351 (IL-12p35(I171A)); xxxv) SEQ ID NO: 279 (IL-12p35(N71Q)); xxxvi) SEQ ID NO: 280 (IL-12p35(N85Q)); xxxvii) SEQ ID NO: 281 (IL-12p35(N195Q)); xxxviii) SEQ ID NO: 282 (IL-12p35(N71Q / N85Q)); xxxix) SEQ ID NO: 283 (IL-12p35(N71Q / N195Q)); xl) SEQ ID NO: 284 (IL-12p35(N85Q / N195Q); xli) SEQ ID NO: 285 (IL-12p35(N71Q / N85Q / N195Q)); xlii) SEQ ID NO: 286 (IL-12p35(N71D / N85D)); xliii) SEQ ID NO: 287 (IL-12p35(N71D / N195D); xliv) SEQ ID NO: 288 (IL-12p35(N85D / N195D)); xlv) SEQ ID NO: 333 (IL-12p35(D55Q)) and xlvi) SEQ ID NO: 334 (IL-12p35(D55K)).

[0102] In some embodiments, the present application provides a heterodimeric Fc fusion protein, wherein the heterodimeric Fc fusion protein comprises: 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).

[0103] In some embodiments, the present application provides a heterodimeric Fc fusion protein, wherein the heterodimeric Fc fusion protein is XENP27204.

[0104] In some embodiments, the present application provides a composition comprising a heterodimeric Fc fusion protein for use in treating cancer in a subject.

[0105] In some embodiments, the present application provides one or more nucleic acids encoding a heterodimeric Fc fusion protein.

[0106] In some embodiments, the present application provides a host cell comprising the one or more nucleic acids encoding a heterodimeric Fc fusion protein.

[0107] In some embodiments, the present application provides a method of making a heterodimeric Fc fusion protein, the method comprising culturing a host cell under conditions wherein the heterodimeric Fc fusion protein is produced.

[0108] In some embodiments, the present application provides a method of 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.

[0109] In another aspect, the present application 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 connected to the C-terminus of the first Fc domain, and wherein the second protein domain is covalently connected to the first protein domain; and b) a second Fc domain; wherein the first Fc domain and the second Fc domain comprise modifications that promote heterodimerization of the first Fc domain and the second Fc domain, and wherein the first protein domain comprises an IL-12 p40 subunit and the second protein domain comprises an IL-12 p35 subunit.

[0110] In some embodiments, the present application provides a heterodimeric Fc fusion protein wherein the modification that promotes heterodimerization of the first Fc domain and the second Fc domain is 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 according to EU numbering; L368E / K370S and S364K, K370S and S364K / E357Q, and T366S / L368A / Y407V:T366W (optionally comprising a bridging disulfide T366S / L368A / Y407V / Y349C:T366W / S354C).

[0111] In some embodiments, the present application provides a heterodimeric Fc fusion protein wherein the first protein domain is connected to the second protein domain using a first domain linker and / or the second protein domain is connected to the first Fc domain using a second domain linker.

[0112] In some embodiments, the present application provides a heterodimeric Fc fusion protein wherein the first Fc domain and / or the second Fc domain has an additional set of amino acid substitutions comprising Q295E / N384D / Q418E / N421D according to EU numbering.

[0113] In some embodiments, the present application provides a heterodimeric Fc fusion protein wherein the first Fc domain and / or the 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.

[0114] In some embodiments, the present application provides a heterodimeric Fc fusion protein, wherein the variant IL-12 p40 subunit has a polypeptide sequence selected from the group consisting of SEQ ID NO: 3 (human IL-12 subunit beta (IL-12 p40) precursor sequence) and SEQ ID NO: 4 (human IL-12 subunit beta (IL-12 p40) mature form sequence), and / or the IL-12 p35 subunit has a polypeptide sequence selected from the group consisting of SEQ ID NO: 1 (human IL-12 subunit alpha (IL-12 p35) precursor sequence) and SEQ ID NO: 2 (human IL-12 subunit alpha (IL-12 p35) mature form sequence).

[0115] In some embodiments, the present application provides a heterodimeric Fc fusion protein, wherein the first Fc domain and / or the second Fc domain further comprises the amino acid substitutions M428L / N424S.

[0116] In some embodiments, the present application provides a heterodimeric Fc fusion protein, wherein the IL-12 p40 subunit is a variant IL-12 p40 subunit and / or the IL-12 p35 subunit is a variant IL-12 p35 subunit.

[0117] In some embodiments, the present application provides a heterodimeric Fc fusion protein, wherein the IL-12 p40 subunit is a variant IL-12 p40 subunit having altered affinity for IL-12 receptor subunit beta-1 (IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2), and / or IL-12 receptor complex; and / or the IL-12 p35 subunit is a variant IL-12 p35 subunit having altered affinity for IL-12 receptor subunit beta-1 (IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2), and / or IL-12 receptor complex.

[0118] In some embodiments, the present application provides a heterodimeric Fc fusion protein, wherein the IL-12 p40 subunit has one or more amino acid modifications at an amino acid residue 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, 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.

[0119] In some embodiments, the present application provides a heterodimeric Fc fusion protein, wherein the IL-12 p40 subunit has one or more amino acid substitutions selected from the group consisting of D18N, D18K, E32Q, E33Q, D34N, D34K, Q42E, S43E, S43K, E45Q, Q56E, E59Q, E59K, D62N, E73Q, D87N, K99E, K99Y, E100Q, N103D, N103Q, N113D, N113Q, Q144E, D161N, R159E, K163E, E187Q, N200D, N200Q, N218Q, Q229E, E235Q, C252S, Q256N, K258E, K260E, E262Q, K264E, N281D, N281Q, and E299Q.

[0120] In some embodiments, the present application provides a heterodimeric Fc fusion protein wherein the IL-12p40 subunit has an amino acid substitution selected from the group consisting 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, 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, 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 / C252S / K264E, E59K / K99E / N113Q / C252S / K264E, E59K / K99E / N200Q / 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 / 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.

[0121] In some embodiments, the present application provides a heterodimeric Fc fusion protein, wherein the IL-12p40 subunit has a polypeptide sequence 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)); 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 (S43K); xxxiii) SEQ ID NO: 379 (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: 326 (IL-12p40 (D34N / E59K)); 1) SEQ ID NO: 325 (IL-12p40 (E59K / K99E)); li) SEQ ID NO: 339 (IL-12p40 (D18K / E59K / K99E)); lii) SEQ ID NO: 343 (IL-12p40 (E59K / K99E / K264E)); liii) SEQ ID NO: 336 (IL-12p40 (E59K / K99Y)); liv) SEQ ID NO: 335 (IL-12p40 (E59Y / K99E));lv) SEQ ID NO: 338 (IL-12p40 (E45K / E59K / K99E)); lvi) SEQ ID NO: 340 (IL-12p40 (E59K / K99E / Q144E)); lvii) SEQ ID NO: 341 (IL-12p40 (E59K / K99E / Q144K)); lviii) SEQ ID NO: 342 (IL-12p40 (E59K / K99E / R159E)); Ix) SEQ ID NO: 343 (IL-12p40 (E59K / K99E / K264E)); lx) SEQ ID NO: 344 (IL-12p40 (D18K / E59K / K99E / K264E)); lxi) SEQ ID NO: 360 (IL-12p40 (C252S)); lxii) SEQ ID NO: 361 (IL-12p40 (D18K / E59K / K99E / C252S)); lxiii) SEQ ID NO: 362 (IL-12p40 (D18K / E59K / K99E / C252S / K264E)); lxiv) SEQ ID NO: 363 (IL-12p40 (E59K / K99Y / C252S)); lxv) SEQ ID NO: 364 (IL-12p40 (E59K / K99E / C252S / K264E)); lxvi) SEQ ID NO: 365 (IL-12p40 (E59K / K99E / C252S)); lxvii) SEQ ID NO: 254 (IL-12p40 (N103D / N113D)); lxviii) SEQ ID NO: 255 (IL-12p40 (N103D / N200D)); lxix) SEQ ID NO: 256 (IL-12p40 (N103D / N281D)); lxx) SEQ ID NO: 257 (IL-12p40 (N113D / N200D)); lxxi) SEQ ID NO: 258 (IL-12p40 (N113D / N281D)); lxxii) SEQ ID NO: 259 (IL-12p40 (N200D / N281D)); lxxiii) SEQ ID NO: 260 (IL-12p40 (N103D / N113D / N200D)); lxxiv) SEQ ID NO: 261 (IL-12p40 (N103D / N113D / N281D)); lxxv) SEQ ID NO: 262 (IL-12p40 (N103D / N200D / N281D));lxxvi) SEQ ID NO: 263 (IL-12p40 (N113D / N200D / N281D)); lxxvii) SEQ ID NO: 264 (IL-12p40 (N103Q)); lxxviii) SEQ ID NO: 265 (IL-12p40 (N113Q)); lxxix) SEQ ID NO: 266 (IL-12p40 (N200Q)); lxxx) SEQ ID NO: 267 (IL-12p40 (N281Q)); lxxxi) SEQ ID NO: 268 (IL-12p40 (N103Q / N113Q)); lxxxii) SEQ ID NO: 269 (IL-12p40 (N103Q / N200Q)); lxxxiii) SEQ ID NO: 270 (IL-12p40 (N103Q / N281Q)); lxxxiv) SEQ ID NO: 271 (IL-12p40 (N113Q / N200Q)); lxxxv) SEQ ID NO: 272 (IL-12p40 (N113Q / N281Q)); lxxxvi) SEQ ID NO: 273 (IL-12p40 (N200Q / N281Q)); lxxxvii) SEQ ID NO: 274 (IL-12p40 (N103Q / N113Q / N200Q)); lxxxviii) SEQ ID NO: 275 (IL-12p40 (N103Q / N113Q / N281Q)); lxxxix) SEQ ID NO: 276 (IL-12p40 (N103Q / N200Q / N281Q)); xc) SEQ ID NO: 277 (IL-12p40 (N113Q / N200Q / N281Q)); xci) SEQ ID NO: 278 (IL-12p40 (N103Q / N113Q / N200Q / N281Q)); xcii) SEQ ID NO: 327 (IL-12p40 (D34N / E59K / K99E)); xciii) SEQ ID NO: 328 (IL-12p40 (D34K / E59K / K99E)); xciv) SEQ ID NO: 329 (IL-12p40 (E32Q / D34N / E59K / K99E)); xcv) SEQ ID NO: 331 (IL-12p40 (E32K / D34N / E59K / K99E)); xcvi) SEQ ID NO: 337 (IL-12p40 (E59Y / K99Y));xcvii) SEQ ID NO: 366 (IL-12p40 (E59K / K99E / N103Q / C252S / K264E)); xcviii) SEQ ID NO: 367 (IL-12p40 (E59K / K99E / N113Q / C252S / K264E)); xcix) SEQ ID NO: 368 (IL-12p40 (E59K / K99E / N200Q / C252S / K264E)); c) SEQ ID NO: 369 (IL-12p40 (E59K / K99E / N281Q / C252S / K264E)); ci) SEQ ID NO: 370 (IL-12p40 (E59K / K99E / N103Q / N113Q / C252S / K264E)); cii) SEQ ID NO: 371 (IL-12p40 (E59K / K99E / N103Q / N200Q / C252S / K264E)); ciii) SEQ ID NO: 372 (IL-12p40 (E59K / K99E / N103Q / N281Q / C252S / K264E)); civ) SEQ ID NO: 373 (IL-12p40 (E59K / K99E / N113Q / N200Q / C252S / K264E)); cv) SEQ ID NO: 374 (IL-12p40 (E59K / K99E / N113Q / N281Q / C252S / K264E)); cvi) SEQ ID NO: 375 (IL-12p40 (E59K / K99E / N200Q / N281Q / C252S / K264E)); cvii) SEQ ID NO: 376 (IL-12p40 (E59K / K99E / N103Q / N113Q / N200Q / C252S / K264E)); cviii) SEQ ID NO: 377 (IL-12p40 (E59K / K99E / N103Q / N200Q / N281Q / C252S / K264E)); and cx) SEQ ID NO: 378 (IL-12p40 (E59K / K99E / N113Q / N200Q / N281Q / C252S / K264E)).

[0122] In some embodiments, the present application provides a heterodimeric Fc fusion protein, wherein the IL-12 p35 subunit has one or more amino acid modifications 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, Q135, N136, E143, Q146, N151, E153, K158, E162, E163, D165, I171, R181, I182, R183, V185, T186, D188, R189, V190, S192, Y193, N195, and A196 as amino acid residues.

[0123] In some embodiments, the present application provides a heterodimeric Fc fusion protein, wherein the IL-12 p35 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.

[0124] In some embodiments, the present application provides a heterodimeric Fc fusion protein, wherein the IL-12 p35 subunit has one or more 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.

[0125] In some embodiments, the present application provides a heterodimeric Fc fusion protein wherein the IL-12 p35 subunit has a polypeptide sequence 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: 333 (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: 345 (IL-12p35(F96A)); xxxiv) SEQ ID NO: 346 (IL-12p35(M97A)); xxxv) SEQ ID NO: 347 (IL-12p35(L89A)); xxxvi) SEQ ID NO: 348 (IL-12p35(L124A)); xxxvii) SEQ ID NO: 349 (IL-12p35(M125A)); xxxviii) SEQ ID NO: 350 (IL-12p35(L75A)); xxxiv) SEQ ID NO: 351 (IL-12p35(I171A)); xxxv) SEQ ID NO: 279 (IL-12p35(N71Q)); xxxvi) SEQ ID NO: 280 (IL-12p35(N85Q)); xxxvii) SEQ ID NO: 281 (IL-12p35(N195Q)); xxxviii) SEQ ID NO: 282 (IL-12p35(N71Q / N85Q)); xxxix) SEQ ID NO: 283 (IL-12p35(N71Q / N195Q)); xl) SEQ ID NO: 284 (IL-12p35(N85Q / N195Q); xli) SEQ ID NO: 285 (IL-12p35(N71Q / N85Q / N195Q)); xlii) SEQ ID NO: 286 (IL-12p35(N71D / N85D)); xliii) SEQ ID NO: 287 (IL-12p35(N71D / N195D); xliv) SEQ ID NO: 288 (IL-12p35(N85D / N195D)); xlv) SEQ ID NO: 333 (IL-12p35(D55Q)) and xlvi) SEQ ID NO: 334 (IL-12p35(D55K)).

[0126] In some embodiments, the present application provides a composition comprising a heterodimeric Fc fusion protein for use in treating cancer in a subject.

[0127] In some embodiments, the present application provides one or more nucleic acids encoding a heterodimeric Fc fusion protein.

[0128] In some embodiments, the present application provides a host cell comprising the one or more nucleic acids encoding a heterodimeric Fc fusion protein.

[0129] In some embodiments, the present application provides a method of making a heterodimeric Fc fusion protein, the method comprising culturing a host cell under conditions wherein the heterodimeric Fc fusion protein is produced.

[0130] In some embodiments, the present application provides a method of 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 a fraction comprising the heterodimeric Fc fusion protein.

[0131] In another aspect, the present application 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 connected to the C-terminus of the first Fc domain, and wherein the second protein domain is covalently connected to the first protein domain; and b) a second Fc domain; wherein the first Fc domain and the second Fc domain comprise modifications that promote heterodimerization of the first Fc domain and the second Fc domain, and wherein the first protein domain comprises an IL-12 p35 subunit and the second protein domain comprises an IL-12 p40 subunit.

[0132] In some embodiments, the present application provides a heterodimeric Fc fusion protein, wherein the modifications that promote heterodimerization of the first Fc domain and the second Fc domain are a set of amino acid substitutions selected from the group consisting of L368D / K370S and S364K, L368D / K370S and S364K / E357L, L368D / K370S and S364K / E357Q, T411E / K360E / Q362E and D401K, L368E / K370S and S364K, K370S and S364K / E357Q, and T366S / L368A / Y407V:T366W (optionally including a bridging disulfide T366S / L368A / Y407V / Y349C:T366W / S354C) according to EU numbering.

[0133] In some embodiments, the present application provides a heterodimeric Fc fusion protein, wherein the first protein domain is connected to the second protein domain using a first domain linker and / or the second protein domain is connected to the first Fc domain using a second domain linker.

[0134] In some embodiments, the present application provides a heterodimeric Fc fusion protein, wherein the first Fc domain and / or the second Fc domain has a further set of amino acid substitutions comprising Q295E / N384D / Q418E / N421D, according to EU numbering.

[0135] In some embodiments, the present application provides a heterodimeric Fc fusion protein, wherein the first Fc domain and / or the second Fc domain has a further 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.

[0136] In some embodiments, the present application provides a heterodimeric Fc fusion protein, wherein the variant IL-12 p40 subunit has a polypeptide sequence selected from the group consisting of SEQ ID NO: 3 (human IL-12 subunit beta (IL-12 p40) precursor sequence) and SEQ ID NO: 4 (human IL-12 subunit beta (IL-12 p40) mature form sequence), and / or the IL-12 p35 subunit has a polypeptide sequence selected from the group consisting of SEQ ID NO: 1 (human IL-12 subunit alpha (IL-12 p35) precursor sequence) and SEQ ID NO: 2 (human IL-12 subunit alpha (IL-12 p35) mature form sequence).

[0137] In some embodiments, the present application provides a heterodimeric Fc fusion protein, wherein the first Fc domain and / or the second Fc domain further comprises the amino acid substitutions M428L / N424S.

[0138] In some embodiments, the present application provides a heterodimeric Fc fusion protein, wherein the IL-12 p40 subunit is a variant IL-12 p40 subunit and / or the IL-12 p35 subunit is a variant IL-12 p35 subunit.

[0139] In some embodiments, the present application provides a heterodimeric Fc fusion protein, wherein the IL-12p40 subunit is a variant IL-12p40 subunit having altered affinity for IL-12 receptor subunit beta-1 (IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2), and / or IL-12 receptor complex; and / or the IL-12p35 subunit is a variant IL-12p35 subunit having altered affinity for IL-12 receptor subunit beta-1 (IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2), and / or IL-12 receptor complex.

[0140] In some embodiments, the present application provides a heterodimeric Fc fusion protein, wherein the IL-12p40 subunit has one or more amino acid modifications at an amino acid residue 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, 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.

[0141] In some embodiments, the present application provides a heterodimeric Fc fusion protein wherein the IL-12p40 subunit has one or more amino acid substitutions selected from the group consisting of D18N, D18K, E32Q, E33Q, D34N, D34K, Q42E, S43E, S43K, E45Q, Q56E, E59Q, E59K, D62N, E73Q, D87N, K99E, K99Y, E100Q, N103D, N103Q, N113D, N113Q, Q144E, D161N, R159E, K163E, E187Q, N200D, N200Q, N218Q, Q229E, E235Q, C252S, Q256N, K258E, K260E, E262Q, K264E, N281D, N281Q, and E299Q.

[0142] In some embodiments, the present application provides a heterodimeric Fc fusion protein wherein the IL-12p40 subunit has an amino acid substitution selected from the group consisting 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, 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, 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 / C252S / K264E, E59K / K99E / N113Q / C252S / K264E, E59K / K99E / N200Q / 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 / 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.

[0143] In some embodiments, the present application provides a heterodimeric Fc fusion protein, wherein the IL-12p40 subunit has a polypeptide sequence 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)); 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 (S43K); xxxiii) SEQ ID NO: 379 (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: 326 (IL-12p40 (D34N / E59K)); 1) SEQ ID NO: 325 (IL-12p40 (E59K / K99E)); li) SEQ ID NO: 339 (IL-12p40 (D18K / E59K / K99E)); lii) SEQ ID NO: 343 (IL-12p40 (E59K / K99E / K264E)); liii) SEQ ID NO: 336 (IL-12p40 (E59K / K99Y)); liv) SEQ ID NO: 335 (IL-12p40 (E59Y / K99E));lv) SEQ ID NO: 338 (IL-12p40 (E45K / E59K / K99E)); lvi) SEQ ID NO: 340 (IL-12p40 (E59K / K99E / Q144E)); lvii) SEQ ID NO: 341 (IL-12p40 (E59K / K99E / Q144K)); lviii) SEQ ID NO: 342 (IL-12p40 (E59K / K99E / R159E)); Ix) SEQ ID NO: 343 (IL-12p40 (E59K / K99E / K264E)); lx) SEQ ID NO: 344 (IL-12p40 (D18K / E59K / K99E / K264E)); lxi) SEQ ID NO: 360 (IL-12p40 (C252S)); lxii) SEQ ID NO: 361 (IL-12p40 (D18K / E59K / K99E / C252S)); lxiii) SEQ ID NO: 362 (IL-12p40 (D18K / E59K / K99E / C252S / K264E)); lxiv) SEQ ID NO: 363 (IL-12p40 (E59K / K99Y / C252S)); lxv) SEQ ID NO: 364 (IL-12p40 (E59K / K99E / C252S / K264E)); lxvi) SEQ ID NO: 365 (IL-12p40 (E59K / K99E / C252S)); lxvii) SEQ ID NO: 254 (IL-12p40 (N103D / N113D)); lxviii) SEQ ID NO: 255 (IL-12p40 (N103D / N200D)); lxix) SEQ ID NO: 256 (IL-12p40 (N103D / N281D)); lxx) SEQ ID NO: 257 (IL-12p40 (N113D / N200D)); lxxi) SEQ ID NO: 258 (IL-12p40 (N113D / N281D)); lxxii) SEQ ID NO: 259 (IL-12p40 (N200D / N281D)); lxxiii) SEQ ID NO: 260 (IL-12p40 (N103D / N113D / N200D)); lxxiv) SEQ ID NO: 261 (IL-12p40 (N103D / N113D / N281D)); lxxv) SEQ ID NO: 262 (IL-12p40 (N103D / N200D / N281D));lxxvi) SEQ ID NO: 263 (IL-12p40 (N113D / N200D / N281D)); lxxvii) SEQ ID NO: 264 (IL-12p40 (N103Q)); lxxviii) SEQ ID NO: 265 (IL-12p40 (N113Q)); lxxix) SEQ ID NO: 266 (IL-12p40 (N200Q)); lxxx) SEQ ID NO: 267 (IL-12p40 (N281Q)); lxxxi) SEQ ID NO: 268 (IL-12p40 (N103Q / N113Q)); lxxxii) SEQ ID NO: 269 (IL-12p40 (N103Q / N200Q)); lxxxiii) SEQ ID NO: 270 (IL-12p40 (N103Q / N281Q)); lxxxiv) SEQ ID NO: 271 (IL-12p40 (N113Q / N200Q)); lxxxv) SEQ ID NO: 272 (IL-12p40 (N113Q / N281Q)); lxxxvi) SEQ ID NO: 273 (IL-12p40 (N200Q / N281Q)); lxxxvii) SEQ ID NO: 274 (IL-12p40 (N103Q / N113Q / N200Q)); lxxxviii) SEQ ID NO: 275 (IL-12p40 (N103Q / N113Q / N281Q)); lxxxix) SEQ ID NO: 276 (IL-12p40 (N103Q / N200Q / N281Q)); xc) SEQ ID NO: 277 (IL-12p40 (N113Q / N200Q / N281Q)); xci) SEQ ID NO: 278 (IL-12p40 (N103Q / N113Q / N200Q / N281Q)); xcii) SEQ ID NO: 327 (IL-12p40 (D34N / E59K / K99E)); xciii) SEQ ID NO: 328 (IL-12p40 (D34K / E59K / K99E)); xciv) SEQ ID NO: 329 (IL-12p40 (E32Q / D34N / E59K / K99E)); xcv) SEQ ID NO: 331 (IL-12p40 (E32K / D34N / E59K / K99E)); xcvi) SEQ ID NO: 337 (IL-12p40 (E59Y / K99Y));xcvii) SEQ ID NO: 366 (IL-12p40 (E59K / K99E / N103Q / C252S / K264E)); xcviii) SEQ ID NO: 367 (IL-12p40 (E59K / K99E / N113Q / C252S / K264E)); xcix) SEQ ID NO: 368 (IL-12p40 (E59K / K99E / N200Q / C252S / K264E)); c) SEQ ID NO: 369 (IL-12p40 (E59K / K99E / N281Q / C252S / K264E)); ci) SEQ ID NO: 370 (IL-12p40 (E59K / K99E / N103Q / N113Q / C252S / K264E)); cii) SEQ ID NO: 371 (IL-12p40 (E59K / K99E / N103Q / N200Q / C252S / K264E)); ciii) SEQ ID NO: 372 (IL-12p40 (E59K / K99E / N103Q / N281Q / C252S / K264E)); civ) SEQ ID NO: 373 (IL-12p40 (E59K / K99E / N113Q / N200Q / C252S / K264E)); cv) SEQ ID NO: 374 (IL-12p40 (E59K / K99E / N113Q / N281Q / C252S / K264E)); cvi) SEQ ID NO: 375 (IL-12p40 (E59K / K99E / N200Q / N281Q / C252S / K264E)); cvii) SEQ ID NO: 376 (IL-12p40 (E59K / K99E / N103Q / N113Q / N200Q / C252S / K264E)); cviii) SEQ ID NO: 377 (IL-12p40 (E59K / K99E / N103Q / N200Q / N281Q / C252S / K264E)); and cx) SEQ ID NO: 378 (IL-12p40 (E59K / K99E / N113Q / N200Q / N281Q / C252S / K264E)).

