Use and methods of IL-2, IL-13 and IL-4 cytokine bifunctional molecules

Abstract

Human interleukin 2 (IL-2), human interleukin 13 (IL-13), and / or human interleukin 4 (IL-4) cytokine fusions are provided. In particular, IL-2, IL-4, and / or IL-13 cytokine fusions are provided for use in monotherapeutic applications as well as in combination therapies to treat cancer. Also provided are pharmaceutical compositions comprising such IL-2, IL-4, and / or IL-13 cytokine fusions.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 317,876, filed on March 8, 2022, U.S. Provisional Application No. 63 / 328,689, filed on April 7, 2022, U.S. Provisional Application No. 63 / 375,132, filed on September 9, 2022, U.S. Provisional Application No. 63 / 375,299, filed on September 12, 2022, and U.S. Provisional Application No. 63 / 381,072, filed on October 26, 2022, the disclosures of which are incorporated herein by reference in their entirety. Background Art

[0003] Interleukin-2 (IL-2) is a multipotent cytokine produced primarily by activated CD4+ T cells that plays a vital role in generating a normal immune response. IL-2 promotes the proliferation and expansion of activated T lymphocytes, enhances B cell growth, and activates monocytes and natural killer cells. It is precisely because of these activities that IL-2 has been tested and approved for cancer treatment (aldesleukin, In eukaryotic cells, human IL-2 is synthesized as a 153 amino acid precursor polypeptide, from which 20 amino acids are removed to produce mature, secreted IL-2 (Taniguchi 1983). Recombinant human IL-2 has been produced in Escherichia coli (E. coli) (Rosenberg 1984), insect cells (Smith 1985), and mammalian COS cells (Taniguchi 1983).

[0004] Interleukin 2 (IL-2) is a four alpha-helical bundle, type I cytokine that was originally identified as a T cell growth factor (Morgan et al., Science 193:1007 (1976)), but has subsequently been shown to have a wide range of effects. IL-2 promotes the differentiation of CD4+ T helper cells (Zhu et al., Annual review of immunology 28:445 (2010); Liao et al., Nat Immunol 9:1288 (2008); and Liao et al., Nat Immunol 12:551 (2011)) and the development of regulatory T (Treg) cells (Cheng et al., Immunol Rev 241:63 (2011)), induces natural killer cells and cytotoxic CD8+ T cells (Liao et al., Immunity 38:13 (2013)), and mediates activation-induced cell death (AICD) (Lenardo et al., Nature 353:858 (1991)).

[0005] IL-2 exerts its effects by interacting with three different receptors: interleukin-2 receptor alpha (IL-2Rα; CD25), interleukin-2 receptor beta (IL-2Rβ; CD122), and interleukin-2 receptor gamma (IL-2Rγ; CD132; common gamma chain). The first receptor identified was IL-2Rα, a 55 kD polypeptide (p55) that appears upon T cell activation and was originally called Tac (for T activation) antigen. IL-2Rα is expressed in approximately 10 -8 M's K d Binds to IL-2 and is also known as the "high affinity" IL-2 receptor. IL-2 does not produce any detectable biological response when it binds to cells that only express IL-2Rα. In most cases, IL-2 acts through three different receptors: IL-2Rα, IL-2Rβ, and IL-2Rγ. Most cells, such as resting T cells, do not respond to IL-2 because they only express IL-2Rβ and IL-2Rγ, which have low affinity for IL-2. After stimulation, resting T cells express the IL-2 receptor IL-2Rα with relatively high affinity. IL-2 binds to IL-2Rα, causing the receptor to engage IL-2Rβ and IL-2Rγ in turn, thereby causing T cell activation. IL-2 "super factors" have been previously developed, and the effects of these super factors have been enhanced due to the enhanced binding affinity to IL-2Rβ (Levin et al., Nature 484:529 (2012)).

[0006] Despite the wealth of knowledge about IL-2 (including IL-2 superagonists), there remains a need in the art for bispecific IL-2 cytokine fusions. The present invention addresses this need by providing IL-2 superagonists or agonists as fusions with another protein. In some embodiments, the IL-2 mutein portion of the bispecific fusion comprises the substitutions L80F, R81D, L85V, I86V, and I92F, numbered according to wild-type IL-2. Summary of the Invention

[0007] IL-2 has a broad impact on the immune system and plays a crucial role in regulating immune activation and homeostasis. As immune system stimulators, bispecific IL-2 cytokine fusions have been described and are used as monotherapy and in combination with anti-PD-1 antibodies or other immune checkpoint inhibitors and / or therapeutics to treat cancer.

[0008] In some embodiments, the present invention provides a bifunctional molecule comprising (i) an IL-2-based amino acid sequence of Table 2 or Table 4, and (ii) an amino acid sequence of any one of Table 3, Table 8, Table 9, or Table 10.

[0009] In some embodiments, the present invention provides a bifunctional molecule comprising (i) an IL-4-based amino acid sequence of Table 9, and (ii) an amino acid sequence of any one of Table 2, Table 3, Table 4, Table 8, or Table 10.

[0010] In some embodiments, the present invention provides a bifunctional molecule comprising (i) an IL-13-based amino acid sequence of Table 8, and (ii) an amino acid sequence of any one of Table 2, Table 3, Table 4, Table 9, or Table 10.

[0011] In some embodiments, the invention provides a bifunctional molecule comprising (i) an amino acid sequence based on IL-7, IL-12, IL-15, IL-18 or IL-33 of Table 10, and (ii) an amino acid sequence of any one of Table 2, Table 3, Table 4, Table 8 or Table 9.

[0012] In some embodiments, the present invention provides a bifunctional molecule comprising the amino acid sequence of SEQ ID NO: 395, 484, 501, 502, 503, 504, 505, 506, 507 or 508, and the IL-2-based amino acid sequence of Table 2.

[0013] In some embodiments, the present invention provides a bifunctional molecule comprising an amino acid sequence of SEQ ID NO: 395, 484, 501, 502, 503, 504, 505, 506, 507 or 508, and an amino acid sequence based on IL-7, IL-12, IL-15 or IL-18, IL-33 in Table 10.

[0014] In some embodiments, the present invention provides a bifunctional molecule comprising the amino acid sequence of SEQ ID NO: 395, 484, 501, 502, 503, 504, 505, 506, 507 or 508, and the amino acid sequence of any one of Table 2, Table 3, Table 4, Table 5, Table 6, Table 7, Table 8, Table 9, Table 10, Table 11, Table 12, Table 13, Table 15 or Table 39.

[0015] In some embodiments, the present invention provides a bifunctional molecule comprising the amino acid sequence of SEQ ID NO: 395, 484, 501, 502, 503, 504, 505, 506, 507 or 508, and an amino acid sequence selected from the group consisting of SEQ ID NO: 6 (H9-F42A), SEQ ID NO: 7 (H9-K43N), SEQ ID NO: 8 (H9-F42A / Y45A; H9-FYAA), SEQ ID NO: 9 (H9-F42A / E62A; H9-FEAA), SEQ ID NO: 10 (H9-F42A / Y45A / E62A; H9-FYEAAA), SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31 (MDNA109 or H9), SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35 and SEQ ID NO: 146 (F42A, E62A, L80F, R81D, L85V, I86V, I92F and C125S).

[0016] In some embodiments, the bifunctional molecule comprises the following substitutions compared to wild-type IL-13: L10H, E15R, R86T, D87G, T88R, and R108K.

[0017] In some embodiments, the bifunctional molecule comprises the following substitutions compared to wild-type IL-13: L10V, E12A, V18I, R65D, D87S, T88S, L101F, K104R, and K105T.

[0018] In some embodiments, the bifunctional molecule further comprises an R39 polymorphism and / or a Q111 polymorphism.

[0019] In some embodiments, the bifunctional molecule comprises the following substitutions compared to wild-type IL-2: L80F, R81D, L85V, I86V, I92F.

[0020] In some embodiments, the bifunctional molecule further comprises the following substitutions compared to wild-type IL-2: F42A and E62A.

[0021] In some embodiments, the bifunctional molecule further comprises the following substitution compared to wild-type IL-2: C125S.

[0022] In some embodiments, the bifunctional molecule comprises the following substitutions compared to wild-type IL-4: R121K, Y124F, S125R.

[0023] In some embodiments, the bifunctional molecule comprises the following substitutions compared to wild-type IL-4: K117R, T118V, R121Q, D122S, Y124W, S125F, S128G, S129A.

[0024] In some embodiments, the present invention provides a bifunctional molecule comprising one or more amino acid sequences of any one of Table 2, Table 3, Table 4, Table 8, Table 9 or Table 10, including one or more cytokine binding portions of Table 2, Table 3, Table 4, Table 8, Table 9 or Table 10.

[0025] In some embodiments, the present invention provides a bifunctional molecule comprising one or more amino acid sequences of any one of Table 5, Table 6, Table 7, Table 11, Table 12, Table 13, Table 15, or Table 39, including one or more cytokine binding portions of Table 5, Table 6, Table 7, Table 11, Table 12, Table 13, Table 15, or Table 39.

[0026] In some embodiments, the present invention provides a bifunctional molecule comprising any one or more of the following amino acid sequences: NO:1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 ,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,8 1, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145、146、147、148、149、150、151、152、153、154、155、156、157、158、159、160、161、162、163、164、165、166、167、168、169、170、171、172、173、174、175、176、177、178、179、180、181、182、183、184、185、186、187、188、189、190、191、192、193、194、195、196、197、198、199、200、201、202、203、 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262,263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 3 23, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383 , 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 501, 502, 503, 504, 505, 506, 507, and / or 508.

[0027] In some embodiments, the bifunctional molecule further comprises an Fc domain, albumin, an anti-PD1 antibody, or an anti-CD3 antibody.

[0028] In some embodiments, the bifunctional molecule is mPD1 IgG-MDNA132L39 / Q111(KiH), huPD1 IgG-MDNA132 L39 / Q111(KiH), mPD1 IgG-MDNA109FEAAS125(KiH), huPD1 IgG-MDNA109FEAAS125(KiH), mPD1 IgG-MDNA413R39 / Q111, huPD1 IgG-MDNA413 R39 / Q111, MDNA413R39 / Q111-Fc(1:1KIH), mPD1 IgG-MDNA109FEAAS125(KiH), huPD1 IgG-MDNA109FEAAS125(KiH), mPD1 IgG-MDNA413R39 / Q111, huPD1 IgG-MDNA413 R39 / Q111, MDNA413R39 / Q111-Fc(1:1KIH), huPD1 IgG-MDNA109FEAAC125(KiH), mPD1 IgG-MDNA109FEAAC125(KiH), m anti-PD1-MDNA132.15(1:1KIH), hu anti-PD1-MDNA109FEAA-T3A-C125S(1:1KIH), huPD1-MDNA109FEAA(KiH), mPD1-MDNA109FEAA(KiH), MDNA109FEAA -Fc-MDNA132.15(2:1:1KIH), MDNA132.15-Fc-MDNA413(1:1:2KIH), huPD1-MDNA109F EAA(KiH)*, mPD1-MDNA109FEAA(KiH)*, anti-mPD1-MDNA109(KIH) or anti-huPD1-MDNA109(KIH).

[0029] In some embodiments, the bifunctional molecule comprises SEQ ID NO: 395 (MDNA132.15).

[0030] In some embodiments, the bifunctional molecule comprises SEQ ID NO: 484 (MDNA132R.15).

[0031] In some embodiments, the bifunctional molecule comprises SEQ ID NO: 501 (MDNA132-Q111).

[0032] In some embodiments, the bifunctional molecule comprises SEQ ID NO: 502 (MDNA132-R111).

[0033] In some embodiments, the bifunctional molecule comprises SEQ ID NO: 503 (cpMDNA132.15-Q111).

[0034] In some embodiments, the bifunctional molecule comprises SEQ ID NO: 504 (cpMDNA132.15-R111).

[0035] In some embodiments, the bifunctional molecule comprises SEQ ID NO: 505 (cpMDNA132.15-Q111-PE).

[0036] In some embodiments, the bifunctional molecule comprises SEQ ID NO: 506 (cpMDNA132.15-R111-PE).

[0037] In some embodiments, the bifunctional molecule comprises SEQ ID NO: 507 (MDNA132.15-Q111-PE).

[0038] In some embodiments, the bifunctional molecule comprises SEQ ID NO: 508 (MDNA132.15-R111-PE).

[0039] In some embodiments, the bifunctional molecule comprises an IL-2 based sequence that exhibits increased binding affinity to CD122 (IL-2Rβ) compared to wild-type human IL-2.

[0040] In some embodiments, the bifunctional molecule comprises an IL-2 based sequence that exhibits increased binding to IL-2Rβ compared to wild-type human IL-2.

[0041] In some embodiments, the bifunctional molecule comprises an IL-2 based sequence that exhibits abolished and / or no IL2Rα binding.

[0042] In some embodiments, the IL-2-based sequence further comprises the following amino acid substitutions: F42A and / or E62A, wherein the numbering is according to wild-type human IL-2 of SEQ ID NO: 2.

[0043] In some embodiments, the bifunctional molecule exhibits reduced binding affinity for CD25 (IL-2Rα), induces expansion of immune cells (including CD8 T cells and NK cells), and / or induces activation of effector immune cells (including CD8 T cells and NK cells).

[0044] In some embodiments, the bifunctional molecule comprises an IL-2 based sequence that exhibits reduced affinity for CD25 compared to wild-type human IL-2.

[0045] In some embodiments, the bifunctional molecule induces limited and / or no activity in the expansion and / or activation of immunosuppressive regulatory T cells (Tregs).

[0046] In some embodiments, the bifunctional molecule binds to IL-2R and PD1 on a target cell.

[0047] In some embodiments, the bifunctional molecule comprises a cytokine binding portion and an anti-PD1 antibody, and:

[0048] i) induce activation of tumor-infiltrating CD8+ T cells; and

[0049] ii) preventing the depletion of the same tumor-infiltrating CD8+ T cells as in i).

[0050] In some embodiments, the cytokine binding moiety and the anti-PD1 antibody are covalently linked.

[0051] In some embodiments, tumor-infiltrating CD8+ T cells are analyzed for expression of one or more of the following markers: inhibitory PD1 receptor, TIM3, and / or cytotoxic granzyme B.

[0052] In some embodiments, the bifunctional molecule induces a decrease in expression of the inhibitory PD1 receptor and / or induces a decrease in TIM3 expression in CD8+ T cells compared to untreated cells and / or cells treated with the cytokine binding portion and the anti-PD1 antibody that are not covalently linked.

[0053] In some embodiments, the bifunctional molecule induces an increase in granzyme expression in tumor-infiltrating CD8+ T cells compared to untreated cells and / or cells treated with the cytokine binding moiety and the anti-PD1 antibody that are not covalently linked.

[0054] In some embodiments, the bifunctional molecule binds to IL-2R and CD3 on a target cell.

[0055] In some embodiments, the bifunctional molecule comprises a cytokine binding moiety and an anti-CD3 antibody.

[0056] In some embodiments, the bifunctional molecule comprises an IL-13 based sequence that exhibits increased binding affinity to IL-13Rα1 and decreased binding affinity to IL-13Rα2.

[0057] In some embodiments, the bifunctional molecule exhibits an increased binding affinity for IL-13Rα1 of at least 5-fold, 8-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 110-fold, 120-fold or more, and wherein the decreased binding affinity for IL-13Rα2 is at least 30-fold, 40-fold, 50-fold or 60-fold or more.

[0058] In some embodiments, the bifunctional molecule blocks pSTAT6 signaling by at least 20%, at least 30%, at least 40%, or at least 50%.

[0059] In some embodiments, the bifunctional molecule blocks IL-13-induced TF-I proliferation by at least 20%, at least 30%, at least 40%, or at least 50%.

[0060] In some embodiments, the bifunctional molecule blocks IL-4 and / or IL-13-induced M2 macrophage polarization by at least 20%, at least 30%, at least 40%, or at least 50%.

[0061] In some embodiments, the bifunctional molecule comprises an IL-4-based sequence that exhibits at least 5-fold, 8-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 110-fold, 120-fold increased specific binding to type I or type II IL-4R compared to native IL-4.

[0062] In some embodiments, the bifunctional molecule comprises an IL-2 mutein or an IL-2 based sequence comprising a sequence selected from the group consisting of:

[0063] a. SEQ ID NO: 271, 272 and 273;

[0064] b. SEQ ID NO: 274, 275 and 276;

[0065] c. SEQ ID NO: 283, 284 and 285; and

[0066] d. SEQ ID NO: 365, 366 and 367.

[0067] In some embodiments, the bifunctional molecule is covalently linked to an antibody selected from the group consisting of dupilumab, nivolumab, BMS-936558, MDX-1106, ONO-4538, AMP224, CT-011, and MK-3475 (pembrolizumab or ), cemiplimab (REGN2810), SHR-1210 (CTR20160175 and CTR20170090), SHR-1210 (CTR20170299 and CTR20170322), JS-001 (CTR20160274), IBI308 (CTR20160735) and / or BGB-A317 (CTR20160872).

[0068] In some embodiments, the bifunctional molecule is covalently linked to an antibody selected from the group consisting of: anti-CTLA4 mAb, such as ipilimumab, tremelimumab; anti-PD-L1 antagonist antibodies, such as BMS-936559 / MDX-1105, MEDI4736, RG-7446 / MPDL3280A; anti-LAG-3, such as IMP-321; agonistic antibodies targeting immunostimulatory proteins, including anti-CD40 mAb, such as CP-870,893, lucatumumab, dacetuzumab; anti-CD137 mAbs (anti-4-1-BB antibodies), such as BMS-663513 urelumab (anti-4-1BB antibody; see, e.g., U.S. Pat. Nos. 7,288,638 and 8,962,804, herein incorporated by reference in their entireties), lirilumab (anti-KIR mAB; IPH2102 / BMS-986015; blocks NK cell inhibitory receptors), and PF-05082566 (utomilumab; see, e.g., U.S. Pat. Nos. 8,821,867, 8,337,850, and 9,468,678, and International Patent Application Publication No. WO 2012 / 032433, herein incorporated by reference in their entireties); anti-OX40 mAbs (see, e.g., WO 2006 / 029879 or WO 2010 / 096418, which is incorporated herein by reference in its entirety); anti-GITR mAbs, such as TRX518 (see, e.g., U.S. Patent No. 7,812,135, which is incorporated herein by reference in its entirety); anti-CD27 mAbs, such as varlilumab CDX-1127 (see, e.g., WO 2016 / 145085 and U.S. Patent Publication Nos. US2011 / 0274685 and US2012 / 0213771, which are incorporated herein by reference in their entirety); anti-ICOS mAbs (e.g., MEDI-570, JTX-2011; and anti-TIM-3 antibodies (see, e.g., WO 2013 / 006490 or U.S. Patent Publication No. US2016 / 0257758, which are incorporated herein by reference in their entirety), Herceptin, anti-EGFR, anti-VEGF, anti-TIGIT, anti-LAG3, anti-CD8, anti-CD47, anti-sirs-α and / or anti-CD112R.

[0069] In some embodiments, the present invention provides a composition comprising any one or more amino acid sequences of SEQ ID NO: 395, 484, 501, 502, 503, 504, 505, 506, 507 or 508.

[0070] In some embodiments, the present invention provides a composition comprising the amino acid sequence of SEQ ID NO: 395 (MDNA132.15).

[0071] In some embodiments, the present invention provides a composition comprising the amino acid sequence of SEQ ID NO: 484 (MDNA132R.15).

[0072] In some embodiments, the present invention provides a composition comprising the amino acid sequence of SEQ ID NO: 501 (MDNA132-Q111).

[0073] In some embodiments, the present invention provides a composition comprising the amino acid sequence of SEQ ID NO: 502 (MDNA132-R111).

[0074] In some embodiments, the present invention provides a composition comprising the amino acid sequence of SEQ ID NO: 503 (cpMDNA132.15-Q111).

[0075] In some embodiments, the present invention provides a composition comprising the amino acid sequence of SEQ ID NO: 504 (cpMDNA132.15-R111).

[0076] In some embodiments, the present invention provides a composition comprising the amino acid sequence of SEQ ID NO: 505 (cpMDNA132.15-Q111-PE).

[0077] In some embodiments, the present invention provides a composition comprising the amino acid sequence of SEQ ID NO: 506 (cpMDNA132.15-R111-PE).

[0078] In some embodiments, the present invention provides a composition comprising the amino acid sequence of SEQ ID NO: 507 (MDNA132.15-Q111-PE).

[0079] In some embodiments, the present invention provides a composition comprising the amino acid sequence of SEQ ID NO: 508 (MDNA132.15-R111-PE).

[0080] In some embodiments, the invention provides a nucleic acid encoding a bifunctional molecule or composition as described herein.

[0081] In some embodiments, the present invention provides a vector comprising a nucleic acid as described herein.

[0082] In some embodiments, the present invention provides a method of treating cancer in a subject in need thereof, comprising administering a bifunctional molecule or composition as described herein.

[0083] In some embodiments, the present invention provides a method of treating cancer in a subject in need thereof, the method comprising administering a nucleic acid encoding a bifunctional molecule or composition as described herein.

[0084] In some embodiments, the present invention provides a method of treating cancer in a subject in need thereof, the method comprising administering a vector comprising a nucleic acid encoding a bifunctional molecule or composition as described herein.

[0085] In some embodiments, the cancer is a solid tumor.

[0086] In some embodiments, the cancer is selected from the group consisting of sarcoma, carcinoma, head and neck cancer, glioblastoma, bladder cancer, oral cancer, mesothelioma, pancreatic cancer, liver cancer, colorectal cancer, lung cancer, skin cancer, lymphoma, gastrointestinal cancer, prostate cancer, ovarian cancer, breast cancer, basaloid breast tumor, endometrial cancer, multiple myeloma, melanoma, lymphoma, lung cancer (including small cell lung cancer), kidney cancer, gastric cancer, brain cancer, and CNS tumors.

[0087] In some embodiments, the cancer is colon cancer.

[0088] In some embodiments, the present invention provides a method of treating a viral disease in a subject in need thereof, the method comprising administering a vector comprising a nucleic acid encoding a bifunctional molecule or composition as described herein.

[0089] In some embodiments, the viral disease is human papillomavirus (HPV) and / or hepatitis, such as hepatitis A, hepatitis B, hepatitis C, and / or hepatitis D.

[0090] A method of treating cancer comprising administering a combination therapy comprising:

[0091] (i) therapeutic antibodies, and

[0092] (ii) a bifunctional molecule or composition as described herein, optionally wherein the bifunctional molecule comprises an IL-2, IL-4 or IL-13 based sequence as described herein.

[0093] In some embodiments, the therapeutic antibody is an anti-PD-1 antibody or inhibitor, or an anti-PD-L1 antibody or inhibitor.

[0094] In some embodiments, the therapeutic antibody or inhibitor is selected from the group consisting of: dupilumab, nivolumab BMS-936558, MDX-1106, ONO-4538, AMP224, CT-011, and MK-3475 (pembrolizumab or ), seplizumab (REGN2810), SHR-1210 (CTR20160175 and CTR20170090), SHR-1210 (CTR20170299 and CTR20170322), JS-001 (CTR20160274), IBI308 (CTR20160735) and / or BGB-A317 (CTR20160872); anti-CTLA4 mAbs, such as ipilimumab, tremelimumab; anti-PD-L1 antagonist antibodies, such as BMS-936559 / MDX-1105, MEDI4736, RG-7446 / MPDL3280A; anti-LAG-3, such as IMP-321; agonistic antibodies targeting immunostimulatory proteins, including anti-CD40 mAbs, such as CP-870,893, rukatumomab, dacitumomab; anti-CD137 mAbs (anti-4-1-BB antibodies), such as BMS-663513, urelumab (anti-4-1BB antibody; see, e.g., U.S. Pat. Nos. 7,288,638 and 8,962,804, herein incorporated by reference in their entireties), lirelumab (anti-KIR mAB; IPH2102 / BMS-986015; blocks NK cell inhibitory receptors), and PF-05082566 (utemilumab; see, e.g., U.S. Pat. Nos. 8,821,867, 8,337,850, and 9,468,678, and International Patent Application Publication No. WO 2012 / 032433, herein incorporated by reference in their entireties); anti-OX40 mAbs (see, e.g., WO 2006 / 029879 or WO 2010 / 096418, which are incorporated herein by reference in their entireties); anti-GITR mAbs, such as TRX518 (see, e.g., U.S. Patent No. 7,812,135, which is incorporated herein by reference in its entirety); anti-CD27 mAbs, such as valirumab CDX-1127 (see, e.g., WO 2016 / 145085 and U.S. Patent Publication Nos. US2011 / 0274685 and US2012 / 0213771, which are incorporated herein by reference in their entireties); anti-ICOS mAb (e.g., MEDI-570, JTX-2011; and anti-TIM-3 antibodies (see, e.g., WO2013 / 006490 or U.S. Patent Publication No. US2016 / 0257758, which are incorporated herein by reference in their entirety), Herceptin, anti-EGFR, anti-VEGF, anti-TIGIT, anti-LAG3, and / or anti-CD112R).