[0144] In some embodiments, the present application provides a heterodimeric Fc fusion protein, wherein the IL-12 p35 subunit has one or more amino acid modifications 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, Q135, N136, E143, Q146, N151, E153, K158, E162, E163, D165, I171, R181, I182, R183, V185, T186, D188, R189, V190, S192, Y193, N195, and A196 as amino acid residues.

[0145] In some embodiments, the present application provides a heterodimeric Fc fusion protein, wherein the IL-12 p35 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.

[0146] In some embodiments, the present application provides a heterodimeric Fc fusion protein, wherein the IL-12 p35 subunit has one or more 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.

[0147] In some embodiments, the present application provides a heterodimeric Fc fusion protein wherein the IL-12 p35 subunit has a polypeptide sequence 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: 333 (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: 345 (IL-12p35(F96A)); xxxiv) SEQ ID NO: 346 (IL-12p35(M97A)); xxxv) SEQ ID NO: 347 (IL-12p35(L89A)); xxxvi) SEQ ID NO: 348 (IL-12p35(L124A)); xxxvii) SEQ ID NO: 349 (IL-12p35(M125A)); xxxviii) SEQ ID NO: 350 (IL-12p35(L75A)); xxxiv) SEQ ID NO: 351 (IL-12p35(I171A)); xxxv) SEQ ID NO: 279 (IL-12p35(N71Q)); xxxvi) SEQ ID NO: 280 (IL-12p35(N85Q)); xxxvii) SEQ ID NO: 281 (IL-12p35(N195Q)); xxxviii) SEQ ID NO: 282 (IL-12p35(N71Q / N85Q)); xxxix) SEQ ID NO: 283 (IL-12p35(N71Q / N195Q)); xl) SEQ ID NO: 284 (IL-12p35(N85Q / N195Q); xli) SEQ ID NO: 285 (IL-12p35(N71Q / N85Q / N195Q)); xlii) SEQ ID NO: 286 (IL-12p35(N71D / N85D)); xliii) SEQ ID NO: 287 (IL-12p35(N71D / N195D); xliv) SEQ ID NO: 288 (IL-12p35(N85D / N195D)); xlv) SEQ ID NO: 333 (IL-12p35(D55Q)) and xlvi) SEQ ID NO: 334 (IL-12p35(D55K)).

[0148] In some embodiments, the present application provides a composition comprising a heterodimeric Fc fusion protein for use in treating cancer in a subject.

[0149] In some embodiments, the present application provides one or more nucleic acids encoding a heterodimeric Fc fusion protein.

[0150] In some embodiments, the present application provides a host cell comprising the one or more nucleic acids encoding a heterodimeric Fc fusion protein.

[0151] In some embodiments, the present application provides a method of making a heterodimeric Fc fusion protein, the method comprising culturing a host cell under conditions wherein the heterodimeric Fc fusion protein is produced.

[0152] In some embodiments, the present application provides a method of 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 a fraction containing the heterodimeric Fc fusion protein.

[0153] In another aspect, the present application provides a heterodimeric complex comprising: a) an IL-12 p40 subunit, and b) an IL-12 p35 subunit.

[0154] In some embodiments, the present application provides a heterodimeric complex, wherein the IL-12 p40 subunit is covalently linked to the IL-12 p35 subunit using a domain linker.

[0155] In some embodiments, the present application provides a heterodimeric complex, wherein the variant IL-12 p40 subunit has a polypeptide sequence selected from the group consisting of SEQ ID NO: 3 (human IL-12 subunit beta (IL-12 p40) precursor sequence) and SEQ ID NO: 4 (human IL-12 subunit beta (IL-12 p40) mature form sequence), and / or the IL-12 p35 subunit has a polypeptide sequence selected from the group consisting of SEQ ID NO: 1 (human IL-12 subunit alpha (IL-12 p35) precursor sequence) and SEQ ID NO: 2 (human IL-12 subunit alpha (IL-12 p35) mature form sequence).

[0156] In some embodiments, the present application provides a heterodimeric complex, wherein the first Fc domain and / or the second Fc domain further comprises the amino acid substitutions M428L / N424S.

[0157] In some embodiments, the present application provides a heterodimeric complex, wherein the IL-12 p40 subunit is a variant IL-12 p40 subunit and / or the IL-12 p35 subunit is a variant IL-12 p35 subunit.

[0158] In some embodiments, the present application provides a heterodimeric complex, wherein the IL-12p40 subunit is a variant IL-12p40 subunit having altered affinity for IL-12 receptor subunit beta-1 (IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2), and / or IL-12 receptor complex; and / or the IL-12p35 subunit is a variant IL-12p35 subunit having altered affinity for IL-12 receptor subunit beta-1 (IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2), and / or IL-12 receptor complex.

[0159] In some embodiments, the present application provides a heterodimeric complex, wherein the IL-12p40 subunit has one or more amino acid modifications at an amino acid residue 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, 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.

[0160] In some embodiments, the present application provides a heterodimeric complex, wherein the IL-12p40 subunit has one or more amino acid substitutions selected from the group consisting of D18N, D18K, E32Q, E33Q, D34N, D34K, Q42E, S43E, S43K, E45Q, Q56E, E59Q, E59K, D62N, E73Q, D87N, K99E, K99Y, E100Q, N103D, N103Q, N113D, N113Q, Q144E, D161N, R159E, K163E, E187Q, N200D, N200Q, N218Q, Q229E, E235Q, C252S, Q256N, K258E, K260E, E262Q, K264E, N281D, N281Q, and E299Q.

[0161] In some embodiments, the present application provides a heterodimeric complex, wherein the IL-12p40 subunit has an amino acid substitution selected from the group consisting 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, 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, 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 / C252S / K264E, E59K / K99E / N113Q / C252S / K264E, E59K / K99E / N200Q / 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 / 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.

[0162] In some embodiments, the present application provides a heterodimeric complex, wherein the IL-12p40 subunit has a polypeptide sequence 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)); 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 (S43K); xxxiii) SEQ ID NO: 379 (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: 326 (IL-12p40 (D34N / E59K)); 1) SEQ ID NO: 325 (IL-12p40 (E59K / K99E)); li) SEQ ID NO: 339 (IL-12p40 (D18K / E59K / K99E)); lii) SEQ ID NO: 343 (IL-12p40 (E59K / K99E / K264E)); liii) SEQ ID NO: 336 (IL-12p40 (E59K / K99Y)); liv) SEQ ID NO: 335 (IL-12p40 (E59Y / K99E));lv) SEQ ID NO: 338 (IL-12p40 (E45K / E59K / K99E)); lvi) SEQ ID NO: 340 (IL-12p40 (E59K / K99E / Q144E)); lvii) SEQ ID NO: 341 (IL-12p40 (E59K / K99E / Q144K)); lviii) SEQ ID NO: 342 (IL-12p40 (E59K / K99E / R159E)); Ix) SEQ ID NO: 343 (IL-12p40 (E59K / K99E / K264E)); lx) SEQ ID NO: 344 (IL-12p40 (D18K / E59K / K99E / K264E)); lxi) SEQ ID NO: 360 (IL-12p40 (C252S)); lxii) SEQ ID NO: 361 (IL-12p40 (D18K / E59K / K99E / C252S)); lxiii) SEQ ID NO: 362 (IL-12p40 (D18K / E59K / K99E / C252S / K264E)); lxiv) SEQ ID NO: 363 (IL-12p40 (E59K / K99Y / C252S)); lxv) SEQ ID NO: 364 (IL-12p40 (E59K / K99E / C252S / K264E)); lxvi) SEQ ID NO: 365 (IL-12p40 (E59K / K99E / C252S)); lxvii) SEQ ID NO: 254 (IL-12p40 (N103D / N113D)); lxviii) SEQ ID NO: 255 (IL-12p40 (N103D / N200D)); lxix) SEQ ID NO: 256 (IL-12p40 (N103D / N281D)); lxx) SEQ ID NO: 257 (IL-12p40 (N113D / N200D)); lxxi) SEQ ID NO: 258 (IL-12p40 (N113D / N281D)); lxxii) SEQ ID NO: 259 (IL-12p40 (N200D / N281D)); lxxiii) SEQ ID NO: 260 (IL-12p40 (N103D / N113D / N200D)); lxxiv) SEQ ID NO: 261 (IL-12p40 (N103D / N113D / N281D)); lxxv) SEQ ID NO: 262 (IL-12p40 (N103D / N200D / N281D));lxxvi) SEQ ID NO: 263 (IL-12p40 (N113D / N200D / N281D)); lxxvii) SEQ ID NO: 264 (IL-12p40 (N103Q)); lxxviii) SEQ ID NO: 265 (IL-12p40 (N113Q)); lxxix) SEQ ID NO: 266 (IL-12p40 (N200Q)); lxxx) SEQ ID NO: 267 (IL-12p40 (N281Q)); lxxxi) SEQ ID NO: 268 (IL-12p40 (N103Q / N113Q)); lxxxii) SEQ ID NO: 269 (IL-12p40 (N103Q / N200Q)); lxxxiii) SEQ ID NO: 270 (IL-12p40 (N103Q / N281Q)); lxxxiv) SEQ ID NO: 271 (IL-12p40 (N113Q / N200Q)); lxxxv) SEQ ID NO: 272 (IL-12p40 (N113Q / N281Q)); lxxxvi) SEQ ID NO: 273 (IL-12p40 (N200Q / N281Q)); lxxxvii) SEQ ID NO: 274 (IL-12p40 (N103Q / N113Q / N200Q)); lxxxviii) SEQ ID NO: 275 (IL-12p40 (N103Q / N113Q / N281Q)); lxxxix) SEQ ID NO: 276 (IL-12p40 (N103Q / N200Q / N281Q)); xc) SEQ ID NO: 277 (IL-12p40 (N113Q / N200Q / N281Q)); xci) SEQ ID NO: 278 (IL-12p40 (N103Q / N113Q / N200Q / N281Q)); xcii) SEQ ID NO: 327 (IL-12p40 (D34N / E59K / K99E)); xciii) SEQ ID NO: 328 (IL-12p40 (D34K / E59K / K99E)); xciv) SEQ ID NO: 329 (IL-12p40 (E32Q / D34N / E59K / K99E)); xcv) SEQ ID NO: 331 (IL-12p40 (E32K / D34N / E59K / K99E)); xcvi) SEQ ID NO: 337 (IL-12p40 (E59Y / K99Y));xcvii) SEQ ID NO: 366 (IL-12p40 (E59K / K99E / N103Q / C252S / K264E)); xcviii) SEQ ID NO: 367 (IL-12p40 (E59K / K99E / N113Q / C252S / K264E)); xcix) SEQ ID NO: 368 (IL-12p40 (E59K / K99E / N200Q / C252S / K264E)); c) SEQ ID NO: 369 (IL-12p40 (E59K / K99E / N281Q / C252S / K264E)); ci) SEQ ID NO: 370 (IL-12p40 (E59K / K99E / N103Q / N113Q / C252S / K264E)); cii) SEQ ID NO: 371 (IL-12p40 (E59K / K99E / N103Q / N200Q / C252S / K264E)); ciii) SEQ ID NO: 372 (IL-12p40 (E59K / K99E / N103Q / N281Q / C252S / K264E)); civ) SEQ ID NO: 373 (IL-12p40 (E59K / K99E / N113Q / N200Q / C252S / K264E)); cv) SEQ ID NO: 374 (IL-12p40 (E59K / K99E / N113Q / N281Q / C252S / K264E)); cvi) SEQ ID NO: 375 (IL-12p40 (E59K / K99E / N200Q / N281Q / C252S / K264E)); cvii) SEQ ID NO: 376 (IL-12p40 (E59K / K99E / N103Q / N113Q / N200Q / C252S / K264E)); cviii) SEQ ID NO: 377 (IL-12p40 (E59K / K99E / N103Q / N200Q / N281Q / C252S / K264E)); and cx) SEQ ID NO: 378 (IL-12p40 (E59K / K99E / N113Q / N200Q / N281Q / C252S / K264E)).

[0163] In some embodiments, the present application provides a heterodimeric complex, wherein the IL-12 p35 subunit has one or more amino acid modifications as amino acid residues 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, Q135, N136, E143, Q146, N151, E153, K158, E162, E163, D165, I171, R181, I182, R183, V185, T186, D188, R189, V190, S192, Y193, N195, and A196.

[0164] In some embodiments, the present application provides a heterodimeric complex, wherein the IL-12 p35 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.

[0165] In some embodiments, the present application provides a heterodimeric complex, wherein the IL-12 p35 subunit has one or more 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.

[0166] In some embodiments, the present application provides a heterodimeric complex, wherein the IL-12p35 subunit has a polypeptide sequence 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: 333 (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: 345 (IL-12p35(F96A)); xxxiv) SEQ ID NO: 346 (IL-12p35(M97A)); xxxv) SEQ ID NO: 347 (IL-12p35(L89A)); xxxvi) SEQ ID NO: 348 (IL-12p35(L124A)); xxxvii) SEQ ID NO: 349 (IL-12p35(M125A)); xxxviii) SEQ ID NO: 350 (IL-12p35(L75A)); xxxiv) SEQ ID NO: 351 (IL-12p35(I171A)); xxxv) SEQ ID NO: 279 (IL-12p35(N71Q)); xxxvi) SEQ ID NO: 280 (IL-12p35(N85Q)); xxxvii) SEQ ID NO: 281 (IL-12p35(N195Q)); xxxviii) SEQ ID NO: 282 (IL-12p35(N71Q / N85Q)); xxxix) SEQ ID NO: 283 (IL-12p35(N71Q / N195Q)); xl) SEQ ID NO: 284 (IL-12p35(N85Q / N195Q); xli) SEQ ID NO: 285 (IL-12p35(N71Q / N85Q / N195Q)); xlii) SEQ ID NO: 286 (IL-12p35(N71D / N85D)); xliii) SEQ ID NO: 287 (IL-12p35(N71D / N195D); xliv) SEQ ID NO: 288 (IL-12p35(N85D / N195D)); xlv) SEQ ID NO: 333 (IL-12p35(D55Q)) and xlvi) SEQ ID NO: 334 (IL-12p35(D55K)).

[0167] In some embodiments, the present application provides a composition comprising a heterodimeric complex for use in treating a cancer in a subject.

[0168] In some embodiments, the present application provides one or more nucleic acids encoding a heterodimeric complex.

[0169] In some embodiments, the present application provides a host cell comprising the one or more nucleic acids encoding a heterodimeric complex.

[0170] In some embodiments, the present application provides a method of making a heterodimeric complex, the method comprising culturing a host cell under conditions wherein the heterodimeric complex is produced.

[0171] In some embodiments, the present application provides a method of purifying a heterodimeric complex, the method comprising: a) providing a composition comprising the heterodimeric complex; b) loading the composition onto an ion exchange column; and c) collecting fractions containing the heterodimeric complex.

[0172] In another aspect, the present application provides a variant IL-12p40 subunit having altered affinity for IL-12 receptor subunit beta-1 (IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2), and / or IL-12 receptor complex.

[0173] In some embodiments, the present application provides a variant IL-12p40 subunit, wherein the variant IL-12p40 subunit has one or more amino acid modifications at an amino acid residue 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, 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.

[0174] In some embodiments, the present application provides a variant IL-12p40 subunit, wherein the variant IL-12p40 subunit has one or more amino acid substitutions selected from the group consisting of D18N, D18K, E32Q, E33Q, D34N, D34K, Q42E, S43E, S43K, E45Q, Q56E, E59Q, E59K, D62N, E73Q, D87N, K99E, K99Y, E100Q, N103D, N103Q, N113D, N113Q, Q144E, D161N, R159E, K163E, E187Q, N200D, N200Q, N218Q, Q229E, E235Q, C252S, Q256N, K258E, K260E, E262Q, K264E, N281D, N281Q, and E299Q.

[0175] In some embodiments, the present application provides a variant IL-12p40 subunit, wherein the variant IL-12p40 subunit has an amino acid substitution selected from the group consisting 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, 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, 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 / C252S / K264E, E59K / K99E / N113Q / C252S / K264E, E59K / K99E / N200Q / 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 / 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.

[0176] In some embodiments, the present application provides a variant IL-12p40 subunit, wherein the variant IL-12p40 subunit has a polypeptide sequence 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)); 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 (S43K); xxxiii) SEQ ID NO: 379 (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: 326 (IL-12p40 (D34N / E59K)); 1) SEQ ID NO: 325 (IL-12p40 (E59K / K99E)); li) SEQ ID NO: 339 (IL-12p40 (D18K / E59K / K99E)); lii) SEQ ID NO: 343 (IL-12p40 (E59K / K99E / K264E)); liii) SEQ ID NO: 336 (IL-12p40 (E59K / K99Y)); liv) SEQ ID NO: 335 (IL-12p40 (E59Y / K99E));lv) SEQ ID NO: 338 (IL-12p40 (E45K / E59K / K99E)); lvi) SEQ ID NO: 340 (IL-12p40 (E59K / K99E / Q144E)); lvii) SEQ ID NO: 341 (IL-12p40 (E59K / K99E / Q144K)); lviii) SEQ ID NO: 342 (IL-12p40 (E59K / K99E / R159E)); Ix) SEQ ID NO: 343 (IL-12p40 (E59K / K99E / K264E)); lx) SEQ ID NO: 344 (IL-12p40 (D18K / E59K / K99E / K264E)); lxi) SEQ ID NO: 360 (IL-12p40 (C252S)); lxii) SEQ ID NO: 361 (IL-12p40 (D18K / E59K / K99E / C252S)); lxiii) SEQ ID NO: 362 (IL-12p40 (D18K / E59K / K99E / C252S / K264E)); lxiv) SEQ ID NO: 363 (IL-12p40 (E59K / K99Y / C252S)); lxv) SEQ ID NO: 364 (IL-12p40 (E59K / K99E / C252S / K264E)); lxvi) SEQ ID NO: 365 (IL-12p40 (E59K / K99E / C252S)); lxvii) SEQ ID NO: 254 (IL-12p40 (N103D / N113D)); lxviii) SEQ ID NO: 255 (IL-12p40 (N103D / N200D)); lxix) SEQ ID NO: 256 (IL-12p40 (N103D / N281D)); lxx) SEQ ID NO: 257 (IL-12p40 (N113D / N200D)); lxxi) SEQ ID NO: 258 (IL-12p40 (N113D / N281D)); lxxii) SEQ ID NO: 259 (IL-12p40 (N200D / N281D)); lxxiii) SEQ ID NO: 260 (IL-12p40 (N103D / N113D / N200D)); lxxiv) SEQ ID NO: 261 (IL-12p40 (N103D / N113D / N281D)); lxxv) SEQ ID NO: 262 (IL-12p40 (N103D / N200D / N281D));lxxvi) SEQ ID NO: 263 (IL-12p40 (N113D / N200D / N281D)); lxxvii) SEQ ID NO: 264 (IL-12p40 (N103Q)); lxxviii) SEQ ID NO: 265 (IL-12p40 (N113Q)); lxxix) SEQ ID NO: 266 (IL-12p40 (N200Q)); lxxx) SEQ ID NO: 267 (IL-12p40 (N281Q)); lxxxi) SEQ ID NO: 268 (IL-12p40 (N103Q / N113Q)); lxxxii) SEQ ID NO: 269 (IL-12p40 (N103Q / N200Q)); lxxxiii) SEQ ID NO: 270 (IL-12p40 (N103Q / N281Q)); lxxxiv) SEQ ID NO: 271 (IL-12p40 (N113Q / N200Q)); lxxxv) SEQ ID NO: 272 (IL-12p40 (N113Q / N281Q)); lxxxvi) SEQ ID NO: 273 (IL-12p40 (N200Q / N281Q)); lxxxvii) SEQ ID NO: 274 (IL-12p40 (N103Q / N113Q / N200Q)); lxxxviii) SEQ ID NO: 275 (IL-12p40 (N103Q / N113Q / N281Q)); lxxxix) SEQ ID NO: 276 (IL-12p40 (N103Q / N200Q / N281Q)); xc) SEQ ID NO: 277 (IL-12p40 (N113Q / N200Q / N281Q)); xci) SEQ ID NO: 278 (IL-12p40 (N103Q / N113Q / N200Q / N281Q)); xcii) SEQ ID NO: 327 (IL-12p40 (D34N / E59K / K99E)); xciii) SEQ ID NO: 328 (IL-12p40 (D34K / E59K / K99E)); xciv) SEQ ID NO: 329 (IL-12p40 (E32Q / D34N / E59K / K99E)); xcv) SEQ ID NO: 331 (IL-12p40 (E32K / D34N / E59K / K99E)); xcvi) SEQ ID NO: 337 (IL-12p40 (E59Y / K99Y));xcvii) SEQ ID NO: 366 (IL-12p40 (E59K / K99E / N103Q / C252S / K264E)); xcviil) SEQ ID NO: 367 (IL-12p40 (E59K / K99E / N113Q / C252S / K264E)); xcix) SEQ ID NO: 368 (IL-12p40 (E59K / K99E / N200Q / C252S / K264E)); c) SEQ ID NO: 369 (IL-12p40 (E59K / K99E / N281Q / C252S / K264E)); ci) SEQ ID NO: 370 (IL-12p40 (E59K / K99E / N103Q / N113Q / C252S / K264E)); cii) SEQ ID NO: 371 (IL-12p40 (E59K / K99E / N103Q / N200Q / C252S / K264E)); ciii) SEQ ID NO: 372 (IL-12p40 (E59K / K99E / N103Q / N281Q / C252S / K264E)); civ) SEQ ID NO: 373 (IL-12p40 (E59K / K99E / N113Q / N200Q / C252S / K264E)); cv) SEQ ID NO: 374 (IL-12p40 (E59K / K99E / N113Q / N281Q / C252S / K264E)); cvi) SEQ ID NO: 375 (IL-12p40 (E59K / K99E / N200Q / N281Q / C252S / K264E)); cvii) SEQ ID NO: 376 (IL-12p40 (E59K / K99E / N103Q / N113Q / N200Q / C252S / K264E)); cviii) SEQ ID NO: 377 (IL-12p40 (E59K / K99E / N103Q / N200Q / N281Q / C252S / K264E)); and cx) SEQ ID NO: 378 (IL-12p40 (E59K / K99E / N113Q / N200Q / N281Q / C252S / K264E)).

[0177] In some embodiments, the present application provides a composition comprising a variant IL-12p40 subunit.

[0178] In some embodiments, the present application provides a nucleic acid encoding a variant IL-12p40 subunit.

[0179] In some embodiments, the present application provides a host cell comprising the nucleic acid encoding a variant IL-12 p40 subunit.

[0180] In some embodiments, the present application provides a method of making a variant IL-12 p40 subunit, the method comprising culturing the host cell of claim 18 under conditions wherein the variant IL-12 p40 subunit is produced.

[0181] In another aspect, the present application provides a variant IL-12 p35 subunit having altered affinity for IL-12 receptor subunit beta-1 (IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2), and / or IL-12 receptor complex.

[0182] In some embodiments, the present application provides a variant IL-12 p35 subunit, wherein the variant IL-12 p35 subunit has one or more amino acid modifications as amino acid residues 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, Q135, N136, E143, Q146, N151, E153, K158, E162, E163, D165, I171, R181, I182, R183, V185, T186, D188, R189, V190, S192, Y193, N195, and A196.

[0183] In some embodiments, the present application provides a variant IL-12 p35 subunit, wherein the variant IL-12 p35 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.

[0184] In some embodiments, the present application provides a variant IL-12 p35 subunit, wherein the variant IL-12 p35 subunit has the amino acid substitutions N71D / N85D / N195D.

[0185] In some embodiments, the present application provides a variant IL-12 p35 subunit, wherein the variant IL-12 p35 subunit has the amino acid substitutions N71D / N85D / N195D.

[0186] In some embodiments, the present application provides a variant IL-12p35 subunit, wherein the variant IL-12p35 subunit has a polypeptide sequence 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: 333 (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: 345 (IL-12p35(F96A)); xxxiv) SEQ ID NO: 346 (IL-12p35(M97A)); xxxv) SEQ ID NO: 347 (IL-12p35(L89A)); xxxvi) SEQ ID NO: 348 (IL-12p35(L124A)); xxxvii) SEQ ID NO: 349 (IL-12p35(M125A)); xxxviii) SEQ ID NO: 350 (IL-12p35(L75A)); xxxiv) SEQ ID NO: 351 (IL-12p35(I171A)); xxxv) SEQ ID NO: 279 (IL-12p35(N71Q)); xxxvi) SEQ ID NO: 280 (IL-12p35(N85Q)); xxxvii) SEQ ID NO: 281 (IL-12p35(N195Q)); xxxviii) SEQ ID NO: 282 (IL-12p35(N71Q / N85Q)); xxxix) SEQ ID NO: 283 (IL-12p35(N71Q / N195Q)); xl) SEQ ID NO: 284 (IL-12p35(N85Q / N195Q); xli) SEQ ID NO: 285 (IL-12p35(N71Q / N85Q / N195Q)); xlii) SEQ ID NO: 286 (IL-12p35(N71D / N85D)); xliii) SEQ ID NO: 287 (IL-12p35(N71D / N195D); xliv) SEQ ID NO: 288 (IL-12p35(N85D / N195D)); xlv) SEQ ID NO: 333 (IL-12p35(D55Q)) and xlvi) SEQ ID NO: 334 (IL-12p35(D55K)).

[0187] In some embodiments, the present application provides a composition comprising a variant IL-12p35 subunit.

[0188] In some embodiments, the present application provides a nucleic acid encoding a variant IL-12 p35 subunit.

[0189] In some embodiments, the present application provides a host cell comprising the nucleic acid encoding a variant IL-12 p35 subunit.

[0190] In some embodiments, the present application provides a variant IL-12 p35 subunit, the method comprising culturing a host cell under conditions to produce the variant IL-12 p35 subunit.

[0191] In another aspect, the present application provides a heterodimeric protein comprising: a) a first fusion protein comprising an IL-12 p40 subunit domain covalently linked to a first Fc domain; and b) a second fusion protein comprising an IL-12 p35 subunit domain covalently linked to a second Fc domain; wherein the first Fc domain and the second Fc domain are selected from the group consisting of the following sets of amino acid substitutions: 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; and T366S / L368A / Y407V / Y349C and T366W / S354C, according to EU numbering.

[0192] In some embodiments, the present application provides a heterodimeric protein wherein the IL-12 p40 subunit domain is linked to the N-terminus of the first Fc domain and the IL-12 p35 subunit domain is linked to the N-terminus of the second Fc domain.

[0193] In some embodiments, the present application provides a heterodimeric protein wherein the IL-12 p40 subunit domain is linked to the C-terminus of the first Fc domain and the IL-12 p35 subunit domain is linked to the C-terminus of the second Fc domain.

[0194] In some embodiments, the present application provides a heterodimeric protein wherein the IL-12 p40 subunit domain is linked to the first Fc domain using a domain linker and / or the IL-12 p35 subunit domain is linked to the second Fc domain using a domain linker.

[0195] In some embodiments, the present application provides a heterodimeric protein, wherein the first Fc domain and the second Fc domain comprise an IgGl hinge-CH2-CH3.

[0196] In some embodiments, the present application provides a heterodimeric protein, wherein the first fusion protein comprises, from N- to C-terminus, an IL-12p40 subunit domain-domain linker-hinge-CH2-CH3, and the second fusion protein comprises, from N- to C-terminus, an IL-12p35 subunit domain-domain linker-hinge-CH2-CH3.

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

[0198] In some embodiments, the present application provides a heterodimeric protein, wherein the IL-12p40 subunit has a sequence 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)); 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 (S43K); xxxiii) SEQ ID NO: 379 (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: 326 (IL-12p40 (D34N / E59K)); 1) SEQ ID NO: 325 (IL-12p40 (E59K / K99E)); li) SEQ ID NO: 339 (IL-12p40 (D18K / E59K / K99E)); lii) SEQ ID NO: 343 (IL-12p40 (E59K / K99E / K264E)); liii) SEQ ID NO: 336 (IL-12p40 (E59K / K99Y)); liv) SEQ ID NO: 335 (IL-12p40 (E59Y / K99E));lv) SEQ ID NO: 338 (IL-12p40 (E45K / E59K / K99E)); lvi) SEQ ID NO: 340 (IL-12p40 (E59K / K99E / Q144E)); lvii) SEQ ID NO: 341 (IL-12p40 (E59K / K99E / Q144K)); lviii) SEQ ID NO: 342 (IL-12p40 (E59K / K99E / R159E)); Ix) SEQ ID NO: 343 (IL-12p40 (E59K / K99E / K264E)); lx) SEQ ID NO: 344 (IL-12p40 (D18K / E59K / K99E / K264E)); lxi) SEQ ID NO: 360 (IL-12p40 (C252S)); lxii) SEQ ID NO: 361 (IL-12p40 (D18K / E59K / K99E / C252S)); lxiii) SEQ ID NO: 362 (IL-12p40 (D18K / E59K / K99E / C252S / K264E)); lxiv) SEQ ID NO: 363 (IL-12p40 (E59K / K99Y / C252S)); lxv) SEQ ID NO: 364 (IL-12p40 (E59K / K99E / C252S / K264E)); lxvi) SEQ ID NO: 365 (IL-12p40 (E59K / K99E / C252S)); lxvii) SEQ ID NO: 254 (IL-12p40 (N103D / N113D)); lxviii) SEQ ID NO: 255 (IL-12p40 (N103D / N200D)); lxix) SEQ ID NO: 256 (IL-12p40 (N103D / N281D)); lxx) SEQ ID NO: 257 (IL-12p40 (N113D / N200D)); lxxi) SEQ ID NO: 258 (IL-12p40 (N113D / N281D)); lxxii) SEQ ID NO: 259 (IL-12p40 (N200D / N281D)); lxxiii) SEQ ID NO: 260 (IL-12p40 (N103D / N113D / N200D)); lxxiv) SEQ ID NO: 261 (IL-12p40 (N103D / N113D / N281D)); lxxv) SEQ ID NO: 262 (IL-12p40 (N103D / N200D / N281D));lxxvi) SEQ ID NO: 263 (IL-12p40 (N113D / N200D / N281D)); lxxvii) SEQ ID NO: 264 (IL-12p40 (N103Q)); lxxviii) SEQ ID NO: 265 (IL-12p40 (N113Q)); lxxix) SEQ ID NO: 266 (IL-12p40 (N200Q)); lxxx) SEQ ID NO: 267 (IL-12p40 (N281Q)); lxxxi) SEQ ID NO: 268 (IL-12p40 (N103Q / N113Q)); lxxxii) SEQ ID NO: 269 (IL-12p40 (N103Q / N200Q)); lxxxiii) SEQ ID NO: 270 (IL-12p40 (N103Q / N281Q)); lxxxiv) SEQ ID NO: 271 (IL-12p40 (N113Q / N200Q)); lxxxv) SEQ ID NO: 272 (IL-12p40 (N113Q / N281Q)); lxxxvi) SEQ ID NO: 273 (IL-12p40 (N200Q / N281Q)); lxxxvii) SEQ ID NO: 274 (IL-12p40 (N103Q / N113Q / N200Q)); lxxxviii) SEQ ID NO: 275 (IL-12p40 (N103Q / N113Q / N281Q)); lxxxix) SEQ ID NO: 276 (IL-12p40 (N103Q / N200Q / N281Q)); xc) SEQ ID NO: 277 (IL-12p40 (N113Q / N200Q / N281Q)); xci) SEQ ID NO: 278 (IL-12p40 (N103Q / N113Q / N200Q / N281Q)); xcii) SEQ ID NO: 327 (IL-12p40 (D34N / E59K / K99E)); xciii) SEQ ID NO: 328 (IL-12p40 (D34K / E59K / K99E)); xciv) SEQ ID NO: 329 (IL-12p40 (E32Q / D34N / E59K / K99E)); xcv) SEQ ID NO: 331 (IL-12p40 (E32K / D34N / E59K / K99E)); xcvi) SEQ ID NO: 337 (IL-12p40 (E59Y / K99Y)); xcvi i) SEQ ID NO: 366 (IL-12p40 (E59K / K99E / N103Q / C252S / K264E));xcviii) SEQ ID NO: 367 (IL-12p40 (E59K / K99E / N113Q / C252S / K264E)); xcix) SEQ ID NO: 368 (IL-12p40 (E59K / K99E / N200Q / C252S / K264E)); c) SEQ ID NO: 369 (IL-12p40 (E59K / K99E / N281Q / C252S / K264E)); ci) SEQ ID NO: 370 (IL-12p40 (E59K / K99E / N103Q / N113Q / C252S / K264E)); cii) SEQ ID NO: 371 (IL-12p40 (E59K / K99E / N103Q / N200Q / C252S / K264E)); ciii) SEQ ID NO: 372 (IL-12p40 (E59K / K99E / N103Q / N281Q / C252S / K264E)); civ) SEQ ID NO: 373 (IL-12p40 (E59K / K99E / N113Q / N200Q / C252S / K264E)); cv) SEQ ID NO: 374 (IL-12p40 (E59K / K99E / N113Q / N281Q / C252S / K264E)); cvi) SEQ ID NO: 375 (IL-12p40 (E59K / K99E / N200Q / N281Q / C252S / K264E)); cvii) SEQ ID NO: 376 (IL-12p40 (E59K / K99E / N103Q / N113Q / N200Q / C252S / K264E)); cviii) SEQ ID NO: 377 (IL-12p40 (E59K / K99E / N103Q / N200Q / N281Q / C252S / K264E)); and cx) SEQ ID NO: 378 (IL-12p40 (E59K / K99E / N113Q / N200Q / N281Q / C252S / K264E)).

[0199] In some embodiments, the present application provides a heterodimeric protein, wherein the IL-12 p35 subunit has a sequence selected from the group consisting of: i) SEQ ID NO: 113 (IL-12 p35 (N71D)); ii) SEQ ID NO: 114 (IL-12 p35 (N85D)); iii) SEQ ID NO: 115 (IL-12 p35 (N195D)); iv) SEQ ID NO: 116 (IL-12 p35 (N71D / N85D / N195D)); v) SEQ ID NO: 117 (IL-12 p35 (E153Q)); vi) SEQ ID NO: 118 (IL-12 p35 (E38Q)); vii) SEQ ID NO: 119 (IL-12 p35 (N151D)); viii) SEQ ID NO: 120 (IL-12 p35 (Q135E)); ix) SEQ ID NO: 121 (IL-12 p35 (Q35D)); x) SEQ ID NO: 122 (IL-12 p35 (Q146E)); xi) SEQ ID NO: 123 (IL-12 p35 (N76D)); xii) SEQ ID NO: 124 (IL-12 p35 (E162Q)); xiii) SEQ ID NO: 125 (IL-12 p35 (E163Q)); xiv) IL-12 p35 (N21D); xv) SEQ ID NO: 333 (IL-12 p35 (D55Q)); xvi) IL-12 p35 (E79Q); xvii) IL-12 p35 (Q130E); xviii) IL-12 p35 (N136D); xix) IL-12 p35 (E143Q); xx) SEQ ID NO: 227 (IL-12 p35 (N151K)); xxi) SEQ ID NO: 226 (IL-12 p35 (E153K)); xxii) (IL-12 p35 (K158E)); xxiii) IL-12 p35 (D165N); xxiv) SEQ ID NO: 225 (IL-12 p35 (N151D / E153Q)); xxv) SEQ ID NO: 228 (IL-12 p35 (N151D / D165N)); xxvi) SEQ ID NO: 229 (IL-12 p35 (Q130E / N151D)); xxvii) SEQ ID NO: 230 (IL-12 p35 (N151D / K158E)); xxviii) SEQ ID NO: 231 (IL-12 p35 (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: 345 (IL-12p35(F96A)); xxxiv) SEQ ID NO: 346 (IL-12p35(M97A)); xxxv) SEQ ID NO: 347 (IL-12p35(L89A)); xxxvi) SEQ ID NO: 348 (IL-12p35(L124A)); xxxvii) SEQ ID NO: 349 (IL-12p35(M125A)); xxxviii) SEQ ID NO: 350 (IL-12p35(L75A)); xxxiv) SEQ ID NO: 351 (IL-12p35(I171A)); xxxv) SEQ ID NO: 279 (IL-12p35(N71Q)); xxxvi) SEQ ID NO: 280 (IL-12p35(N85Q)); xxxvii) SEQ ID NO: 281 (IL-12p35(N195Q)); xxxviii) SEQ ID NO: 282 (IL-12p35(N71Q / N85Q)); xxxix) SEQ ID NO: 283 (IL-12p35(N71Q / N195Q)); lx) SEQ ID NO: 284 (IL-12p35(N85Q / N195Q), lxi) SEQ ID NO: 285 (IL-12p35(N71Q / N85Q / N195Q)); lxii) SEQ ID NO: 286 (IL-12p35(N71D / N85D)); lxiii) SEQ ID NO: 287 (IL-12p35(N71D / N195D), lxiv) SEQ ID NO: 288 (IL-12p35(N85D / N195D)); lxv) SEQ ID NO: 333 (IL-12p35(D55Q)) and lxvi) SEQ ID NO: 334 (IL-12p35(D55K)).

[0200] In another aspect, the application includes a heterodimeric protein comprising: a) a first Fc domain; and b) a fusion protein comprising: i) a second Fc domain; ii) an IL-12 p40 subunit domain; and iii) an IL-12 p35 subunit domain; wherein the first Fc domain and the second Fc domain 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; and T366S / L368A / Y407V / Y349C and T366W / S354C, according to EU numbering.

[0201] In some embodiments, the application provides a heterodimeric protein, wherein the fusion protein comprises, from N-terminal to C-terminal: IL-12 p40 subunit domain-domain linker-IL-12 p35 subunit domain-domain linker-hinge-CH2-CH3.

[0202] In some embodiments, the application provides a heterodimeric protein, wherein the fusion protein comprises, from N-terminal to C-terminal: IL-12 p35 subunit domain-domain linker-IL-12 p40 subunit domain-domain linker-hinge-CH2-CH3.

[0203] In some embodiments, the application provides a heterodimeric protein, wherein the fusion protein comprises, from N-terminal to C-terminal: hinge-CH2-CH3-domain linker-IL-12 p35 subunit domain-domain linker-IL-12 p40 subunit domain.

[0204] In some embodiments, the application provides a heterodimeric protein, wherein the fusion protein comprises, from N-terminal to C-terminal: hinge-CH2-CH3-domain linker-IL-12 p40 subunit domain-domain linker-IL-12 p35 subunit domain.