[0095] In some embodiments, the anti-PD-1 antibody or inhibitor is selected from the group consisting of: nivolumab BMS-936558, MDX-1106, ONO-4538, AMP224, CT-011, and MK-3475 (pembrolizumab or ), seplizumab (REGN2810), SHR-1210 (CTR20160175 and CTR20170090), SHR-1210 (CTR20170299 and CTR20170322), JS-001 (CTR20160274), IBI308 (CTR20160735), BGB-A317 (CTR20160872), and PD-1 antibodies as listed in Table 38.

[0096] In some embodiments, the anti-PD-L1 antibody or inhibitor is selected from the group consisting of atezolizumab, avelumab, and durvalumab.

[0097] In some embodiments, the cancer is selected from the group consisting of sarcoma, carcinoma, head and neck cancer, glioblastoma, bladder cancer, oral cancer, mesothelioma, pancreatic cancer, liver cancer, colorectal cancer, lung cancer, skin cancer, lymphoma, gastrointestinal cancer, prostate cancer, ovarian cancer, breast cancer, basaloid breast tumor, endometrial cancer, multiple myeloma, melanoma, lymphoma, lung cancer (including small cell lung cancer), kidney cancer, stomach cancer, and brain cancer.

[0098] In some embodiments, the cancer is colon cancer.

[0099] In some embodiments, the present invention provides a pharmaceutical composition comprising a bifunctional molecule as described herein and a pharmaceutically acceptable carrier.

[0100] A pharmaceutical composition comprising an anti-PD-1 antibody or inhibitor, a bifunctional molecule or composition as described herein, and a pharmaceutically acceptable carrier, wherein optionally the anti-PD-1 antibody or inhibitor and the bifunctional molecule are covalently linked.

[0101] A pharmaceutical composition comprising an anti-PD-L1 antibody or inhibitor, a bifunctional molecule or composition as described herein, and a pharmaceutically acceptable carrier, wherein optionally the anti-PD-L1 antibody or inhibitor and the bifunctional molecule are covalently linked.

[0102] A pharmaceutical composition comprising an anti-CD3 antibody or inhibitor, a bifunctional molecule or composition as described herein, and a pharmaceutically acceptable carrier, wherein optionally the anti-CD3 antibody or inhibitor and the bifunctional molecule are covalently linked.

[0103] A pharmaceutical composition comprising a therapeutic antibody or inhibitor, a bifunctional molecule or composition as described herein, and a pharmaceutically acceptable carrier, wherein optionally the therapeutic antibody or inhibitor and the bifunctional molecule are covalently linked.

[0104] In some embodiments, the present invention provides a use according to any of the preceding method paragraphs comprising administering a bifunctional molecule or composition as described herein for treating cancer in a subject in need thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0105] Figure 1: provides examples of IgG1, IgG2, IgG3 and IgG4 sequences.

[0106] Figure 2 : Exemplary H9-Fc fusion sequences are provided.

[0107] Figure 3 : Exemplary oncolytic viruses.

[0108] Figure 4 : Targeting immunologically “cold tumors” by modulating the TME with an IL-2 / IL-13 bispecific superkine. “Cold” tumors are unresponsive to checkpoint inhibitors due to a pro-tumor TME: 1) Low CD8 + and NK cell counts; high Treg counts, and 2) high numbers of immunosuppressive myeloid cells (ie, TAMs and MDSCs).

[0111] 5A to 5T : Bispecific sequence information for various construct embodiments.

[0112] Figure 6 : Role of IL-4 and IL-13 receptors in cancer.

[0113] Figure 7 :The mechanism of action of the dual-specific cytokine (DUCK Cancer) MDNA109FEAA-Fc-MDNA413.

[0114] Figure 8 :Mouse anti-PD1-MDNA109FEAA S125 Results of SPR analysis of binding to human IL-2Rα (CD25) and human IL-2Rβ (CD122).

[0115] Figure 9 :Mouse anti-PD1-MDNA109FEAA S125 SPR analysis results of binding to human PD-1 and mouse PD-1.

[0116] Figure 10A: Human anti-PD1-MDNA109FEAA S125 SPR analysis results of binding to human PD-1 and mouse PD-1.

[0117] Figure 10B :IL2-Fc, mouse anti-PD1-MDNA109FEAA C125 and human anti-PD1-MDNA109FEAA C125 Results of SPR analysis of binding to human IL-2Rα (CD25) and human IL-2Rβ (CD122).

[0118] Figures 11A to 11C : Results of PD-1 reporter assay.

[0119] 12A to 12E : Results of pSTAT5 phosphorylation reporter assay.

[0120] Figure 13 : Results of the Jurkat IL2Rβγ bioassay.

[0121] Figure 14 : Schematic diagram of the study design for the IT treatment study in the CT26 model.

[0122] Figure 15 : Tumor growth inhibition in the CT26 colon cancer model via IT treatment.

[0123] Figure 16 : Tumor growth inhibition in IP treatment studies in the CT26 colon cancer model.

[0124] Figure 17 :Mouse anti-PD1-MDNA109FEAA C125 Survival curves of CT26 colon cancer treated intraperitoneally (IP).

[0125] Figure 18 : Schematic diagram of the study design for IP treatment studies in the B16F10 model.

[0126] Figure 19 : Tumor growth inhibition in a B16F10 melanoma model tumor growth inhibition study.

[0127] Figure 20 : Survival curves in a treatment study of the B16F10 melanoma cell line.

[0128] Figure 21 : Results of CTLL-2 assay.

[0129] Figure 22 : Research overview of in vivo CT26 colon tumor efficacy studies.

[0130] Figure 23 : Results of an in vivo CT26 colon tumor efficacy study.

[0131] Figure 24 : Results of an in vivo CT26 colon tumor efficacy study.

[0132] FIG. 25A to FIG. 25B :Fc-MDNA413 R39 / Q111 SPR analysis of the binding of (1:2) to IL-13Rα1 and IL-13Rα2 ( Figure 25A ), and Fc-MDNA413 R39 / Q111 SPR analysis of (1:2) binding to mouse and cynomolgus monkey IL-13Rα1 ( Figure 25B ).

[0133] Figure 26 :MDNA413 R39 / Q111 -Fc KIH, MDNA109FEAA C125 -Fc-MDNA413 R39 / Q111 (2:1:2), Fc-MDNA413 R39 / Q111 (1:2) and MDNA413 R39 / Q111 -Fc-MDNA132 L39 / Q111 HEK Blue IL-4 competition assay by KIH.

[0134] Figure 27 :MDNA413 R39 / Q111 -Fc KIH, MDNA109FEAA C125 -Fc-MDNA413 R39 / Q111 、Fc-MDNA413 R39 / Q111 (1:2) and MDNA413 R39 / Q111 -Fc-MDNA132 L39 / Q111 HEK Blue IL-13 competition assay by KIH.

[0135] Figure 28 :Fc-MDNA413 R39 / Q111 (1:2) showed dose-dependent inhibition of TF-1 proliferation at both EC50 (upper panel) and EC80 (lower panel) of rhIL-13.

[0136] Figure 29 :Fc-MDNA413 R39 / Q111 Phenotypic analysis of macrophages treated with IL-4 in the presence of (1:2). Dotted line represents: IL-4 only control. Dashed line represents: M0 macrophage control.

[0137] Figure 30 :Fc-MDNA413R39 / Q111 Phenotypic analysis of macrophages treated with IL-13 in the presence of (1:2). Dotted line represents: IL-13 only control. Dashed line represents: M0 macrophage control.

[0138] Figures 31A to 31E :Fc-MDNA413 R39 / Q111 (1:2) growth inhibition of various tumor types.

[0139] Figure 32: Fc-MDNA413 R39 / Q111 (1:2), MDNA19 / MDNA109FEAA C125 -Fc, Fc-MDNA413 R39 / Q111 (1:2)+MDNA19 / MDNA109FEAA C125 -Fc combination or Fc-MDNA413 R39 / Q111 (1:2)+MDNA109FEAA C125 -Fc-MDNA413 R39 / Q111 (2:1:2) combination inhibited tumor growth in the B16F10 melanoma model.

[0140] Figures 33A to 33B :Fc-MDNA413 R39 / Q111 (1:2), Fc-MDNA413 R39 / Q111 (1:2)+MDNA19 / MDNA109FEAA C125 -Fc combination, Fc-MDNA413 R39 / Q111 (1:2)+anti-PD-1 antibody combination, MDNA19 / MDNA109FEAA C125 B16F10 melanoma tumor growth inhibition by -Fc and anti-PD-1 antibodies.

[0141] Figure 34 : Results of HEK Blue IL-2 assay.

[0142] Figure 35 : Results of the Jurkat IL-2 bioassay.

[0143] Figure 36 :Mouse anti-PD1-MDNA109FEAA C125 PK properties upon IP (intraperitoneal) administration.

[0144] Figures 37A to 37C :Mouse anti-PD1-MDNA109FEAA C125 PD characteristics upon IP (intraperitoneal) administration.

[0145] Figure 38 : Results of E0771 tumor growth inhibition studies.

[0146] Figure 39 : Body weights of animals during the course of the Fc-MDNA413R39 / Q111 (1:2) MTD study.

[0147] Figure 40 : MTD study of Fc-MDNA413 after first and second doses as indicated R39 / Q111 (1:2) PK properties.

[0148] Figures 41A to 41B :Fc-MDNA413 R39 / Q111 (1:2) for B16F10 melanoma ( Figure 41A ) and CT26 colon cancer ( Figure 41B ) Inhibition of tumor growth.

[0149] Figures 42A to 42C : Results of the SPR studies described in Example 5.

[0150] Figures 43A to 43B : Results of the IL13Ra1 and IL13Ra2 binding assay described in Example 5.

[0151] Figures 44A to 44C : Results of the CD3 epitope and CD3ε / δ binding assay described in Example 5.

[0152] Figures 45A to 45C : Results of the IL2Rα and IL2Rβ binding assay described in Example 5.

[0153] Figure 46 : Results of the in vivo imaging analysis described in Example 5.

[0154] Figures 47A to 47C : Results of the PTNG binding affinity analysis described in Example 5.

[0155] Figures 48A to 48C : Results of the MDNA132 BiSKIT binding affinity analysis described in Example 5.

[0156] Figure 49 : Results of the Fc-MDNA132L39 / Q111 (1:1 KIH) binding assay described in Example 5.

[0157] Figure 50 : Results of the mouse anti-CD3-MDNA132L39 / Q111 (1:1 KIH) binding assay described in Example 5.

[0158] Figure 51 : Results of the receptor internalization assay described in Example 5.

[0159] Figure 52 : Results of the Jurkat IL2Rβγ bioassay described in Example 5.

[0160] Figure 53 : Results of the PD1 reporter assay described in Example 5.

[0161] Figure 54 Schematic diagram of MDNA11. The IL-2 portion contains mutations to enhance affinity for CD122 and prevent binding to CD25, while the albumin portion extends in vivo half-life and promotes tumor accumulation.

[0162] Figure 55 : Body weight of BALB / c mice treated with MDNA11 according to SUD or fixed dosing schedule. Each line corresponds to a single mouse. SUD (Groups #1-4) and fixed dose (Group #5). Arrows indicate administration of MDNA11 at the indicated dose.

[0163] Figure 56 : Body weight of MDNA11 mice treated with MDNA11 by SC injection. (A) Average body weight of treatment groups. Individual body weights of mice in each treatment group are shown in (B)-(F). Dotted vertical lines (study days 1 and 8) indicate MDNA11 administration.

[0164] Figure 57 : Survival curves of mice treated by SC injection of MDNA11. Kaplan-Meier plots show the overall survival rate of each group. Mice surviving the end of the study were censored on day 15. N=3 per group.

[0165] Figure 58 : Binding of MDNA11 and MDNA19 to CD122. Sensorgrams of MDNA11 (top) and MDNA19 (bottom) show binding to human CD122. Unlabeled (left) and Vivo Tag800-labeled constructs (right) show highly similar binding properties to CD122.

[0166] Figure 59 : Binding of MDNA11, MDNA19, and rhIL-2 to CD25. Sensorgrams of MDNA11 (top), MDNA19 (center), and rhIL-2 binding to human CD25. Unlabeled (left) and Vivo Tag800-labeled (right) MDNA11 and MDNA19 showed no binding to CD25.

[0167] Figure 60: In vivo and ex vivo IVIS imaging of CT26 tumor-bearing mice. (A) In vivo imaging after administration of VivoTag800-labeled MDNA19 (left) or MDNA11 (center). In all figures, the mouse on the far right is a PBS-treated control animal. (B) Ex vivo IVIS imaging of CT26 tumors in mice treated with VivoTag800-MDNA19 (left) and VivoTag800-MDNA11 (right) collected 144 hours after dosing (end of study). White arrows indicate tumors in mice treated with MDNA19; black arrows indicate tumors in control mice treated with PBS.

[0168] Figure 61 : Proposed mechanism of action of MDNA223 (anti-PD1-MDNA109FEAA): MDNA109FEAA binds to a medium-affinity receptor (CD122) for cis-activation, leading to selective stimulation of effector T cells, and anti-PD1 simultaneously blocks the co-inhibitory PD1 on the same cell.

[0169] Figure 62 : Survival curves for all groups as indicated in the subcutaneous MTD study in Balb / c mice.

[0170] Figure 63 Figure 2: Body weights of animals at the MTD of MDNA223. All animals were treated with 0.5 mg / kg in week 1 and 1 mg / kg in week 2. Animals were treated as indicated in the figure in weeks 3 and 4. In week 5, all animals were treated with 8 mg / kg. Data are presented as mean ± SEM.

[0171] Figure 64 : B16F10 melanoma model (a) Average tumor measurements for groups 1-6 treated as indicated in a tumor growth inhibition study (IP treatment). The downward black arrow indicates the dosing schedule (once a week x 3 weeks). Data are presented as mean + SEM, (b) percent inhibition of tumor growth for the indicated groups on day 14 of the study, (c) survival curves for the indicated groups in the study, and (d) a table showing percent survival at the end of the study.

[0172] Figure 65 : E0771 breast cancer model (a) Average tumor measurements for groups 1-5 treated as indicated in a tumor growth inhibition study (IP treatment). The downward black arrow indicates the dosing schedule (once a week x 2 weeks). Data are represented by mean+SEM, (b) percent inhibition of tumor growth for the indicated group on day 15 of the study, (c) survival curves for the indicated groups in the study, and (d) a table showing percent survival at the end of the study.

[0173] Figure 66: CT26 Colon Model (a) Tumor measurements for all indicated groups in the CT26 colon cancer tumor growth inhibition study. Data are presented as mean ± SEM. Tumor volume was measured for any animal euthanized prematurely or found dead to determine the group mean. (b) Percent inhibition of tumor growth for the indicated groups on day 15 of the study, (c) Survival curves for the indicated groups in the study, and (d) Table showing percent survival at the end of the study.

[0174] Figure 67 PK ELISA analysis: Time and concentration data are presented on the log10 y-axis. Error bars represent the standard error of the mean of technical replicates. To allow for plotting on a logarithmic scale, zero values ​​were replaced with a value of 1.0 ng / mL. (a) SQ route of administration, (b) IV route of administration, (c) IP route of administration.

[0175] Figure 68 Flow cytometric analysis of CD4, CD8, NK, and Treg proliferation marker Ki67: After treatment with MDNA223 (IV, IP, or SQ), samples were taken on the indicated days, and cells were isolated and stained with markers for analysis. Data are presented as mean ± SEM.

[0176] Figure 69 Flow cytometric analysis of the absolute number of CD4, CD8, NK, and Treg cells (as a percentage of CD45+ cells): After treatment with MDNA223 (IV, IP, or SQ), samples were taken on the indicated days, and cells were isolated and stained with markers for analysis. Data are presented as mean ± SEM.

[0177] Figure 70 Figure 2: Flow cytometric analysis of TILs in B16F10 tumors. Tumors were harvested and processed for flow cytometry on day 7 post-dose as indicated. Data are presented as mean ± SEM: (a) percentage of CD45+ cells in tumor, (b) intratumoral CD8+ T cells per gram of tumor, (c) intratumoral CD4+ T cells per gram of tumor, (d) intratumoral NK cells per gram of tumor, and (e) intratumoral Tregs per gram of tumor.

[0178] Figure 71 Figure 2: Flow cytometric analysis of TILs in B16F10 tumors. Tumors were harvested on day 7 post-dose and processed for flow cytometry as indicated. Data are presented as mean + SEM of CD8+ T cell to Treg ratio.

[0179] Figure 72Figure 2: Flow cytometric analysis of TILs in B16F10 tumors. Tumors were harvested and processed for flow cytometry on day 7 post-dose as indicated. Data are presented as mean ± SEM for the following: (a) CD8+PD1+ T cell population, (b) CD8+Tim3-GrzB+ T cell population, and (c) CD8+Tim3+GrzB- T cell population.

[0180] Figure 73 : (a) Mean tumor growth graphs for the indicated groups in the E0771 tumor growth inhibition study. Data are presented as mean + SEM, (b) Survival curves for the indicated treatments.

[0181] Figure 74 : Principles of IL13R SPR studies of mouse and cynomolgus monkey receptors.

[0182] Figure 75 Figure 2: Plot of Fc-MDNA413 (R39) plasma concentrations after each dose. Mean Fc-MDNA413 concentrations for each group and time point after administration are presented on a logarithmic scale. Bars represent the standard error of the mean. Values ​​below the limit of quantification are assigned a value of 1 ng / mL for logarithmic plotting.

[0183] Figure 76 Figure 2: Fc-MDNA413 (R39) drug exposure combination plot. Mean Fc-MDNA413 concentrations for each group and time point are presented on a logarithmic scale. Bars represent the standard error of the mean. Values ​​below the limit of quantification were assigned a value of 1 ng / mL to facilitate plotting on a logarithmic scale.

[0184] Figure 77 : Body weight of CT26 colon cancer animals: Animals were treated as indicated and body weights were measured twice weekly throughout the study. Data are presented as mean and standard deviation.

[0185] Figure 78 Figure 26: Tumor growth curves for the CT26 colon cancer model (a) vehicle and Fc-MDNA413 groups. Data points represent the mean values ​​for each group. Error bars represent the standard error of each data point. Any animals euthanized prematurely or found dead undergo tumor volume measurements to determine the group mean value. The downward arrows represent the dosing day, (b) the percentage inhibition of tumor growth on the 18th and 21st days of the study, and (c) the survival curves for each group as indicated.

[0186] Figure 79 Animal body weights in the B16F10 melanoma model: Animals were treated as indicated and body weights were measured twice weekly throughout the study. Data are presented as mean and standard deviation. (a) Experiment 1, (b) Experiment 2.

[0187] Figure 80Figure 2: B16F10 melanoma model. (a) Tumor measurements for the indicated groups. Data points represent the mean for each group. Error bars represent the standard error for each data point. Tumor volume was measured for any animal euthanized prematurely or found dead to determine the group mean. (b) Percent inhibition of tumor growth on study day 22 (experiment #1).

[0188] Figure 81 : B16F10 melanoma model: Survival curves for the groups as indicated (Experiment #1). The table shows the percentage of survival for each group at the end of the study.

[0189] Figure 82 Figure 2: B16F10 melanoma model (experiment #2). Tumor measurements for the indicated groups. Data points represent the mean for each group. Error bars represent the standard error for each data point. Tumor volume was measured for any animal euthanized prematurely or found dead to determine the group mean. (a) Combination of Fc-MDNA413 and MDNA19, (b) Combination of Fc-MDNA413 and anti-PD-1, (c) Percentage inhibition of tumor growth on day 15 of the study.

[0190] Figure 83 : B16F10 melanoma model (experiment #2). Survival curves for animals treated in the study are as indicated. (a) Fc-MDNA413 in combination with MDNA19, (b) Fc-MDNA413 in combination with anti-PD-1, (c) The table shows the percentage of survival for each group at the end of the study.

[0191] Figure 84 : Principle of IL-13R SPR studies to test binding affinity.

[0192] Figure 85 : Representative sensorgrams of various constructs as indicated, showing binding affinity to human IL-13Rα1 and IL-13Rα2.

[0193] Figure 86 : Flow cytometry data showing the transduction efficiency of IL-13Rα2 in EMT6 cells. A375 cells were used as a positive control for flow cytometry analysis because they constitutively express IL-13Rα2.

[0194] Figure 87 : Flow cytometric analysis of IL-13 decoy receptor expression in EMT6 and EMT6 / IL-13Rα2 tumors of Balb / c mice.

[0195] Figure 88 : Receptor internalization data plotted against MFI data over time. Cells were treated with ligands as indicated and tested for surface ligand binding using FITC-conjugated anti-Fc antibodies at various time points.

[0196] Figure 89 : Principle of IL13R SPR studies of the human IL-13 receptor. The left figure shows the principle used to test Fc fusions, while the right figure shows the principle used to test antibody fusions.

[0197] Figure 90 : Assay principle: The construct active Fc-MDNA132.15 was captured directly on a CM5 chip (right panel) or via an anti-human IgG (Fc) antibody (left panel). The analyte was human IL-13 receptor (left panel) or human Fc receptor (right panel).

[0198] Figure 91 : Representative sensorgrams of various constructs showing binding affinity to human IL13Rα1 and IL13Rα2.

[0199] Figure 92 : Representative sensorgrams of various constructs showing binding affinity to human CD25 and CD122.

[0200] Figure 93 : Representative sensorgrams of various constructs showing binding affinity to mouse PD1.

[0201] Figure 94 : Representative sensorgrams. The binding affinity of active Fc-MDNA132.15 to human IL-13Rα1 (left) and human IL-13Rα2 (right) was tested.

[0202] Figure 95 : Representative sensorgrams. The binding affinity of active Fc-MDNA132.15 to human CD32b / c (left) and human CD16a (right) was tested.

[0203] Figure 96 Figure 2: hIL-13 dose-response plot generated in a surrogate antagonist assay. OD650nm is plotted as a function of hIL-13 concentration on a semi-logarithmic plot. The four-parameter logistic curve fit is shown as a solid line. Error bars represent the standard error of the mean for replicate wells.

[0204] Figure 97 : Representative sensorgrams of unlabeled and labeled Fc-MDNA132.15 showing binding affinity to mouse IL13Rα2.

[0205] Figure 98 : In vivo imaging data at the indicated time points. In all figures, the two mice on the far right were not treated with Fc-MDNA132.15 and served as controls. As observed, EMT6 and EMT6 / IL13Ra2 tumors were located on the left and right sides of the abdomen, respectively.

[0206] Figure 99 : In vivo imaging data at the indicated time points. In all figures, the two mice on the far right were not treated with Fc-MDNA132.15 and served as controls. As observed, A549 and U87 tumors were located on the left and right sides of the abdomen, respectively.

[0207] Figure 100 In the TRAMP-C1 prostate tumor model, Fc-MDNA413 exhibited tumor growth similar to the vehicle control, while MDNA19 exhibited moderate tumor growth inhibition. However, the combination of Fc-MDNA413 and MDNA19 exhibited superior tumor growth inhibition compared to either agent alone.

[0208] Figure 101 Figure 2: Dose-response plots for A375 and U87 cells. Percent viability in each data set was normalized, with 0% defined as the minimum mean and 100% defined as the maximum mean. This was then plotted as a function of construct concentration (pM). The mean viability of the positive control wells is represented by a dotted line in the bar graph.

[0209] Figure 102 : Dose response diagram of EMT6-IL13Rα2 and EMT6 wild-type cells. The percentage of activity in each data set of EMT6-IL13Rα2 was normalized, where 0% was defined as the minimum mean and 100% was defined as the maximum mean. It was then plotted as a function of construct concentration (pM). For EMT6 wild-type cells, the average activity at each concentration was plotted as a bar graph, while the positive control wells were represented by dotted lines.

[0210] Figure 103 Figure 2: Dose-response plots for A375 and U87 cells. Percent viability in each data set was normalized, with 0% defined as the minimum mean and 100% defined as the maximum mean. This was then plotted as a function of construct concentration (pM). The mean viability of the positive control wells is represented by a dotted line in the bar graph.

[0211] Figure 104 : Dose response diagram of EMT6-IL13Rα2 and EMT6 wild-type cells. The percentage of activity in each data set of EMT6-IL13Rα2 was normalized, where 0% was defined as the minimum mean and 100% was defined as the maximum mean. It was then plotted as a function of construct concentration (pM). For EMT6 wild-type cells, the average activity at each concentration was plotted as a bar graph, while the positive control wells were represented by dotted lines. DETAILED DESCRIPTION

[0212] To facilitate understanding of this disclosure, certain terms and phrases are defined below and throughout the specification.

[0213] definition

[0214] All references cited herein are incorporated by reference in their entirety as if fully incorporated herein. Unless otherwise defined, technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton et al., Dictionary of Microbiology and Molecular Biology, 3rd ed., J. Wiley & Sons (New York, NY 2001); March, Advanced Organic Chemistry Reactions, Mechanisms and Structure, 5th ed., J. Wiley & Sons (New York, NY 2001); and Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY 2001), provide those skilled in the art with a general guide to many of the terms used in this disclosure. Where appropriate, procedures including the use of commercially available kits and reagents are generally performed according to the manufacturer's protocols and / or parameters unless otherwise noted.

[0215] As used herein, "IL-2" means wild-type IL-2, whether natural or recombinant. As described by Fujita et al., PNAS USA, 80, 7437-7441 (1983), mature human IL-2 appears as a 133 amino acid sequence (less a signal peptide, consisting of an additional 20 N-terminal amino acids). The amino acid sequence of human IL-2 (SEQ ID NO: 1; full length) is found in Genbank under accession locator NP_000577.2. The amino acid sequence of mature human IL-2 is depicted in SEQ ID NO: 2 (human wild-type mature; substituted position numbering is based on this sequence). The mouse (Mus musculus) IL-2 amino acid sequence is found in Genbank under accession locator (SEQ ID NO: 3). The amino acid sequence of mature mouse IL-2 is depicted in SEQ ID NO: 4.

[0216]

[0217]

[0218] As used herein, "IL-2 mutein" refers to an IL-2 polypeptide in which specific substitutions have been made to the interleukin-2 protein. The IL-2 mutein is characterized by amino acid insertions, deletions, substitutions, and modifications at one or more sites within or at other residues in the native IL-2 polypeptide chain. According to the present disclosure, any such insertions, deletions, substitutions, and modifications result in an IL-2 mutein that retains IL-2Rβ binding activity. Exemplary muteins may include substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids.

[0219] Mutants also include conservative modifications and substitutions at other positions of IL-2 (i.e., those that have minimal effect on the secondary or tertiary structure of the mutant protein). Such conservative substitutions include those described by Dayhoff in The Atlas of Protein Sequence and Structure 5 (1978) and Argos in EMBO J., 8:779-785 (1989). For example, amino acids belonging to one of the following categories represent conservative changes: Class I: ala, pro, gly, gln, asn, ser, thr; Class II: cys, ser, tyr, thr; Class III: val, ile, leu, met, ala, phe; Class IV: lys, arg, his; Class V: phe, tyr, trp, his; and Class VI: asp, glu.

[0220] "IL-2 numbering" means identifying a selected amino acid by reference to the position at which it typically occurs in the mature sequence of wild-type IL-2, e.g., R81 refers to the 81st amino acid appearing in SEQ ID NO:2, i.e., arginine. L80 refers to the 80th amino acid appearing in SEQ ID NO:2, i.e., leucine. L85 refers to the 85th amino acid appearing in SEQ ID NO:2, i.e., leucine. I86 refers to the 86th amino acid appearing in SEQ ID NO:2, i.e., isoleucine. I92 refers to the 92nd amino acid appearing in SEQ ID NO:2, i.e., isoleucine. F42 refers to the 42nd amino acid appearing in SEQ ID NO:2, i.e., phenylalanine. K43 refers to the 43rd amino acid appearing in SEQ ID NO:2, i.e., lysine.

[0221] As used herein, the abbreviations for the genetically encoded L-enantiomeric amino acids used in the disclosed methods are conventional and are shown in Table 1 below.

[0222] Table 1: Amino acid abbreviations

[0223] amino acids Single-letter symbols Common abbreviations Alanine A Ala Arginine R Arg Asparagine N Asn Aspartic acid D Asp Cysteine C Cys glutamine Q Gln glutamate E Glu Glycine G Gly Histidine H His Isoleucine I Ile Leucine L Leu Lysine K Lys Methionine M Met Phenylalanine F Phe Proline P Pro Serine S Ser Threonine T Thr Tryptophan W Trp Tyrosine Y Tyr Valine V Val

[0224] "Hydrophilic amino acids" refer to amino acids that exhibit a hydrophobicity of less than zero according to the normalized consensus hydrophobicity scale of Eisenberg et al., 1984, J. Mol. Biol. 179: 125-142. Genetically encoded hydrophilic amino acids include Thr (T), Ser (S), His (H), Glu (E), Asn (N), Gln (Q), Asp (D), Lys (K), and Arg (R).

[0225] The term "cell type having an IL-2Rαβγ receptor" means cells known to have this receptor type, i.e., T cells, activated T cells, B cells, activated monocytes, and activated NK cells. The term "cell type having an IL-2Rβγ receptor" means cells known to have this receptor type, i.e., B cells, resting monocytes, and resting NK cells.

[0226] As used herein with respect to polypeptides or DNA sequences, the term "identity" refers to the subunit sequence identity between two molecules. When a subunit position in two molecules is occupied by the same monomeric subunit (i.e., the same amino acid residue or nucleotide), the molecules are identical at that position. The similarity between two amino acid or two nucleotide sequences is a direct function of the number of identical positions. In general, sequences are aligned to obtain the highest order match. If necessary, identity can be calculated using disclosed techniques and widely available computer programs such as the GCS program package (Devereux et al., Nucleic Acids Res. 12: 387, 1984), BLASTP, BLASTN, FASTA (Atschul et al., J. Molecular Biol. 215: 403, 1990). Sequence identity can be measured using sequence analysis software, such as the Sequence Analysis Software Package of the Genetics Computer Group from the University of Wisconsin Biotechnology Center (1710 University Avenue, Madison, Wis. 53705) with its default parameters.

[0227] The terms "polypeptide," "protein," or "peptide" refer to any chain of amino acid residues, regardless of length or post-translational modification (eg, glycosylation or phosphorylation).

[0228] If the mutant IL-2 polypeptides of the present disclosure are "substantially pure," they can be at least about 60% by weight (dry weight) of the polypeptide of interest (e.g., a polypeptide comprising a mutant IL-2 amino acid sequence). For example, the polypeptide can be at least about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% by weight of the polypeptide of interest. Purity can be measured by any suitable standard method, such as column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

[0229] An "agonist" is a compound that interacts with a target to cause or promote increased activation of the target.

[0230] A "partial agonist" is a compound that interacts with the same target as an agonist, but does not produce the same magnitude of biochemical and / or physiological effects as the agonist, even at increased doses of the partial agonist.

[0231] A "superagonist" (also called a "superkine") is an agonist that produces a maximal response greater than the endogenous agonist of the target receptor, and therefore has an efficacy exceeding 100%.

[0232] "Operably linked" is intended to mean that the target nucleotide sequence (i.e., the sequence encoding the IL-2 mutant protein) is linked to the regulatory sequence in a manner that allows the expression of the nucleotide sequence (e.g., expression in an in vitro transcription / translation system or expression in a host cell when the vector is introduced into the host cell). "Regulatory sequences" include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). See, for example, Goeddel (1990), Gene Expression Technology: Methods in Enzymology 185 (Academic Press, San Diego, Calif.). Regulatory sequences include those that direct constitutive expression of the nucleotide sequence in many types of host cells and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). Those skilled in the art will recognize that the design of the expression vector may depend on factors such as the choice of host cell to be transformed, the expression level of the desired protein, and the like. The expression constructs of the present invention can be introduced into host cells to produce the human IL-2 mutant protein disclosed herein or a biologically active variant thereof.

[0233] The terms "host cell" and "recombinant host cell" are used interchangeably herein. It should be understood that such terms refer not only to the particular subject cell, but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in subsequent generations due to mutations or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the terms as used herein.

[0234] As used herein, the terms "transformation" and "transfection" refer to various techniques recognized in the art for introducing exogenous nucleic acid (e.g., DNA) into host cells, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, particle gun or electroporation.

[0235] As used herein, the term "pharmaceutically acceptable carrier" includes, but is not limited to, saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc., compatible with pharmaceutical administration. Supplementary active compounds (e.g., antibiotics) can also be incorporated into the composition.

[0236] As used herein, the term "anti-PD-1 antibody" refers to any antibody that binds to PD-1, including inhibitory antibodies. An "anti-PD-1 inhibitor" refers to an inhibitor that binds to and inhibits PD-1. Such anti-PD-1 antibodies and / or inhibitors include, but are not limited to, nivolumab, BMS-936558, MDX-1106, ONO-4538, AMP224, CT-011, and MK-3475.

[0237] As used herein, the terms "cancer" (or "cancerous"), "hyperproliferative," "tumor," and / or "neoplastic" refer to cells that have the capacity for autonomous growth (i.e., an abnormal state or condition characterized by rapidly proliferative cell growth). Hyperproliferative and neoplastic disease states can be classified as pathological (i.e., characterizing or constituting a disease state), or they can be classified as non-pathological (i.e., deviations from normal but not associated with a disease state). These terms are intended to include all types of cancerous growths or oncogenic processes, metastatic tissues, or malignantly transformed cells, tissues, or organs, regardless of histopathological type or invasive stage. "Pathological hyperproliferative" cells occur in disease states characterized by malignant tumor growth. Examples of non-pathological hyperproliferative cells include cell proliferation associated with wound repair. The terms "cancer" or "neoplasm" are used to refer to malignancies of various organ systems, including those affecting the lungs, breasts, thyroid glands, lymph nodes and lymphoid tissues, reproductive system, gastrointestinal organs, and genitourinary tract, as well as adenocarcinomas, which are generally considered to include malignancies such as most colon cancers, renal cell carcinomas, prostate cancer, and / or testicular tumors, non-small cell lung cancer, small intestine cancer, and esophageal cancer. Cancers generally can include solid tumors, as well as sarcomas, carcinomas, head and neck cancers, glioblastomas, bladder cancer, oral cancer, mesothelioma, pancreatic cancer, liver cancer, colorectal cancer, lung cancer, skin cancer, lymphomas, gastrointestinal cancer, prostate cancer, ovarian cancer, breast cancer, basaloid breast tumors, endometrial cancer, multiple myeloma, melanoma, lymphomas, lung cancer (including small cell lung cancer), kidney cancer, stomach cancer, brain cancer, and CNS tumors. CNS tumors include glioma, glioblastoma, glioblastoma multiforme (GBM), refractory glioblastoma multiforme (rGBM), recurrent glioblastoma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, meningioma, neuroblastoma, retinoblastoma, medulloblastoma, adult pituitary adenoma, O6-methylguanine methyltransferase (MGMT)-positive or -negative CNS tumors, and furin-positive CNS tumors.

[0238] The term "carcinoma" is art-recognized and refers to malignant tumors of epithelial or endocrine tissue, including cancers of the respiratory system, gastrointestinal system, genitourinary system, testicular cancer, breast cancer, prostate cancer, endocrine system cancer, and melanoma. "Adenocarcinoma" refers to a cancer that arises from glandular tissue or in which the tumor cells form recognizable glandular structures.

[0239] As used herein, the term "hematopoietic neoplastic disorder" refers to a disease involving hyperplastic / neoplastic cells of hematopoietic origin, such as a disease caused by bone marrow, lymphoid or erythroid cell lineages or their precursor cells. Preferably, the disease is caused by a poorly differentiated acute leukemia (e.g., erythroblastic leukemia and acute megakaryoblastic leukemia). Other exemplary bone marrow disorders include, but are not limited to, acute promyelocytic leukemia (APML), acute myeloid leukemia (AML), and chronic myeloid leukemia (CML) (reviewed in Vaickus, L. (1991) Crit Rev. in Oncol. / Hemotol. 11: 267-97); lymphoid malignancies, including but not limited to acute lymphoblastic leukemia (ALL) (including B lineage ALL and T lineage ALL), chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), hairy cell leukemia (HLL), and Waldenstrom's macroglobulinemia (WM). Other forms of malignant lymphoma include, but are not limited to, non-Hodgkin lymphoma and its variants, peripheral T-cell lymphoma, adult T-cell leukemia / lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), large granular lymphocytic leukemia (LGF), Hodgkin's disease, and Reed-Stemberg disease.

[0240] As used herein, the term "treat" and the like refers to obtaining a desired pharmacological and / or physiological effect. This effect may be preventive in terms of completely or partially preventing a disease or its symptoms, and / or therapeutic in terms of partially or completely curing a disease and / or the adverse effects caused by the disease. As used herein, "treatment" covers any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the disease from occurring in a subject who is susceptible to the disease or is at risk of acquiring the disease but has not yet been diagnosed with the disease; (b) inhibiting the disease, i.e., preventing its development; and (c) alleviating the disease, i.e., causing the disease to regress. A therapeutically effective amount may be an amount that reduces the number of tumors, reduces the size of tumors, and / or increases survival.

[0241] The terms "individual," "subject," and "patient" are used interchangeably herein and refer to mammals, including but not limited to humans and non-human primates, including monkeys and humans; mammalian sports animals (e.g., horses); mammalian livestock (e.g., sheep, goats, etc.); mammalian companion animals (dogs, cats, etc.); and rodents (e.g., mice, rats, etc.).

[0242] The terms "pharmaceutically acceptable" and "physiologically acceptable" mean a biologically acceptable formulation, its gaseous, liquid or solid state, or a mixture thereof, suitable for one or more routes of administration, in vivo delivery or contact. A "pharmaceutically acceptable" or "physiologically acceptable" composition is a material that is not biologically or otherwise undesirable, for example, the material can be administered to a subject without causing substantial undesirable biological effects. Thus, such pharmaceutical compositions can be used, for example, to administer IL-2 mutant proteins to a subject. In particular, IL-2 mutant proteins comprising substitutions L80F, R81D, L85V, I86V and I92F are administered in combination with anti-PD-1 to a subject with cancer. In some embodiments, the administered IL-2 mutant protein further comprises a substitution at position F42A. In some embodiments, the administered IL-2 mutant protein further comprises a substitution at position K43N.

[0243] As used herein, the phrase "unit dosage form" refers to a physically discrete unit suitable as a unit dose for a subject to be treated; each unit contains a predetermined amount, optionally in combination with a pharmaceutical carrier (excipient, diluent, vehicle, or filler), which, when administered in one or more doses, produces a desired effect (e.g., a prophylactic or therapeutic effect). In some embodiments, the therapeutic effect is a reduction in the number of tumors. In some embodiments, the therapeutic effect is a reduction in tumor size. In some embodiments, the therapeutic effect is an increase in survival.

[0244] In some embodiments, the unit dosage form can be in, for example, ampoules and vials, including liquid compositions, or compositions in a freeze-dried or lyophilized state; for example, a sterile liquid carrier can be added prior to administration or in vivo delivery. A single unit dosage form can be included in a multi-dose kit or container. IL-2 mutant proteins combined with anti-PD-1 antibodies and pharmaceutical compositions thereof can be packaged in single or multiple unit dosage forms for ease of administration and uniformity of dosage.

[0245] A "therapeutically effective amount" will fall within a relatively broad range that can be determined experimentally and / or clinically. For example, for in vivo injection, for example, injection directly into a subject's tissue or vasculature (e.g., liver tissue or vein). Other effective doses can be readily determined by one of ordinary skill in the art through routine experiments establishing dose-response curves.

[0246] By "effective amount" or "sufficient amount" is meant an amount that, in single or multiple doses, alone or in combination with one or more other compositions (therapeutics, such as drugs), treatments, regimens or therapeutic regimen agents (including, for example, vaccine regimens), provides a detectable response of any duration (long or short), any measurable or detectable level in a subject, or an expected or desired result or benefit of any duration (e.g., minutes, hours, days, months, years, or a cure).

[0247] Although reducing, decreasing, inhibiting, suppressing, limiting or controlling the progression or worsening of a disease is also a satisfactory result, an "effective amount" or "sufficient amount" for treating (e.g., improving or providing a therapeutic benefit or improvement) a dose is generally effective to provide a measurable degree of one, multiple or all adverse symptoms, consequences or complications of the disease, one or more adverse symptoms, conditions, illnesses, pathologies or complications caused by or associated with the disease. In some embodiments, an effective amount is an amount sufficient to reduce the number of tumors. In some embodiments, an effective amount is an amount sufficient to reduce the size of a tumor. In some embodiments, an effective amount is an amount sufficient to increase survival.

[0248] "Prevention" and its grammatical variations mean a method of contacting, administering or delivering the drug to a subject in vivo before the disease. Administration or in vivo delivery to a subject can be performed before the development of adverse symptoms, conditions, complications, etc. caused by or associated with the disease. For example, screening (e.g., genetic screening) can be used to identify such subjects as candidates for the methods and uses, but the subject may not exhibit the disease. Thus, such subjects include those that screen positive for insufficient or absent amounts of functional gene products (proteins), or for the production of abnormal, partially functional or non-functional gene products (proteins) that cause the disease; and subjects that screen positive for abnormal or defective (mutated) gene products (proteins) that cause the disease, even if such subjects do not exhibit symptoms of the disease.

[0249] I. Detailed Description

[0250] Described herein are bispecific IL-2 cytokine fusions (also referred to herein as bifunctional molecules) comprising an IL-2 mutant protein fused to a second cytokine. Also described herein are IL-2 mutant proteins comprising substitutions of L80F, R81D, L85V, I86V, and I92F, which have increased binding capacity to IL-2Rβ receptors and can be included in bispecific IL-2 cytokine fusions. Also described herein are uses of bispecific IL-2 cytokine fusions for monotherapy and combination therapy with anti-PD-1 antibodies. In some embodiments, an IL-2 mutant protein comprising L80F, R81D, L85V, I86V, and I92F numbered according to wild-type human IL-2 (SEQ ID NO: 2; wild-type hIL-2) is referred to as H9. For example, when combined with an anti-PD-1 antibody to treat cancer, such IL-2 mutant proteins play a role. Also provided are nucleic acids encoding such IL-2 muteins, methods for preparing such IL-2 muteins, pharmaceutical compositions comprising such IL-2 muteins, and therapeutic methods using such IL-2 muteins.

[0251] A. IL-2 muteins for bispecific IL-2 cytokine fusions

[0252] The substituted amino acid residues may be, but are not necessarily, conservative substitutions and generally include substitutions in the following categories: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. These mutations may be at amino acid residues that contact IL-2Rβ and / or IL-2Rγ.

[0253] More specifically, mutations (whether conservative or non-conservative, by addition or deletion) can be made at one or more positions. For example, the mutations can be: I24V, P65H, Q74R, Q74H, Q74N, Q74S, L80F, L80V, R81I, R81T, R81D, L85V, I86V, I89V, I92F, V93I. The sequences of exemplary IL-2 mutant proteins are as follows: S-1 SEQ ID NO: 5, S-2 SEQ ID NO: 6, 6-6 SEQ ID NO: 7, A2 SEQ ID NO: 8, B1 SEQ ID NO: 9, B11 SEQ ID NO: 10, C5 SEQ ID NO: 11, D10 SEQ ID NO: 12, E10 SEQ ID NO: 13, G8 SEQ ID NO: 14, H4 SEQ ID NO: 15, and H9 SEQ ID NO: 16.

[0254] In some embodiments, the substitutions in the IL-2 mutein include L80F, R81D, L85V, I86V, and I92F, numbered according to wild-type human IL-2 of SEQ ID NO: 2. In some embodiments, the IL-2 mutein further comprises an F42A substitution, wherein the numbering is according to wild-type human IL-2 of SEQ ID NO: 2. In some embodiments, the IL-2 mutein further comprises an Y45A substitution, wherein the numbering is according to wild-type human IL-2 of SEQ ID NO: 2. In some embodiments, the IL-2 mutein further comprises an E62A substitution, wherein the numbering is according to wild-type human IL-2 of SEQ ID NO: 2. In some embodiments, the substitutions in the IL-2 mutein include F42A, L80F, R81D, L85V, I86V, and I92F, wherein the numbering is according to wild-type human IL-2 of SEQ ID NO: 2. In some embodiments, the substitutions in the IL-2 mutein include F42A, Y45A, L80F, R81D, L85V, I86V, and I92F as numbered according to the wild-type human IL-2 of SEQ ID NO: 2. In some embodiments, the substitutions in the IL-2 mutein include F42A, E62A, L80F, R81D, L85V, I86V, and I92F as numbered according to the wild-type human IL-2 of SEQ ID NO: 2. In some embodiments, the substitutions in the IL-2 mutein include F42A, Y45A, E62A, L80F, R81D, L85V, I86V, and I92F as numbered according to the wild-type human IL-2 of SEQ ID NO: 2. In some embodiments, the substitutions in the IL-2 mutein include E62A, L80F, R81D, L85V, I86V, and I92F as numbered according to the wild-type human IL-2 of SEQ ID NO: 2. In some embodiments, the substitutions in the IL-2 mutein include Y45A, E62A, L80F, R81D, L85V, I86V, and I92F as numbered according to the wild-type human IL-2 of SEQ ID NO: 2. In some embodiments, the substitutions in the IL-2 mutein include Y45A and E62A as numbered according to the wild-type human IL-2 of SEQ ID NO: 2.

[0255] In some embodiments, the substitutions in the IL-2 mutant protein that result in increased and / or enhanced IL-2Rβ binding include L80F, R81D, L85V, I86V, and I92F, numbered according to wild-type human IL-2 of SEQ ID NO: 2. In some embodiments, the IL-2 mutant protein used in the present invention comprises L80F, R81D, L85V, I86V, and I92F, and exhibits increased IL-2Rβ binding. In some embodiments, the IL-2 mutant protein used in the present invention further comprises a substitution at position F42A. In some embodiments, the IL-2 mutant protein used in the present invention further comprises a substitution at position K43N. In some embodiments, all of the mutant proteins comprise the substitutions L80F, R81D, L85V, I86V, and I92F, and one or more substitutions selected from the group consisting of F42A, Y45A, and E62A, compared to wild-type human IL-2 (SEQ ID NO: 2).

[0256] In some embodiments, amino acid substitutions that increase IL-2Rβ binding affinity include: L80F, R81D, L85V, I86V, and I92F. In some embodiments, amino acid substitutions that increase IL-2Rβ binding affinity include: L80F, R81D, L85V, I86V, and I92F.

[0257] In some embodiments, the subject IL-2 muteins having a stronger binding affinity for IL-2Rβ than wild-type human IL-2 include amino acid substitutions L80F, R81D, L85V, I86V, and I92F. In some embodiments, the IL-2 muteins have the following amino acid sequence:

[0258] (SEQ ID NO: 5 or 16; H9 as used in the Examples).

[0259] In some embodiments, IL-2 mutein stimulation is dependent on IL-2Rβ / IL-2Rγ cIn some embodiments, the ability of the subject IL-2 mutant protein to stimulate STAT5 phosphorylation in IL-2Rβ+ cells is increased compared to wild-type human IL-2. In some embodiments, the IL-2 mutant protein stimulates STAT5 phosphorylation in IL-2Rβ+ cells at a level that is 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of the level of STAT5 phosphorylation stimulated by wild-type IL-2 in the same cells. In some embodiments, IL-2 mutant proteins stimulate STAT5 phosphorylation in IL-2Rβ+ cells at a level that is 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 155%, 160%, 165%, 170%, 175%, 180%, 185%, 190%, 195% or higher of the level of STAT5 phosphorylation stimulated by wild-type IL-2 in the same cells. In some embodiments, IL-2Rβ+ cells are T cells. In specific embodiments, T cells are CD8+T cells. In some embodiments, CD8+T cells are freshly isolated CD8+T cells. In other embodiments, CD8+T cells T cells are activated CD8+T cells. In other embodiments, IL-2Rβ+ cells are natural killer (NK) cells. In some embodiments, the IL-2 mutein comprises substitutions L80F, R81D, L85V, I86V, and I92F compared to wild-type human IL-2 (SEQ ID NO: 2).

[0260] In some embodiments, the mutant protein has an increased ability to stimulate ERK1 / ERK2 signaling in IL-2Rβ+ cells compared to wild-type human IL-2. In some embodiments, the IL-2 mutant protein stimulates pERK1 / ERK2 signaling in IL-2Rβ+ cells at a level that is 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of the level that wild-type IL-2 stimulates pERK1 / ERK2 signaling in the same cells. In some embodiments, the IL-2 mutant protein stimulates pERK1 / ERK2 phosphorylation in IL-2Rβ+ cells at a level that is 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 155%, 160%, 165%, 170%, 175%, 180%, 185%, 190%, 195% or higher of the level of pERK1 / ERK2 phosphorylation stimulated by wild-type IL-2 in the same cells. In some embodiments, the IL-2Rβ+ cells are T cells. In specific embodiments, the T cells are CD8+T cells. In some embodiments, the CD8+T cells are freshly isolated CD8+T cells. In other embodiments, the CD8+T cells are activated CD8+T cells. In other embodiments, the IL-2Rβ+ cells are natural killer (NK) cells. In some embodiments, the IL-2 mutein comprises substitutions L80F, R81D, L85V, I86V, and I92F compared to wild-type human IL-2 (SEQ ID NO: 2).