[0205] In some embodiments, the present application provides a heterodimeric protein, wherein the IL-12p40 subunit has a sequence 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)); 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 (S43K); xxxiii) SEQ ID NO: 379 (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: 326 (IL-12p40 (D34N / E59K)); 1) SEQ ID NO: 325 (IL-12p40 (E59K / K99E)); li) SEQ ID NO: 339 (IL-12p40 (D18K / E59K / K99E)); lii) SEQ ID NO: 343 (IL-12p40 (E59K / K99E / K264E)); liii) SEQ ID NO: 336 (IL-12p40 (E59K / K99Y)); liv) SEQ ID NO: 335 (IL-12p40 (E59Y / K99E));lv) SEQ ID NO: 338 (IL-12p40 (E45K / E59K / K99E)); lvi) SEQ ID NO: 340 (IL-12p40 (E59K / K99E / Q144E)); lvii) SEQ ID NO: 341 (IL-12p40 (E59K / K99E / Q144K)); lviii) SEQ ID NO: 342 (IL-12p40 (E59K / K99E / R159E)); Ix) SEQ ID NO: 343 (IL-12p40 (E59K / K99E / K264E)); lx) SEQ ID NO: 344 (IL-12p40 (D18K / E59K / K99E / K264E)); lxi) SEQ ID NO: 360 (IL-12p40 (C252S)); lxii) SEQ ID NO: 361 (IL-12p40 (D18K / E59K / K99E / C252S)); lxiii) SEQ ID NO: 362 (IL-12p40 (D18K / E59K / K99E / C252S / K264E)); lxiv) SEQ ID NO: 363 (IL-12p40 (E59K / K99Y / C252S)); lxv) SEQ ID NO: 364 (IL-12p40 (E59K / K99E / C252S / K264E)); lxvi) SEQ ID NO: 365 (IL-12p40 (E59K / K99E / C252S)); lxvii) SEQ ID NO: 254 (IL-12p40 (N103D / N113D)); lxviii) SEQ ID NO: 255 (IL-12p40 (N103D / N200D)); lxix) SEQ ID NO: 256 (IL-12p40 (N103D / N281D)); lxx) SEQ ID NO: 257 (IL-12p40 (N113D / N200D)); lxxi) SEQ ID NO: 258 (IL-12p40 (N113D / N281D)); lxxii) SEQ ID NO: 259 (IL-12p40 (N200D / N281D)); lxxiii) SEQ ID NO: 260 (IL-12p40 (N103D / N113D / N200D)); lxxiv) SEQ ID NO: 261 (IL-12p40 (N103D / N113D / N281D)); lxxv) SEQ ID NO: 262 (IL-12p40 (N103D / N200D / N281D));lxxvi) SEQ ID NO: 263 (IL-12p40 (N113D / N200D / N281D)); lxxvii) SEQ ID NO: 264 (IL-12p40 (N103Q)); lxxviii) SEQ ID NO: 265 (IL-12p40 (N113Q)); lxxix) SEQ ID NO: 266 (IL-12p40 (N200Q)); lxxx) SEQ ID NO: 267 (IL-12p40 (N281Q)); lxxxi) SEQ ID NO: 268 (IL-12p40 (N103Q / N113Q)); lxxxii) SEQ ID NO: 269 (IL-12p40 (N103Q / N200Q)); lxxxiii) SEQ ID NO: 270 (IL-12p40 (N103Q / N281Q)); lxxxiv) SEQ ID NO: 271 (IL-12p40 (N113Q / N200Q)); lxxxv) SEQ ID NO: 272 (IL-12p40 (N113Q / N281Q)); lxxxvi) SEQ ID NO: 273 (IL-12p40 (N200Q / N281Q)); lxxxvii) SEQ ID NO: 274 (IL-12p40 (N103Q / N113Q / N200Q)); lxxxviii) SEQ ID NO: 275 (IL-12p40 (N103Q / N113Q / N281Q)); lxxxix) SEQ ID NO: 276 (IL-12p40 (N103Q / N200Q / N281Q)); xc) SEQ ID NO: 277 (IL-12p40 (N113Q / N200Q / N281Q)); xci) SEQ ID NO: 278 (IL-12p40 (N103Q / N113Q / N200Q / N281Q)); xcii) SEQ ID NO: 327 (IL-12p40 (D34N / E59K / K99E)); xciii) SEQ ID NO: 328 (IL-12p40 (D34K / E59K / K99E)); xciv) SEQ ID NO: 329 (IL-12p40 (E32Q / D34N / E59K / K99E)); xcv) SEQ ID NO: 331 (IL-12p40 (E32K / D34N / E59K / K99E)); xcvi) SEQ ID NO: 337 (IL-12p40 (E59Y / K99Y)); xcvi i) SEQ ID NO: 366 (IL-12p40 (E59K / K99E / N103Q / C252S / K264E));xcviii) SEQ ID NO: 367 (IL-12p40 (E59K / K99E / N113Q / C252S / K264E)); xcix) SEQ ID NO: 368 (IL-12p40 (E59K / K99E / N200Q / C252S / K264E)); c) SEQ ID NO: 369 (IL-12p40 (E59K / K99E / N281Q / C252S / K264E)); ci) SEQ ID NO: 370 (IL-12p40 (E59K / K99E / N103Q / N113Q / C252S / K264E)); cii) SEQ ID NO: 371 (IL-12p40 (E59K / K99E / N103Q / N200Q / C252S / K264E)); ciii) SEQ ID NO: 372 (IL-12p40 (E59K / K99E / N103Q / N281Q / C252S / K264E)); civ) SEQ ID NO: 373 (IL-12p40 (E59K / K99E / N113Q / N200Q / C252S / K264E)); cv) SEQ ID NO: 374 (IL-12p40 (E59K / K99E / N113Q / N281Q / C252S / K264E)); cvi) SEQ ID NO: 375 (IL-12p40 (E59K / K99E / N200Q / N281Q / C252S / K264E)); cvii) SEQ ID NO: 376 (IL-12p40 (E59K / K99E / N103Q / N113Q / N200Q / C252S / K264E)); cviii) SEQ ID NO: 377 (IL-12p40 (E59K / K99E / N103Q / N200Q / N281Q / C252S / K264E)); and cx) SEQ ID NO: 378 (IL-12p40 (E59K / K99E / N113Q / N200Q / N281Q / C252S / K264E)).

[0206] In some embodiments, the present application provides a heterodimeric protein, wherein the IL-12 p35 subunit has a sequence selected from the group consisting of: i) SEQ ID NO: 113 (IL-12 p35 (N71D)); ii) SEQ ID NO: 114 (IL-12 p35 (N85D)); iii) SEQ ID NO: 115 (IL-12 p35 (N195D)); iv) SEQ ID NO: 116 (IL-12 p35 (N71D / N85D / N195D)); v) SEQ ID NO: 117 (IL-12 p35 (E153Q)); vi) SEQ ID NO: 118 (IL-12 p35 (E38Q)); vii) SEQ ID NO: 119 (IL-12 p35 (N151D)); viii) SEQ ID NO: 120 (IL-12 p35 (Q135E)); ix) SEQ ID NO: 121 (IL-12 p35 (Q35D)); x) SEQ ID NO: 122 (IL-12 p35 (Q146E)); xi) SEQ ID NO: 123 (IL-12 p35 (N76D)); xii) SEQ ID NO: 124 (IL-12 p35 (E162Q)); xiii) SEQ ID NO: 125 (IL-12 p35 (E163Q)); xiv) IL-12 p35 (N21D); xv) SEQ ID NO: 333 (IL-12 p35 (D55Q)); xvi) IL-12 p35 (E79Q); xvii) IL-12 p35 (Q130E); xviii) IL-12 p35 (N136D); xix) IL-12 p35 (E143Q); xx) SEQ ID NO: 227 (IL-12 p35 (N151K)); xxi) SEQ ID NO: 226 (IL-12 p35 (E153K)); xxii) IL-12 p35 (K158E); xxiii) IL-12 p35 (D165N); xxiv) SEQ ID NO: 225 (IL-12 p35 (N151D / E153Q)); xxv) SEQ ID NO: 228 (IL-12 p35 (N151D / D165N)); xxvi) SEQ ID NO: 229 (IL-12 p35 (Q130E / N151D)); xxvii) SEQ ID NO: 230 (IL-12 p35 (N151D / K158E)); xxviii) SEQ ID NO: 231 (IL-12 p35 (E79Q / N151D)); xxix) SEQ ID NO: 232 (IL-12 p35 (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: 345 (IL-12p35(F96A)); xxxiv) SEQ ID NO: 346 (IL-12p35(M97A)); xxxv) SEQ ID NO: 347 (IL-12p35(L89A)); xxxvi) SEQ ID NO: 348 (IL-12p35(L124A)); xxxvii) SEQ ID NO: 349 (IL-12p35(M125A)); xxxviii) SEQ ID NO: 350 (IL-12p35(L75A)); xxxiv) SEQ ID NO: 351 (IL-12p35(I171A)); xxxv) SEQ ID NO: 279 (IL-12p35(N71Q)); xxxvi) SEQ ID NO: 280 (IL-12p35(N85Q)); xxxvii) SEQ ID NO: 281 (IL-12p35(N195Q)); xxxviii) SEQ ID NO: 282 (IL-12p35(N71Q / N85Q)); xxxix) SEQ ID NO: 283 (IL-12p35(N71Q / N195Q)); lx) SEQ ID NO: 284 (IL-12p35(N85Q / N195Q), lxi) SEQ ID NO: 285 (IL-12p35(N71Q / N85Q / N195Q)); lxii) SEQ ID NO: 286 (IL-12p35(N71D / N85D)); lxiii) SEQ ID NO: 287 (IL-12p35(N71D / N195D), lxiv) SEQ ID NO: 288 (IL-12p35(N85D / N195D)); lxv) SEQ ID NO: 333 (IL-12p35(D55Q)) and lxvi) SEQ ID NO: 334 (IL-12p35(D55K)).

[0207] In another aspect, the present application provides a homodimeric Fc fusion protein comprising a first monomer and a second monomer, each comprising, from N- to C-terminus, an IL-12 p40 subunit domain - an optional first domain linker - an IL-12 p35 subunit domain - an optional second domain linker - an Fc domain.

[0208] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the modification that promotes homodimerization of the Fc domain is 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.

[0209] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the first domain linker and the second domain linker have the same amino acid sequence.

[0210] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the Fc domain has a further set of amino acid substitutions comprising Q295E / N384D / Q418E / N421D, according to EU numbering.

[0211] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the Fc domain has a further 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.

[0212] In some embodiments, the present application provides a homodimeric Fc fusion protein, 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).

[0213] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein each of the Fc domains further comprises the amino acid substitutions M428L / N424S.

[0214] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the IL-12p40 subunit is a variant IL-12p40 subunit and / or the IL-12p35 subunit is a variant IL-12p35 subunit.

[0215] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the IL-12p40 subunit is a variant IL-12p40 subunit having altered affinity for IL-12 receptor subunit beta-1 (IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2), and / or IL-12 receptor complex; and / or the IL-12p35 subunit is a variant IL-12p35 subunit having altered affinity for IL-12 receptor subunit beta-1 (IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2), and / or IL-12 receptor complex.

[0216] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the IL-12p40 subunit has one or more amino acid modifications at an amino acid residue 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, D209, 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.

[0217] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the IL-12p40 subunit has one or more amino acid substitutions selected from the group consisting of D18N, D18K, E32Q, E33Q, D34N, D34K, Q42E, S43E, S43K, E45Q, Q56E, E59Q, E59K, D62N, E73Q, D87N, K99E, K99Y, E100Q, N103D, N103Q, N113D, N113Q, Q144E, D161N, R159E, K163E, E187Q, N200D, N200Q, N218Q, Q229E, E235Q, C252S, Q256N, K258E, K260E, E262Q, K264E, N281D, N281Q, and E299Q.

[0218] In some embodiments, the present application provides a homodimeric Fc fusion protein wherein the IL-12p40 subunit has an amino acid substitution selected from the group consisting 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, 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, 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 / C252S / K264E, E59K / K99E / N113Q / C252S / K264E, E59K / K99E / N200Q / 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 / 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.

[0219] In some embodiments, the present application provides a homodimeric Fc fusion protein wherein the IL-12p40 subunit has a polypeptide sequence 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)); 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 (S43K); xxxiii) SEQ ID NO: 379 (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: 326 (IL-12p40 (D34N / E59K)); 1) SEQ ID NO: 325 (IL-12p40 (E59K / K99E)); li) SEQ ID NO: 339 (IL-12p40 (D18K / E59K / K99E)); lii) SEQ ID NO: 343 (IL-12p40 (E59K / K99E / K264E)); liii) SEQ ID NO: 336 (IL-12p40 (E59K / K99Y)); liv) SEQ ID NO: 335 (IL-12p40 (E59Y / K99E));lv) SEQ ID NO: 338 (IL-12p40 (E45K / E59K / K99E)); lvi) SEQ ID NO: 340 (IL-12p40 (E59K / K99E / Q144E)); lvii) SEQ ID NO: 341 (IL-12p40 (E59K / K99E / Q144K)); lviii) SEQ ID NO: 342 (IL-12p40 (E59K / K99E / R159E)); Ix) SEQ ID NO: 343 (IL-12p40 (E59K / K99E / K264E)); lx) SEQ ID NO: 344 (IL-12p40 (D18K / E59K / K99E / K264E)); lxi) SEQ ID NO: 360 (IL-12p40 (C252S)); lxii) SEQ ID NO: 361 (IL-12p40 (D18K / E59K / K99E / C252S)); lxiii) SEQ ID NO: 362 (IL-12p40 (D18K / E59K / K99E / C252S / K264E)); lxiv) SEQ ID NO: 363 (IL-12p40 (E59K / K99Y / C252S)); lxv) SEQ ID NO: 364 (IL-12p40 (E59K / K99E / C252S / K264E)); lxvi) SEQ ID NO: 365 (IL-12p40 (E59K / K99E / C252S)); lxvii) SEQ ID NO: 254 (IL-12p40 (N103D / N113D)); lxviii) SEQ ID NO: 255 (IL-12p40 (N103D / N200D)); lxix) SEQ ID NO: 256 (IL-12p40 (N103D / N281D)); lxx) SEQ ID NO: 257 (IL-12p40 (N113D / N200D)); lxxi) SEQ ID NO: 258 (IL-12p40 (N113D / N281D)); lxxii) SEQ ID NO: 259 (IL-12p40 (N200D / N281D)); lxxiii) SEQ ID NO: 260 (IL-12p40 (N103D / N113D / N200D)); lxxiv) SEQ ID NO: 261 (IL-12p40 (N103D / N113D / N281D)); lxxv) SEQ ID NO: 262 (IL-12p40 (N103D / N200D / N281D));lxxvi) SEQ ID NO: 263 (IL-12p40 (N113D / N200D / N281D)); lxxvii) SEQ ID NO: 264 (IL-12p40 (N103Q)); lxxviii) SEQ ID NO: 265 (IL-12p40 (N113Q)); lxxix) SEQ ID NO: 266 (IL-12p40 (N200Q)); lxxx) SEQ ID NO: 267 (IL-12p40 (N281Q)); lxxxi) SEQ ID NO: 268 (IL-12p40 (N103Q / N113Q)); lxxxii) SEQ ID NO: 269 (IL-12p40 (N103Q / N200Q)); lxxxiii) SEQ ID NO: 270 (IL-12p40 (N103Q / N281Q)); lxxxiv) SEQ ID NO: 271 (IL-12p40 (N113Q / N200Q)); lxxxv) SEQ ID NO: 272 (IL-12p40 (N113Q / N281Q)); lxxxvi) SEQ ID NO: 273 (IL-12p40 (N200Q / N281Q)); lxxxvii) SEQ ID NO: 274 (IL-12p40 (N103Q / N113Q / N200Q)); lxxxviii) SEQ ID NO: 275 (IL-12p40 (N103Q / N113Q / N281Q)); lxxxix) SEQ ID NO: 276 (IL-12p40 (N103Q / N200Q / N281Q)); xc) SEQ ID NO: 277 (IL-12p40 (N113Q / N200Q / N281Q)); xci) SEQ ID NO: 278 (IL-12p40 (N103Q / N113Q / N200Q / N281Q)); xcii) SEQ ID NO: 327 (IL-12p40 (D34N / E59K / K99E)); xciii) SEQ ID NO: 328 (IL-12p40 (D34K / E59K / K99E)); xciv) SEQ ID NO: 329 (IL-12p40 (E32Q / D34N / E59K / K99E)); xcv) SEQ ID NO: 331 (IL-12p40 (E32K / D34N / E59K / K99E)); xcvi) SEQ ID NO: 337 (IL-12p40 (E59Y / K99Y));xcvii) SEQ ID NO: 366 (IL-12p40 (E59K / K99E / N103Q / C252S / K264E)); xcviii) SEQ ID NO: 367 (IL-12p40 (E59K / K99E / N113Q / C252S / K264E)); xcix) SEQ ID NO: 368 (IL-12p40 (E59K / K99E / N200Q / C252S / K264E)); c) SEQ ID NO: 369 (IL-12p40 (E59K / K99E / N281Q / C252S / K264E)); ci) SEQ ID NO: 370 (IL-12p40 (E59K / K99E / N103Q / N113Q / C252S / K264E)); cii) SEQ ID NO: 371 (IL-12p40 (E59K / K99E / N103Q / N200Q / C252S / K264E)); ciii) SEQ ID NO: 372 (IL-12p40 (E59K / K99E / N103Q / N281Q / C252S / K264E)); civ) SEQ ID NO: 373 (IL-12p40 (E59K / K99E / N113Q / N200Q / C252S / K264E)); cv) SEQ ID NO: 374 (IL-12p40 (E59K / K99E / N113Q / N281Q / C252S / K264E)); cvi) SEQ ID NO: 375 (IL-12p40 (E59K / K99E / N200Q / N281Q / C252S / K264E)); cvii) SEQ ID NO: 376 (IL-12p40 (E59K / K99E / N103Q / N113Q / N200Q / C252S / K264E)); cviii) SEQ ID NO: 377 (IL-12p40 (E59K / K99E / N103Q / N200Q / N281Q / C252S / K264E)); and cx) SEQ ID NO: 378 (IL-12p40 (E59K / K99E / N113Q / N200Q / N281Q / C252S / K264E)).

[0220] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the IL-12 p35 subunit has one or more amino acid modifications 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, Q135, N136, E143, Q146, N151, E153, K158, E162, E163, D165, I171, R181, I182, R183, V185, T186, D188, R189, V190, S192, Y193, N195, and A196 as amino acid residues.

[0221] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the IL-12 p35 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.

[0222] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the IL-12 p35 subunit has one or more 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.

[0223] In some embodiments, the present application provides a homodimeric Fc fusion protein wherein the IL-12 p35 subunit has a polypeptide sequence 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: 333 (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: 345 (IL-12p35(F96A)); xxxiv) SEQ ID NO: 346 (IL-12p35(M97A)); xxxv) SEQ ID NO: 347 (IL-12p35(L89A)); xxxvi) SEQ ID NO: 348 (IL-12p35(L124A)); xxxvii) SEQ ID NO: 349 (IL-12p35(M125A)); xxxviii) SEQ ID NO: 350 (IL-12p35(L75A)); xxxiv) SEQ ID NO: 351 (IL-12p35(I171A)); xxxv) SEQ ID NO: 279 (IL-12p35(N71Q)); xxxvi) SEQ ID NO: 280 (IL-12p35(N85Q)); xxxvii) SEQ ID NO: 281 (IL-12p35(N195Q)); xxxviii) SEQ ID NO: 282 (IL-12p35(N71Q / N85Q)); xxxix) SEQ ID NO: 283 (IL-12p35(N71Q / N195Q)); xl) SEQ ID NO: 284 (IL-12p35(N85Q / N195Q); xli) SEQ ID NO: 285 (IL-12p35(N71Q / N85Q / N195Q)); xlii) SEQ ID NO: 286 (IL-12p35(N71D / N85D)); xliii) SEQ ID NO: 287 (IL-12p35(N71D / N195D); xliv) SEQ ID NO: 288 (IL-12p35(N85D / N195D)); xlv) SEQ ID NO: 333 (IL-12p35(D55Q)) and xlvi) SEQ ID NO: 334 (IL-12p35(D55K)).

[0224] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the first monomer and the second monomer are XENP31289.

[0225] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the first monomer and the second monomer are XENP31291.

[0226] In some embodiments, the present application provides a composition of homodimeric Fc fusion proteins for use in treating cancer in a subject.

[0227] In some embodiments, the present application provides one or more nucleic acids encoding a homodimeric Fc fusion protein.

[0228] In some embodiments, the present application provides a host cell comprising the one or more nucleic acids encoding a homodimeric Fc fusion protein.

[0229] In some embodiments, the present application provides a method of making a homodimeric Fc fusion protein, the method comprising culturing a host cell under conditions wherein the homodimeric Fc fusion protein is produced.

[0230] In some embodiments, the present application provides a method of 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 a fraction containing the homodimeric Fc fusion protein.

[0231] In another aspect, the present application provides a homodimeric Fc fusion protein comprising a first monomer and a second monomer, each comprising, from N- to C-terminus, an IL-12 p35 subunit domain - an optional first domain linker - an IL-12 p40 subunit domain - an optional second domain linker - an Fc domain.

[0232] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the modification that promotes homodimerization of the Fc domain is 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.

[0233] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the first domain linker and the second domain linker have the same amino acid sequence.

[0234] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the Fc domain has an additional set of amino acid substitutions comprising Q295E / N384D / Q418E / N421D, according to EU numbering.

[0235] In some embodiments, the present application provides a homodimeric Fc fusion protein, 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.

[0236] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the variant IL-12 p40 subunit has a polypeptide sequence selected from the group consisting of SEQ ID NO: 3 (human IL-12 subunit beta (IL-12 p40) precursor sequence) and SEQ ID NO: 4 (human IL-12 subunit beta (IL-12 p40) mature form sequence), and the IL-12 p35 subunit has a polypeptide sequence selected from the group consisting of SEQ ID NO: 1 (human IL-12 subunit alpha (IL-12 p35) precursor sequence) and SEQ ID NO: 2 (human IL-12 subunit alpha (IL-12 p35) mature form sequence).

[0237] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein each of the Fc domains further comprises the amino acid substitutions M428L / N424S.

[0238] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the IL-12 p40 subunit is a variant IL-12 p40 subunit and / or the IL-12 p35 subunit is a variant IL-12 p35 subunit.

[0239] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the IL-12p40 subunit is a variant IL-12p40 subunit having altered affinity for IL-12 receptor subunit beta-1 (IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2), and / or IL-12 receptor complex; and / or the IL-12p35 subunit is a variant IL-12p35 subunit having altered affinity for IL-12 receptor subunit beta-1 (IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2), and / or IL-12 receptor complex.

[0240] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the IL-12p40 subunit has one or more amino acid modifications at an amino acid residue 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, D209, 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.

[0241] In some embodiments, the present application provides a homodimeric Fc fusion protein wherein the IL-12p40 subunit has one or more amino acid substitutions selected from the group consisting of D18N, D18K, E32Q, E33Q, D34N, D34K, Q42E, S43E, S43K, E45Q, Q56E, E59Q, E59K, D62N, E73Q, D87N, K99E, K99Y, E100Q, N103D, N103Q, N113D, N113Q, Q144E, D161N, R159E, K163E, E187Q, N200D, N200Q, N218Q, Q229E, E235Q, C252S, Q256N, K258E, K260E, E262Q, K264E, N281D, N281Q, and E299Q.

[0242] In some embodiments, the present application provides a homodimeric Fc fusion protein wherein the IL-12p40 subunit has an amino acid substitution selected from the group consisting 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, 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, 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 / 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, E59 K / K99E / N103Q / N281Q / C252S / K264E, E59K / K99E / N113Q / N200Q / C252S / K264E, E59K / K99E / N113Q / N281Q / C252S / K264E, E 59K / K99E / N200Q / 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.