[0261] STAT5 and ERK1 / 2 signaling can be measured, for example, by phosphorylation of STAT5 and ERK1 / 2 using any suitable method known in the art. For example, antibodies specific for the phosphorylation patterns of these molecules can be used in combination with flow cytometry analysis as described herein to measure phosphorylation of STAT5 and ERK1 / 2. In some embodiments, the mutant protein has an enhanced ability to stimulate PI 3 kinase signaling in IL-2Rβ+ cells compared to wild-type human IL-2. In some embodiments, the IL-2 mutant protein stimulates PI 3-kinase signaling in IL-2Rβ+ cells at a level that is 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or less of the level at which wild-type IL-2 stimulates PI 3-kinase signaling in the same cells. In some embodiments, the IL-2 mutant protein stimulates PI 3-kinase signaling in IL-2Rβ+ cells at a level that is 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 155%, 160%, 165%, 170%, 175%, 180%, 185%, 190%, 195% or more of the level of PI 3-kinase signaling in the same cells stimulated by wild-type IL-2. In some embodiments, the IL-2Rβ+ cells are T cells. In specific embodiments, the T cells are CD8+T cells. In some embodiments, the CD8+T cells are activated CD8+T cells. In other embodiments, the IL-2Rβ+ cells are natural killer (NK) cells. In some embodiments, the IL-2 mutant protein comprises substitutions L80F, R81D, L85V, I86V, and I92F compared to wild-type human IL-2 (SEQ ID NO: 2). PI3-kinase signaling can be measured using any suitable method known in the art. For example, PI 3-kinase signaling can be measured using antibodies specific for phospho-S6 ribosomal protein in combination with flow cytometry analysis as described herein.

[0262] In some embodiments, IL-2 mutant protein is a stimulator of IL-2 and / or IL-15STAT5 phosphorylation in CD8+T cells. In some embodiments, mutant protein is a promoter of CD8+T cell proliferation induced by IL-2 and / or IL-15. In some embodiments, mutant protein is a stimulator of IL-2-dependent, TCR-induced cell proliferation. In some embodiments, compared to wild-type human IL-2 (SEQ ID NO: 2), IL-2 mutant protein comprises substitutions L80F, R81D, L85V, I86V and I92F.

[0263] IL-2 promotes Th1, Th9 and Treg T cell differentiation, and suppresses Th17 differentiation. Therefore, it is not subject to any particular operating theory, it is believed that the IL-2 mutant protein that plays the role of IL-2 super agonist can promote Th1, Th9 and / or Treg cell differentiation or suppress Th17 cell differentiation. In some embodiments, IL-2 mutant protein is a promoter of IL-2 dependent Th1, Th9 and / or Treg differentiation. In some embodiments, the mutant protein is an inhibitor of Th17 differentiation. In some embodiments, compared with wild-type human IL-2 (SEQ ID NO: 2), IL-2 mutant protein includes substitutions L80F, R81D, L85V, I86V and I92F.

[0264] In some embodiments, compared with wild-type human IL-2, IL-2 mutant protein signaling is less and / or independent of CD25 (for example, CD25 combines to reduce or eliminate). In some embodiments, about CD25 reduction and / or independent signaling allows preferential activation effector T cells, while limiting the stimulation to Treg. In some embodiments, about CD25 reduction and / or independent signaling reduces toxicity. In some embodiments, all compared with wild-type human IL-2 (SEQ ID NO: 2), mutant protein includes replacement L80F, R81D, L85V, I86V and I92F, and one or more replacements selected from the group consisting of F42A, Y45A and E62A.

[0265] In some embodiments, IL-2 mutant proteins can increase and / or restore the responsiveness to anergic NK cells. In some embodiments, IL-2 mutant proteins can increase and / or restore the responsiveness to anergic NK cells in the tumor microenvironment. In some embodiments, compared to wild-type human IL-2 (SEQ ID NO: 2), IL-2 mutant proteins comprise substitutions L80F, R81D, L85V, I86V, and I92F.

[0266] In some embodiments, the mutant protein is an inhibitor of IL-2 dependent activation of natural killer (NK) cells. As described herein, IL-2 activation of NK cells can be measured by any suitable method known in the art, for example, by measuring IL-2 induced CD69 expression and / or cytotoxicity.

[0267] In some embodiments, the increase in IL-2Rβ binding affinity is any IL-2Rβ binding affinity that is greater than the binding affinity of wild-type human IL-2 for IL-2Rβ. In some embodiments, the binding affinity is an increase of 2-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 120-fold, 150-fold, 170-fold, 190-fold, 200-fold, 220-fold, 240-fold or more in binding affinity for IL-2Rβ compared to the binding affinity of wild-type human IL-2 for IL-2Rβ.

[0268] In some embodiments, the increase in IL-2Rβ binding is any IL-2Rβ binding that is greater than the binding of wild-type human IL-2 to IL-2Rβ. In some embodiments, the binding is an increase of 2-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 120-fold, 150-fold, 170-fold, 190-fold, 200-fold, 220-fold, 240-fold or more compared to the binding of wild-type human IL-2 to IL-2Rβ.

[0269] In some embodiments, the subject IL-2 mutant proteins having a stronger binding affinity for IL-2Rβ than wild-type human IL-2 also exhibit reduced binding to CD25 and include amino acid substitutions F42A, L80F, R81D, L85V, I86V, and I92F. In some embodiments, the binding affinity is reduced by about 220-fold, i.e., from a Kd of about 6.6 nM for wild-type human IL-2 to about 1.4 μM for the mutant protein comprising F42A, L80F, R81D, L85V, I86V, and I92F. In some embodiments, the IL-2 mutant protein has the following amino acid sequence:

[0270] (SEQ ID NO:6; also known as H9-F42A).

[0271] In some embodiments, the subject IL-2 mutant proteins having a stronger binding affinity for IL-2Rβ than wild-type human IL-2 also exhibit reduced binding to CD25 and include amino acid substitutions K43N, L80F, R81D, L85V, I86V, and I92F. In some embodiments, the reduced binding affinity is due to allowing glycosylation at position 43 with the K43N substitution. By replacing asparagine (K43N) with lysine, CD25 binding is reduced and / or eliminated in IL-2 mutant proteins comprising amino acid substitutions K43N, L80F, R81D, L85V, I86V, and I92F. In some embodiments, the IL-2 mutant protein has the following amino acid sequence:

[0272] (SEQ ID NO:7; also known as H9-K43N).

[0273] In some embodiments, the reduction in binding affinity for CD25 is any CD25 binding affinity that is less than the binding affinity of wild-type human IL-2. In some embodiments, the binding affinity is a 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 120-fold, 150-fold, 170-fold, 190-fold, 200-fold, 220-fold, 240-fold or more reduction in binding affinity for CD25 compared to the binding affinity of wild-type human IL-2 for CD25.

[0274] In some embodiments, the subject IL-2 muteins having a stronger binding affinity for IL-2Rβ and a reduced binding affinity for CD25 compared to wild-type human IL-2 include amino acid substitutions F42A, Y45A, L80F, R81D, L85V, I86V, and I92F. In some embodiments, the IL-2 mutein has the following amino acid sequence:

[0275]

[0276] In some embodiments, the subject IL-2 muteins having enhanced binding affinity for IL-2Rβ and reduced binding affinity for CD25 compared to wild-type human IL-2 include amino acid substitutions F42A, E62A, L80F, R81D, L85V, I86V, and I92F. In some embodiments, the IL-2 mutein has the following amino acid sequence:

[0277]

[0278] In some embodiments, the subject IL-2 muteins having a stronger binding affinity for IL-2Rβ and a reduced binding affinity for CD25 compared to wild-type human IL-2 include amino acid substitutions F42A, Y45A, E62A, L80F, R81D, L85V, I86V, and I92F. In some embodiments, the IL-2 mutein has the following amino acid sequence:

[0279]

[0280] In some embodiments, the IL-2 mutein sequence is 90% identical to SEQ ID NO: 2 or any one of SEQ ID NO: 6 to SEQ ID NO: 10 or SEQ ID NO: 16. In some embodiments, the IL-2 mutein sequence is 95% identical to SEQ ID NO: 2 or any one of SEQ ID NO: 6 to SEQ ID NO: 10. In some embodiments, the IL-2 mutein sequence is 98% identical to SEQ ID NO: 2 or any one of SEQ ID NO: 6 to SEQ ID NO: 10. In some embodiments, the IL-2 mutein sequence is 99% identical to SEQ ID NO: 2 or any one of SEQ ID NO: 6 to SEQ ID NO: 10.

[0281] Additional exemplary IL-2 sequences are provided in the table below.

[0282] Table 2: List of exemplary IL-2 muteins

[0283]

[0284]

[0285]

[0286]

[0287] B. IL-2 mutein fusion protein

[0288] IL-2 mutant proteins can be prepared as fusions or chimeric polypeptides comprising the subject IL-2 mutant protein and a heterologous polypeptide (i.e., a polypeptide that is not IL-2 or a mutant thereof) (see, e.g., U.S. Patent No. 6,451,308), including, for example, bispecific IL-2 cytokine fusions. Exemplary heterologous polypeptides can increase the circulating half-life of the chimeric polypeptide in vivo, thereby further enhancing the properties of the mutant IL-2 polypeptide. In various embodiments, the polypeptide that increases the circulating half-life can be serum albumin (such as human serum albumin), PEG, a PEG derivative, or an Fc region of an antibody of the IgG subclass lacking the IgG heavy chain variable region. Exemplary Fc regions can comprise mutations that inhibit complement fixation and Fc receptor binding, or they can be lytic, i.e., able to bind complement, or lyse cells via another mechanism such as antibody-dependent complement lysis (ADCC; USSN 08 / 355,502, filed December 12, 1994).

[0289] An "Fc region" may be a naturally occurring or synthetic polypeptide homologous to the C-terminal domain of IgG produced by papain digestion of IgG. The molecular weight of IgG Fc is approximately 50 kDa. A mutant IL-2 polypeptide may include the entire Fc region, or a smaller portion that retains the ability to extend the circulating half-life of the chimeric polypeptide of which it is a part. Additionally, a full-length or fragmented Fc region may be a variant of the wild-type molecule. In some embodiments, an IL-2 mutein fusion protein (e.g., an IL-2 mutein as described herein) includes an IgG1, IgG2, IgG3, or IgG4 Fc region (see, e.g., Figure 2 A to Figure 2 B). In some embodiments, the Fc region comprises the substitution N297A.

[0290] In some embodiments, the IL-2 mutein is linked directly or indirectly to a heterologous fusion polypeptide.

[0291] In some embodiments, IL-2 mutant protein is directly connected to the Fc region. In some embodiments, IL-2 mutant protein is connected to the Fc region via a linker peptide such as GGGGS. In some embodiments, the linker is (GGGGS)n, wherein n is an integer between 1 and 10. In some embodiments, the linker is GGGGS (SEQ ID NO: 496). In some embodiments, the linker is GGGGSGGGGS (SEQ ID NO: 497). In some embodiments, the linker is GGGGSGGGGSGGGGS (SEQ ID NO: 498). In some embodiments, the linker is GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 499). In some embodiments, the linker is GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 500). In some embodiments, the linker contains one or more protease cleavage sites (for example, a linker cleavable by a protease). The linker can also contain one or more protease cleavage sites or be sensitive to cleavage via oxidation and / or reduction. In one example, a peptide linker that is susceptible to cleavage by an enzyme of the complement system, urokinase, tissue plasminogen activator, trypsin, plasmin, caspase, kallikrein, cathepsin, legumin, MMP, thrombin, urokinase-type plasminogen activator (uPA), matriptase, or another enzyme with proteolytic activity can be used. According to another example, the linker can comprise a disulfide bond (e.g., a disulfide bond on a cysteine ​​molecule). According to another example, the linker can include a protease-cleavable Val-Cit (VC) linker, a Phe-Arg linker, a Val-Lys linker, a Val-Ala linker, a Val-Arg linker, a Val-Leu-Lys linker, a Gly-Phe-Leu-Gly linker, an Ala-Phe-Lys linker, a poly-L-lysine linker, a β-Ala-Leu-Ala-Leu linker, an Arg-Arg-Ala-Leu-Ala-Leu linker, a peptidomimetic linker, a leguminous cleavable Ala-Ala-Asn tripeptide linker, a peptide linker cleaved by cathepsin B and other lysosomal proteases (such as Gly-Phe-Leu-Gly and Ala-Leu-Ala-Leu), a caspase 3DEVD sequence, or a self-immolative linker. For example, the present disclosure encompasses the linkers disclosed in Poreba, M, FEBS J. 287(10): 1936-1969 (2020), which is incorporated herein by reference. Since many tumors naturally release high levels of glutathione (a reducing agent), this can reduce disulfide bonds and subsequently release the cargo moiety at the delivery site. In some embodiments, the linker is a protease-cleavable linker, which is a linker that can be cleaved by matrix metalloproteinases (MMPs).MMPs are overexpressed in situ in tumors, and the present disclosure encompasses linkers that are cleavable in this context. For example, the present disclosure encompasses linkers disclosed in Hsu, EJ et al., Nat. Commun. 12(2768): 1-13(2021), which is incorporated herein by reference. In some embodiments, the MMP linker sequence is selected from the group consisting of SGARYRWLTA (SEQ ID NO: 234), SGRSYAILTA (SEQ ID NO: 235), SRSGRSPAIFTATG (SEQ ID NO: 236), GSSGRSPAIFTAGS (SEQ ID NO: 237), and SGFIANPVTA (SEQ ID NO: 238). In some embodiments, the MMP linker sequence is SGARYRWLTA. In some embodiments, the MMP linker sequence is SGRSYAILTA. In some embodiments, the MMP linker sequence is SRSGRSPAIFTATG. In some embodiments, the MMP linker sequence is GSSGRSPAIFTAGS. In some embodiments, the MMP linker sequence is SGFIANPVTA.

[0292] Fc regions can be either "soluble" or "insoluble," but are typically insoluble. Insoluble Fc regions typically lack the high-affinity Fc receptor binding site and the C'1q binding site. The high-affinity Fc receptor binding site of mouse IgG Fc includes the Leu residue at position 235 of the IgG Fc. Therefore, the Fc receptor binding site can be disrupted by mutating or deleting Leu 235. For example, replacing Leu 235 with Glu inhibits the Fc region's ability to bind to high-affinity Fc receptors. The mouse C'1q binding site can be functionally disrupted by mutating or deleting Glu 318, Lys 320, and Lys 322 of IgG. For example, replacing Glu 318, Lys 320, and Lys 322 with Ala residues renders the IgG1 Fc incapable of inducing antibody-dependent complement lysis. In contrast, soluble IgG Fc regions possess both the high-affinity Fc receptor binding site and the C'1q binding site. The high affinity Fc receptor binding site includes the Leu residue at position 235 of IgG Fc, and the C'1q binding site includes the Glu 318, Lys 320, and Lys 322 residues of IgG1. Solubility IgG Fc has wild-type residues or conservative amino acid substitutions at these sites. Solubility IgG Fc can target cells to produce antibody-dependent cellular toxicity or complement-directed cell lysis (CDC). Suitable mutations of human IgG are also known (see, for example, Morrison et al., The Immunologist 2: 119-124, 1994; and Brekke et al., The Immunologist 2: 125, 1994).

[0293] In other embodiments, the chimeric polypeptide may include a subject IL-2 mutant protein and a polypeptide that acts as an antigenic tag, such as a FLAG sequence. As described herein, the FLAG sequence is recognized by a biotinylated, highly specific anti-FLAG antibody (see also Blanar et al., Science 256:1014, 1992; LeClair et al., Proc. Natl. Acad. Sci. USA 89:8145, 1992). In some embodiments, the chimeric polypeptide further comprises a C-terminal c-myc epitope tag.

[0294] In other embodiments, the chimeric polypeptide comprises a mutant IL-2 polypeptide and a heterologous polypeptide that serves to enhance expression or direct cellular localization of the mutant IL-2 polypeptide, such as an Aga2p lectin subunit (see, e.g., Boder and Wittrup, Nature Biotechnol. 15:553-7, 1997).

[0295] In other embodiments, chimeric polypeptides comprising a mutant IL-2 and an antibody or antigen-binding portion thereof can be produced. The antibody or antigen-binding component of the chimeric protein can be used as a targeting moiety. For example, it can be used to localize the chimeric protein to a specific cell subset or target molecule. Methods for producing cytokine-antibody chimeric polypeptides are described, for example, in U.S. Patent No. 6,617,135.

[0296] In some embodiments, the chimeric polypeptide comprises a fusion with an antibody or antigen-binding portion thereof that disrupts the interaction between the PD-1 receptor and its ligand PD-L1 and / or is an antibody directed against a component of the PD-1 / PD-L1 signaling pathway. Antibodies known in the art that bind to PD-1 and disrupt the interaction between PD-1 and its ligand PD-L1 and stimulate an anti-tumor immune response are suitable for use in the chimeric polypeptides disclosed herein. In some embodiments, the antibody or antigen-binding portion thereof specifically binds to PD-1. For example, antibodies that target PD-1 and can be used in the present invention include, for example, but are not limited to, nivolumab (BMS-936558, Bristol-Myers Squibb), pembrolizumab (lambrolizumab, MK03475 or MK-3475, Merck), humanized anti-PD-1 antibody JS001 (Shanghai JunShi), monoclonal anti-PD-1 antibody TSR-042 (Tesaro, Inc.), Pidilizumab (anti-PD-1 mAb CT-011, Medivation), anti-PD-1 monoclonal antibody BGB-A317 (BeiGene) and / or anti-PD-1 antibody SHR-1210 (Shanghai HengRui), human monoclonal antibody REGN2810 (seplimab, Regeneron), human monoclonal antibody MDX-1106 (Bristol-Myers Squibb) and / or humanized anti-PD-1 IgG4 antibody PDR001 (Novartis). In some embodiments, the PD-1 antibody is from the following clone: ​​RMP1-14 (rat IgG)-BioXcell cat#BP0146. Other suitable antibodies include the anti-PD-1 antibodies disclosed in U.S. Patent No. 8,008,449, which is incorporated herein by reference. In some embodiments, the antibody or its antigen-binding portion specifically binds to PD-L1 and inhibits its interaction with PD-1, thereby enhancing immune activity. Any antibody known in the art that binds to PD-L1 and disrupts the interaction between PD-1 and PD-L1, and stimulates an anti-tumor immune response is suitable for use in the chimeric polypeptide disclosed herein. For example, antibodies targeting PD-L1 and in clinical trials include BMS-936559 (Bristol-Myers Squibb) and MPDL3280A (Genetech). Other suitable antibodies targeting PD-L1 are disclosed in U.S. Pat. No. 7,943,743, which is incorporated herein by reference.Those of ordinary skill will understand that any antibody that binds to PD-1 or PD-L1, disrupts the PD-1 / PD-L1 interaction, and stimulates an anti-tumor immune response is suitable for use in the chimeric polypeptides disclosed herein. In some embodiments, the chimeric polypeptide comprises a fusion with an anti-PD-1 antibody. In some embodiments, the chimeric polypeptide comprises a fusion with an anti-PD-L1 antibody.

[0297] In some embodiments, the chimeric polypeptide comprises a fusion of an antibody or antigen-binding portion thereof that targets CTLA-4 and disrupts its interaction with CD80 and CD86. Exemplary antibodies targeting CTLA-4 include FDA-approved ipilimumab (MDX-010, MDX-101, Bristol-Myers Squibb) and tremelimumab (ticilimumab, CP-675, 206, Pfizer), which is currently undergoing human trials. Other suitable antibodies targeting CTLA-4 are disclosed in WO 2012 / 120125, U.S. Patent Nos. 6,984720 and 6,682,7368, and U.S. Patent Application Nos. 2002 / 0039581, 2002 / 0086014, and 2005 / 0201994, which are incorporated herein by reference. Those of ordinary skill will understand that any antibody that binds to CTLA-4, disrupts its interaction with CD80 and CD86, and stimulates an anti-tumor immune response is suitable for use in the chimeric polypeptides disclosed herein. In some embodiments, the chimeric polypeptide comprises a fusion with an anti-CTLA-4 antibody.

[0298] In some embodiments, the chimeric polypeptide comprises a fusion with an antibody, or an antigen-binding portion thereof, that targets LAG-3 and disrupts its interaction with MHC class II molecules. An exemplary antibody targeting LAG-3 is IMP321 (Immutep), which is currently undergoing human trials. Other suitable antibodies targeting LAG-3 are disclosed in U.S. Patent Application 2011 / 0150892, which is incorporated herein by reference. One of ordinary skill will appreciate that any antibody that binds to LAG-3, disrupts its interaction with MHC class II molecules, and stimulates an anti-tumor immune response is suitable for use in the chimeric polypeptides disclosed herein. In some embodiments, the chimeric polypeptide comprises a fusion with an anti-LAG-3 antibody.

[0299] In some embodiments, the chimeric polypeptide comprises a fusion with an antibody, or an antigen-binding portion thereof, that targets TIGIT and disrupts its interaction with CD155 (PVR) and / or CD112 (PVRL2, adhesion protein 2). One of ordinary skill will understand that any antibody that binds to TIGIT, disrupts its interaction with CD155 (PVR) and / or CD112 (PVRL2, adhesion protein 2) and stimulates an anti-tumor immune response or immunostimulatory response that produces an overall anti-tumor activity is suitable for use in the chimeric polypeptides disclosed herein. In some embodiments, the chimeric polypeptide comprises a fusion with an anti-TIGIT antibody.

[0300] In some embodiments, the chimeric polypeptide comprises a fusion with an antibody or antigen-binding portion thereof that targets CD112R (also known as PVRIG) and disrupts its interaction with CD112 and / or PVRL2 / nectin 2. One of ordinary skill will appreciate that any antibody that binds to CD112R, disrupts its interaction with CD112 and / or PVRL2 / nectin 2, and stimulates an anti-tumor immune response or immunostimulatory response that generally produces anti-tumor activity is suitable for use in the chimeric polypeptides disclosed herein. In some embodiments, the chimeric polypeptide comprises a fusion with an anti-CD112R antibody.

[0301] In some embodiments, the chimeric polypeptide comprises a fusion with an antibody or antigen-binding portion thereof that targets B7-H3 or B7-H4. The B7 family does not have any defined receptors, but these ligands are upregulated on tumor cells or tumor-infiltrating cells. An exemplary antibody targeting B7-H3 is MGA271 (Macrogenics), which is currently undergoing human trials. Other suitable antibodies targeting B7 family members are disclosed in U.S. Patent Application 2013 / 0149236, which is incorporated herein by reference. One of ordinary skill will appreciate that any antibody that binds to B7-H3 or H4 and stimulates an anti-tumor immune response is suitable for use in the chimeric polypeptides disclosed herein. In some embodiments, the chimeric polypeptide comprises a fusion with an anti-B7-H3 or B7-H4 antibody.

[0302] In some embodiments, the chimeric polypeptide comprises a fusion with an antibody, or an antigen-binding portion thereof, that targets TIM-3 and disrupts its interaction with galectin-9. Suitable antibodies targeting TIM-3 are disclosed in U.S. Patent Application 2013 / 0022623, which is incorporated herein by reference. A person of ordinary skill will appreciate that any antibody that binds to TIM-3, disrupts its interaction with galectin-9, and stimulates an anti-tumor immune response is suitable for use in the chimeric polypeptides disclosed herein. In some embodiments, the chimeric polypeptide comprises a fusion with an anti-TIM-3 antibody.

[0303] In some embodiments, the chimeric polypeptide comprises a fusion of an antibody or antigen binding portion thereof that targets 4-1BB / CD137 and disrupts its interaction with CD137L. One of ordinary skill will appreciate that any antibody that binds to 4-1BB / CD137, disrupts its interaction with CD137L or another ligand, and stimulates an overall anti-tumor immune response or immunostimulatory response that produces anti-tumor activity is suitable for use in the chimeric polypeptides disclosed herein. In some embodiments, the chimeric polypeptide comprises a fusion of an anti-4-1BB / CD137 antibody.

[0304] In some embodiments, the chimeric polypeptide comprises a fusion with an antibody, or antigen-binding portion thereof, that targets GITR and disrupts its interaction with a ligand. One of ordinary skill will appreciate that any antibody that binds to GITR, disrupts its interaction with GITRL or another ligand, and stimulates an anti-tumor immune response or immunostimulatory response that generally produces anti-tumor activity is suitable for use in the chimeric polypeptides disclosed herein. In some embodiments, the chimeric polypeptide comprises a fusion with an anti-GITR antibody.

[0305] In some embodiments, the chimeric polypeptide comprises a fusion with an antibody, or antigen-binding portion thereof, that targets OX40 and disrupts its interaction with a ligand. One of ordinary skill will appreciate that any antibody that binds to OX40, disrupts its interaction with OX40L or another ligand, and stimulates an anti-tumor immune response or immunostimulatory response that generally produces anti-tumor activity is suitable for use in the chimeric polypeptides disclosed herein. In some embodiments, the chimeric polypeptide comprises a fusion with an anti-OX40 antibody.

[0306] In some embodiments, the chimeric polypeptide comprises a fusion with an antibody, or antigen-binding portion thereof, that targets CD40 and disrupts its interaction with a ligand. One of ordinary skill will appreciate that any antibody that binds to CD40, disrupts its interaction with a ligand, and stimulates an anti-tumor immune response or immunostimulatory response that generally produces anti-tumor activity is suitable for use in the chimeric polypeptides disclosed herein. In some embodiments, the chimeric polypeptide comprises a fusion with an anti-CD40 antibody.

[0307] In some embodiments, the chimeric polypeptide comprises a fusion with an antibody, or an antigen-binding portion thereof, that targets ICOS and disrupts its interaction with a ligand. One of ordinary skill will appreciate that any antibody that binds to ICOS, disrupts its interaction with a ligand, and stimulates an anti-tumor immune response or immunostimulatory response that generally produces anti-tumor activity is suitable for use in the chimeric polypeptides disclosed herein. In some embodiments, the chimeric polypeptide comprises a fusion with an anti-ICOS antibody.

[0308] In some embodiments, the chimeric polypeptide comprises a fusion with an antibody or antigen binding portion thereof that targets CD28 and disrupts its interaction with a ligand. One of ordinary skill will appreciate that any antibody that binds to CD28, disrupts its interaction with a ligand, and stimulates an anti-tumor immune response or immunostimulatory response that generally produces anti-tumor activity is suitable for use in the chimeric polypeptides disclosed herein. In some embodiments, the chimeric polypeptide comprises a fusion with an anti-CD28 antibody. In some embodiments, the chimeric polypeptide comprises a fusion with an anti-CD3 antibody, including a T cell engager anti-CD3 antibody.

[0309] In some embodiments, the chimeric polypeptide comprises a fusion with an antibody or antigen-binding portion thereof that targets IFNα and disrupts its interaction with a ligand. One of ordinary skill will appreciate that any antibody that binds to IFNα, disrupts its interaction with a ligand, and stimulates an anti-tumor immune response or immunostimulatory response that generally produces anti-tumor activity is suitable for use in the chimeric polypeptides disclosed herein. In some embodiments, the chimeric polypeptide comprises a fusion with an anti-IFNα antibody.

[0310] In some embodiments, the chimeric polypeptide comprises a fusion with a tumor antigen or a polypeptide targeting a tumor antigen. Typically, tumor antigens allow tumor cells to be distinguished from their normal cell counterparts and may include, for example, tumor-specific antigens (TSAs) and tumor-associated antigens (TAAs). In some embodiments, tumor antigens are proto-oncogenes and / or tumor suppressors, as well as overexpressed or abnormally expressed cellular proteins, tumor antigens produced by oncogenic viruses, tumor-fetal antigens, altered cell surface glycolipids and glycoproteins, and / or cell type-specific differentiation antigens. Such tumor antigens may include melanoma antigens, cancer-testis antigens, epithelial tumor antigens, cell cycle regulatory proteins, prostate-specific antigens (including prostate cancer antigens, such as those disclosed in, for example, U.S. Patent No. 5,538,866), lymphomas (U.S. Patent Nos. 4,816,249, 5,068,177, and 5,227,159). Tumor antigens can include, for example, but are not limited to, HMW mucins that bind to 2G3 and 369F10, c-erbB-2 related tumor antigen (a glycoprotein of approximately 42 kD or 55 kD), antigens of approximately 40, 60, 100, and 200 kD that bind to 113F1, 9-O-acetyl GD3, p97, alpha-fetoprotein (AFP) (e.g., for germ cell tumors and / or hepatocellular carcinoma), carcinoembryonic antigen (CEA) (e.g., for colorectal cancer, sporadic lung cancer, or breast cancer), CA-125 (e.g., for ovarian cancer), MUC- 1 (e.g., for breast cancer), epithelial tumor antigen (ETA) (e.g., for breast cancer), tyrosinase (e.g., for malignant melanoma), melanoma-associated antigen (MAGE) (e.g., for malignant melanoma), cancer / testis antigen 1 (CTAG1B), melanoma-associated antigen 1 (MAGEA1), aberrant Ras products, aberrant p53 products, overexpressed cyclins (including, for example, cyclin B1), mutant fibronectin, post-translationally altered MUC1 glycoprotein, secreted tumor antigens (including, for example, gangliosides).

[0311] Other fusions can include fusions with pro-apoptotic payloads. Such exemplary sequences are provided in the following table. In some embodiments, IL-2 mutant proteins as described herein are fused with pro-apoptotic payloads, such as BAD, BAX, BAK, BIK and / or BID sequences. In some embodiments, the pro-apoptotic payload is a peptide containing a Bcl-2 domain and / or a sequence of BAD, BAX, BAK, BIK and / or BID sequences. Exemplary pro-apoptotic fusions are provided in Table 3 below.

[0312] Table 3: List of selected pro-apoptotic fusion partners

[0313]

[0314] In some specific embodiments, IL-2 antagonists can be fused with a pro-apoptotic payload to treat cancer." Antagonist" is a compound that, for example, resists the action of an agonist by preventing, reducing, inhibiting or neutralizing the activity of an agonist. Even in the absence of an identified agonist, "antagonist" can also prevent, inhibit or reduce the constitutive activity of a target (e.g., a target receptor). Although IL-2 mutant proteins with agonist or superagonist activity compared to wild-type IL-2 are generally used together with the cancer treatment methods of the present invention, when such antagonists are fused with a pro-apoptotic payload, IL-2 mutant proteins with antagonistic properties can also be used. In some embodiments, compared to the wild-type IL-2 of SEQ ID NO: 2, IL-2 antagonists include the following amino acid substitutions: L18R, Q22E, Q126T, and S130R. In some embodiments, the IL-2 antagonist comprises the following amino acid substitutions L18R, Q22E, L80F, R81D, L85V, I86V, and Q126T compared to the wild-type IL-2 of SEQ ID NO: 2. In some embodiments, the IL-2 antagonist comprises the following amino acid substitutions L18R, Q22E, L80F, R81D, L85V, I86V, Q126T, and S130R compared to the wild-type IL-2 of SEQ ID NO: 2. Exemplary antagonists that can be fused to pro-apoptotic payloads, such as those provided above, are provided in Table 4 below.

[0315] Table 4: IL-2 antagonists for fusion with pro-apoptotic payloads

[0316]

[0317]

[0318]

[0319] Other fusions may include fusions with anti-apoptotic payloads for prolonged activation of CD8 cells, NK cells, and anergic NK cells, and exemplary sequences of such are provided in the table below. Such prolonged activation of T cells may prove beneficial in cancer therapy methods.

[0320] Table 5: List of exemplary IL-2 anti-apoptotic fusion amino acid sequences

[0321]

[0322]

[0323]

[0324] Other exemplary IL-2 fusions include those listed in the following table:

[0325] Table 6: List of exemplary half-life extended IL-2 fusion amino acid sequences

[0326]

[0327]

[0328]

[0329]

[0330]

[0331]

[0332]

[0333]

[0334]

[0335]

[0336]

[0337]

[0338] In some embodiments, the IL-2 mutein-Fc fusion comprises one of the following sequences:

[0339] Table 7: List of IL-2 amino acid sequences

[0340]

[0341]

[0342] In some embodiments, the IL-2 mutein sequence is 90% identical to any one of SEQ ID NO: 12 to SEQ ID NO: 15 and / or SEQ ID NO: 20 to SEQ ID NO: 80 (e.g., any IL-2 sequence provided herein). In some embodiments, the IL-2 mutein sequence is 95% identical to any one of SEQ ID NO: 12 to SEQ ID NO: 15 and / or SEQ ID NO: 20 to SEQ ID NO: 80 (e.g., any IL-2 sequence provided herein). In some embodiments, the IL-2 mutein sequence is 98% identical to any one of SEQ ID NO: 12 to SEQ ID NO: 15 and / or SEQ ID NO: 20 to SEQ ID NO: 80 (e.g., any IL-2 sequence provided herein). In some embodiments, the IL-2 mutein sequence is 99% identical to any one of SEQ ID NO: 12 to SEQ ID NO: 15 and / or SEQ ID NO: 20 to SEQ ID NO: 80 (e.g., any IL-2 sequence provided herein).

[0343] C. IL-4, IL-7, IL-13, IL-12, IL15, IL-18, or IL-33 for Bispecific IL-2 Cytokine Fusions

[0344] In some embodiments, IL-2 mutant protein can be fused with IL-4 mutant protein as described herein. In some embodiments, IL-2 mutant protein can be fused with IL-13 mutant protein as described herein. In some embodiments, IL-2, IL-4 or IL-13 mutant protein can be fused with IL-7. In some embodiments, IL-2, IL-4 or IL-13 mutant protein can be fused with IL-10. In some embodiments, IL-2, IL-4 or IL-13 mutant protein can be fused with IL-12. In some embodiments, IL-2, IL-4 or IL-13 mutant protein can be fused with IL-15. In some embodiments, IL-2, IL-4 or IL-13 mutant protein can be fused with IL-18. In some embodiments, IL-2, IL-4 or IL-13 mutant protein can be fused with IL-33. In some embodiments, such fusions play a role in specifically targeting cancer cells and / or cancer stem cells and reducing or inhibiting cancer stem cell growth, as well as targeting the immunosuppressive cells in the tumor microenvironment (TME).

[0345] Any IL-13 sequence or variant thereof can be used in combination with an IL-2 mutein as described herein. In some embodiments, the IL-2 mutein comprises any one of S-1 SEQ ID NO:5, S-2 SEQ ID NO:6, 6-6 SEQ ID NO:7, A2 SEQ ID NO:8, B1 SEQ ID NO:9, B11 SEQ ID NO:10, C5 SEQ ID NO:11, D10 SEQ ID NO:12, E10 SEQ ID NO:13, G8 SEQ ID NO:14, H4 SEQ ID NO:15, and H9 SEQ ID NO:16. Exemplary IL-13 polypeptide sequences are provided in SEQ ID NO:81-SEQ ID NO:128 and in the table below. In some embodiments, the IL-13 polypeptide sequence is provided in any one of SEQ ID NO:81-SEQ ID NO:128. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO:81. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO: 82. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO: 83. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO: 84. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO: 85. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO: 86. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO: 87. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO: 88. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO: 89. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO: 90. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO: 91. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO: 92. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO: 93. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO: 94. In some embodiments, the polypeptide sequence is SEQ ID NO: 95. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO: 96. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO: 97. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO: 98. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO: 99. In some embodiments, the polypeptide sequence is SEQ ID NO: 100.In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO: 101. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO: 102. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO: 103. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO: 104. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO: 105. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO: 106. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO: 107. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO: 108. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO: 109. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO: 110. In some embodiments, the polypeptide sequence is SEQ ID NO: 111. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO: 112. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO: 113. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO: 114. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO: 115. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO: 116. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO: 117. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO: 118. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO: 119. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO: 120. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO: 121. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO: 122. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO: 123. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO: 124. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO: 125. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO: 126. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO: 127. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO: 128. In some embodiments, the IL-13 polypeptide sequence is 90% identical to any one of SEQ ID NO: 81 to SEQ ID NO: 128.In some embodiments, the IL-13 polypeptide sequence is 95% identical to any one of SEQ ID NO: 81 to SEQ ID NO: 128. In some embodiments, the IL-13 polypeptide sequence is 98% identical to any one of SEQ ID NO: 81 to SEQ ID NO: 128. In some embodiments, the IL-13 polypeptide sequence is 99% identical to any one of SEQ ID NO: 81 to SEQ ID NO: 128.

[0346] In some embodiments, any one of SEQ ID NO: 81-SEQ ID NO: 128 is linked to an IL-2 or IL-2 mutant protein as described herein. In some embodiments, SEQ ID NO: 81 is linked to an IL-2 or IL-2 mutant protein as described herein. In some embodiments, SEQ ID NO: 82 is linked to an IL-2 or IL-2 mutant protein as described herein. In some embodiments, SEQ ID NO: 83 is linked to an IL-2 or IL-2 mutant protein as described herein. In some embodiments, SEQ ID NO: 84 is linked to an IL-2 or IL-2 mutant protein as described herein. In some embodiments, SEQ ID NO: 85 is linked to an IL-2 or IL-2 mutant protein as described herein. In some embodiments, SEQ ID NO: 86 is linked to an IL-2 or IL-2 mutant protein as described herein. In some embodiments, SEQ ID NO: 87 is linked to an IL-2 or IL-2 mutant protein as described herein. In some embodiments, SEQ ID NO: 88 is linked to an IL-2 or IL-2 mutant protein as described herein. In some embodiments, SEQ ID NO: 89 is linked to an IL-2 or IL-2 mutant protein as described herein. In some embodiments, SEQ ID NO: 90 is linked to an IL-2 or IL-2 mutant protein as described herein. In some embodiments, SEQ ID NO: 91 is linked to an IL-2 or IL-2 mutant protein as described herein. In some embodiments, SEQ ID NO: 92 is linked to an IL-2 or IL-2 mutant protein as described herein. In some embodiments, SEQ ID NO: 93 is linked to an IL-2 or IL-2 mutant protein as described herein. In some embodiments, SEQ ID NO: 94 is linked to an IL-2 or IL-2 mutant protein as described herein. In some embodiments, SEQ ID NO: 94 is linked to an IL-2 or IL-2 mutant protein as described herein. In some embodiments, SEQ ID NO: 96 is linked to an IL-2 or IL-2 mutant protein as described herein. In some embodiments, SEQ ID NO: 97 is linked to an IL-2 or IL-2 mutant protein as described herein. In some embodiments, SEQ ID NO: 98 is linked to an IL-2 or IL-2 mutant protein as described herein. In some embodiments, SEQ ID NO: 99 is linked to an IL-2 or IL-2 mutant protein as described herein. In some embodiments, SEQ ID NO: 100 is linked to an IL-2 or IL-2 mutant protein as described herein. In some embodiments, SEQ ID NO: 101 is linked to an IL-2 or IL-2 mutant protein as described herein.In some embodiments, SEQ ID NO: 102 is linked to an IL-2 or IL-2 mutant protein as described herein. In some embodiments, SEQ ID NO: 103 is linked to an IL-2 or IL-2 mutant protein as described herein. In some embodiments, SEQ ID NO: 104 is linked to an IL-2 or IL-2 mutant protein as described herein. In some embodiments, SEQ ID NO: 105 is linked to an IL-2 or IL-2 mutant protein as described herein. In some embodiments, SEQ ID NO: 106 is linked to an IL-2 or IL-2 mutant protein as described herein. In some embodiments, SEQ ID NO: 107 is linked to an IL-2 or IL-2 mutant protein as described herein. In some embodiments, SEQ ID NO: 108 is linked to an IL-2 or IL-2 mutant protein as described herein. In some embodiments, SEQ ID NO: 109 is linked to an IL-2 or IL-2 mutant protein as described herein. In some embodiments, SEQ ID NO: 110 is linked to an IL-2 or IL-2 mutant protein as described herein. In some embodiments, SEQ ID NO: 111 is linked to an IL-2 or IL-2 mutant protein as described herein. In some embodiments, SEQ ID NO: 112 is linked to an IL-2 or IL-2 mutant protein as described herein. In some embodiments, SEQ ID NO: 113 is linked to an IL-2 or IL-2 mutant protein as described herein. In some embodiments, SEQ ID NO: 114 is linked to an IL-2 or IL-2 mutant protein as described herein. In some embodiments, SEQ ID NO: 115 is linked to an IL-2 or IL-2 mutant protein as described herein. In some embodiments, SEQ ID NO: 116 is linked to an IL-2 or IL-2 mutant protein as described herein. In some embodiments, SEQ ID NO: 117 is linked to an IL-2 or IL-2 mutant protein as described herein. In some embodiments, SEQ ID NO: 118 is linked to an IL-2 or IL-2 mutant protein as described herein. In some embodiments, SEQ ID NO: 119 is linked to an IL-2 or IL-2 mutant protein as described herein. In some embodiments, SEQ ID NO: 120 is linked to an IL-2 or IL-2 mutein as described herein. In some embodiments, SEQ ID NO: 121 is linked to an IL-2 or IL-2 mutein as described herein. In some embodiments, SEQ ID NO: 122 is linked to an IL-2 or IL-2 mutein as described herein. In some embodiments, SEQ ID NO: 123 is linked to an IL-2 or IL-2 mutein as described herein.In some embodiments, SEQ ID NO: 124 is linked to an IL-2 or IL-2 mutant protein as described herein. In some embodiments, SEQ ID NO: 125 is linked to an IL-2 or IL-2 mutant protein as described herein. In some embodiments, SEQ ID NO: 126 is linked to an IL-2 or IL-2 mutant protein as described herein. In some embodiments, SEQ ID NO: 127 is linked to an IL-2 or IL-2 mutant protein as described herein. In some embodiments, SEQ ID NO: 128 is linked to an IL-2 or IL-2 mutant protein as described herein. In some embodiments, the IL-2 mutein comprises any one of 5-1 SEQ ID NO:5, 5-2 SEQ ID NO:6, 6-6 SEQ ID NO:7, A2 SEQ ID NO:8, B1 SEQ ID NO:9, B11 SEQ ID NO:10, C5 SEQ ID NO:11, D10 SEQ ID NO:12, E10 SEQ ID NO:13, G8 SEQ ID NO:14, H4 SEQ ID NO:15, and H9 SEQ ID NO:16.

[0347] In some embodiments, the IL-13 peptides of the present invention comprise one or more of the following amino acid substitutions: (1) L10F, L10I, L10V, L10A, L10D, L10T, L10H; (2) R11S, R11N, R11H, R11L, R11I; (3) I14L, I14F, I14V, I14M; (4) V18L, V18F, V18I; (5) E12A, (6) R65D, (7) R86K, R86T, R86M; (8) D87E, D87K, D87R, D87G, D87S; (9) T88I, T88K, T88R; (10) K89R, K89T, K89M; (11) L101 F, L101I, L101Y, L101H, L101N; (12) K104R, K104T, K104M; (13) K105T, K105A, K105R, K105E; (14) F107L, F107I, F107V, F107M; (15) R108K, R108T, R108M; and (16) E15R, which substitutions result in altered affinity for one or both of IL-13Rα1 and IL-13Rα2. In other embodiments, the modified residues are at two or more, three or more, four or more, five or more, and no more than 14 amino acids within the combined set of contact residues defined above. As described in International Patent Publication WO 2013 / 112871, the disclosure of which is incorporated herein by reference in its entirety. In some embodiments, amino acid substitutions include, but are not limited to Figure 4 Those provided in .

[0348] The set of modifications may include the following specific changes: (1) L10H; L10A; (2) R11L; (4) V18I; (7) R86M; R86K; R86T; (8) D87K; D87G; (9) T88R; T88S; T88K; (10) K89R; (11) L101N; (12) K104R; (13) K105A; K105E; (14) R108K; (15) E15R. In some embodiments, the modification includes any of the listed specific changes. In some embodiments, the modification includes L10H. In some embodiments, the modification includes L10A. In some embodiments, the modification includes R11L. In some embodiments, the modification includes E15R. In some embodiments, the modification includes V18I. In some embodiments, the modification includes R86M. In some embodiments, the modification includes R86K. In some embodiments, the modification comprises R86T. In some embodiments, the modification comprises D87K. In some embodiments, the modification comprises D87G. In some embodiments, the modification comprises T88R. In some embodiments, the modification comprises T88S. In some embodiments, the modification comprises T88K. In some embodiments, the modification comprises K89R. In some embodiments, the modification comprises L101N. In some embodiments, the modification comprises K104R. In some embodiments, the modification comprises K105A. In some embodiments, the modification comprises K105E. In some embodiments, the modification comprises R108K. In some embodiments, the polypeptide comprising one or more modifications is linked to an IL-2 or IL-2 mutant protein as described herein. In some embodiments, amino acid substitutions include but are not limited to Figure 4 In some embodiments, the IL-2 mutein comprises any one of S-1 SEQ ID NO: 5, S-2 SEQ ID NO: 6, 6-6 SEQ ID NO: 7, A2 SEQ ID NO: 8, B1 SEQ ID NO: 9, B11 SEQ ID NO: 10, C5 SEQ ID NO: 11, D10 SEQ ID NO: 12, E10 SEQ ID NO: 13, G8 SEQ ID NO: 14, H4 SEQ ID NO: 15, and H9 SEQ ID NO: 16.

[0349] Specific sets of modifications that provide increased selectivity for binding to IL-13Rα2 over IL-13Rα1 relative to the native IL-13 sequence may include, but are not limited to:

[0350] [L10D, R11I, V18I, R86K, D87K, k89R, R108K] (e.g., C2, e.g., SEQ ID NO: 109)

[0351] [L10A, R86T, D87G, T88K, K89R, L101N, K104R, K105A, R108K] (e.g., C3, e.g., SEQ ID NO: 110)

[0352] [L10V, K89R, L101N, K105E, R108T] (e.g., C4, e.g., SEQ ID NO: 111 or SEQ ID NO: 93)

[0353] [R11S, I14M, T88S, L101N, K105A, R108K] (e.g., C7, e.g., SEQ ID NO: 112 or SEQ ID NO: 94)

[0354] [L10H, R11L, V18I, R86K, D87E, K89R, L101N, K105T, R108K] (C9, e.g., SEQ ID NO: 113)

[0355] [L10H, R86T, D87G, T88R, R108K] (C11, e.g., SEQ ID NO: 98 or SEQ ID NO: 115)

[0356] [L10H, E15R, R86T, D87G, T88R, R108K] (MDNA132+E15R, e.g., SEQ ID NO: 395)

[0357] [L10A, V18F, R86K, D87K, K89R, L101I, K104R, R108K] (D7, e.g., SEQ ID NO: 117)

[0358] ·[L10T / D; R11I; V18I; R86K; D87K / G; T88S; K89R; L101Y; K104R; K105T; R108K]

[0359] ·[L10A / V; R86T; D87G; T88K; K89R; L101N; K104R; K105A / E; R108K / T]

[0360] In some embodiments, the set of modifications comprises L10V, K89R, L101N, K105E, R108T. In some embodiments, the set of modifications comprises R11S, I14M, T88S, L101N, K105A, and R108K (C7, e.g., SEQ ID NO: 112 or SEQ ID NO: 94). In some embodiments, the set of modifications comprises L10H, R11L, V18I, R86K, D87E, K89R, L101N, K105T, and R108K (C9, e.g., SEQ ID NO: 113). In some embodiments, the set of modifications comprises L10H, R86T, D87G, T88R, and R108K (C11, e.g., SEQ ID NO: 98 or SEQ ID NO: 115). In some embodiments, the set of modifications comprises L10H, E15R, R86T, D87G, T88R, and R108K (MDNA132+E15R, e.g., SEQ ID NO: 395). In some embodiments, the set of modifications comprises L10A, V18F, R86K, D87K, K89R, L101I, K104R, and R108K (D7, e.g., SEQ ID NO: 117). In some embodiments, the set of modifications comprises L10T / D, R11I, V18I, R86K, D87K / G, T88S, K89R, L101Y, K104R, K105T, and R108K. In some embodiments, the set of modifications comprises L10T, R11I, V18I, R86K, D87K, T88S, K89R, L101Y, K104R, K105T, and R108K. In some embodiments, the set of modifications comprises L10T, R11I, V18I, R86K, D87G, T88S, K89R, L101Y, K104R, K105T, and R108K. In some embodiments, the set of modifications comprises L10D, R11I, V18I, R86K, D87K, T88S, K89R, L101Y, K104R, K105T, and R108K. In some embodiments, the set of modifications comprises L10D, R11I, V18I, R86K, D87G, T88S, K89R, L101Y, K104R, K105T, R108K. In some embodiments, the set of modifications comprises L10A / V, R86T, D87G, T88K, K89R, L101N, K104R, K105A / E, and R108K / T. In some embodiments, the set of modifications comprises L10A, R86T, D87G, T88K, K89R, L101N, K104R, K105A, and R108K.In some embodiments, the set of modifications comprises L10A, R86T, D87G, T88K, K89R, L101N, K104R, K105E, and R108K. In some embodiments, the set of modifications comprises L10A, R86T, D87G, T88K, K89R, L101N, K104R, K105A, and R108T. In some embodiments, the set of modifications comprises L10A, R86T, D87G, T88K, K89R, L101N, K104R, K105E, and R108T. In some embodiments, the set of modifications comprises L10V, R86T, D87G, T88K, K89R, L101N, K104R, K105A, and R108K. In some embodiments, the set of modifications comprises L10V, R86T, D87G, T88K, K89R, L101N, K104R, K105E, and R108K. In some embodiments, the set of modifications comprises L10V, R86T, D87G, T88K, K89R, L101N, K104R, K105A, and R108T. In some embodiments, the set of modifications comprises L10V, R86T, D87G, T88K, K89R, L101N, K104R, K105E, and R108T. In some embodiments, the amino acid sequences are 90% identical. In some embodiments, the amino acid sequences are 95% identical. In some embodiments, the amino acid sequences are 98% identical. In some embodiments, the amino acid sequences are 99% identical. In some embodiments, the polypeptide comprising one or more of the modifications is linked to an IL-2 or IL-2 mutein as described herein. In some embodiments, amino acid substitutions include, but are not limited to. Figure 4 In some embodiments, the IL-2 mutein comprises any one of 5-1 SEQ ID NO: 5, 5-2 SEQ ID NO: 6, 6-6 SEQ ID NO: 7, A2 SEQ ID NO: 8, B1 SEQ ID NO: 9, B11 SEQ ID NO: 10, C5 SEQ ID NO: 11, D10 SEQ ID NO: 12, E10 SEQ ID NO: 13, G8 SEQ ID NO: 14, H4 SEQ ID NO: 15, and H9 SEQ ID NO: 16.