[0243] In some embodiments, the present invention provides a homodimeric Fc fusion protein, wherein the IL-12p40 subunit has a polypeptide sequence selected from the group consisting of:

[0244] 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 (S43K); xxxiii) SEQ ID NO: 379 (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: 326 (IL-12p40 (D34N / E59K)); I) SEQ ID NO: 325 (IL-12p40 (E59K / K99E)); li) SEQ ID NO: 339 (IL-12p40 (D18K / E59K / K99E)); lii) SEQ ID NO: 343 (IL-12p40 (E59K / K99E / K264E)); liii) SEQ ID NO: 336 (IL-12p40 (E59K / K99Y)); liv) SEQ ID NO: 335 (IL-12p40 (E59Y / K99E)); lv) SEQ ID NO: 338 (IL-12p40 (E45K / E59K / K99E)); lvi) SEQ ID NO: 340 (IL-12p40 (E59K / K99E / Q144E));lxvii) SEQ ID NO: 341 (IL-12p40 (E59K / K99E / Q144K)); lviii) SEQ ID NO: 342 (IL-12p40 (E59K / K99E / R159E)); Ix) SEQ ID NO: 343 (IL-12p40 (E59K / K99E / K264E)); lx) SEQ ID NO: 344 (IL-12p40 (D18K / E59K / K99E / K264E)); lxi) SEQ ID NO: 360 (IL-12p40 (C252S)); lxii) SEQ ID NO: 361 (IL-12p40 (D18K / E59K / K99E / C252S)); lxiii) SEQ ID NO: 362 (IL-12p40 (D18K / E59K / K99E / C252S / K264E)); lxiv) SEQ ID NO: 363 (IL-12p40 (E59K / K99Y / C252S)); lxv) SEQ ID NO: 364 (IL-12p40 (E59K / K99E / C252S / K264E)); lxvi) SEQ ID NO: 365 (IL-12p40 (E59K / K99E / C252S)); lxvii) SEQ ID NO: 254 (IL-12p40 (N103D / N113D)); lxviii) SEQ ID NO: 255 (IL-12p40 (N103D / N200D)); lxix) SEQ ID NO: 256 (IL-12p40 (N103D / N281D)); lxx) SEQ ID NO: 257 (IL-12p40 (N113D / N200D)); lxxi) SEQ ID NO: 258 (IL-12p40 (N113D / N281D)); lxxii) SEQ ID NO: 259 (IL-12p40 (N200D / N281D)); lxxiii) SEQ ID NO: 260 (IL-12p40 (N103D / N113D / N200D)); lxxiv) SEQ ID NO: 261 (IL-12p40 (N103D / N113D / N281D)); lxxv) SEQ ID NO: 262 (IL-12p40 (N103D / N200D / N281D)); lxxvi) SEQ ID NO: 263 (IL-12p40 (N113D / N200D / N281D)); lxxvii) SEQ ID NO: 264 (IL-12p40 (N103Q)); lxxviii) SEQ ID NO: 265 (IL-12p40 (N113Q));lxxix) SEQ ID NO: 266 (IL-12p40 (N200Q)); lxxx) SEQ ID NO: 267 (IL-12p40 (N281Q)); lxxxi) SEQ ID NO: 268 (IL-12p40 (N103Q / N113Q)); lxxxii) SEQ ID NO: 269 (IL-12p40 (N103Q / N200Q)); lxxxiii) SEQ ID NO: 270 (IL-12p40 (N103Q / N281Q)); lxxxiv) SEQ ID NO: 271 (IL-12p40 (N113Q / N200Q)); lxxxv) SEQ ID NO: 272 (IL-12p40 (N113Q / N281Q)); lxxxvi) SEQ ID NO: 273 (IL-12p40 (N200Q / N281Q)); lxxxvii) SEQ ID NO: 274 (IL-12p40 (N103Q / N113Q / N200Q)); lxxxviii) SEQ ID NO: 275 (IL-12p40 (N103Q / N113Q / N281Q)); lxxxix) SEQ ID NO: 276 (IL-12p40 (N103Q / N200Q / N281Q)); xc) SEQ ID NO: 277 (IL-12p40 (N113Q / N200Q / N281Q)); xci) SEQ ID NO: 278 (IL-12p40 (N103Q / N113Q / N200Q / N281Q)); xcii) SEQ ID NO: 327 (IL-12p40 (D34N / E59K / K99E)); xciii) SEQ ID NO: 328 (IL-12p40 (D34K / E59K / K99E)); xciv) SEQ ID NO: 328 (IL-12p40 (E32Q / D34N / E59K / K99E)); xcv) SEQ ID NO: 331 (IL-12p40 (E32K / D34N / E59K / K99E)); xcvi) SEQ ID NO: 337 (IL-12p40 (E59Y / K99Y)); xcvi i) SEQ ID NO: 366 (IL-12p40 (E59K / K99E / N103Q / C252S / K264E)); xcvi i i) SEQ ID NO: 367 (IL-12p40 (E59K / K99E / N113Q / C252S / K264E)); xcix) SEQ ID NO: 368 (IL-12p40 (E59K / K99E / N200Q / C252S / K264E));c) SEQ ID NO: 369 (IL-12p40 (E59K / K99E / N281Q / C252S / K264E)); ci) SEQ ID NO: 370 (IL-12p40 (E59K / K99E / N103Q / N113Q / C252S / K264E)); cii) SEQ ID NO: 371 (IL-12p40 (E59K / K99E / N103Q / N200Q / C252S / K264E)); ciii) SEQ ID NO: 372 (IL-12p40 (E59K / K99E / N103Q / N281Q / C252S / K264E)); civ) SEQ ID NO: 373 (IL-12p40 (E59K / K99E / N113Q / N200Q / C252S / K264E)); cv) SEQ ID NO: 374 (IL-12p40 (E59K / K99E / N113Q / N281Q / C252S / K264E)); cvi) SEQ ID NO: 375 (IL-12p40 (E59K / K99E / N200Q / N281Q / C252S / K264E)); cvii) SEQ ID NO: 376 (IL-12p40 (E59K / K99E / N103Q / N113Q / N200Q / C252S / K264E)); cviii) SEQ ID NO: 377 (IL-12p40 (E59K / K99E / N103Q / N200Q / N281Q / C252S / K264E)); and cix) SEQ ID NO: 378 (IL-12p40 (E59K / K99E / N113Q / N200Q / N281Q / C252S / K264E)).

[0245] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the IL-12 p35 subunit has one or more amino acid modifications as amino acid residues 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, Q135, N136, E143, Q146, N151, E153, K158, E162, E163, D165, I171, R181, I182, R183, V185, T186, D188, R189, V190, S192, Y193, N195, and A196.

[0246] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the IL-12 p35 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.

[0247] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the IL-12 p35 subunit has one or more 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.

[0248] In some embodiments, the present application provides a homodimeric Fc fusion protein wherein the IL-12 p35 subunit has a polypeptide sequence 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: 333 (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: 345 (IL-12p35(F96A)); xxxiv) SEQ ID NO: 346 (IL-12p35(M97A)); xxxv) SEQ ID NO: 347 (IL-12p35(L89A)); xxxvi) SEQ ID NO: 348 (IL-12p35(L124A)); xxxvii) SEQ ID NO: 349 (IL-12p35(M125A)); xxxviii) SEQ ID NO: 350 (IL-12p35(L75A)); xxxiv) SEQ ID NO: 351 (IL-12p35(I171A)); xxxv) SEQ ID NO: 279 (IL-12p35(N71Q)); xxxvi) SEQ ID NO: 280 (IL-12p35(N85Q)); xxxvii) SEQ ID NO: 281 (IL-12p35(N195Q)); xxxviii) SEQ ID NO: 282 (IL-12p35(N71Q / N85Q)); xxxix) SEQ ID NO: 283 (IL-12p35(N71Q / N195Q)); xl) SEQ ID NO: 284 (IL-12p35(N85Q / N195Q); xli) SEQ ID NO: 285 (IL-12p35(N71Q / N85Q / N195Q)); xlii) SEQ ID NO: 286 (IL-12p35(N71D / N85D)); xliii) SEQ ID NO: 287 (IL-12p35(N71D / N195D); xliv) SEQ ID NO: 288 (IL-12p35(N85D / N195D)); xlv) SEQ ID NO: 333 (IL-12p35(D55Q)) and xlvi) SEQ ID NO: 334 (IL-12p35(D55K)).

[0249] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the first monomer and the second monomer are XENP31289.

[0250] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the first monomer and the second monomer are XENP31291.

[0251] In some embodiments, the present application provides a composition of homodimeric Fc fusion proteins for use in treating cancer in a subject.

[0252] In some embodiments, the present application provides one or more nucleic acids encoding a homodimeric Fc fusion protein.

[0253] In some embodiments, the present application provides a host cell comprising the one or more nucleic acids encoding a homodimeric Fc fusion protein.

[0254] In some embodiments, the present application provides a method of making a homodimeric Fc fusion protein, the method comprising culturing a host cell under conditions wherein the homodimeric Fc fusion protein is produced.

[0255] In some embodiments, the present application provides a method of 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 a fraction containing the homodimeric Fc fusion protein.

[0256] In another aspect, the present application provides a homodimeric Fc fusion protein comprising a first monomer and a second monomer, each comprising, from N- to C-terminus, an Fc domain - an optional first domain linker - an IL-12 p40 subunit domain - an optional second domain linker - an IL-12 p35 subunit domain.

[0257] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the modification that promotes homodimerization of the Fc domain is 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.

[0258] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the first domain linker and the second domain linker have the same amino acid sequence.

[0259] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the Fc domain has an additional set of amino acid substitutions comprising Q295E / N384D / Q418E / N421D, according to EU numbering.

[0260] In some embodiments, the present application provides a homodimeric Fc fusion protein, 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.

[0261] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the variant IL-12 p40 subunit has a polypeptide sequence selected from the group consisting of SEQ ID NO: 3 (human IL-12 subunit beta (IL-12 p40) precursor sequence) and SEQ ID NO: 4 (human IL-12 subunit beta (IL-12 p40) mature form sequence), and the IL-12 p35 subunit has a polypeptide sequence selected from the group consisting of SEQ ID NO: 1 (human IL-12 subunit alpha (IL-12 p35) precursor sequence) and SEQ ID NO: 2 (human IL-12 subunit alpha (IL-12 p35) mature form sequence).

[0262] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein each of the Fc domains further comprises the amino acid substitutions M428L / N424S.

[0263] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the IL-12 p40 subunit is a variant IL-12 p40 subunit and / or the IL-12 p35 subunit is a variant IL-12 p35 subunit.

[0264] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the IL-12p40 subunit is a variant IL-12p40 subunit having altered affinity for IL-12 receptor subunit beta-1 (IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2), and / or IL-12 receptor complex; and / or the IL-12p35 subunit is a variant IL-12p35 subunit having altered affinity for IL-12 receptor subunit beta-1 (IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2), and / or IL-12 receptor complex.

[0265] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the IL-12p40 subunit has one or more amino acid modifications at an amino acid residue 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, D209, 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.

[0266] In some embodiments, the present application provides a homodimeric Fc fusion protein wherein the IL-12p40 subunit has one or more amino acid substitutions selected from the group consisting of D18N, D18K, E32Q, E33Q, D34N, D34K, Q42E, S43E, S43K, E45Q, Q56E, E59Q, E59K, D62N, E73Q, D87N, K99E, K99Y, E100Q, N103D, N103Q, N113D, N113Q, Q144E, D161N, R159E, K163E, E187Q, N200D, N200Q, N218Q, Q229E, E235Q, C252S, Q256N, K258E, K260E, E262Q, K264E, N281D, N281Q, and E299Q.

[0267] In some embodiments, the present application provides a homodimeric Fc fusion protein wherein the IL-12p40 subunit has an amino acid substitution selected from the group consisting 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, 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, 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 / C252S / K264E, E59K / K99E / N113Q / C252S / K264E, E59K / K99E / N200Q / 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 / 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.

[0268] In some embodiments, the present application provides a homodimeric Fc fusion protein wherein the IL-12p40 subunit has a polypeptide sequence 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)); 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 (S43K); xxxiii) SEQ ID NO: 379 (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: 326 (IL-12p40 (D34N / E59K)); 1) SEQ ID NO: 325 (IL-12p40 (E59K / K99E)); li) SEQ ID NO: 339 (IL-12p40 (D18K / E59K / K99E)); lii) SEQ ID NO: 343 (IL-12p40 (E59K / K99E / K264E)); liii) SEQ ID NO: 336 (IL-12p40 (E59K / K99Y)); liv) SEQ ID NO: 335 (IL-12p40 (E59Y / K99E));lv) SEQ ID NO: 338 (IL-12p40 (E45K / E59K / K99E)); lvi) SEQ ID NO: 340 (IL-12p40 (E59K / K99E / Q144E)); lvii) SEQ ID NO: 341 (IL-12p40 (E59K / K99E / Q144K)); lviii) SEQ ID NO: 342 (IL-12p40 (E59K / K99E / R159E)); Ix) SEQ ID NO: 343 (IL-12p40 (E59K / K99E / K264E)); lx) SEQ ID NO: 344 (IL-12p40 (D18K / E59K / K99E / K264E)); lxi) SEQ ID NO: 360 (IL-12p40 (C252S)); lxii) SEQ ID NO: 361 (IL-12p40 (D18K / E59K / K99E / C252S)); lxiii) SEQ ID NO: 362 (IL-12p40 (D18K / E59K / K99E / C252S / K264E)); lxiv) SEQ ID NO: 363 (IL-12p40 (E59K / K99Y / C252S)); lxv) SEQ ID NO: 364 (IL-12p40 (E59K / K99E / C252S / K264E)); lxvi) SEQ ID NO: 365 (IL-12p40 (E59K / K99E / C252S)); lxvii) SEQ ID NO: 254 (IL-12p40 (N103D / N113D)); lxviii) SEQ ID NO: 255 (IL-12p40 (N103D / N200D)); lxix) SEQ ID NO: 256 (IL-12p40 (N103D / N281D)); lxx) SEQ ID NO: 257 (IL-12p40 (N113D / N200D)); lxxi) SEQ ID NO: 258 (IL-12p40 (N113D / N281D)); lxxii) SEQ ID NO: 259 (IL-12p40 (N200D / N281D)); lxxiii) SEQ ID NO: 260 (IL-12p40 (N103D / N113D / N200D)); lxxiv) SEQ ID NO: 261 (IL-12p40 (N103D / N113D / N281D)); lxxv) SEQ ID NO: 262 (IL-12p40 (N103D / N200D / N281D));lxxvi) SEQ ID NO: 263 (IL-12p40 (N113D / N200D / N281D)); lxxvii) SEQ ID NO: 264 (IL-12p40 (N103Q)); lxxviii) SEQ ID NO: 265 (IL-12p40 (N113Q)); lxxix) SEQ ID NO: 266 (IL-12p40 (N200Q)); lxxx) SEQ ID NO: 267 (IL-12p40 (N281Q)); lxxxi) SEQ ID NO: 268 (IL-12p40 (N103Q / N113Q)); lxxxii) SEQ ID NO: 269 (IL-12p40 (N103Q / N200Q)); lxxxiii) SEQ ID NO: 270 (IL-12p40 (N103Q / N281Q)); lxxxiv) SEQ ID NO: 271 (IL-12p40 (N113Q / N200Q)); lxxxv) SEQ ID NO: 272 (IL-12p40 (N113Q / N281Q)); lxxxvi) SEQ ID NO: 273 (IL-12p40 (N200Q / N281Q)); lxxxvii) SEQ ID NO: 274 (IL-12p40 (N103Q / N113Q / N200Q)); lxxxviii) SEQ ID NO: 275 (IL-12p40 (N103Q / N113Q / N281Q)); lxxxix) SEQ ID NO: 276 (IL-12p40 (N103Q / N200Q / N281Q)); xc) SEQ ID NO: 277 (IL-12p40 (N113Q / N200Q / N281Q)); xci) SEQ ID NO: 278 (IL-12p40 (N103Q / N113Q / N200Q / N281Q)); xcii) SEQ ID NO: 327 (IL-12p40 (D34N / E59K / K99E)); xciii) SEQ ID NO: 328 (IL-12p40 (D34K / E59K / K99E)); xciv) SEQ ID NO: 329 (IL-12p40 (E32Q / D34N / E59K / K99E)); xcv) SEQ ID NO: 331 (IL-12p40 (E32K / D34N / E59K / K99E)); xcvi) SEQ ID NO: 337 (IL-12p40 (E59Y / K99Y));xcvii) SEQ ID NO: 366 (IL-12p40 (E59K / K99E / N103Q / C252S / K264E)); xcviii) SEQ ID NO: 367 (IL-12p40 (E59K / K99E / N113Q / C252S / K264E)); xcix) SEQ ID NO: 368 (IL-12p40 (E59K / K99E / N200Q / C252S / K264E)); c) SEQ ID NO: 369 (IL-12p40 (E59K / K99E / N281Q / C252S / K264E)); ci) SEQ ID NO: 370 (IL-12p40 (E59K / K99E / N103Q / N113Q / C252S / K264E)); cii) SEQ ID NO: 371 (IL-12p40 (E59K / K99E / N103Q / N200Q / C252S / K264E)); ciii) SEQ ID NO: 372 (IL-12p40 (E59K / K99E / N103Q / N281Q / C252S / K264E)); civ) SEQ ID NO: 373 (IL-12p40 (E59K / K99E / N113Q / N200Q / C252S / K264E)); cv) SEQ ID NO: 374 (IL-12p40 (E59K / K99E / N113Q / N281Q / C252S / K264E)); cvi) SEQ ID NO: 375 (IL-12p40 (E59K / K99E / N200Q / N281Q / C252S / K264E)); cvii) SEQ ID NO: 376 (IL-12p40 (E59K / K99E / N103Q / N113Q / N200Q / C252S / K264E)); cviii) SEQ ID NO: 377 (IL-12p40 (E59K / K99E / N103Q / N200Q / N281Q / C252S / K264E)); and cx) SEQ ID NO: 378 (IL-12p40 (E59K / K99E / N113Q / N200Q / N281Q / C252S / K264E)).

[0269] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the IL-12 p35 subunit has one or more amino acid modifications 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, Q135, N136, E143, Q146, N151, E153, K158, E162, E163, D165, I171, R181, I182, R183, V185, T186, D188, R189, V190, S192, Y193, N195, and A196 as amino acid residues.

[0270] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the IL-12 p35 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.

[0271] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the IL-12 p35 subunit has one or more 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.

[0272] In some embodiments, the present application provides a homodimeric Fc fusion protein wherein the IL-12 p35 subunit has a polypeptide sequence 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: 333 (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: 345 (IL-12p35(F96A)); xxxiv) SEQ ID NO: 346 (IL-12p35(M97A)); xxxv) SEQ ID NO: 347 (IL-12p35(L89A)); xxxvi) SEQ ID NO: 348 (IL-12p35(L124A)); xxxvii) SEQ ID NO: 349 (IL-12p35(M125A)); xxxviii) SEQ ID NO: 350 (IL-12p35(L75A)); xxxiv) SEQ ID NO: 351 (IL-12p35(I171A)); xxxv) SEQ ID NO: 279 (IL-12p35(N71Q)); xxxvi) SEQ ID NO: 280 (IL-12p35(N85Q)); xxxvii) SEQ ID NO: 281 (IL-12p35(N195Q)); xxxviii) SEQ ID NO: 282 (IL-12p35(N71Q / N85Q)); xxxix) SEQ ID NO: 283 (IL-12p35(N71Q / N195Q)); xl) SEQ ID NO: 284 (IL-12p35(N85Q / N195Q); xli) SEQ ID NO: 285 (IL-12p35(N71Q / N85Q / N195Q)); xlii) SEQ ID NO: 286 (IL-12p35(N71D / N85D)); xliii) SEQ ID NO: 287 (IL-12p35(N71D / N195D); xliv) SEQ ID NO: 288 (IL-12p35(N85D / N195D)); xlv) SEQ ID NO: 333 (IL-12p35(D55Q)) and xlvi) SEQ ID NO: 334 (IL-12p35(D55K)).

[0273] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the first monomer and the second monomer are XENP31289.

[0274] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the first monomer and the second monomer are XENP31291.

[0275] In some embodiments, the present application provides a composition of homodimeric Fc fusion proteins for use in treating cancer in a subject.

[0276] In some embodiments, the present application provides one or more nucleic acids encoding a homodimeric Fc fusion protein.

[0277] In some embodiments, the present application provides a host cell comprising the one or more nucleic acids encoding a homodimeric Fc fusion protein.

[0278] In some embodiments, the present application provides a method of making a homodimeric Fc fusion protein, the method comprising culturing a host cell under conditions wherein the homodimeric Fc fusion protein is produced.

[0279] In some embodiments, the present application provides a method of 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 a fraction containing the homodimeric Fc fusion protein.

[0280] In another aspect, the present application provides a homodimeric Fc fusion protein comprising a first monomer and a second monomer, each comprising, from N- to C-terminus, an Fc domain - optional first domain linker - IL-12 p35 subunit domain - optional second domain linker - IL-12 p40 subunit domain.

[0281] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the modification that promotes homodimerization of the Fc domain is 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.

[0282] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the first domain linker and the second domain linker have the same amino acid sequence.

[0283] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the Fc domain has an additional set of amino acid substitutions comprising Q295E / N384D / Q418E / N421D, according to EU numbering.

[0284] In some embodiments, the present application provides a homodimeric Fc fusion protein, 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.

[0285] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the variant IL-12 p40 subunit has a polypeptide sequence selected from the group consisting of SEQ ID NO: 3 (human IL-12 subunit beta (IL-12 p40) precursor sequence) and SEQ ID NO: 4 (human IL-12 subunit beta (IL-12 p40) mature form sequence), and the IL-12 p35 subunit has a polypeptide sequence selected from the group consisting of SEQ ID NO: 1 (human IL-12 subunit alpha (IL-12 p35) precursor sequence) and SEQ ID NO: 2 (human IL-12 subunit alpha (IL-12 p35) mature form sequence).

[0286] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein each of the Fc domains further comprises the amino acid substitutions M428L / N424S.

[0287] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the IL-12 p40 subunit is a variant IL-12 p40 subunit and / or the IL-12 p35 subunit is a variant IL-12 p35 subunit.

[0288] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the IL-12p40 subunit is a variant IL-12p40 subunit having altered affinity for IL-12 receptor subunit beta-1 (IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2), and / or IL-12 receptor complex; and / or the IL-12p35 subunit is a variant IL-12p35 subunit having altered affinity for IL-12 receptor subunit beta-1 (IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2), and / or IL-12 receptor complex.

[0289] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the IL-12p40 subunit has one or more amino acid modifications at an amino acid residue 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, D209, 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.

[0290] In some embodiments, the present application provides a homodimeric Fc fusion protein wherein the IL-12p40 subunit has one or more amino acid substitutions selected from the group consisting of D18N, D18K, E32Q, E33Q, D34N, D34K, Q42E, S43E, S43K, E45Q, Q56E, E59Q, E59K, D62N, E73Q, D87N, K99E, K99Y, E100Q, N103D, N103Q, N113D, N113Q, Q144E, D161N, R159E, K163E, E187Q, N200D, N200Q, N218Q, Q229E, E235Q, C252S, Q256N, K258E, K260E, E262Q, K264E, N281D, N281Q, and E299Q.

[0291] In some embodiments, the present application provides a homodimeric Fc fusion protein wherein the IL-12p40 subunit has an amino acid substitution selected from the group consisting 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, 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, 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 / C252S / K264E, E59K / K99E / N113Q / C252S / K264E, E59K / K99E / N200Q / 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 / 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.