[0361] Specific sets of modifications that provide increased selectivity for binding to IL-13Rα1 over IL-13Rα2 relative to the native IL-13 sequence may include, but are not limited to:

[0362] ·[L10V, V18I, D87S, D88S, L101F, K104R, K105T];

[0363] ·[R11S, V18I, R86K, D87G, T88S, K89M, L101Y, K104R, K105T];

[0364] ·[L10V, V18I, D87S, T88S, L101F, K104R, K105T];

[0365] ·[L10V / I; D87S; T88S; K89R; L101H / F; K104R; K105T];

[0366] ·[L10I; V18I; R86T; D87G; T88S; K89R; L101Y / H; K104R; K105A];

[0367] ·[L10V; V18I; D87S; T88S; L101F; K104R; K105T];

[0368] ·[V18I, R86T, D87G, T88S, L101Y, K104R, K105A];

[0369] ·[R11I, V18I, R86K, D87G, T88S, L101H, K104R, K105A, F107M];

[0370] These substitutions are optionally combined with the substitutions [E12A / G / S, R65D / E];

[0371] [L10V, V18I, D87S, T88S, L101F, K104R, K105T, and R39 polymorphisms];

[0372] [L10V, V18I, D87S, T88S, L101F, K104R, K105T, and Q111 polymorphisms];

[0373] [L10V, V18I, D87S, T88S, L101F, K104R, K105T, R39 and Q111 polymorphisms].

[0374] [E15R]

[0375] ·[L10V, V18I, D87S, D88S, L101F, K104R, K105T, E15R];

[0376] ·[R11S, V18I, R86K, D87G, T88S, K89M, L101Y, K104R, K105T, E15R];

[0377] ·[L10V, V18I, D87S, T88S, L101F, K104R, K105T, E15R];

[0378] ·[L10V / I; D87S; T88S; K89R; L101H / F; K104R; K105T, E15R];

[0379] ·[L10I; V18I; R86T; D87G; T88S; K89R; L101Y / H; K104R; K105A, E15R];

[0380] ·[L10V; V18I; D87S; T88S; L101F; K104R; K105T, E15R];

[0381] ·[V18I, R86T, D87G, T88S, L101Y, K104R, K105A, E15R];

[0382] ·[R11I, V18I, R86K, D87G, T88S, L101H, K104R, K105A, F107M, E15R];

[0383] These substitutions are optionally combined with substitutions [E12A / G / S, R65D / E] and / or E15R];

[0384] [L10V, E15R, V18I, D87S, T88S, L101F, K104R, K105T, and R39 polymorphisms];

[0385] [L10V, E15R, V18I, D87S, T88S, L101F, K104R, K105T, and Q111 polymorphisms]; and

[0386] [L10V, E15R, V18I, D87S, T88S, L101F, K104R, K105T, and R39 and Q111 polymorphisms].

[0387] In some embodiments, the modified set comprises L10V, V18I, D87S, D88S, L101F, K104R, and K105T. In some embodiments, the modified set comprises R11S, V18I, R86K, D87G, T88S, K89M, L101Y, K104R, and K105T. In some embodiments, the modified set comprises L10V, V18I, D87S, T88S, L101F, K104R, and K105T. In some embodiments, the modified set comprises L10V, V18I, D87S, T88S, L101F, K104R, K105T, and R39 polymorphisms. In some embodiments, the modified set comprises L10V, V18I, D87S, T88S, L101F, K104R, K105T, and Q111 polymorphisms. In some embodiments, the modified set comprises L10V, V18I, D87S, T88S, L101F, K104R, K105T, and Q111 polymorphisms. In some embodiments, the modified set comprises L10V, V18I, D87S, T88S, L101F, K104R, K105T, R39 polymorphisms, and Q111 polymorphisms. In some embodiments, the modified set comprises L10V / I, D87S, T88S, K89R, L101H / F, K104R, and K105T. In some embodiments, the modification set comprises L10I, V18I, R86T, D87G, T88S, K89R, L101Y / H, K104R, and K105A. In some embodiments, the modification set comprises L10V, V18I, D87S, T88S, L101F, K104R, and K105T. In some embodiments, the modification set comprises V18I, R86T, D87G, T88S, L101Y, K104R, and K105A. In some embodiments, the modification set comprises R11I, V18I, R86K, D87G, T88S, L101H, K104R, K105A, and F107M. In some embodiments, the set of modifications comprises L10V, V18I, D87S, D88S, L101F, K104R, K105T, E12A / G / S, and R65D / E. In some embodiments, the set of modifications comprises R11S, V18I, R86K, D87G, T88S, K89M, L101Y, K104R, K105T, E12A / G / S, and R65D / E. In some embodiments, the set of modifications comprises L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12A / G / S, and R65D / E.In some embodiments, the set of modifications comprises L10V / I, D87S, T88S, K89R, L101H / F, K104R, K105T, E12A / G / S, and R65D / E. In some embodiments, the set of modifications comprises L10I, V18I, R86T, D87G, T88S, K89R, L101Y / H, K104R, K105A, E12A / G / S, and R65D / E. In some embodiments, the set of modifications comprises L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12A / G / S, and R65D / E. In some embodiments, the set of modifications comprises V18I, R86T, D87G, T88S, L101Y, K104R, K105A, E12A / G / S, and R65D / E. In some embodiments, the set of modifications comprises R11I, V18I, R86K, D87G, T88S, L101H, K104R, K105A, F107M, E12A / G / S, and R65D / E. In some embodiments, the set of modifications comprises L10V, V18I, D87S, D88S, L101F, K104R, K105T, E12A, and R65D / E. In some embodiments, the set of modifications comprises R11S, V18I, R86K, D87G, T88S, K89M, L101Y, K104R, K105T, E12A, and R65D / E. In some embodiments, the set of modifications comprises L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12A, and R65D / E. In some embodiments, the set of modifications comprises L10V / I, D87S, T88S, K89R, L101H / F, K104R, K105T, E12A, and R65D / E. In some embodiments, the set of modifications comprises L10I, V18I, R86T, D87G, T88S, K89R, L101Y / H, K104R, K105A, E12A, and R65D / E. In some embodiments, the set of modifications comprises L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12A, and R65D / E. In some embodiments, the set of modifications comprises V18I, R86T, D87G, T88S, L101Y, K104R, K105A, E12A, and R65D / E. In some embodiments, the set of modifications comprises R11I, V18I, R86K, D87G, T88S, L101H, K104R, K105A, F107M, E12A, and R65D / E. In some embodiments, the set of modifications comprises L10V, V18I, D87S, D88S, L101F, K104R, K105T, E12G, and R65D / E.In some embodiments, the set of modifications comprises R11S, V18I, R86K, D87G, T88S, K89M, L101Y, K104R, K105T, E12G, and R65D / E. In some embodiments, the set of modifications comprises L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12A / G / S, and R65D / E. In some embodiments, the set of modifications comprises L10V / I, D87S, T88S, K89R, L101H / F, K104R, K105T, E12G, and R65D / E. In some embodiments, the set of modifications comprises L10I, V18I, R86T, D87G, T88S, K89R, L101Y / H, K104R, K105A, E12G, and R65D / E. In some embodiments, the set of modifications comprises L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12G, and R65D / E. In some embodiments, the set of modifications comprises V18I, R86T, D87G, T88S, L101Y, K104R, K105A, E12G, and R65D / E. In some embodiments, the set of modifications comprises R11I, V18I, R86K, D87G, T88S, L101H, K104R, K105A, F107M, E12G, and R65D / E. In some embodiments, the set of modifications comprises L10V, V18I, D87S, D88S, L101F, K104R, K105T, E12S, and R65D / E. In some embodiments, the set of modifications comprises R11S, V18I, R86K, D87G, T88S, K89M, L101Y, K104R, K105T, E12A / G / S, and R65D / E. In some embodiments, the modified set comprises L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12S, and R65D / E. In some embodiments, the modified set comprises L10V / I, D87S, T88S, K89R, L101H / F, K104R, K105T, E12S, and R65D / E. In some embodiments, the modified set comprises L10I, V18I, R86T, D87G, T88S, K89R, L101Y / H, K104R, K105A, E12S, and R65D / E. In some embodiments, the modified set comprises L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12S, and R65D / E. In some embodiments, the set of modifications comprises V18I, R86T, D87G, T88S, L101Y, K104R, K105A, E12S, and R65D / E.In some embodiments, the modification set comprises R11I, V18I, R86K, D87G, T88S, L101H, K104R, K105A, F107M, E12S, and R65D / E. In some embodiments, the modification set comprises L10V, V18I, D87S, D88S, L101F, K104R, K105T, E12A, and R65D. In some embodiments, the modification set comprises R11S, V18I, R86K, D87G, T88S, K89M, L101Y, K104R, K105T, E12A, and R65E. In some embodiments, the modification set comprises L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12A, and R65D. In some embodiments, the modified set comprises L10V / I, D87S, T88S, K89R, L101H / F, K104R, K105T, E12A, and R65D. In some embodiments, the modified set comprises L10I, V18I, R86T, D87G, T88S, K89R, L101Y / H, K104R, K105A, E12A, and R65D. In some embodiments, the modified set comprises L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12A, and R65D. In some embodiments, the modified set comprises V18I, R86T, D87G, T88S, L101Y, K104R, K105A, E12A, and R65D. In some embodiments, the modification set comprises R11I, V18I, R86K, D87G, T88S, L101H, K104R, K105A, F107M, E12A, and R65D. In some embodiments, the modification set comprises L10V, V18I, D87S, D88S, L101F, K104R, K105T, E12G, and R65D. In some embodiments, the modification set comprises R11S, V18I, R86K, D87G, T88S, K89M, L101Y, K104R, K105T, E12G, and R65D. In some embodiments, the modification set comprises L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12A / G / S, and R65D. In some embodiments, the set of modifications comprises L10V / I, D87S, T88S, K89R, L101H / F, K104R, K105T, E12G, and R65D. In some embodiments, the set of modifications comprises L10I, V18I, R86T, D87G, T88S, K89R, L101Y / H, K104R, K105A, E12G, and R65D.In some embodiments, the modified set comprises L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12G, and R65D. In some embodiments, the modified set comprises V18I, R86T, D87G, T88S, L101Y, K104R, K105A, E12G, and R65D. In some embodiments, the modified set comprises R11I, V18I, R86K, D87G, T88S, L101H, K104R, K105A, F107M, E12G, and R65D. In some embodiments, the modified set comprises L10V, V18I, D87S, D88S, L101F, K104R, K105T, E12S, and R65D. In some embodiments, the modified set comprises R11S, V18I, R86K, D87G, T88S, K89M, L101Y, K104R, K105T, E12S, and R65D. In some embodiments, the modified set comprises L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12S, and R65D. In some embodiments, the modified set comprises L10V / I, D87S, T88S, K89R, L101H / F, K104R, K105T, E12S, and R65D. In some embodiments, the modified set comprises L10I, V18I, R86T, D87G, T88S, K89R, L101Y / H, K104R, K105A, E12S, and R65D. In some embodiments, the modified set comprises L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12S, and R65D. In some embodiments, the modified set comprises V18I, R86T, D87G, T88S, L101Y, K104R, K105A, E12S, and R65D. In some embodiments, the modified set comprises R11I, V18I, R86K, D87G, T88S, L101H, K104R, K105A, F107M, E12S, and R65D. In some embodiments, the modified set comprises L10V, V18I, D87S, D88S, L101F, K104R, K105T, E12A, and R65E. In some embodiments, the set of modifications comprises R11S, V18I, R86K, D87G, T88S, K89M, L101Y, K104R, K105T, E12A, and R65E. In some embodiments, the set of modifications comprises L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12A, and R65E.In some embodiments, the modification set comprises L10V / I, D87S, T88S, K89R, L101H / F, K104R, K105T, E12A, and R65E. In some embodiments, the modification set comprises L10I, V18I, R86T, D87G, T88S, K89R, L101Y / H, K104R, K105A, E12A, and R65E. In some embodiments, the modification set comprises L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12A, and R65E. In some embodiments, the modification set comprises V18I, R86T, D87G, T88S, L101Y, K104R, K105A, E12A, and R65E. In some embodiments, the modification set comprises R11I, V18I, R86K, D87G, T88S, L101H, K104R, K105A, F107M, E12A, and R65E. In some embodiments, the modification set comprises L10V, V18I, D87S, D88S, L101F, K104R, K105T, E12G, and R65E. In some embodiments, the modification set comprises R11S, V18I, R86K, D87G, T88S, K89M, L101Y, K104R, K105T, E12G, and R65E. In some embodiments, the modification set comprises L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12A / G / S, and R65E. In some embodiments, the modification set comprises L10V / I, D87S, T88S, K89R, L101H / F, K104R, K105T, E12G, and R65E. In some embodiments, the modification set comprises L10I, V18I, R86T, D87G, T88S, K89R, L101Y / H, K104R, K105A, E12G, and R65E. In some embodiments, the modification set comprises L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12G, and R65E. In some embodiments, the modification set comprises V18I, R86T, D87G, T88S, L101Y, K104R, K105A, E12G, and R65E. In some embodiments, the set of modifications comprises R11I, V18I, R86K, D87G, T88S, L101H, K104R, K105A, F107M, E12G, and R65E. In some embodiments, the set of modifications comprises L10V, V18I, D87S, D88S, L101F, K104R, K105T, E12S, and R65E.In some embodiments, the set of modifications comprises R11S, V18I, R86K, D87G, T88S, K89M, L101Y, K104R, K105T, E12A / G / S, and R65E. In some embodiments, the set of modifications comprises L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12S, and R65E. In some embodiments, the set of modifications comprises L10V / I, D87S, T88S, K89R, L101H / F, K104R, K105T, E12S, and R65E. In some embodiments, the modification set comprises L10I, V18I, R86T, D87G, T88S, K89R, L101Y / H, K104R, K105A, E12S, and R65E. In some embodiments, the modification set comprises L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12S, and R65E. In some embodiments, the modification set comprises V18I, R86T, D87G, T88S, L101Y, K104R, K105A, E12S, and R65E. In some embodiments, the modification set comprises R11I, V18I, R86K, D87G, T88S, L101H, K104R, K105A, F107M, E12S, and R65E. In some embodiments, the set of modifications comprises L10V, E12A, V18I, R65D, D87S, T88S, L101F, K104R, and K105T (see, e.g., IL-13dn; SEQ ID NO: 118). In some embodiments, the set of modifications further comprises E15R. In some embodiments, the amino acid sequences are 90% identical. In some embodiments, the amino acid sequences are 95% identical. In some embodiments, the amino acid sequences are 98% identical. In some embodiments, the amino acid sequences are 99% identical. In some embodiments, the polypeptide comprising one or more modifications is linked to an IL-2 or IL-2 mutein as described herein. In some embodiments, amino acid substitutions include, but are not limited to. Figure 3 In some embodiments, the IL-2 mutein comprises any one of 5-1 SEQ ID NO:5, 5-2 SEQ ID NO:6, 6-6 SEQ ID NO:7, A2 SEQ ID NO:8, B1 SEQ ID NO:9, B11 SEQ ID NO:10, C5 SEQ ID NO:11, D10 SEQ ID NO:12, E10 SEQ ID NO:13, G8 SEQ ID NO:14, H4 SEQ ID NO:15, and H9 SEQ ID NO:16.

[0388] A table of IL-13 sequences is provided below.

[0389] Table 8: List of IL-13 amino acid sequences

[0390]

[0391]

[0392]

[0393]

[0394]

[0395]

[0396]

[0397] Any IL-4 sequence or variant thereof can be fused with an IL-2 mutant protein or variant (including those described herein). In some embodiments, the IL-2 mutant protein comprises any one of 5-1 SEQ ID NO: 5, 5-2 SEQ ID NO: 6, 6-6 SEQ ID NO: 7, A2 SEQ ID NO: 8, B1 SEQ ID NO: 9, B11 SEQ ID NO: 10, C5 SEQ ID NO: 11, D10 SEQ ID NO: 12, E10 SEQ ID NO: 13, G8 SEQ ID NO: 14, H4 SEQ ID NO: 15, and H9 SEQ ID NO: 16. Exemplary polypeptide sequences are provided in SEQ ID NO: 130-SEQ ID NO: 135, including any one of those provided herein. In some embodiments, the IL-4 polypeptide sequence is provided as any one of SEQ ID NO: 130 to SEQ ID NO: 135. In some embodiments, the IL-4 polypeptide sequence is SEQ ID NO: 130. In some embodiments, the IL-4 polypeptide sequence is SEQ ID NO: 131. In some embodiments, the IL-4 polypeptide sequence is SEQ ID NO: 132. In some embodiments, the IL-4 polypeptide sequence is SEQ ID NO: 133. In some embodiments, the IL-4 polypeptide sequence is SEQ ID NO: 134. In some embodiments, the IL-4 polypeptide sequence is SEQ ID NO: 135. In some embodiments, the IL-4 polypeptide sequence is 98% identical to any one of SEQ ID NO: 130 to SEQ ID NO: 135. In some embodiments, the IL-4 polypeptide sequence is 99% identical to any one of SEQ ID NO: 130 to SEQ ID NO: 135. In some embodiments, any one of SEQ ID NO: 130 to SEQ ID NO: 135 is linked to an IL-2 or IL-2 mutein as described herein. In some embodiments, SEQ ID NO: 130 is linked to an IL-2 or IL-2 mutein as described herein. In some embodiments, SEQ ID NO: 131 is linked to an IL-2 or IL-2 mutein as described herein. In some embodiments, SEQ ID NO: 132 is linked to an IL-2 or IL-2 mutein as described herein. In some embodiments, SEQ ID NO: 133 is linked to an IL-2 or IL-2 mutein as described herein. In some embodiments, SEQ ID NO: 134 is linked to an IL-2 or IL-2 mutein as described herein.In some embodiments, SEQ ID NO: 135 is linked to an IL-2 or IL-2 mutant protein as described herein. In some embodiments, the IL-2 mutant protein comprises any one of 5-1 SEQ ID NO: 5, 5-2 SEQ ID NO: 6, 6-6 SEQ ID NO: 7, A2 SEQ ID NO: 8, B1 SEQ ID NO: 9, B11 SEQ ID NO: 10, C5 SEQ ID NO: 11, D10 SEQ ID NO: 12, E10 SEQ ID NO: 13, G8 SEQ ID NO: 14, H4 SEQ ID NO: 15, and H9 SEQ ID NO: 16. In some embodiments, the IL-4 component comprises the following substitutions compared to wild-type IL-4: R121K, Y124F, S125R. In some embodiments, the IL-4 component comprises the following substitutions compared to wild-type IL-4: K117R, T118V, R121Q, D122S, Y124W, S125F, S128G, S129A.

[0398] A table of IL-4 sequences is provided below.

[0399] Table 9: List of IL-4 amino acid sequences

[0400]

[0401] In some embodiments, IL-2 mutant proteins can be fused with IL-7, IL-12, IL-15, IL-18 and / or IL-33 sequences. In some embodiments, such fusions serve to specifically target the fusion construct to NK cells and / or CD8+ cells. In some embodiments, the IL-2 mutant protein comprises 5-1 SEQ ID NO:5, 5-2 SEQ ID NO:6, 6-6 SEQ ID NO:7, A2 SEQ ID NO:8, B1 SEQ ID NO:9, B11 SEQ ID NO:10, C5 SEQ ID NO:11, D10 SEQ ID NO:12, E10 SEQ ID NO:13, G8 SEQ ID NO:14, H4 SEQ ID NO:15 and H9 SEQ ID NO:16. In some embodiments, SEQ ID NO: 136 is connected to IL-2 or IL-2 mutant proteins as described herein. In some embodiments, SEQ ID NO: 137 is linked to IL-2 or IL-2 mutant proteins as described herein. In some embodiments, SEQ ID NO: 138 is linked to IL-2 or IL-2 mutant proteins as described herein. In some embodiments, SEQ ID NO: 139 is linked to IL-2 or IL-2 mutant proteins as described herein. In some embodiments, SEQ ID NO: 140 is linked to IL-2 or IL-2 mutant proteins as described herein. In some embodiments, SEQ ID NO: 141 is linked to IL-2 or IL-2 mutant proteins as described herein. In some embodiments, the IL-2 mutant proteins can be fused to the IL-IL-7, IL-12, IL-15, IL-18, and / or IL-33 sequences provided in the following table in SEQ ID NOs: 136-141.

[0402] Table 10: Exemplary IL-10, IL-12, IL-15 and / or IL-18 sequences

[0403]

[0404]

[0405] The sequences of exemplary IL-2 muteins include any one of 5-1 SEQ ID NO:5, 5-2 SEQ ID NO:6, 6-6 SEQ ID NO:7, A2 SEQ ID NO:8, B1 SEQ ID NO:9, B11 SEQ ID NO:10, C5 SEQ ID NO:11, D10 SEQ ID NO:12, E10 SEQ ID NO:13, G8 SEQ ID NO:14, H4 SEQ ID NO:15, and H9 SEQ ID NO:16.

[0406] In some embodiments, the cytokine-cytokine fusion is one of those included in the table below.

[0407] Table 11: List of exemplary IL-2 fusion amino acid sequences

[0408]

[0409]

[0410]

[0411] In some embodiments, the cytokine-cytokine fusion is one of those included in the following table (see Table 12, and WO2021258213 (incorporated herein by reference in its entirety) Figure 54 ).

[0412] Table 12: List of long-acting and bifunctional fusion constructs

[0413]

[0414]

[0415]

[0416]

[0417]

[0418]

[0419]

[0420]

[0421]

[0422]

[0423] Table 13: List of fusion constructs with indicated mutations

[0424]

[0425]

[0426]

[0427]

[0428]

[0429]

[0430]

[0431]

[0432]

[0433]

[0434]

[0435]

[0436]

[0437]

[0438]

[0439]

[0440]

[0441]

[0442]

[0443]

[0444]

[0445]

[0446]

[0447]

[0448]

[0449]

[0450]

[0451]

[0452]

[0453]

[0454]

[0455]

[0456]

[0457]

[0458]

[0459]

[0460]

[0461]

[0462]

[0463]

[0464]

[0465]

[0466]

[0467]

[0468]

[0469]

[0470]

[0471]

[0472]

[0473]

[0474]

[0475]

[0476]

[0477]

[0478]

[0479]

[0480] D. Recombinant Expression of IL-2, IL-4, or IL-13 Mutant Bifunctional Molecules, Expression Vectors, and Host Cells

[0481] In various embodiments, the polypeptides used in the practice of the present invention are synthesized or produced by expression of a recombinant nucleic acid molecule. If the polypeptide is a chimera (e.g., a fusion protein comprising at least a mutant IL-2 polypeptide and a heterologous polypeptide, including bispecific IL-2 cytokine fusions), it can be encoded by a hybrid nucleic acid molecule comprising a sequence encoding all or part of the IL-2, IL-4, or IL-13 mutein bifunctional molecule and a second sequence encoding all or part of the heterologous polypeptide. For example, the subject IL-2, IL-4, or IL-13 mutein bifunctional molecules described herein can be fused to a hexahistidine tag to facilitate purification of bacterially expressed proteins, or to a hemagglutinin tag to facilitate purification of proteins expressed in eukaryotic cells.