[0292] In some embodiments, the present application provides a homodimeric Fc fusion protein wherein the IL-12p40 subunit has a polypeptide sequence 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)); 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 (S43K); xxxiii) SEQ ID NO: 379 (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: 326 (IL-12p40 (D34N / E59K)); 1) SEQ ID NO: 325 (IL-12p40 (E59K / K99E)); li) SEQ ID NO: 339 (IL-12p40 (D18K / E59K / K99E)); lii) SEQ ID NO: 343 (IL-12p40 (E59K / K99E / K264E)); liii) SEQ ID NO: 336 (IL-12p40 (E59K / K99Y)); liv) SEQ ID NO: 335 (IL-12p40 (E59Y / K99E));lv) SEQ ID NO: 338 (IL-12p40 (E45K / E59K / K99E)); lvi) SEQ ID NO: 340 (IL-12p40 (E59K / K99E / Q144E)); lvii) SEQ ID NO: 341 (IL-12p40 (E59K / K99E / Q144K)); lviii) SEQ ID NO: 342 (IL-12p40 (E59K / K99E / R159E)); Ix) SEQ ID NO: 343 (IL-12p40 (E59K / K99E / K264E)); lx) SEQ ID NO: 344 (IL-12p40 (D18K / E59K / K99E / K264E)); lxi) SEQ ID NO: 360 (IL-12p40 (C252S)); lxii) SEQ ID NO: 361 (IL-12p40 (D18K / E59K / K99E / C252S)); lxiii) SEQ ID NO: 362 (IL-12p40 (D18K / E59K / K99E / C252S / K264E)); lxiv) SEQ ID NO: 363 (IL-12p40 (E59K / K99Y / C252S)); lxv) SEQ ID NO: 364 (IL-12p40 (E59K / K99E / C252S / K264E)); lxvi) SEQ ID NO: 365 (IL-12p40 (E59K / K99E / C252S)); lxvii) SEQ ID NO: 254 (IL-12p40 (N103D / N113D)); lxviii) SEQ ID NO: 255 (IL-12p40 (N103D / N200D)); lxix) SEQ ID NO: 256 (IL-12p40 (N103D / N281D)); lxx) SEQ ID NO: 257 (IL-12p40 (N113D / N200D)); lxxi) SEQ ID NO: 258 (IL-12p40 (N113D / N281D)); lxxii) SEQ ID NO: 259 (IL-12p40 (N200D / N281D)); lxxiii) SEQ ID NO: 260 (IL-12p40 (N103D / N113D / N200D)); lxxiv) SEQ ID NO: 261 (IL-12p40 (N103D / N113D / N281D)); lxxv) SEQ ID NO: 262 (IL-12p40 (N103D / N200D / N281D));lxxvi) SEQ ID NO: 263 (IL-12p40 (N113D / N200D / N281D)); lxxvii) SEQ ID NO: 264 (IL-12p40 (N103Q)); lxxviii) SEQ ID NO: 265 (IL-12p40 (N113Q)); lxxix) SEQ ID NO: 266 (IL-12p40 (N200Q)); lxxx) SEQ ID NO: 267 (IL-12p40 (N281Q)); lxxxi) SEQ ID NO: 268 (IL-12p40 (N103Q / N113Q)); lxxxii) SEQ ID NO: 269 (IL-12p40 (N103Q / N200Q)); lxxxiii) SEQ ID NO: 270 (IL-12p40 (N103Q / N281Q)); lxxxiv) SEQ ID NO: 271 (IL-12p40 (N113Q / N200Q)); lxxxv) SEQ ID NO: 272 (IL-12p40 (N113Q / N281Q)); lxxxvi) SEQ ID NO: 273 (IL-12p40 (N200Q / N281Q)); lxxxvii) SEQ ID NO: 274 (IL-12p40 (N103Q / N113Q / N200Q)); lxxxviii) SEQ ID NO: 275 (IL-12p40 (N103Q / N113Q / N281Q)); lxxxix) SEQ ID NO: 276 (IL-12p40 (N103Q / N200Q / N281Q)); xc) SEQ ID NO: 277 (IL-12p40 (N113Q / N200Q / N281Q)); xci) SEQ ID NO: 278 (IL-12p40 (N103Q / N113Q / N200Q / N281Q)); xcii) SEQ ID NO: 327 (IL-12p40 (D34N / E59K / K99E)); xciii) SEQ ID NO: 328 (IL-12p40 (D34K / E59K / K99E)); xciv) SEQ ID NO: 329 (IL-12p40 (E32Q / D34N / E59K / K99E)); xcv) SEQ ID NO: 331 (IL-12p40 (E32K / D34N / E59K / K99E)); xcvi) SEQ ID NO: 337 (IL-12p40 (E59Y / K99Y));xcvii) SEQ ID NO: 366 (IL-12p40 (E59K / K99E / N103Q / C252S / K264E)); xcviii) SEQ ID NO: 367 (IL-12p40 (E59K / K99E / N113Q / C252S / K264E)); xcix) SEQ ID NO: 368 (IL-12p40 (E59K / K99E / N200Q / C252S / K264E)); c) SEQ ID NO: 369 (IL-12p40 (E59K / K99E / N281Q / C252S / K264E)); ci) SEQ ID NO: 370 (IL-12p40 (E59K / K99E / N103Q / N113Q / C252S / K264E)); cii) SEQ ID NO: 371 (IL-12p40 (E59K / K99E / N103Q / N200Q / C252S / K264E)); ciii) SEQ ID NO: 372 (IL-12p40 (E59K / K99E / N103Q / N281Q / C252S / K264E)); civ) SEQ ID NO: 373 (IL-12p40 (E59K / K99E / N113Q / N200Q / C252S / K264E)); cv) SEQ ID NO: 374 (IL-12p40 (E59K / K99E / N113Q / N281Q / C252S / K264E)); cvi) SEQ ID NO: 375 (IL-12p40 (E59K / K99E / N200Q / N281Q / C252S / K264E)); cvii) SEQ ID NO: 376 (IL-12p40 (E59K / K99E / N103Q / N113Q / N200Q / C252S / K264E)); cviii) SEQ ID NO: 377 (IL-12p40 (E59K / K99E / N103Q / N200Q / N281Q / C252S / K264E)); and cx) SEQ ID NO: 378 (IL-12p40 (E59K / K99E / N113Q / N200Q / N281Q / C252S / K264E)).

[0293] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the IL-12 p35 subunit has one or more amino acid modifications 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, Q135, N136, E143, Q146, N151, E153, K158, E162, E163, D165, I171, R181, I182, R183, V185, T186, D188, R189, V190, S192, Y193, N195, and A196 as amino acid residues.

[0294] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the IL-12 p35 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.

[0295] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the IL-12 p35 subunit has one or more 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.

[0296] In some embodiments, the present application provides a homodimeric Fc fusion protein wherein the IL-12 p35 subunit has a polypeptide sequence 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: 333 (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: 345 (IL-12p35(F96A)); xxxiv) SEQ ID NO: 346 (IL-12p35(M97A)); xxxv) SEQ ID NO: 347 (IL-12p35(L89A)); xxxvi) SEQ ID NO: 348 (IL-12p35(L124A)); xxxvii) SEQ ID NO: 349 (IL-12p35(M125A)); xxxviii) SEQ ID NO: 350 (IL-12p35(L75A)); xxxiv) SEQ ID NO: 351 (IL-12p35(I171A)); xxxv) SEQ ID NO: 279 (IL-12p35(N71Q)); xxxvi) SEQ ID NO: 280 (IL-12p35(N85Q)); xxxvii) SEQ ID NO: 281 (IL-12p35(N195Q)); xxxviii) SEQ ID NO: 282 (IL-12p35(N71Q / N85Q)); xxxix) SEQ ID NO: 283 (IL-12p35(N71Q / N195Q)); xl) SEQ ID NO: 284 (IL-12p35(N85Q / N195Q); xli) SEQ ID NO: 285 (IL-12p35(N71Q / N85Q / N195Q)); xlii) SEQ ID NO: 286 (IL-12p35(N71D / N85D)); xliii) SEQ ID NO: 287 (IL-12p35(N71D / N195D); xliv) SEQ ID NO: 288 (IL-12p35(N85D / N195D)); xlv) SEQ ID NO: 333 (IL-12p35(D55Q)) and xlvi) SEQ ID NO: 334 (IL-12p35(D55K)).

[0297] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the first monomer and the second monomer are XENP31289.

[0298] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the first monomer and the second monomer are XENP31291.

[0299] In some embodiments, the present application provides a composition of homodimeric Fc fusion proteins for use in treating cancer in a subject.

[0300] In some embodiments, the present application provides one or more nucleic acids encoding a homodimeric Fc fusion protein.

[0301] In some embodiments, the present application provides a host cell comprising the one or more nucleic acids encoding a homodimeric Fc fusion protein.

[0302] In some embodiments, the present application provides a method of making a homodimeric Fc fusion protein, the method comprising culturing a host cell under conditions wherein the homodimeric Fc fusion protein is produced.

[0303] In some embodiments, the present application provides a method of 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 a fraction containing the homodimeric Fc fusion protein.

[0304] In another aspect, the present application provides a homodimeric Fc fusion protein comprising a first monomer and a second monomer, each comprising, from N- to C-terminus, an IL-12 p40 subunit domain - a first domain linker - an IL-12 p35 subunit domain - a second domain linker - an Fc domain.

[0305] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the modification that promotes homodimerization of the Fc domain is 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.

[0306] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the first domain linker and the second domain linker have the same amino acid sequence.

[0307] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the Fc domain has an additional set of amino acid substitutions comprising Q295E / N384D / Q418E / N421D, according to EU numbering.

[0308] In some embodiments, the present application provides a homodimeric Fc fusion protein, 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.

[0309] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the variant IL-12 p40 subunit has a polypeptide sequence selected from the group consisting of SEQ ID NO: 3 (human IL-12 subunit beta (IL-12 p40) precursor sequence) and SEQ ID NO: 4 (human IL-12 subunit beta (IL-12 p40) mature form sequence), and the IL-12 p35 subunit has a polypeptide sequence selected from the group consisting of SEQ ID NO: 1 (human IL-12 subunit alpha (IL-12 p35) precursor sequence) and SEQ ID NO: 2 (human IL-12 subunit alpha (IL-12 p35) mature form sequence).

[0310] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein each of the Fc domains further comprises the amino acid substitutions M428L / N424S.

[0311] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the IL-12 p40 subunit is a variant IL-12 p40 subunit and / or the IL-12 p35 subunit is a variant IL-12 p35 subunit.

[0312] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the IL-12p40 subunit is a variant IL-12p40 subunit having altered affinity for IL-12 receptor subunit beta-1 (IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2), and / or IL-12 receptor complex; and / or the IL-12p35 subunit is a variant IL-12p35 subunit having altered affinity for IL-12 receptor subunit beta-1 (IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2), and / or IL-12 receptor complex.

[0313] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the IL-12p40 subunit has one or more amino acid modifications at an amino acid residue 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, D209, 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.

[0314] In some embodiments, the present application provides a homodimeric Fc fusion protein wherein the IL-12p40 subunit has one or more amino acid substitutions selected from the group consisting of D18N, D18K, E32Q, E33Q, D34N, D34K, Q42E, S43E, S43K, E45Q, Q56E, E59Q, E59K, D62N, E73Q, D87N, K99E, K99Y, E100Q, N103D, N103Q, N113D, N113Q, Q144E, D161N, R159E, K163E, E187Q, N200D, N200Q, N218Q, Q229E, E235Q, C252S, Q256N, K258E, K260E, E262Q, K264E, N281D, N281Q, and E299Q.

[0315] In some embodiments, the present application provides a homodimeric Fc fusion protein wherein the IL-12p40 subunit has an amino acid substitution selected from the group consisting 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, 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, 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 / 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, E59 K / K99E / N103Q / N281Q / C252S / K264E, E59K / K99E / N113Q / N200Q / C252S / K264E, E59K / K99E / N113Q / N281Q / C252S / K264E, E 59K / K99E / N200Q / 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.

[0316] In some embodiments, the present invention provides a homodimeric Fc fusion protein, wherein the IL-12p40 subunit has a polypeptide sequence selected from the group consisting of:

[0317] 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 (S43K); xxxiii) SEQ ID NO: 379 (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: 326 (IL-12p40 (D34N / E59K)); I) SEQ ID NO: 325 (IL-12p40 (E59K / K99E)); li) SEQ ID NO: 339 (IL-12p40 (D18K / E59K / K99E)); lii) SEQ ID NO: 343 (IL-12p40 (E59K / K99E / K264E)); liii) SEQ ID NO: 336 (IL-12p40 (E59K / K99Y)); liv) SEQ ID NO: 335 (IL-12p40 (E59Y / K99E)); lv) SEQ ID NO: 338 (IL-12p40 (E45K / E59K / K99E)); lvi) SEQ ID NO: 340 (IL-12p40 (E59K / K99E / Q144E));lxvii) SEQ ID NO: 341 (IL-12p40 (E59K / K99E / Q144K)); lviii) SEQ ID NO: 342 (IL-12p40 (E59K / K99E / R159E)); Ix) SEQ ID NO: 343 (IL-12p40 (E59K / K99E / K264E)); lx) SEQ ID NO: 344 (IL-12p40 (D18K / E59K / K99E / K264E)); lxi) SEQ ID NO: 360 (IL-12p40 (C252S)); lxii) SEQ ID NO: 361 (IL-12p40 (D18K / E59K / K99E / C252S)); lxiii) SEQ ID NO: 362 (IL-12p40 (D18K / E59K / K99E / C252S / K264E)); lxiv) SEQ ID NO: 363 (IL-12p40 (E59K / K99Y / C252S)); lxv) SEQ ID NO: 364 (IL-12p40 (E59K / K99E / C252S / K264E)); lxvi) SEQ ID NO: 365 (IL-12p40 (E59K / K99E / C252S)); lxvii) SEQ ID NO: 254 (IL-12p40 (N103D / N113D)); lxviii) SEQ ID NO: 255 (IL-12p40 (N103D / N200D)); lxix) SEQ ID NO: 256 (IL-12p40 (N103D / N281D)); lxx) SEQ ID NO: 257 (IL-12p40 (N113D / N200D)); lxxi) SEQ ID NO: 258 (IL-12p40 (N113D / N281D)); lxxii) SEQ ID NO: 259 (IL-12p40 (N200D / N281D)); lxxiii) SEQ ID NO: 260 (IL-12p40 (N103D / N113D / N200D)); lxxiv) SEQ ID NO: 261 (IL-12p40 (N103D / N113D / N281D)); lxxv) SEQ ID NO: 262 (IL-12p40 (N103D / N200D / N281D)); lxxvi) SEQ ID NO: 263 (IL-12p40 (N113D / N200D / N281D)); lxxvii) SEQ ID NO: 264 (IL-12p40 (N103Q)); lxxviii) SEQ ID NO: 265 (IL-12p40 (N113Q));lxxix) SEQ ID NO: 266 (IL-12p40 (N200Q)); lxxx) SEQ ID NO: 267 (IL-12p40 (N281Q)); lxxxi) SEQ ID NO: 268 (IL-12p40 (N103Q / N113Q)); lxxxii) SEQ ID NO: 269 (IL-12p40 (N103Q / N200Q)); lxxxiii) SEQ ID NO: 270 (IL-12p40 (N103Q / N281Q)); lxxxiv) SEQ ID NO: 271 (IL-12p40 (N113Q / N200Q)); lxxxv) SEQ ID NO: 272 (IL-12p40 (N113Q / N281Q)); lxxxvi) SEQ ID NO: 273 (IL-12p40 (N200Q / N281Q)); lxxxvii) SEQ ID NO: 274 (IL-12p40 (N103Q / N113Q / N200Q)); lxxxviii) SEQ ID NO: 275 (IL-12p40 (N103Q / N113Q / N281Q)); lxxxix) SEQ ID NO: 276 (IL-12p40 (N103Q / N200Q / N281Q)); xc) SEQ ID NO: 277 (IL-12p40 (N113Q / N200Q / N281Q)); xci) SEQ ID NO: 278 (IL-12p40 (N103Q / N113Q / N200Q / N281Q)); xcii) SEQ ID NO: 327 (IL-12p40 (D34N / E59K / K99E)); xciii) SEQ ID NO: 328 (IL-12p40 (D34K / E59K / K99E)); xciv) SEQ ID NO: 329 (IL-12p40 (E32Q / D34N / E59K / K99E)); xcv) SEQ ID NO: 331 (IL-12p40 (E32K / D34N / E59K / K99E)); xcvi) SEQ ID NO: 337 (IL-12p40 (E59Y / K99Y)); xcvi i) SEQ ID NO: 366 (IL-12p40 (E59K / K99E / N103Q / C252S / K264E)); xcvi i) SEQ ID NO: 367 (IL-12p40 (E59K / K99E / N113Q / C252S / K264E)); xcix) SEQ ID NO: 368 (IL-12p40 (E59K / K99E / N200Q / C252S / K264E));c) SEQ ID NO: 369 (IL-12p40 (E59K / K99E / N281Q / C252S / K264E)); ci) SEQ ID NO: 370 (IL-12p40 (E59K / K99E / N103Q / N113Q / C252S / K264E)); cii) SEQ ID NO: 371 (IL-12p40 (E59K / K99E / N103Q / N200Q / C252S / K264E)); ciii) SEQ ID NO: 372 (IL-12p40 (E59K / K99E / N103Q / N281Q / C252S / K264E)); civ) SEQ ID NO: 373 (IL-12p40 (E59K / K99E / N113Q / N200Q / C252S / K264E)); cv) SEQ ID NO: 374 (IL-12p40 (E59K / K99E / N113Q / N281Q / C252S / K264E)); cvi) SEQ ID NO: 375 (IL-12p40 (E59K / K99E / N200Q / N281Q / C252S / K264E)); cvii) SEQ ID NO: 376 (IL-12p40 (E59K / K99E / N103Q / N113Q / N200Q / C252S / K264E)); cviii) SEQ ID NO: 377 (IL-12p40 (E59K / K99E / N103Q / N200Q / N281Q / C252S / K264E)); and cix) SEQ ID NO: 378 (IL-12p40 (E59K / K99E / N113Q / N200Q / N281Q / C252S / K264E)).

[0318] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the IL-12 p35 subunit has one or more amino acid modifications as amino acid residues 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, Q135, N136, E143, Q146, N151, E153, K158, E162, E163, D165, I171, R181, I182, R183, V185, T186, D188, R189, V190, S192, Y193, N195, and A196.

[0319] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the IL-12 p35 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.

[0320] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the IL-12 p35 subunit has one or more 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.

[0321] In some embodiments, the present application provides a homodimeric Fc fusion protein wherein the IL-12 p35 subunit has a polypeptide sequence 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: 333 (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: 345 (IL-12p35(F96A)); xxxiv) SEQ ID NO: 346 (IL-12p35(M97A)); xxxv) SEQ ID NO: 347 (IL-12p35(L89A)); xxxvi) SEQ ID NO: 348 (IL-12p35(L124A)); xxxvii) SEQ ID NO: 349 (IL-12p35(M125A)); xxxviii) SEQ ID NO: 350 (IL-12p35(L75A)); xxxiv) SEQ ID NO: 351 (IL-12p35(I171A)); xxxv) SEQ ID NO: 279 (IL-12p35(N71Q)); xxxvi) SEQ ID NO: 280 (IL-12p35(N85Q)); xxxvii) SEQ ID NO: 281 (IL-12p35(N195Q)); xxxviii) SEQ ID NO: 282 (IL-12p35(N71Q / N85Q)); xxxix) SEQ ID NO: 283 (IL-12p35(N71Q / N195Q)); xl) SEQ ID NO: 284 (IL-12p35(N85Q / N195Q); xli) SEQ ID NO: 285 (IL-12p35(N71Q / N85Q / N195Q)); xlii) SEQ ID NO: 286 (IL-12p35(N71D / N85D)); xliii) SEQ ID NO: 287 (IL-12p35(N71D / N195D); xliv) SEQ ID NO: 288 (IL-12p35(N85D / N195D)); xlv) SEQ ID NO: 333 (IL-12p35(D55Q)) and xlvi) SEQ ID NO: 334 (IL-12p35(D55K)).

[0322] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the first monomer and the second monomer are XENP31289.

[0323] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the first monomer and the second monomer are XENP31291.

[0324] In some embodiments, the present application provides a composition of homodimeric Fc fusion proteins for use in treating cancer in a subject.

[0325] In some embodiments, the present application provides one or more nucleic acids encoding a homodimeric Fc fusion protein.

[0326] In some embodiments, the present application provides a host cell comprising the one or more nucleic acids encoding a homodimeric Fc fusion protein.

[0327] In some embodiments, the present application provides a method of making a homodimeric Fc fusion protein, the method comprising culturing a host cell under conditions wherein the homodimeric Fc fusion protein is produced.

[0328] In some embodiments, the present application provides a method of 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 a fraction containing the homodimeric Fc fusion protein.

[0329] In another aspect, the present application provides a homodimeric Fc fusion protein comprising a first monomer and a second monomer, each comprising, from N- to C-terminus, an IL-12 p35 subunit domain - a first domain linker - an IL-12 p40 subunit domain - a second domain linker - an Fc domain.

[0330] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the modification that promotes homodimerization of the Fc domain is 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.

[0331] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the first domain linker and the second domain linker have the same amino acid sequence.

[0332] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the Fc domain has an additional set of amino acid substitutions comprising Q295E / N384D / Q418E / N421D, according to EU numbering.

[0333] In some embodiments, the present application provides a homodimeric Fc fusion protein, 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.

[0334] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the variant IL-12 p40 subunit has a polypeptide sequence selected from the group consisting of SEQ ID NO: 3 (human IL-12 subunit beta (IL-12 p40) precursor sequence) and SEQ ID NO: 4 (human IL-12 subunit beta (IL-12 p40) mature form sequence), and the IL-12 p35 subunit has a polypeptide sequence selected from the group consisting of SEQ ID NO: 1 (human IL-12 subunit alpha (IL-12 p35) precursor sequence) and SEQ ID NO: 2 (human IL-12 subunit alpha (IL-12 p35) mature form sequence).

[0335] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein each of the Fc domains further comprises the amino acid substitutions M428L / N424S.

[0336] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the IL-12 p40 subunit is a variant IL-12 p40 subunit and / or the IL-12 p35 subunit is a variant IL-12 p35 subunit.

[0337] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the IL-12p40 subunit is a variant IL-12p40 subunit having altered affinity for IL-12 receptor subunit beta-1 (IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2), and / or IL-12 receptor complex; and / or the IL-12p35 subunit is a variant IL-12p35 subunit having altered affinity for IL-12 receptor subunit beta-1 (IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2), and / or IL-12 receptor complex.

[0338] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the IL-12p40 subunit has one or more amino acid modifications at an amino acid residue 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, D209, 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.

[0339] In some embodiments, the present application provides a homodimeric Fc fusion protein wherein the IL-12p40 subunit has one or more amino acid substitutions selected from the group consisting of D18N, D18K, E32Q, E33Q, D34N, D34K, Q42E, S43E, S43K, E45Q, Q56E, E59Q, E59K, D62N, E73Q, D87N, K99E, K99Y, E100Q, N103D, N103Q, N113D, N113Q, Q144E, D161N, R159E, K163E, E187Q, N200D, N200Q, N218Q, Q229E, E235Q, C252S, Q256N, K258E, K260E, E262Q, K264E, N281D, N281Q, and E299Q.

[0340] In some embodiments, the present application provides a homodimeric Fc fusion protein wherein the IL-12p40 subunit has an amino acid substitution selected from the group consisting 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, 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, 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 / 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, E59 K / K99E / N103Q / N281Q / C252S / K264E, E59K / K99E / N113Q / N200Q / C252S / K264E, E59K / K99E / N113Q / N281Q / C252S / K264E, E 59K / K99E / N200Q / 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.

[0341] In some embodiments, the present invention provides a homodimeric Fc fusion protein, wherein the IL-12p40 subunit has a polypeptide sequence selected from the group consisting of:

[0342] 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 (S43K); xxxiii) SEQ ID NO: 379 (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: 326 (IL-12p40 (D34N / E59K)); I) SEQ ID NO: 325 (IL-12p40 (E59K / K99E)); li) SEQ ID NO: 339 (IL-12p40 (D18K / E59K / K99E)); lii) SEQ ID NO: 343 (IL-12p40 (E59K / K99E / K264E)); liii) SEQ ID NO: 336 (IL-12p40 (E59K / K99Y)); liv) SEQ ID NO: 335 (IL-12p40 (E59Y / K99E)); lv) SEQ ID NO: 338 (IL-12p40 (E45K / E59K / K99E)); lvi) SEQ ID NO: 340 (IL-12p40 (E59K / K99E / Q144E));lxvii) SEQ ID NO: 341 (IL-12p40 (E59K / K99E / Q144K)); lviii) SEQ ID NO: 342 (IL-12p40 (E59K / K99E / R159E)); Ix) SEQ ID NO: 343 (IL-12p40 (E59K / K99E / K264E)); lx) SEQ ID NO: 344 (IL-12p40 (D18K / E59K / K99E / K264E)); lxi) SEQ ID NO: 360 (IL-12p40 (C252S)); lxii) SEQ ID NO: 361 (IL-12p40 (D18K / E59K / K99E / C252S)); lxiii) SEQ ID NO: 362 (IL-12p40 (D18K / E59K / K99E / C252S / K264E)); lxiv) SEQ ID NO: 363 (IL-12p40 (E59K / K99Y / C252S)); lxv) SEQ ID NO: 364 (IL-12p40 (E59K / K99E / C252S / K264E)); lxvi) SEQ ID NO: 365 (IL-12p40 (E59K / K99E / C252S)); lxvii) SEQ ID NO: 254 (IL-12p40 (N103D / N113D)); lxviii) SEQ ID NO: 255 (IL-12p40 (N103D / N200D)); lxix) SEQ ID NO: 256 (IL-12p40 (N103D / N281D)); lxx) SEQ ID NO: 257 (IL-12p40 (N113D / N200D)); lxxi) SEQ ID NO: 258 (IL-12p40 (N113D / N281D)); lxxii) SEQ ID NO: 259 (IL-12p40 (N200D / N281D)); lxxiii) SEQ ID NO: 260 (IL-12p40 (N103D / N113D / N200D)); lxxiv) SEQ ID NO: 261 (IL-12p40 (N103D / N113D / N281D)); lxxv) SEQ ID NO: 262 (IL-12p40 (N103D / N200D / N281D)); lxxvi) SEQ ID NO: 263 (IL-12p40 (N113D / N200D / N281D)); lxxvii) SEQ ID NO: 264 (IL-12p40 (N103Q)); lxxviii) SEQ ID NO: 265 (IL-12p40 (N113Q));lxxix) SEQ ID NO: 266 (IL-12p40 (N200Q)); lxxx) SEQ ID NO: 267 (IL-12p40 (N281Q)); lxxxi) SEQ ID NO: 268 (IL-12p40 (N103Q / N113Q)); lxxxii) SEQ ID NO: 269 (IL-12p40 (N103Q / N200Q)); lxxxiii) SEQ ID NO: 270 (IL-12p40 (N103Q / N281Q)); lxxxiv) SEQ ID NO: 271 (IL-12p40 (N113Q / N200Q)); lxxxv) SEQ ID NO: 272 (IL-12p40 (N113Q / N281Q)); lxxxvi) SEQ ID NO: 273 (IL-12p40 (N200Q / N281Q)); lxxxvii) SEQ ID NO: 274 (IL-12p40 (N103Q / N113Q / N200Q)); lxxxviii) SEQ ID NO: 275 (IL-12p40 (N103Q / N113Q / N281Q)); lxxxix) SEQ ID NO: 276 (IL-12p40 (N103Q / N200Q / N281Q)); xc) SEQ ID NO: 277 (IL-12p40 (N113Q / N200Q / N281Q)); xci) SEQ ID NO: 278 (IL-12p40 (N103Q / N113Q / N200Q / N281Q)); xcii) SEQ ID NO: 327 (IL-12p40 (D34N / E59K / K99E)); xciii) SEQ ID NO: 328 (IL-12p40 (D34K / E59K / K99E)); xciv) SEQ ID NO: 329 (IL-12p40 (E32Q / D34N / E59K / K99E)); xcv) SEQ ID NO: 331 (IL-12p40 (E32K / D34N / E59K / K99E)); xcvi) SEQ ID NO: 337 (IL-12p40 (E59Y / K99Y)); xcvi i) SEQ ID NO: 366 (IL-12p40 (E59K / K99E / N103Q / C252S / K264E)); xcvi i) SEQ ID NO: 367 (IL-12p40 (E59K / K99E / N113Q / C252S / K264E)); xcix) SEQ ID NO: 368 (IL-12p40 (E59K / K99E / N200Q / C252S / K264E));c) SEQ ID NO: 369 (IL-12p40 (E59K / K99E / N281Q / C252S / K264E)); ci) SEQ ID NO: 370 (IL-12p40 (E59K / K99E / N103Q / N113Q / C252S / K264E)); cii) SEQ ID NO: 371 (IL-12p40 (E59K / K99E / N103Q / N200Q / C252S / K264E)); ciii) SEQ ID NO: 372 (IL-12p40 (E59K / K99E / N103Q / N281Q / C252S / K264E)); civ) SEQ ID NO: 373 (IL-12p40 (E59K / K99E / N113Q / N200Q / C252S / K264E)); cv) SEQ ID NO: 374 (IL-12p40 (E59K / K99E / N113Q / N281Q / C252S / K264E)); cvi) SEQ ID NO: 375 (IL-12p40 (E59K / K99E / N200Q / N281Q / C252S / K264E)); cvii) SEQ ID NO: 376 (IL-12p40 (E59K / K99E / N103Q / N113Q / N200Q / C252S / K264E)); cviii) SEQ ID NO: 377 (IL-12p40 (E59K / K99E / N103Q / N200Q / N281Q / C252S / K264E)); and cix) SEQ ID NO: 378 (IL-12p40 (E59K / K99E / N113Q / N200Q / N281Q / C252S / K264E)).

[0343] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the IL-12 p35 subunit has one or more amino acid modifications as amino acid residues 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, Q135, N136, E143, Q146, N151, E153, K158, E162, E163, D165, I171, R181, I182, R183, V185, T186, D188, R189, V190, S192, Y193, N195, and A196.

[0344] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the IL-12 p35 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.

[0345] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the IL-12 p35 subunit has one or more 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.

[0346] In some embodiments, the present application provides a homodimeric Fc fusion protein wherein the IL-12 p35 subunit has a polypeptide sequence 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: 333 (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: 345 (IL-12p35(F96A)); xxxiv) SEQ ID NO: 346 (IL-12p35(M97A)); xxxv) SEQ ID NO: 347 (IL-12p35(L89A)); xxxvi) SEQ ID NO: 348 (IL-12p35(L124A)); xxxvii) SEQ ID NO: 349 (IL-12p35(M125A)); xxxviii) SEQ ID NO: 350 (IL-12p35(L75A)); xxxiv) SEQ ID NO: 351 (IL-12p35(I171A)); xxxv) SEQ ID NO: 279 (IL-12p35(N71Q)); xxxvi) SEQ ID NO: 280 (IL-12p35(N85Q)); xxxvii) SEQ ID NO: 281 (IL-12p35(N195Q)); xxxviii) SEQ ID NO: 282 (IL-12p35(N71Q / N85Q)); xxxix) SEQ ID NO: 283 (IL-12p35(N71Q / N195Q)); xl) SEQ ID NO: 284 (IL-12p35(N85Q / N195Q); xli) SEQ ID NO: 285 (IL-12p35(N71Q / N85Q / N195Q)); xlii) SEQ ID NO: 286 (IL-12p35(N71D / N85D)); xliii) SEQ ID NO: 287 (IL-12p35(N71D / N195D); xliv) SEQ ID NO: 288 (IL-12p35(N85D / N195D)); xlv) SEQ ID NO: 333 (IL-12p35(D55Q)) and xlvi) SEQ ID NO: 334 (IL-12p35(D55K)).

[0347] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the first monomer and the second monomer are XENP31289.

[0348] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the first monomer and the second monomer are XENP31291.

[0349] In some embodiments, the present application provides a composition of homodimeric Fc fusion proteins for use in treating cancer in a subject.

[0350] In some embodiments, the present application provides one or more nucleic acids encoding a homodimeric Fc fusion protein.

[0351] In some embodiments, the present application provides a host cell comprising the one or more nucleic acids encoding a homodimeric Fc fusion protein.

[0352] In some embodiments, the present application provides a method of making a homodimeric Fc fusion protein, the method comprising culturing a host cell under conditions wherein the homodimeric Fc fusion protein is produced.

[0353] In some embodiments, the present application provides a method of 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 a fraction containing the homodimeric Fc fusion protein.

[0354] In another aspect, the present application provides a homodimeric Fc fusion protein comprising a first monomer and a second monomer, each comprising, from N- to C-terminus, an Fc domain - a first domain linker - an IL-12 p40 subunit domain - a second domain linker - an IL-12 p35 subunit domain.

[0355] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the modification that promotes homodimerization of the Fc domain is 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.

[0356] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the first domain linker and the second domain linker have the same amino acid sequence.

[0357] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the Fc domain has an additional set of amino acid substitutions comprising Q295E / N384D / Q418E / N421D, according to EU numbering.

[0358] In some embodiments, the present application provides a homodimeric Fc fusion protein, 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.

[0359] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the variant IL-12 p40 subunit has a polypeptide sequence selected from the group consisting of SEQ ID NO: 3 (human IL-12 subunit beta (IL-12 p40) precursor sequence) and SEQ ID NO: 4 (human IL-12 subunit beta (IL-12 p40) mature form sequence), and the IL-12 p35 subunit has a polypeptide sequence selected from the group consisting of SEQ ID NO: 1 (human IL-12 subunit alpha (IL-12 p35) precursor sequence) and SEQ ID NO: 2 (human IL-12 subunit alpha (IL-12 p35) mature form sequence).

[0360] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein each of the Fc domains further comprises the amino acid substitutions M428L / N424S.

[0361] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the IL-12 p40 subunit is a variant IL-12 p40 subunit and / or the IL-12 p35 subunit is a variant IL-12 p35 subunit.

[0362] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the IL-12p40 subunit is a variant IL-12p40 subunit having altered affinity for IL-12 receptor subunit beta-1 (IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2), and / or IL-12 receptor complex; and / or the IL-12p35 subunit is a variant IL-12p35 subunit having altered affinity for IL-12 receptor subunit beta-1 (IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2), and / or IL-12 receptor complex.

[0363] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the IL-12p40 subunit has one or more amino acid modifications at an amino acid residue 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, D209, 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.

[0364] In some embodiments, the present application provides a homodimeric Fc fusion protein wherein the IL-12p40 subunit has one or more amino acid substitutions selected from the group consisting of D18N, D18K, E32Q, E33Q, D34N, D34K, Q42E, S43E, S43K, E45Q, Q56E, E59Q, E59K, D62N, E73Q, D87N, K99E, K99Y, E100Q, N103D, N103Q, N113D, N113Q, Q144E, D161N, R159E, K163E, E187Q, N200D, N200Q, N218Q, Q229E, E235Q, C252S, Q256N, K258E, K260E, E262Q, K264E, N281D, N281Q, and E299Q.

[0365] In some embodiments, the present application provides a homodimeric Fc fusion protein wherein the IL-12p40 subunit has an amino acid substitution selected from the group consisting 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, 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, 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 / 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, E59 K / K99E / N103Q / N281Q / C252S / K264E, E59K / K99E / N113Q / N200Q / C252S / K264E, E59K / K99E / N113Q / N281Q / C252S / K264E, E 59K / K99E / N200Q / 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.

[0366] In some embodiments, the present invention provides a homodimeric Fc fusion protein, wherein the IL-12p40 subunit has a polypeptide sequence selected from the group consisting of:

[0367] 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 (S43K); xxxiii) SEQ ID NO: 379 (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: 326 (IL-12p40 (D34N / E59K)); I) SEQ ID NO: 325 (IL-12p40 (E59K / K99E)); li) SEQ ID NO: 339 (IL-12p40 (D18K / E59K / K99E)); lii) SEQ ID NO: 343 (IL-12p40 (E59K / K99E / K264E)); liii) SEQ ID NO: 336 (IL-12p40 (E59K / K99Y)); liv) SEQ ID NO: 335 (IL-12p40 (E59Y / K99E)); lv) SEQ ID NO: 338 (IL-12p40 (E45K / E59K / K99E)); lvi) SEQ ID NO: 340 (IL-12p40 (E59K / K99E / Q144E));lxvii) SEQ ID NO: 341 (IL-12p40 (E59K / K99E / Q144K)); lviii) SEQ ID NO: 342 (IL-12p40 (E59K / K99E / R159E)); Ix) SEQ ID NO: 343 (IL-12p40 (E59K / K99E / K264E)); lx) SEQ ID NO: 344 (IL-12p40 (D18K / E59K / K99E / K264E)); lxi) SEQ ID NO: 360 (IL-12p40 (C252S)); lxii) SEQ ID NO: 361 (IL-12p40 (D18K / E59K / K99E / C252S)); lxiii) SEQ ID NO: 362 (IL-12p40 (D18K / E59K / K99E / C252S / K264E)); lxiv) SEQ ID NO: 363 (IL-12p40 (E59K / K99Y / C252S)); lxv) SEQ ID NO: 364 (IL-12p40 (E59K / K99E / C252S / K264E)); lxvi) SEQ ID NO: 365 (IL-12p40 (E59K / K99E / C252S)); lxvii) SEQ ID NO: 254 (IL-12p40 (N103D / N113D)); lxviii) SEQ ID NO: 255 (IL-12p40 (N103D / N200D)); lxix) SEQ ID NO: 256 (IL-12p40 (N103D / N281D)); lxx) SEQ ID NO: 257 (IL-12p40 (N113D / N200D)); lxxi) SEQ ID NO: 258 (IL-12p40 (N113D / N281D)); lxxii) SEQ ID NO: 259 (IL-12p40 (N200D / N281D)); lxxiii) SEQ ID NO: 260 (IL-12p40 (N103D / N113D / N200D)); lxxiv) SEQ ID NO: 261 (IL-12p40 (N103D / N113D / N281D)); lxxv) SEQ ID NO: 262 (IL-12p40 (N103D / N200D / N281D)); lxxvi) SEQ ID NO: 263 (IL-12p40 (N113D / N200D / N281D)); lxxvii) SEQ ID NO: 264 (IL-12p40 (N103Q)); lxxviii) SEQ ID NO: 265 (IL-12p40 (N113Q));lxxix) SEQ ID NO: 266 (IL-12p40 (N200Q)); lxxx) SEQ ID NO: 267 (IL-12p40 (N281Q)); lxxxi) SEQ ID NO: 268 (IL-12p40 (N103Q / N113Q)); lxxxii) SEQ ID NO: 269 (IL-12p40 (N103Q / N200Q)); lxxxiii) SEQ ID NO: 270 (IL-12p40 (N103Q / N281Q)); lxxxiv) SEQ ID NO: 271 (IL-12p40 (N113Q / N200Q)); lxxxv) SEQ ID NO: 272 (IL-12p40 (N113Q / N281Q)); lxxxvi) SEQ ID NO: 273 (IL-12p40 (N200Q / N281Q)); lxxxvii) SEQ ID NO: 274 (IL-12p40 (N103Q / N113Q / N200Q)); lxxxviii) SEQ ID NO: 275 (IL-12p40 (N103Q / N113Q / N281Q)); lxxxix) SEQ ID NO: 276 (IL-12p40 (N103Q / N200Q / N281Q)); xc) SEQ ID NO: 277 (IL-12p40 (N113Q / N200Q / N281Q)); xci) SEQ ID NO: 278 (IL-12p40 (N103Q / N113Q / N200Q / N281Q)); xcii) SEQ ID NO: 327 (IL-12p40 (D34N / E59K / K99E)); xciii) SEQ ID NO: 328 (IL-12p40 (D34K / E59K / K99E)); xciv) SEQ ID NO: 329 (IL-12p40 (E32Q / D34N / E59K / K99E)); xcv) SEQ ID NO: 331 (IL-12p40 (E32K / D34N / E59K / K99E)); xcvi) SEQ ID NO: 337 (IL-12p40 (E59Y / K99Y)); xcvi i) SEQ ID NO: 366 (IL-12p40 (E59K / K99E / N103Q / C252S / K264E)); xcvi i) SEQ ID NO: 367 (IL-12p40 (E59K / K99E / N113Q / C252S / K264E)); xcix) SEQ ID NO: 368 (IL-12p40 (E59K / K99E / N200Q / C252S / K264E));c) SEQ ID NO: 369 (IL-12p40 (E59K / K99E / N281Q / C252S / K264E)); ci) SEQ ID NO: 370 (IL-12p40 (E59K / K99E / N103Q / N113Q / C252S / K264E)); cii) SEQ ID NO: 371 (IL-12p40 (E59K / K99E / N103Q / N200Q / C252S / K264E)); ciii) SEQ ID NO: 372 (IL-12p40 (E59K / K99E / N103Q / N281Q / C252S / K264E)); civ) SEQ ID NO: 373 (IL-12p40 (E59K / K99E / N113Q / N200Q / C252S / K264E)); cv) SEQ ID NO: 374 (IL-12p40 (E59K / K99E / N113Q / N281Q / C252S / K264E)); cvi) SEQ ID NO: 375 (IL-12p40 (E59K / K99E / N200Q / N281Q / C252S / K264E)); cvii) SEQ ID NO: 376 (IL-12p40 (E59K / K99E / N103Q / N113Q / N200Q / C252S / K264E)); cviii) SEQ ID NO: 377 (IL-12p40 (E59K / K99E / N103Q / N200Q / N281Q / C252S / K264E)); and cix) SEQ ID NO: 378 (IL-12p40 (E59K / K99E / N113Q / N200Q / N281Q / C252S / K264E)).

[0368] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the IL-12 p35 subunit has one or more amino acid modifications as amino acid residues 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, Q135, N136, E143, Q146, N151, E153, K158, E162, E163, D165, I171, R181, I182, R183, V185, T186, D188, R189, V190, S192, Y193, N195, and A196.

[0369] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the IL-12 p35 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.

[0370] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the IL-12 p35 subunit has one or more 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.

[0371] In some embodiments, the present application provides a homodimeric Fc fusion protein wherein the IL-12 p35 subunit has a polypeptide sequence 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: 333 (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: 345 (IL-12p35(F96A)); xxxiv) SEQ ID NO: 346 (IL-12p35(M97A)); xxxv) SEQ ID NO: 347 (IL-12p35(L89A)); xxxvi) SEQ ID NO: 348 (IL-12p35(L124A)); xxxvii) SEQ ID NO: 349 (IL-12p35(M125A)); xxxviii) SEQ ID NO: 350 (IL-12p35(L75A)); xxxiv) SEQ ID NO: 351 (IL-12p35(I171A)); xxxv) SEQ ID NO: 279 (IL-12p35(N71Q)); xxxvi) SEQ ID NO: 280 (IL-12p35(N85Q)); xxxvii) SEQ ID NO: 281 (IL-12p35(N195Q)); xxxviii) SEQ ID NO: 282 (IL-12p35(N71Q / N85Q)); xxxix) SEQ ID NO: 283 (IL-12p35(N71Q / N195Q)); xl) SEQ ID NO: 284 (IL-12p35(N85Q / N195Q); xli) SEQ ID NO: 285 (IL-12p35(N71Q / N85Q / N195Q)); xlii) SEQ ID NO: 286 (IL-12p35(N71D / N85D)); xliii) SEQ ID NO: 287 (IL-12p35(N71D / N195D); xliv) SEQ ID NO: 288 (IL-12p35(N85D / N195D)); xlv) SEQ ID NO: 333 (IL-12p35(D55Q)) and xlvi) SEQ ID NO: 334 (IL-12p35(D55K)).

[0372] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the first monomer and the second monomer are XENP31289.

[0373] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the first monomer and the second monomer are XENP31291.

[0374] In some embodiments, the present application provides a composition of homodimeric Fc fusion proteins for use in treating cancer in a subject.

[0375] In some embodiments, the present application provides one or more nucleic acids encoding a homodimeric Fc fusion protein.

[0376] In some embodiments, the present application provides a host cell comprising the one or more nucleic acids encoding a homodimeric Fc fusion protein.

[0377] In some embodiments, the present application provides a method of making a homodimeric Fc fusion protein, the method comprising culturing a host cell under conditions wherein the homodimeric Fc fusion protein is produced.