[0482] Methods for constructing DNA sequences encoding bifunctional molecules of IL-2, IL-4, or IL-13 mutant proteins and expressing those sequences in appropriately transformed hosts include, but are not limited to, PCR-assisted mutagenesis techniques. Mutations consisting of deletions or additions of amino acid residues of the IL-2 polypeptide can also be performed using standard recombinant techniques. In the case of deletions or additions, the nucleic acid molecule encoding IL-2 is optionally digested with an appropriate restriction endonuclease. The resulting fragment can be expressed directly or further manipulated, for example, by ligating it to a second fragment. If the two ends of the nucleic acid molecule contain complementary nucleotides that overlap, this ligation can be facilitated, but blunt-ended fragments can also be ligated. Nucleic acids generated by PCR can also be used to generate various mutant sequences.

[0483] The complete amino acid sequence can be used to construct a reverse-translation gene. DNA oligomers containing the nucleotide sequence encoding the bifunctional IL-2, IL-4, or IL-13 mutein can be synthesized. For example, several small oligonucleotides encoding various portions of the desired polypeptide can be synthesized and then ligated. Individual oligonucleotides typically contain 5' or 3' overhangs to facilitate complementary assembly.

[0484] In addition to producing mutant polypeptides by expressing nucleic acid molecules that have been altered by recombinant molecular biology techniques, the subject IL-2, IL-4 or IL-13 mutant protein bifunctional molecules can also be chemically synthesized. Chemically synthesized polypeptides are routinely generated by those skilled in the art.

[0485] Once assembled (by synthesis, site-directed mutagenesis or other methods), the DNA sequence encoding the IL-2, IL-4 or IL-13 mutein bifunctional molecule is inserted into an expression vector and operably linked to expression control sequences suitable for expressing the IL-2, IL-4 or IL-13 mutein bifunctional molecule in the desired transformed host. Proper assembly can be confirmed by nucleotide sequencing, restriction enzyme mapping, and expression of the biologically active polypeptide in a suitable host. As is well known in the art, in order to achieve high expression levels of the transfected gene in the host, the gene must be operably linked to transcriptional and translational expression control sequences that are functional in the selected expression host.

[0486] Whether the DNA sequence encoding the bifunctional IL-2, IL-4 or IL-13 mutant protein prepared by site-directed mutagenesis, chemical synthesis or other methods can also include a DNA sequence encoding a signal sequence. If present, such a signal sequence should be a signal sequence recognized by the cells selected for expressing the bifunctional IL-2, IL-4 or IL-13 mutant protein. It can be prokaryotic, eukaryotic or a combination of the two. It can also be the signal sequence of natural IL-2. The inclusion of the signal sequence depends on whether it is necessary to secrete the bifunctional IL-2, IL-4 or IL-13 mutant protein from the recombinant cells that produce it. If the selected cell is prokaryotic, it is generally preferred that the DNA sequence does not encode a signal sequence. If the selected cell is eukaryotic, it is generally preferred that an encoding signal sequence and most preferably a wild-type IL-2 signal sequence are used.

[0487] E. Oncolytic Virus Targeting Moiety

[0488] In some instances, bispecific IL-2 cytokine fusions and / or IL-2, IL-4 or IL-13 mutant protein bifunctional molecules described herein can be used to target oncolytic viruses (e.g., see Allen et al., Mol. Ther. 16: 1556-64, 2008). In some instances, oncolytic viruses are armed to tumors or TME by IL-2, IL-4 or IL-13 mutant protein bifunctional molecules. Many viruses can be used as oncolytic viruses, including adenoviruses and self-replicating alphaviruses, and oncolytic vaccinia viruses (see, for example, WO2013038066, which is incorporated herein by reference in its entirety; in particular Figure 17Other oncolytic viruses can include Seneca Valley virus, Newcastle disease virus (also known as Newcastle virus), Maraba virus, vesicular stomatitis virus (VSV), herpes virus (including HSV-1), measles virus, poliovirus, reovirus, coxsackie virus, lentivirus, measles virus, influenza virus, Sinbis virus, myxoma virus and / or retrovirus (see, for example, Twumasi-Boateng et al., "Oncolytic viruses as engineering platforms for combination immunotherapy", Nature Reviews Cancer, 2018), and Kaufman et al., Cancer Immunotherapy, 14: 642-662 (2015), all of which are incorporated herein by reference in their entirety). In some embodiments, the oncolytic virus includes but is not limited to adenovirus, self-replicating alphavirus, vaccinia virus, Seneca Valley virus, Newcastle disease virus, Maraba virus, vesicular stomatitis virus (VSV), herpes virus (including HSV-1 and HSV-2), measles virus, poliovirus, reovirus, coxsackie virus, slow virus, measles virus, influenza virus, Sindbis virus, myxoma virus and / or retrovirus. IL-2 super factor (H9 and IL-2 variants as described herein) can also be used to guide T cells / OV to TME. IL-2 variants (such as H9) can strengthen effector T cells and NK cells, and IL-2 variants can suppress T reg activity. Other oncolytic viruses can include, for example, oncoVex / T-VEC, which involves intratumoral injection of conditional replicative herpes simplex viruses that preferentially infect cancer cells. The virus, which has also been engineered to express GM-CSF, is able to replicate inside cancer cells, causing them to lyse, releasing new viruses and a range of tumor antigens, and in the process secreting GM-CSF. Such oncolytic virus vaccines enhance DC function in the tumor microenvironment to stimulate anti-tumor immune responses. These oncolytic viruses can be used to target or deliver the IL-2, IL-4 or IL-13 mutant proteins described herein to tumors, including the bifunctional molecules described herein. These oncolytic viruses can be used to target or deliver the IL-2, IL-4 or IL-13 mutant protein bifunctional molecules described herein to tumors. In some embodiments, the IL-2, IL-4 or IL-13 mutant protein bifunctional molecules are any of the IL-2,IL-4 or IL-13 mutein bifunctional molecules or variants. In some embodiments, the IL-2 mutein sequence is 90% identical to any one of SEQ ID NO: 2 or SEQ ID NO: 6 to SEQ ID NO: 10 or SEQ ID NO: 16. In some embodiments, the IL-2 mutein or bifunctional molecule comprises any one of 5-1 SEQ ID NO: 5, 5-2 SEQ ID NO: 6, 6-6 SEQ ID NO: 7, A2 SEQ ID NO: 8, B1 SEQ ID NO: 9, B11 SEQ ID NO: 10, C5 SEQ ID NO: 11, D10 SEQ ID NO: 12, E10 SEQ ID NO: 13, G8 SEQ ID NO: 14, H4 SEQ ID NO: 15, and H9 SEQ ID NO: 16. In some embodiments, the substitution in the IL-2 mutant protein or bifunctional molecule includes L80F, R81D, L85V, I86V and I92F according to the wild-type human IL-2 numbering of SEQ ID NO: 2. In some embodiments, the oncolytic virus comprises a transgene capable of expressing an IL-2 mutant protein or bifunctional molecule as described herein. In some embodiments, the oncolytic virus comprises a transgene capable of expressing an IL-2 mutant protein or bifunctional molecule, the IL-2 mutant protein or bifunctional molecule comprising the following amino acid substitutions L80F, R81D, L85V, I86V and I92F according to the wild-type human IL-2 numbering of SEQ ID NO: 2. In some embodiments, the oncolytic virus comprises a nucleic acid encoding an IL-2 mutant protein or bifunctional molecule, the IL-2 mutant protein or bifunctional molecule comprising the following amino acid substitutions L80F, R81D, L85V, I86V and I92F according to the wild-type human IL-2 numbering of SEQ ID NO: 2. In some embodiments, the oncolytic virus comprises a transgene expressed as a therapeutic payload. In some embodiments, the therapeutic payload is 11-2 as described herein. In some embodiments, the therapeutic payload is an IL-2 mutant protein or bifunctional molecule comprising the following amino acid substitutions numbered according to wild-type human IL-2 of SEQ ID NO: 2: L80F, R81D, L85V, I86V, and I92F. In some embodiments, the oncolytic virus can be used to target or deliver a bifunctional molecule comprising (i) an amino acid sequence based on IL-2 of Table 2, and (ii) an amino acid sequence of any one of Table 3, Table 4, Table 8, Table 9, or Table 10. In some embodiments, the oncolytic virus can be used to target or deliver a bifunctional molecule comprising (i) an amino acid sequence based on IL-4 of Table 4 or Table 9, and (ii) an amino acid sequence of any one of Table 2, Table 3,An amino acid sequence of any one of Table 8 or Table 10. In some embodiments, an oncolytic virus can be used to target or deliver a bifunctional molecule comprising (i) an amino acid sequence based on IL-13 of Table 8, and (ii) an amino acid sequence of any one of Table 2, Table 3, Table 4, Table 9, or Table 10. In some embodiments, an oncolytic virus can be used to target or deliver a bifunctional molecule comprising (i) an amino acid sequence based on IL-7, IL-12, IL-15, IL-18, or IL-33 of Table 10, and (ii) an amino acid sequence of one of Table 2, Table 3, Table 4, Table 8, or Table 9. In some embodiments, an oncolytic virus can be used to target or deliver a bifunctional molecule comprising an amino acid sequence of SEQ ID NO: 395, 484, 501, 502, 503, 504, 505, 506, 507, or 508, and an amino acid sequence based on IL-2 of Table 2. In some embodiments, an oncolytic virus can be used to target or deliver a bifunctional molecule comprising an amino acid sequence of SEQ ID NO: 395, 484, 501, 502, 503, 504, 505, 506, 507 or 508, and an amino acid sequence selected from the group consisting of SEQ ID NO: 6 (H9-F42A), SEQ ID NO: 7 (H9-K43N), SEQ ID NO: 8 (H9-F42A / Y45A; H9-FYAA), SEQ ID NO: 9 (H9-F42A / E62A; H9-FEAA), SEQ ID NO: 10; H9-F42A / Y45A / E62A; H9-FYEAAA), SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27 NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 (MDNA109 or H9), SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 146 (F42A, E62A, L80F, R81D, L85V, I86V, I92F and C125S). In some embodiments, oncolytic viruses can be used to target or deliver bifunctional molecules comprising the following substitutions compared to wild-type IL-13: L10H, E15R, R86T, D87G,T88R and R108K; optionally wherein the bifunctional molecule further comprises an R39 polymorphism and / or a Q111 polymorphism. In some embodiments, an oncolytic virus can be used to target or deliver a bifunctional molecule comprising the following substitutions compared to wild-type IL-13: L10V, E12A, V18I, R65D, D87S, T88S, L101F, K104R, and K105T; optionally wherein the bifunctional molecule further comprises an R39 polymorphism and / or a Q111 polymorphism. In some embodiments, an oncolytic virus can be used to target or deliver a bifunctional molecule comprising the following substitutions compared to wild-type IL-2: L80F, R81D, L85V, I86V, I92F; optionally, wherein the bifunctional molecule further comprises the following substitutions compared to wild-type IL-2: F42A and E62A, and / or optionally, wherein the bifunctional molecule further comprises the following substitutions compared to wild-type IL-2: C125S. In some embodiments, an oncolytic virus can be used to target or deliver a bifunctional molecule comprising the following substitutions compared to wild-type IL-4: R121K, Y124F, S125R. In some embodiments, an oncolytic virus can be used to target or deliver a bifunctional molecule comprising the following substitutions compared to wild-type IL-4: K117R, T118V, R121Q, D122S, Y124W, S125F, S128G, S129A. In some embodiments, oncolytic viruses can be used to target or deliver bifunctional molecules comprising one or more amino acid sequences of any one of Table 2, Table 3, Table 4, Table 8, Table 9, or Table 10, including one or more cytokine binding portions of Table 2, Table 3, Table 4, Table 8, Table 9, or Table 10. In some embodiments, oncolytic viruses can be used to target or deliver bifunctional molecules comprising one or more amino acid sequences of any one of Table 5, Table 6, Table 7, Table 11, Table 12, Table 13, Table 15, or Table 39, including one or more cytokine binding portions of Table 5, Table 6, Table 7, Table 11, Table 12, Table 13, Table 15, or Table 39. In some embodiments, an oncolytic virus can be used to target or deliver a bifunctional molecule comprising the amino acid sequence of any one or more of the following: SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158、159、160、161、162、163、164、165、166、167、168、169、170、171、172、173、174、175、176、177、178、179、180、181、182、183、184、185、186、187、188、189、190、191、192、193、194、195、196、197、198、199、200、201、202、203、204、205、206、207、208、209、210、211、212、213、214、215、216、217、218、219、220、221、222、223、224、225、226、227、228、229、230、231、232、233、234、235、236、237、238、239、240、241、242、243、244、245、246、247、248、249、250、251、252、253、254、255、256、257、258、259、260、261、262、263、264、265、266、267、268、269、270、271、272、273、274、275、276、277、278、279、280、281、282、283、284、285、286、287、288、289、290、291、292、293、294、295、296、297、298、299、300、301、302、303、304、305、306、307、308、309、310、311、312、313、314、315、316、317、318、319、320、321、322、323、324、325、326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370 70, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414 , 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 4554, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 501, 502, 503, 504, 505, 506, 507, and / or 508.

[0489] In some embodiments, the oncolytic virus is an oncolytic vaccinia virus. In some embodiments, the oncolytic vaccinia virus vector is characterized in that the virus particles are intracellular mature viruses (IMV), intracellular enveloped viruses (IEV), cell-associated enveloped viruses (CEV) or extracellular enveloped viruses (EEV) types. In some embodiments, the oncolytic vaccinia virus particles are EEV or IMV types. In some embodiments, the oncolytic vaccinia virus particles are EEV types.

[0490] Typically, an oncolytic vaccinia virus recombinant and a cell and a pharmaceutical composition comprising the vector are constructed, wherein the vector preferentially replicates in tumor cells and expresses at least one transgenic (e.g., IL-2, IL-4 or IL-13 mutant protein bifunctional molecules as described herein) to promote anti-tumor efficacy and apoptosis induction, and to regulate the host immune response of the subject. According to the present invention, oncolytic adenovirus and oncolytic vaccinia virus can be combined with IL-2 expression or targeting moiety as described herein to target oncolytic vaccinia virus or oncolytic adenovirus and / or express IL-2, IL-4 or IL-13 mutant protein bifunctional molecules. Oncolysis releases tumor antigens and provides costimulatory danger signals. However, armed viruses can further improve efficacy. For example, it is known that CD40 ligands (CD40L, CD154) induce tumor cell apoptosis and also trigger several immune mechanisms. One of them is type 1 T helper cell (Th1) response, which can result in reduced cytotoxic T cell activation and immunosuppression. The present invention provides oncolytic viruses expressing IL-2, IL-4 or IL-13 mutant protein bifunctional molecules of the present invention. In some embodiments, the present invention provides oncolytic viruses that are targeted (eg, "armed") with a targeting moiety of the invention.

[0491] In some embodiments, the oncolytic virus is a modified vaccinia virus vector, a viral particle, a host cell, a pharmaceutical composition, and a kit comprising a vaccinia virus genome, wherein the thymidine kinase gene is inactivated due to a substitution in the thymidine kinase (TK) gene and / or ablation deletion of at least one nucleotide of the open reading frame, thereby providing a partially deleted thymidine kinase gene, the vaccinia growth factor gene is deleted, and the modified vaccinia virus vector comprises at least one nucleic acid sequence encoding a non-viral protein (e.g., a bifunctional molecule of IL-2, IL-4 or IL-13 mutant proteins capable of expression as described herein). On the other hand, a modified vaccinia virus vector, a viral particle, a pharmaceutical composition, or a kit is provided, which can be used for cancer treatment, for eliciting an immune response in a subject, for inhibiting the proliferation of malignant cells in a mammal, for cancer treatment or prevention, for detecting the presence of a modified vaccinia virus in a subject, and for use as an in situ cancer vaccine, optionally in combination with an adenovirus. In some embodiments, the present invention provides a method for producing a modified vaccinia virus comprising a vaccinia virus genome, wherein the thymidine kinase gene is inactivated due to substitution in the thymidine kinase (TK) gene and / or ablation deletion of at least one nucleotide in the open reading frame, thereby providing a partially deleted thymidine kinase gene, the vaccinia growth factor gene is deleted, and the modified vaccinia virus vector comprises at least one nucleic acid sequence encoding a non-viral protein (e.g., an IL-2, IL-4 or IL-13 bifunctional molecule as described herein), the method comprising the steps of: providing a producer cell capable of maintaining the production of vaccinia virus particles and carrying the modified vaccinia vector; culturing the producer cell under conditions suitable for viral replication and production; and harvesting the viral particles.

[0492] In some embodiments, the present invention provides a method for administering an oncolytic virus, wherein the oncolytic virus is "armed" or includes the nucleic acid encoding the IL-2, IL-4 or IL-13 mutant protein bifunctional molecules as described herein, wherein the IL-2, IL-4 or IL-13 mutant protein bifunctional molecules are expressed in the tumor position or expressed systemically in the subject. In some embodiments, the present invention also provides a method for administering the "armed" or targeted oncolytic virus of the IL-2, IL-4 or IL-13 mutant protein bifunctional molecules as described herein. The route of administration varies naturally with the position and properties of the tumor, and includes, for example, intradermal, transdermal, parenteral, intravenous, intramuscular, intranasal, subcutaneous, regional (e.g., near the tumor, particularly the vasculature of the tumor or adjacent vasculature), percutaneous, intratracheal, intraperitoneal, intraarterial, intravesical, intratumoral, inhalation, perfusion, lavage and oral administration. Compositions are formulated relative to a specific route of administration.

[0493] 1. Oncolytic vaccinia virus

[0494] Vaccinia virus is a member of the genus Orthopoxvirus of the Poxviridae family. It has a large double-stranded DNA genome (about 200kb, about 200 genes), and a complex morphogenetic pathway produces different forms of infectious virions from each infected cell. Viral particles contain a lipid membrane around the nucleus. The viral nucleus contains viral structural proteins, a tightly compressed viral DNA genome, and transcriptases. The size of the vaccinia virus is about 360x270x250nm and weighs about 5-10fg. The genes are tightly packed with a small amount of non-coding DNA and the open reading frame (ORF) lacks introns. There are three types of genes (early, middle, late). Early genes (about 100 genes; immediate early genes and delayed early genes) encode proteins mainly related to immune regulation and viral DNA replication. Middle genes encode regulatory proteins required for the expression of late genes (such as, transcription factors), and late genes encode proteins required for the production of viral particles and enzymes, which are packaged in new virions to start the next round of infection. Vaccinia virus replicates in the cytoplasm.

[0495] Different strains of vaccinia viruses have been identified (e.g., Copenhagen, modified Ankara (MVA), Lister, Tian Tan, Wyeth (New York City Board of Health), Western Reserve (WR)). The genome of WR vaccinia has been sequenced (Accession No. AY243312). In some embodiments, the oncolytic vaccinia virus is Copenhagen, modified Ankara (MVA), Lister, Tian Tan, Wyeth, or Western Reserve (WR) vaccinia virus.

[0496] Different forms of virions play different roles in the viral life cycle. There are several types of virions: intracellular mature virus (IMV), intracellular enveloped virus (IEV), cell-associated enveloped virus (CEV), and extracellular enveloped virus (EEV). EEV particles have an additional membrane derived from the trans-Golgi network. This outer membrane has two important functions: a) protecting the internal IMV from immune attack and b) mediating viral binding to the cell surface.

[0497] CEV and EEV are enclosed in a host-derived membrane that helps the viruses evade host antibodies and complement. IMV and EEV particles have some differences in their biological properties, and they play different roles in the viral life cycle. EEV and IMV bind to different (unknown) receptors (1), and they enter cells through different mechanisms. EEV particles enter cells via endocytosis, and this process is pH-sensitive. After internalization, the outer membrane of EEV ruptures in the acidified endosome, the exposed IMV fuses with the endosomal membrane, and the viral core is released into the cytoplasm. On the other hand, IMV enters the cell through the fusion of the cell membrane and the viral membrane, and this process is independent of pH. In addition, CEV also induces the formation of actin tails on the cell surface, which push the virion into uninfected neighboring cells.

[0498] In addition, EEV can resist neutralization by antibodies (NAbs) and complement toxicity, while IMV cannot. Therefore, EEV mediates long-range transmission in vivo and in vitro. The comet inhibition test has become a method for measuring EEV-specific antibodies because even if EEV NAbs cannot neutralize free EEV, EEV NAbs will prevent EEV from being released from infected cells, and comet-shaped plaques cannot be seen. Compared with IMV particles (lower particle / pfu ratio), EEV has a higher specific infectivity, which makes EEV an interesting candidate for therapeutic use. However, the outer membrane of EEV is an extremely fragile structure and EEV particles need to be handled with caution, which makes it difficult to obtain the amount of EEV particles required for therapeutic applications. The EEV outer membrane ruptures at low pH (pH about 6). Once the EEV outer membrane ruptures, the viral particles within the envelope remain fully infective like IMV.

[0499] Some host cell-derived proteins co-localize with EEV preparations but not with IMV, and the amount of cell-derived proteins depends on the host cell line and virus strain. For example, WR EEV contains more cell-derived proteins than VV IHD-J strain. Host cell-derived proteins can alter the biological effects of EEV particles. For example, the host membrane protein CD55 is incorporated into the EEV surface to render it resistant to complement toxicity. In the present invention, it was demonstrated that proteins derived from human A549 cells on the surface of EEV particles can target the virus to human cancer cells. A similar phenomenon has been demonstrated in studies of human immunodeficiency virus type 1, where the host-derived ICAM-1 glycoprotein increased the infectivity of the virus. The IEV membrane contains at least 9 proteins, two of which are not present in CEV / EEV. The F12L and A36R proteins are involved in the transport of IEV to the cell surface, where they are retained on the cell surface and are not part of CEV / EEV (9, 11). Seven proteins are common among (IEV) / CEV / EEV: F13L, A33R, A34R, A56R, B5R, E2, and (K2L). For the Western Reserve Vaccinia virus strain, typically up to 1% of viral particles are EEV, released into the culture supernatant before oncolysis of producer cells. The International Health Department (IHD)-J vaccinia virus strain releases 50-fold more EEV particles. However, IHD has not been studied for human cancer treatment. The IHD-W phenotype is primarily attributed to a point mutation within the A34R EEV lectin-like protein. Furthermore, deletion of A34R increases EEV release. EEV particles can first be detected on the cell surface (as CEV) 6 hours after infection and in the supernatant (IHD-J strain) 5 hours later. Infection with a low multiplicity of infection (MOI) results in a higher ratio of EEV compared to a high viral dose. The balance between CEV and EEV is influenced by both the host cell and the viral strain.

[0500] Cowpox has been used to eradicate smallpox and later as an expression vector for foreign genes and as a live recombinant vaccine for infectious diseases and cancer. Cowpox virus is the most widely used poxvirus in humans, so safety data for human use are extensive. Millions of people have been safely vaccinated with modified strains of vaccinia virus in smallpox vaccination programs worldwide, and only very rare severe adverse events have been reported. Those adverse events are systemic vaccinia (spread of vaccinia throughout the body), erythema multiforme (toxic / allergic reaction), eczema vaccinatum (widespread infection of the skin), progressive vaccinia (tissue destruction), and post-vaccinia encephalitis.

[0501] A total of 44 melanoma patients were treated with wild-type vaccinia virus in early clinical trials from the 1960s to the 1990s, with an overall objective response rate of 50% in the injected tumor. Some beneficial immune responses were also seen (36). Wild-type vaccinia virus has also been used to treat bladder, lung, and kidney cancers, as well as myeloma, with only mild adverse events. JX-594, an oncolytic Whitworth strain vaccinia virus encoding GM-CSF, has been successfully evaluated in three phase I studies, and preliminary results from a randomized phase II trial have been presented at a scientific conference.

[0502] The vaccinia virus is attractive for cancer gene therapy due to several characteristics. It has a natural tropism for cancer cells, and selectivity can be significantly improved by deleting some viral genes. The present invention relates to the use of a double-deleted vaccinia virus (vvdd), in which two viral genes, viral thymidine kinase (TK) and vaccinia growth factor (VGF), are at least partially deleted. The virus requires the TK and VGF genes to replicate in normal cells but not cancer cells. Partial TK deletions can be engineered in the TK region that confers activity.

[0503] TK-deficient vaccinia virus relies on the cellular nucleotide pool present in dividing cells for DNA synthesis and replication. In some embodiments, TK deficiency significantly limits viral replication in resting cells, thereby allowing only effective viral replication in actively dividing cells (e.g., cancer cells). VGF is secreted from infected cells and produces a paracrine priming effect on surrounding cells by acting as a mitogen. The replication of VGF-deficient vaccinia virus is highly attenuated in resting (non-cancerous) cells. It has been confirmed that the effects of TK and VGF deficiency are synergistic.