[0378] In some embodiments, the present application provides a method of 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 a fraction containing the homodimeric Fc fusion protein.

[0379] In another aspect, the present application provides a homodimeric Fc fusion protein comprising a first monomer and a second monomer, each comprising, from N- to C-terminus, an Fc domain - a first domain linker - an IL-12 p35 subunit domain - a second domain linker - an IL-12 p40 subunit domain.

[0380] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the modification that promotes homodimerization of the Fc domain is 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.

[0381] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the first domain linker and the second domain linker have the same amino acid sequence.

[0382] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the Fc domain has an additional set of amino acid substitutions comprising Q295E / N384D / Q418E / N421D, according to EU numbering.

[0383] In some embodiments, the present application provides a homodimeric Fc fusion protein, 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.

[0384] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the variant IL-12 p40 subunit has a polypeptide sequence selected from the group consisting of SEQ ID NO: 3 (human IL-12 subunit beta (IL-12 p40) precursor sequence) and SEQ ID NO: 4 (human IL-12 subunit beta (IL-12 p40) mature form sequence), and the IL-12 p35 subunit has a polypeptide sequence selected from the group consisting of SEQ ID NO: 1 (human IL-12 subunit alpha (IL-12 p35) precursor sequence) and SEQ ID NO: 2 (human IL-12 subunit alpha (IL-12 p35) mature form sequence).

[0385] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein each of the Fc domains further comprises the amino acid substitutions M428L / N424S.

[0386] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the IL-12 p40 subunit is a variant IL-12 p40 subunit and / or the IL-12 p35 subunit is a variant IL-12 p35 subunit.

[0387] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the IL-12p40 subunit is a variant IL-12p40 subunit having altered affinity for IL-12 receptor subunit beta-1 (IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2), and / or IL-12 receptor complex; and / or the IL-12p35 subunit is a variant IL-12p35 subunit having altered affinity for IL-12 receptor subunit beta-1 (IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2), and / or IL-12 receptor complex.

[0388] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the IL-12p40 subunit has one or more amino acid modifications at an amino acid residue 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, D209, 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.

[0389] In some embodiments, the present application provides a homodimeric Fc fusion protein wherein the IL-12p40 subunit has one or more amino acid substitutions selected from the group consisting of D18N, D18K, E32Q, E33Q, D34N, D34K, Q42E, S43E, S43K, E45Q, Q56E, E59Q, E59K, D62N, E73Q, D87N, K99E, K99Y, E100Q, N103D, N103Q, N113D, N113Q, Q144E, D161N, R159E, K163E, E187Q, N200D, N200Q, N218Q, Q229E, E235Q, C252S, Q256N, K258E, K260E, E262Q, K264E, N281D, N281Q, and E299Q.

[0390] In some embodiments, the present application provides a homodimeric Fc fusion protein wherein the IL-12p40 subunit has an amino acid substitution selected from the group consisting 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, 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, 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 / 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, E59 K / K99E / N103Q / N281Q / C252S / K264E, E59K / K99E / N113Q / N200Q / C252S / K264E, E59K / K99E / N113Q / N281Q / C252S / K264E, E 59K / K99E / N200Q / 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.

[0391] In some embodiments, the present invention provides a homodimeric Fc fusion protein, wherein the IL-12p40 subunit has a polypeptide sequence selected from the group consisting of:

[0392] 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 (S43K); xxxiii) SEQ ID NO: 379 (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: 326 (IL-12p40 (D34N / E59K)); I) SEQ ID NO: 325 (IL-12p40 (E59K / K99E)); li) SEQ ID NO: 339 (IL-12p40 (D18K / E59K / K99E)); lii) SEQ ID NO: 343 (IL-12p40 (E59K / K99E / K264E)); liii) SEQ ID NO: 336 (IL-12p40 (E59K / K99Y)); liv) SEQ ID NO: 335 (IL-12p40 (E59Y / K99E)); lv) SEQ ID NO: 338 (IL-12p40 (E45K / E59K / K99E)); lvi) SEQ ID NO: 340 (IL-12p40 (E59K / K99E / Q144E));lxvii) SEQ ID NO: 341 (IL-12p40 (E59K / K99E / Q144K)); lviii) SEQ ID NO: 342 (IL-12p40 (E59K / K99E / R159E)); Ix) SEQ ID NO: 343 (IL-12p40 (E59K / K99E / K264E)); lx) SEQ ID NO: 344 (IL-12p40 (D18K / E59K / K99E / K264E)); lxi) SEQ ID NO: 360 (IL-12p40 (C252S)); lxii) SEQ ID NO: 361 (IL-12p40 (D18K / E59K / K99E / C252S)); lxiii) SEQ ID NO: 362 (IL-12p40 (D18K / E59K / K99E / C252S / K264E)); lxiv) SEQ ID NO: 363 (IL-12p40 (E59K / K99Y / C252S)); lxv) SEQ ID NO: 364 (IL-12p40 (E59K / K99E / C252S / K264E)); lxvi) SEQ ID NO: 365 (IL-12p40 (E59K / K99E / C252S)); lxvii) SEQ ID NO: 254 (IL-12p40 (N103D / N113D)); lxviii) SEQ ID NO: 255 (IL-12p40 (N103D / N200D)); lxix) SEQ ID NO: 256 (IL-12p40 (N103D / N281D)); lxx) SEQ ID NO: 257 (IL-12p40 (N113D / N200D)); lxxi) SEQ ID NO: 258 (IL-12p40 (N113D / N281D)); lxxii) SEQ ID NO: 259 (IL-12p40 (N200D / N281D)); lxxiii) SEQ ID NO: 260 (IL-12p40 (N103D / N113D / N200D)); lxxiv) SEQ ID NO: 261 (IL-12p40 (N103D / N113D / N281D)); lxxv) SEQ ID NO: 262 (IL-12p40 (N103D / N200D / N281D)); lxxvi) SEQ ID NO: 263 (IL-12p40 (N113D / N200D / N281D)); lxxvii) SEQ ID NO: 264 (IL-12p40 (N103Q)); lxxviii) SEQ ID NO: 265 (IL-12p40 (N113Q));lxxix) SEQ ID NO: 266 (IL-12p40 (N200Q)); lxxx) SEQ ID NO: 267 (IL-12p40 (N281Q)); lxxxi) SEQ ID NO: 268 (IL-12p40 (N103Q / N113Q)); lxxxii) SEQ ID NO: 269 (IL-12p40 (N103Q / N200Q)); lxxxiii) SEQ ID NO: 270 (IL-12p40 (N103Q / N281Q)); lxxxiv) SEQ ID NO: 271 (IL-12p40 (N113Q / N200Q)); lxxxv) SEQ ID NO: 272 (IL-12p40 (N113Q / N281Q)); lxxxvi) SEQ ID NO: 273 (IL-12p40 (N200Q / N281Q)); lxxxvii) SEQ ID NO: 274 (IL-12p40 (N103Q / N113Q / N200Q)); lxxxviii) SEQ ID NO: 275 (IL-12p40 (N103Q / N113Q / N281Q)); lxxxix) SEQ ID NO: 276 (IL-12p40 (N103Q / N200Q / N281Q)); xc) SEQ ID NO: 277 (IL-12p40 (N113Q / N200Q / N281Q)); xci) SEQ ID NO: 278 (IL-12p40 (N103Q / N113Q / N200Q / N281Q)); xcii) SEQ ID NO: 327 (IL-12p40 (D34N / E59K / K99E)); xciii) SEQ ID NO: 328 (IL-12p40 (D34K / E59K / K99E)); xciv) SEQ ID NO: 329 (IL-12p40 (E32Q / D34N / E59K / K99E)); xcv) SEQ ID NO: 331 (IL-12p40 (E32K / D34N / E59K / K99E)); xcvi) SEQ ID NO: 337 (IL-12p40 (E59Y / K99Y)); xcvi i) SEQ ID NO: 366 (IL-12p40 (E59K / K99E / N103Q / C252S / K264E)); xcvi i) SEQ ID NO: 367 (IL-12p40 (E59K / K99E / N113Q / C252S / K264E)); xcix) SEQ ID NO: 368 (IL-12p40 (E59K / K99E / N200Q / C252S / K264E));c) SEQ ID NO: 369 (IL-12p40 (E59K / K99E / N281Q / C252S / K264E)); ci) SEQ ID NO: 370 (IL-12p40 (E59K / K99E / N103Q / N113Q / C252S / K264E)); cii) SEQ ID NO: 371 (IL-12p40 (E59K / K99E / N103Q / N200Q / C252S / K264E)); ciii) SEQ ID NO: 372 (IL-12p40 (E59K / K99E / N103Q / N281Q / C252S / K264E)); civ) SEQ ID NO: 373 (IL-12p40 (E59K / K99E / N113Q / N200Q / C252S / K264E)); cv) SEQ ID NO: 374 (IL-12p40 (E59K / K99E / N113Q / N281Q / C252S / K264E)); cvi) SEQ ID NO: 375 (IL-12p40 (E59K / K99E / N200Q / N281Q / C252S / K264E)); cvii) SEQ ID NO: 376 (IL-12p40 (E59K / K99E / N103Q / N113Q / N200Q / C252S / K264E)); cviii) SEQ ID NO: 377 (IL-12p40 (E59K / K99E / N103Q / N200Q / N281Q / C252S / K264E)); and cix) SEQ ID NO: 378 (IL-12p40 (E59K / K99E / N113Q / N200Q / N281Q / C252S / K264E)).

[0393] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the IL-12 p35 subunit has one or more amino acid modifications as amino acid residues 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, Q135, N136, E143, Q146, N151, E153, K158, E162, E163, D165, I171, R181, I182, R183, V185, T186, D188, R189, V190, S192, Y193, N195, and A196.

[0394] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the IL-12 p35 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.

[0395] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the IL-12 p35 subunit has one or more 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.

[0396] In some embodiments, the present application provides a homodimeric Fc fusion protein wherein the IL-12 p35 subunit has a polypeptide sequence 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: 333 (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: 345 (IL-12p35(F96A)); xxxiv) SEQ ID NO: 346 (IL-12p35(M97A)); xxxv) SEQ ID NO: 347 (IL-12p35(L89A)); xxxvi) SEQ ID NO: 348 (IL-12p35(L124A)); xxxvii) SEQ ID NO: 349 (IL-12p35(M125A)); xxxviii) SEQ ID NO: 350 (IL-12p35(L75A)); xxxiv) SEQ ID NO: 351 (IL-12p35(I171A)); xxxv) SEQ ID NO: 279 (IL-12p35(N71Q)); xxxvi) SEQ ID NO: 280 (IL-12p35(N85Q)); xxxvii) SEQ ID NO: 281 (IL-12p35(N195Q)); xxxviii) SEQ ID NO: 282 (IL-12p35(N71Q / N85Q)); xxxix) SEQ ID NO: 283 (IL-12p35(N71Q / N195Q)); xl) SEQ ID NO: 284 (IL-12p35(N85Q / N195Q); xli) SEQ ID NO: 285 (IL-12p35(N71Q / N85Q / N195Q)); xlii) SEQ ID NO: 286 (IL-12p35(N71D / N85D)); xliii) SEQ ID NO: 287 (IL-12p35(N71D / N195D); xliv) SEQ ID NO: 288 (IL-12p35(N85D / N195D)); xlv) SEQ ID NO: 333 (IL-12p35(D55Q)) and xlvi) SEQ ID NO: 334 (IL-12p35(D55K)).

[0397] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the first monomer and the second monomer are XENP31289.

[0398] In some embodiments, the present application provides a homodimeric Fc fusion protein, wherein the first monomer and the second monomer are XENP31291.

[0399] In some embodiments, the present application provides a composition of homodimeric Fc fusion proteins for use in treating cancer in a subject.

[0400] In some embodiments, the present application provides one or more nucleic acids encoding a homodimeric Fc fusion protein.

[0401] In some embodiments, the present application provides a host cell comprising the one or more nucleic acids encoding a homodimeric Fc fusion protein.

[0402] In some embodiments, the present application provides a method of making a homodimeric Fc fusion protein, the method comprising culturing a host cell under conditions wherein the homodimeric Fc fusion protein is produced.

[0403] In some embodiments, the present application provides a method of 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 a fraction containing the homodimeric Fc fusion protein.

[0404] In another aspect, the present application provides a method of treating cancer in a patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of a heterodimeric Fc fusion protein or a homodimeric Fc fusion protein according to any of the preceding claims.

[0405] In some embodiments, the present application provides a method of treating cancer in a patient in need thereof, the method further comprising administering a therapeutically effective amount of a checkpoint blockade antibody.

[0406] In some further embodiments, the present application provides a method 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.

[0407] In some further embodiments, the present application provides a method of treating cancer in a patient in need thereof, wherein the anti-PD-1 antibody is nivolumab, pembrolizumab, or pidilizumab.

[0408] In some additional embodiments, the present application provides a method of treating cancer in a patient in need thereof, wherein the anti-PD-Ll antibody is atezolizumab, avelumab, or durbalumab.

[0409] In some additional embodiments, the present application provides a method of treating cancer in a patient in need thereof, wherein the patient exhibits an increase in lymphocytes following administration.

[0410] In some additional embodiments, the present application provides a method of treating cancer in a patient in need thereof, wherein the patient exhibits an increase in peripheral CD8+ T cells following administration.

[0411] In some embodiments, the present application provides a method of treating cancer in a patient in need thereof, wherein the IL-12 Fc fusion protein is administered prior to the checkpoint inhibitor to increase checkpoint expression prior to treatment.

[0412] In some embodiments, the present application provides a method of treating cancer in a patient in need thereof, wherein the IL-12 Fc fusion protein is administered prior to the checkpoint inhibitor to increase checkpoint expression prior to treatment.

[0413] In another aspect, the present application provides a method of inducing T cell expansion in a patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of a heterodimeric Fc fusion protein or a homodimeric Fc fusion protein according to any of the preceding claims.

[0414] In some additional embodiments, the present application provides a method of inducing T cell expansion in a patient in need thereof, the method further comprising administering a therapeutically effective amount of a checkpoint blocking antibody.

[0415] In some additional embodiments, the present application provides a method of inducing T cell expansion in a patient in need thereof, wherein the checkpoint blocking antibody is selected from the group consisting of an anti-PD-1 antibody, an anti-PD-Ll antibody, an anti-TIM3 antibody, an anti-TIGIT antibody, an anti-LAG3 antibody, and an anti-CTLA-4 antibody.

[0416] In some additional embodiments, the present application provides a method of inducing T cell expansion in a patient in need thereof, wherein the anti-PD-1 antibody is nivolumab, pembrolizumab, or pidilizumab.

[0417] In some other additional embodiments, the present application provides a method of inducing T cell expansion in a patient in need thereof, wherein the anti-PD-Ll antibody is atezolizumab, avelumab, or durbalumab.

[0418] In some additional embodiments, the present application provides a method of inducing T cell expansion in a patient in need thereof, wherein the T cell expansion is at least a 2-fold increase in T cells.

[0419] In some embodiments, the present application provides a method of inducing T cell expansion in a patient in need thereof, wherein the IL-12 Fc fusion protein is administered prior to a checkpoint inhibitor to increase checkpoint expression prior to treatment.

[0420] In some embodiments, the present application provides a method of inducing T cell expansion in a patient in need thereof, wherein the IL-12 Fc fusion protein is administered prior to a checkpoint inhibitor to increase checkpoint expression prior to treatment.

[0421] In one aspect, the present application provides an IL-12 p40 subunit. In another aspect, the present application provides an IL-12 p35 subunit. In additional aspects, the present application provides a heterodimeric complex comprising a) an IL-12 p40 subunit and b) an IL-12 p35 subunit. BRIEF DESCRIPTION OF DRAWINGS

[0422] Figure 1A and 1B Sequences of IL-12 and its receptor are depicted.

[0423] Figures 2A-2E Useful pairs of Fc heterodimerization variants (including skew variants and pi variants) are depicted. Variants without a corresponding "monomer 2" are pi variants that can be used on either monomer alone.

[0424] Figure 3 A list of homomeric variant antibody constant regions and their corresponding substitutions is depicted. pi_(-) indicates a lower pi variant, while pi_(+) indicates a higher pi variant. These can optionally and independently be combined with other heterodimerization variants of the present application (as well as other variant types, as outlined herein).

[0425] Figure 4 Useful ablation variants (sometimes referred to as "knockout" or "KO" variants) that ablate FcyR binding are depicted. Typically, ablation variants are found on both monomers, but in some cases, the ablation variant can be on only one monomer.

[0426] Figure 5 Particularly useful embodiments of the "non-cytokine" component of the present application are shown.

[0427] Figure 6A number of exemplary domain linkers are depicted. In some embodiments, these linkers can be used to connect the IL-12 p35 subunit, IL-12 p40 subunit, or single chain IL-12 complex to the N-terminus of an Fc region. In some embodiments, these linkers can be used to fuse the IL-12 p35 subunit to the IL-12 p40 subunit in a single chain IL-12 complex. Importantly, it should be noted that the scIL-12 complex can include the IL-12 p35 linked to the IL-12 p40 at the N-terminus or the IL-12 p40 linked to the IL-12 p35 at the N-terminus. Likewise, in some cases described herein, the hinge portion of the Fc domain acts as a domain linker, which can also be combined with any of these linkers.

[0428] Figures 7A-7ESequences are shown for several useful IL-12-Fc fusion backbones based on human IgGl, without cytokine sequences (e.g., IL-12 p35 subunit, IL-12 p40 subunit, or scIL-12 complex). Backbone 1 is based on human IgGl (356E / 358M allotype) and contains C220S, S364K / E357Q:L368D / K370S skew variants on both chains, Q295E / N384D / Q418E / N421D pi variants on the chain with the L368D / K370S skew variants, and E233P / L234V / L235A / G236del / S267K ablation variants on both chains. Backbone 2 is based on human IgGl (356E / 358M allotype) and contains C220S, S364K:L368D / K370S skew variants on both chains, Q295E / N384D / Q418E / N421D pi variants on the chain with the L368D / K370S skew variants, and E233P / L234V / L235A / G236del / S267K ablation variants on both chains. Backbone 3 is based on human IgGl (356E / 358M allotype) and contains C220S, S364K:L368E / K370S skew variants on both chains, Q295E / N384D / Q418E / N421D pi variants on the chain with the L368E / K370S skew variants, and E233P / L234V / L235A / G236del / S267K ablation variants on both chains. Backbone 4 is based on human IgGl (356E / 358M allotype) and contains C220S, D401K:K360E / Q362E / T411E skew variants on both chains, Q295E / N384D / Q418E / N421D pi variants on the chain with the K360E / Q362E / T411E skew variants, and E233P / L234V / L235A / G236del / S267K ablation variants on both chains. Backbone 5 is based on human IgGl (356D / 358L allotype) and contains C220S, S364K / E357Q:L368D / K370S skew variants on both chains, Q295E / N384D / Q418E / N421D pi variants on the chain with the L368D / K370S skew variants, and E233P / L234V / L235A / G236del / S267K ablation variants on both chains.Backbone 6 is based on human IgGl (356E / 358M allotype) and contains C220S on both chains, S364K / E357Q:L368D / K370S skew variants, Q295E / N384D / Q418E / N421D pi variants on the chain with the L368D / K370S skew variants, and E233P / L234V / L235A / G236del / S267K ablation variants on both chains, and N297A variants on both chains. Backbone 7 is identical to 6 except that the mutation is N297S. Alternative versions of 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 S364K / E357Q:L368D / K370S skew variants, Q295E / N384D / Q418E / N421D pi variants on the chain with the L368D / K370S skew variants, and S228P (EU numbering, which is S241P in Kabat) variants on both chains to ablate Fab arm exchange as known in the art. Backbone 9 is based on human IgG2 and contains S364K / E357Q:L368D / K370S skew variants, Q295E / N384D / Q418E / N421D pi variants on the chain with the L368D / K370S skew variants. Backbone 10 is based on human IgG2 and contains S364K / E357Q:L368D / K370S skew variants, Q295E / N384D / Q418E / N421D pi variants on the chain with the L368D / K370S skew variants, and S267K variants on both chains. Backbone 11 is identical to Backbone 1 except that it contains M428L / N434S Xtend mutations. Backbone 12 is based on human IgGl (356E / 358M allotype) and contains C220S on both identical chains, E233P / L234V / L235A / G236del / S267K ablation variants on both identical chains. Backbone 13 is based on human IgGl (356E / 358M allotype)...

Claims

1. A heterodimeric Fc fusion protein consisting of: a) a first fusion protein consisting of an amino acid sequence consisting of the amino acid substitutions M428L / N434S in the amino acid sequence set forth in SEQ ID NO: 382; and b) a second fusion protein consisting of an amino acid sequence consisting of the amino acid substitutions M428L / N434S in the amino acid sequence set forth in SEQ ID NO: 404; wherein numbering is according to EU numbering.

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

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

4. A host cell comprising the set of expression vectors according to claim 3. culturing the host cell according to claim 4 under conditions wherein the heterodimeric Fc fusion protein is expressed; and recovering the protein.

5. A method of producing a heterodimeric Fc fusion protein, the method comprising:

5. The heterodimeric Fc fusion protein according to claim 1, wherein the first fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 383 and the second fusion protein comprises the amino acid sequence set forth in SEQ ID NO:

405.

6. The heterodimeric Fc fusion protein according to claim 1, wherein the first fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 384 and the second fusion protein comprises the amino acid sequence set forth in SEQ ID NO:

406.

7. The heterodimeric Fc fusion protein according to claim 1, wherein the first fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 385 and the second fusion protein comprises the amino acid sequence set forth in SEQ ID NO:

407.

8. The heterodimeric Fc fusion protein according to claim 1, wherein the first fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 386 and the second fusion protein comprises the amino acid sequence set forth in SEQ ID NO:

408.

9. The heterodimeric Fc fusion protein according to claim 1, wherein the first fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 387 and the second fusion protein comprises the amino acid sequence set forth in SEQ ID NO:

409.

10. The heterodimeric Fc fusion protein according to claim 1, wherein the first fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 388 and the second fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 410.

Citation Information

Patent Citations

  • Anti-tim-3 antibody

    EP2581113A1

  • Heterodimeric antibodies that bind CD3 and tumor antigens

    US10259887B2

  • Electrical socket

    US1997270A

  • Optimized Fc variants

    US20060024298A1

  • Fc variants with optimized properties

    US20060235208A1