[0504] 2. Oncolytic adenovirus

[0505] Typically, adenovirus is a 36 kb linear double-stranded DNA virus (Grunhaus and Horwitz, 1992). The term "adenovirus" or "AAV" includes AAV type 1 (AAV1), AAV type 2 (AAV2), AAV type 3 (AAV3), AAV type 4 (AAV4), AAV type 5 (AAV5), AAV type 6 (AAV6), AAV type 7 (AAV7), AAV type 8 (AAV8), AAV type 9 (AAV9), AAV 9-hu14, avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV. "Primate AAV" refers to AAV that can infect primates, "non-primate AAV" refers to AAV that can infect non-primate mammals, "bovine AAV" refers to AAV that can infect bovine mammals, and the like.

[0506] Adenovirus infection of host cells causes adenoviral DNA to remain episomal, thereby reducing the potential genotoxicity associated with the integration vector. Moreover, adenovirus is structurally stable, and no genome rearrangement has been detected after extensive amplification. Adenovirus can infect almost all epithelial cells, regardless of their cell cycle stage. (See, for example, US20060147420, which is incorporated herein by reference in its entirety). Moreover, the E1a and E4 regions of adenovirus are crucial for the effective and efficient infection of human cells. The E1a gene is the first viral gene transcribed in productive infection, and its transcription does not depend on the action of any other viral gene products. However, the transcription of the remaining early viral genes requires E1a gene expression. The E1a promoter, in addition to regulating the expression of the E1a gene, also integrates the signal for packaging the viral genome and the site required for initiating viral DNA replication. See Schmid, SI and Hearing, P., Current Topics in Microbiology and Immunology, Vol. 199: pp. 67–80 (1995).

[0507] In some embodiments, the oncolytic virus is an oncolytic adenovirus. It has been determined that naturally occurring viruses can be engineered to produce oncolysis in tumor cells (Wildner, 2001; Jacotat, 1967; Kim, 2001; Geoerger et al., 2002; Yan et al., 2003; Vile et al., 2002, each of which is incorporated herein by reference). With respect to adenovirus, specific deletions within its adenoviral genome can weaken its ability to replicate in normal dormant cells while retaining the ability to replicate in tumor cells. Fueyo et al. (2000) have described a conditional replication adenovirus Δ24, see also U.S. Patent Application No. 20030138405, each of which is incorporated herein by reference. Δ24 adenovirus is derived from adenovirus type 5 (Ad-5) and contains a deletion of 24 base pairs within the CR2 portion of the E1A gene. See, for example, WO2001036650A2 (incorporated herein by reference in its entirety).

[0508] Oncolytic adenoviruses include conditionally replicating adenoviruses (CRADs), such as Δ24, which have several properties that make them candidates for use as biotherapeutic agents. One such property is the ability to replicate in permissive cells or tissues, which amplifies the original input dose of the oncolytic virus and helps the agent spread to adjacent tumor cells, thereby providing a direct anti-tumor effect.

[0509] In some embodiments, the oncolytic component of Δ24 is combined with a transgenic expression method to produce armed Δ24. Armed Δ24 adenovirus can be used to produce or enhance a bystander effect within a tumor and / or produce or enhance detection / imaging of oncolytic adenovirus in a patient or tumor-related tissue and / or cell. In some embodiments, the combination of oncolytic adenovirus and various transgenic strategies (e.g., expression of IL-2, IL-4 or IL-13 mutant protein bifunctional molecules) will improve therapeutic potential, including, for example, potential for a variety of refractory tumors, and providing improved imaging capabilities. In certain embodiments, an oncolytic adenovirus can be administered together with a replication-defective adenovirus, another oncolytic virus, a replication-competent adenovirus, and / or a wild-type adenovirus. Each of these can be administered simultaneously with other adenoviruses, before or after them.

[0510] In some embodiments, the E1a adenoviral vector involves replacing the basic adenoviral E1a promoter (including the CAAT box, TATA box, and transcription start site) with a basic promoter that exhibits tumor specificity and is preferably E2F-responsive, and more preferably the human E2F-1 promoter. Thus, the virus will be repressed in cells that lack molecules that activate transcription from E2F-responsive promoters (or such molecules are non-functional). Normal non-dividing or dormant cells fall into this category because the transcription factor E2F binds to pRb or retinoblastoma protein, preventing E2F from binding and activating E2F-responsive promoters. In contrast, cells containing free E2F should support E2F-based transcription. Examples of such cells are neoplastic cells that lack pRb function, allowing productive viral infection to occur. In some embodiments, the E1a adenoviral vector is targeted using the IL-2 portion as described herein.

[0511] Retention of the enhancer sequence, packaging signal, and DNA replication origin within the E1a promoter ensures that adenoviral infection proceeds to wild-type levels in neoplastic cells lacking pRb function. In essence, the modified E1a promoter confers tumor-specific transcriptional activation, resulting in substantial tumor-specific killing, but provides enhanced safety in normal cells.

[0512] In some embodiments, an E1a adenoviral vector is prepared by replacing the endogenous E1a promoter with an E2F-responsive promoter, leaving the elements upstream of nucleotide 375 in the adenovirus 5 genome intact. The nucleotide numbering is described in Schmid, SI and Hearing, P. Current Topics in Microbiology and Immunology, Vol. 199: 67–80 (1995). This includes all seven A repeat motifs identified for packaging of the viral genome. The sequence from nucleotide 375 to nucleotide 536 is deleted by restriction initiation sites from BsaAI to BsrBI, while retaining the 23 base pairs upstream of the E1A protein translation start codon. Using known materials and methods, the deleted endogenous E1a promoter sequence is replaced with an E2F-responsive promoter, preferably human E2F-1. The E2F-1 promoter can be isolated as described in the Examples.

[0513] The E4 region has been implicated in many events that occur late in adenovirus infection and is required for efficient viral DNA replication, late mRNA accumulation and protein synthesis, splicing, and host cell protein synthesis shutdown. Adenoviruses lacking most E4 transcription units are severely replication-defective and, in general, must be propagated in E4-complementing cell lines to obtain high titers. The E4 promoter is located near the right end of the viral genome and controls the transcription of multiple open reading frames (ORFs). Many regulatory elements have been characterized in this promoter that are essential for mediating maximal transcriptional activity. In addition to these sequences, the E4 promoter region contains regulatory sequences that are required for viral DNA replication. A description of the E4 promoter and the location of these regulatory sequences can be found in U.S. Patent No. 7,001,596. Figure 2 and Figure 3 , which is incorporated herein by reference in its entirety.

[0514] In some embodiments, the E4 minimal promoter of the adenoviral vector is replaced with a minimal promoter that has been shown to exhibit tumor specificity, preferably an E2F-responsive promoter, and more preferably the human E2F-1 promoter. The reasons for preferring an E2F-responsive promoter to drive E4 expression are the same as those discussed above in the context of replacing the E1a promoter of the E1a adenoviral vector with an E2F-responsive promoter. The tumor suppressor function of pRb is related to its ability to repress E2F-responsive promoters such as the E2F-1 promoter (Adams, PD and WG Kaelin, Jr. 1995, Cancer Biol. 6:99–108; Sellers, WR and WG Kaelin. 1996, published corrigendum to appear in Biochim Biophys Acta, December 9, 1996; 1288(3):E-1, Biochim Biophys Acta. 1288:M1–5. Sellers, WR, JW Rodgers and WG Kaelin, Jr. 1995, Proc Natl Acad Sci USA. 92:11544–8). The human E2F-1 promoter has been extensively characterized and shown to be responsive to the pRb signaling pathway, including pRb / p107, E2F-1 / -2 / -3 and the G1 cyclin / cdk complex, as well as E1A (Johnson, DG, K. Ohtani and JR Nevins. 1994, Genes Dev. 8:1514–25; Neuman, E., EK Flemington, WR Sellers and WG Kaelin, Jr. 1995. Mol Cell Biol. 15:4660; Neuman, E., WR Sellers, JA McNeil, JB Lawrence and WG Kaelin, Jr. 1996, Gene. 173:163–9). Much, if not all, of this regulation has been attributed to the presence of multiple E2F sites within the E2F-1 promoter. Therefore, viruses carrying this modification(s) are expected to be attenuated in normal cells containing an intact (wild-type) pRb pathway, while exhibiting normal infection / replication properties in cells lacking pRb repressor function.To maintain the normal infection / replication properties of the mutant virus, we retained the inverted terminal repeat (ITR) sequence distal to the E4 promoter, as this sequence contains all the regulatory elements required for viral DNA replication (Hatfield, L. and P. Hearing. 1993, J. Virol. 67:3931–9; Rawlins, DR, PJ Rosenfeld, RJ Wides, MD Challberg and TJ Kelly, Jr. 1984, Cell. 37:309–19; Rosenfeld, PJ, EA O'Neill, RJ Wides, and TJ Kelly. 1987, Mol Cell Biol. 7:875–86; Wides, RJ, MD Challberg, DR Rawlins, and TJ Kelly. 1987, Mol Cell Biol. 7:864–74). This facilitates the achievement of wild-type viral levels in pRb pathway-deficient tumor cells infected with the virus.

[0515] In some embodiments, the E4 promoter is located near the right end of the viral genome, and it controls the transcription of multiple open reading frames (ORFs) (Freyer, GA, Y. Katoh and RJ Roberts. 1984, Nucleic Acids Res. 12:3503–19; Tigges, MA and HJ Raskas. 1984. Splice junctions in adenovirus 2 early region 4 mRNAs: multiple splice sites produce 18 to 24 RNAs. J. Virol. 50:106–17; Virtanen, A P Gilardi, A. Naslund, J M Le Moullec, U. Pettersson and M. Perricaudet. 1984, J. Virol. 51:822–31). A number of regulatory elements that mediate transcriptional activity have been characterized in this promoter (Berk, AJ 1986, Annu Rev Genet. 20:45-79; Gilardi, P. and M. Perricaudet. 1986, Nucleic Acids Res. 14:9035-49; Gilardi, P. and M. Perricaudet. 1984, Nucleic Acids Res. 12:7877-88; Hanaka, S., T. Nishigaki, P. A. Sharp and H. Handa. 1987, Mol Cell Biol. 7:2578-87; Jones, C. and K. A. Lee. 1991, Mol Cell Biol. 11:4297-305; Lee, K. A. and M. R. Green. 1987, Embo J. 6:1345-53). In addition to these sequences, the E4 promoter region also contains elements involved in viral DNA replication (Hatfield, L. and P. Hearing. 1993, J Virol. 67:3931-9; Rawlins, DR, PJ Rosenfeld, RJ Wides, MD Challberg and TJ Kelly, Jr. 1984, Cell. 37:309-19; Rosenfeld, PJ, EA O'Neill, RJ Wides and TJ Kelly. 1987, Mol Cell Biol. 7:875-86; Wides, RJ, MD Challberg, DR Rawlins and TJ Kelly. 1987, Mol Cell Biol. 7:864-74).A description of the E4 promoter and the location of these regulatory sequences can be found in Figures 1 and . Figure 2 See also Jones, C. and KA Lee. Mol Cell Biol. 11:4297–305 (1991). Taking these factors into consideration, the E4 promoter shuttle was designed by creating two novel restriction endonuclease sites: an XhoI site at nucleotide 35,576 and an SpeI site at nucleotide 35,815 (see Figure 3 Digestion with both XhoI and SpeI removed nucleotides 35,581 to 35,817. This effectively eliminated bases -208 to +29 relative to the E4 transcription start site, encompassing all sequences shown to have the greatest impact on E4 transcription. Specifically, this encompassed the two inverted repeats of the E4F binding site, which have been shown to have the most significant effect on promoter activation. However, all three Sp1 binding sites, two of the five ATF binding sites, and both the NF1 and NFIII / Oct-1 binding sites, which are critical for viral DNA replication, were retained.

[0516] In some embodiments, the E2F-responsive promoter is the human E2F-1 promoter. Key regulatory elements in the E2F-1 promoter that mediate responses to the pRb pathway have been mapped in vivo and in vitro (Johnson, DG, K. Ohtani, and JR Nevins. 1994, Genes Dev. 8:1514–25; Neuman, E., EK Flemington, WR Sellers, and WG Kaelin, Jr. 1995, Mol Cell Biol. 15:4660; Parr, MJ, Y. Manome, T. Tanaka, P. Wen, DW Kufe, WG Kaelin, Jr., and HAFine. 1997, Nat Med. 3:1145–9). Therefore, we isolated a human E2F-1 promoter fragment from base pairs -218 to +51 relative to the transcription start site by PCR using primers that incorporate SpeI and XhoI sites. This creates the same site present in the E4 promoter shuttle and allows the E2F-1 promoter to directly replace the E4 promoter.

[0517] F. Nucleic Acid Molecules Encoding Mutated IL-2, IL-4, and / or IL-13

[0518] In some embodiments, the subject IL-2, IL-4, or IL-13 muteins, either alone or as part of bifunctional molecules such as those described above (including bispecific IL-2 cytokine fusions), can be expressed by nucleic acid molecules. Just as bifunctional IL-2, IL-4, or IL-13 mutein molecules can be described in terms of their identity to a wild-type IL-2 polypeptide, the nucleic acid molecules encoding them must also have a certain degree of identity to a nucleic acid molecule encoding wild-type IL-2. For example, a nucleic acid molecule encoding a subject IL-2 mutein can be at least 50%, at least 65%, preferably at least 75%, more preferably at least 85%, and most preferably at least 95% (e.g., 99%) identical to a nucleic acid encoding wild-type IL-2 (e.g., SEQ ID NO: 2).

[0519] In some embodiments, the subject IL-2 muteins can be expressed from nucleic acid molecules, either alone or as part of chimeric polypeptides such as those described above (including bispecific IL-4 cytokine fusions). Just as IL-4 muteins can be described in terms of their identity to a wild-type IL-4 polypeptide, the nucleic acid molecules encoding them must also have a certain degree of identity to a nucleic acid molecule encoding wild-type IL-4. For example, a nucleic acid molecule encoding a subject IL-4 mutein can be at least 50%, at least 65%, preferably at least 75%, more preferably at least 85%, and most preferably at least 95% (e.g., 99%) identical to a nucleic acid encoding wild-type IL-4 (e.g., SEQ ID NO: 129).

[0520] In some embodiments, the subject IL-13 muteins can be expressed from nucleic acid molecules, either alone or as part of a chimeric polypeptide such as those described above (including bispecific IL-13 cytokine fusions). Just as IL-13 muteins can be described in terms of their identity to a wild-type IL-13 polypeptide, the nucleic acid molecules encoding them must also have a certain degree of identity to a nucleic acid molecule encoding wild-type IL-13. For example, a nucleic acid molecule encoding a subject IL-13 mutein can be at least 50%, at least 65%, preferably at least 75%, more preferably at least 85%, and most preferably at least 95% (e.g., 99%) identical to a nucleic acid encoding wild-type IL-13 (e.g., SEQ ID NO: 81).

[0521] The nucleic acid molecules provided can contain naturally occurring sequences, or sequences that are different from naturally occurring sequences but that encode the same polypeptide due to the degeneracy of the genetic code. These nucleic acid molecules can be composed of RNA or DNA (e.g., genomic DNA, cDNA, or synthetic DNA, such as DNA produced by phosphoramidite-based synthesis) or combinations or modifications of nucleotides within these types of nucleic acids. Additionally, the nucleic acid molecules can be double-stranded or single-stranded (i.e., sense strand or antisense strand).

[0522] Nucleic acid molecules are not limited to sequences encoding polypeptides; they may also include some or all non-coding sequences upstream or downstream of a coding sequence (e.g., a coding sequence for IL-2, IL-4, or IL-13). Those of ordinary skill in the field of molecular biology are familiar with conventional procedures for isolating nucleic acid molecules. They can be produced, for example, by treating genomic DNA with restriction endonucleases or by performing a polymerase chain reaction (PCR). If the nucleic acid molecule is a ribonucleic acid (RNA), the molecule can be produced, for example, by in vitro transcription.

[0523] Exemplary isolated nucleic acid molecules of the present disclosure may include fragments that are not found in nature as such. Thus, the present disclosure encompasses recombinant molecules, such as those in which a nucleic acid sequence (e.g., a sequence encoding a mutant IL-2, IL-4, or IL-13) is incorporated into a vector (e.g., a plasmid or viral vector) or into the genome of a heterologous cell (or into the genome of a homologous cell at a location other than the native chromosomal location).

[0524] As described above, the subject IL-2, IL-4 or IL-13 mutein bifunctional molecules can exist as part of a chimeric polypeptide. In addition to or in place of the above-mentioned heterologous polypeptides, the subject nucleic acid molecules can contain sequences encoding "markers" or "reporters." Examples of markers or reporter genes include β-lactamase, chloramphenicol acetyltransferase (CAT), adenosine deaminase (ADA), aminoglycoside phosphotransferase (neo), and β-lactamase. r 、G418 r ), dihydrofolate reductase (DHFR), hygromycin-B-phosphotransferase (HPH), thymidine kinase (TK), lacZ (encoding β-galactosidase) and xanthine guanine phosphoribosyltransferase (XGPRT). Those skilled in the art will know other useful reagents, such as other sequences that can function as markers or reporters.

[0525] The subject nucleic acid molecule can be obtained by introducing a mutation into the DNA encoding IL-2 obtained from any biological cell such as a mammalian cell. Therefore, the subject nucleic acid (and its encoded polypeptide) can be those of mouse, rat, guinea pig, cattle, sheep, horse, pig, rabbit, monkey, baboon, dog or cat. In one embodiment, the nucleic acid molecule will be a human nucleic acid molecule.

[0526] G. Chimeric Antigen Receptor (CAR)

[0527] Targeted immunotherapy has become a promising research field for the treatment of malignant tumors and has received great attention in recent years. In fact, the treatment of lymphoma patients with engineered or genetically modified T cells targeting CD19 malignant tumor cells has been reported. This increases the focus on antigens present on cancer cells as targets for gene therapy and immunotherapy. These CARs can be used to target or deliver the bispecific IL-2 cytokine fusions and / or IL-2, IL-4 or IL-13 mutant protein bifunctional molecules described herein to tumors, or even allow systemic IL-2, IL-4 or IL-13 mutant protein bifunctional molecules to be expressed. In some embodiments, the IL-2, IL-4 or IL-13 mutant protein bifunctional molecules are any IL-2, IL-4 or IL-13 mutant protein bifunctional molecules or variants disclosed herein. In some embodiments, the IL-2 mutant protein sequence is 90% identical to any one of SEQ ID NO: 2 or SEQ ID NO: 6 to SEQ ID NO: 10 or SEQ ID NO: 16. In some embodiments, the IL-2 mutein comprises any one of 5-1 SEQ ID NO:5, 5-2 SEQ ID NO:6, 6-6 SEQ ID NO:7, A2 SEQ ID NO:8, B1 SEQ ID NO:9, B11 SEQ ID NO:10, C5 SEQ ID NO:11, D10 SEQ ID NO:12, E10 SEQ ID NO:13, G8 SEQ ID NO:14, H4 SEQ ID NO:15, and H9 SEQ ID NO:16. In some embodiments, the substitutions in the IL-2 mutein comprise L80F, R81D, L85V, I86V, and I92F, numbered according to wild-type human IL-2 of SEQ ID NO:2. In some embodiments, the bifunctional molecule comprises a sequence that is 90% identical to any one of: SEQ ID NO:1 NO:1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 5 3, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102,103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158、159、160、161、162、163、164、165、166、167、168、169、170、171、172、173、174、175、176、177、178、179、180、181、182、183、184、185、186、187、188、189、190、191、192、193、194、195、196、197、198、199、200、201、202、203、204、205、206、207、208、209、210、211、212、213、214、215、216、217、218、219、220、221、222、223、224、225、226、227、228、229、230、231、232、233、234、235、236、237、238、239、240、241、242、243、244、245、246、247、248、249、250、251、252、253、254、255、256、257、258、259、260、261、262、263、264、265、266、267、268、269、270、271、272、273、274、275、276、277、278、279、280、281、282、283、284、285、286、287、288、289、290、291、292、293、294、295、296、297、298、299、300、301、302、303、304、305、306、307、308、309、310、311、312、313、314、315、316、317、318、319、320、321、322、323、324、325、326、327、328、329、330、331、332、333、334、335、336、337、338、339、340、341、342、343、344、345、346、347、348、349、350、351、352、353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390 0, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428 28, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 4554, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466 , 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 501, 502, 503, 504, 505, 506, 507, and / or 508. ,

[0528] Autologous or allogeneic T cells, NK cells, macrophages, γδT cells or NKT cells are genetically manipulated to specifically target specific tumor antigens, providing a strategy for avoiding the failure of most tumor cells to induce cytotoxic immune responses. In some embodiments, these genetically manipulated T cells or NK cells can be used, for example, to target tumors with IL-2, IL-4 or IL-13 mutant protein bifunctional molecules as described herein, so that IL-2, IL-4 or IL-13 mutant protein bifunctional molecules are expressed at the tumor location. These technologies are based on the genetic modification of human immune cells, in which cells can be extracted from patients or donors by leukocyte removal. Specific cells (usually T cells) are purified and engineered to express receptors that target target cancer antigens. Engineering can be transduced using retroviruses, slow viruses, transposons, mRNA electroporation, etc. Immune cells can be expanded to the desired dose and introduced into the patient's body. Engineered cells can specifically kill cancer cells by cell-mediated toxicity (cytotoxic T cells) and / or by immune recognition of tumors, cytokine release, and immune cell recruitment to trigger an immune response to cancer cells.

[0529] For example, the use of chimeric antigen receptors (CARs) for immunogene therapy of malignant tumors is a promising strategy in which an antibody or ligand binding domain is fused to the ζ signaling chain of the T cell receptor. The resulting CAR immune cells are redirected by new specificity to attack tumors expressing surface antigens or receptors recognized by genetically modified T cell receptors, and provide cell therapy that attacks tumors in a highly regulated manner through normal host immune responses. These cells can circulate freely throughout the brain and systemic circulation, making the need for colocalization and bioavailability no longer a problem.

[0530] Multiple generations of CAR immune cells have been developed. CARs are created by fusing a tumor-specific scFv antibody or other extracellular ligand-binding domain with the TCR-associated CD3ζ signaling domain or another intracellular signaling domain from a costimulatory protein receptor. This architecture imparts tumor specificity to the CAR through the B cell antigen receptor and activates T cells through the T cell antigen receptor independently of MHC binding. First-generation CARs contain a single intracellular signaling domain, typically with a CD3ζ signaling domain, to allow TCR signaling. Second-generation CARs have two intracellular signaling domains: a costimulatory domain containing a CD28 or 4-1BB signaling domain, plus a CD3ζ signaling domain. This arrangement enables T cell activation and proliferation following antigen recognition by the scFv region of the CAR. Third-generation CARs have two costimulatory domains and a CD3ζ signaling domain. The first costimulatory domain is a CD28 or 4-1BB domain, while the second costimulatory domain consists of a CD28, 4-1BB, or OX40 domain. The fourth generation of "armored CAR T cells" combines the second generation CAR with an increased variety of genes (including cytokines and co-stimulatory ligands) to enhance the tumoricidal effect of CAR T cells. See, for example, Batlevi et al. (2016) Nature Reviews Clinical Oncology 13: 25-40. See also U.S. Patent No. 7,741,465 and International Patent Publication No. WO2014127261; the patents are incorporated by reference in their entirety.

[0531] Alternative approaches to T cell targeting include T cell antigen couplers, as described in International Application WO 2015 / 117229, entitled “Trifunctional T cell antigen coupler and methods and uses thereof,” which is specifically incorporated herein by reference. The T cell antigen coupler system comprises three linked domains: a target-specific polypeptide ligand; a ligand that binds to a protein associated with the TCR complex, such as a scFv that binds to CD3 (TCR, T cell receptor) to stimulate T cell activation; and a T cell receptor signaling domain that amplifies T cell activation, such as the CD4 transmembrane domain and intracellular domain. By stimulating T cell activation through the TCR, TACs are engineered to work with the basic molecular machinery of T cells.

[0532] Antibody-coupled T-cell receptors (ACTRs) are another approach to T-cell targeting. ACTRs are a hybrid approach combining CARs and established monoclonal antibody oncology therapeutics. ACTRs consist of a typical CAR construct that binds to the heavy chain of an antibody via a high-affinity variant of the Fc receptor CD16. ACTR-T cells can target tumors by binding to a ligand that targets a specific cancer antigen. T-cell activation is performed by the CAR module.

[0533] In some embodiments, directed evolution is used in conjunction with a yeast display platform to produce adjustable super factors. In some embodiments, such a platform produces a wide library of IL-2, IL-4, and IL-13 super factors with unique properties. In some embodiments, MDNA109 is an engineered version of human IL-2 that exhibits enhanced agonist activity. In some embodiments, the MDNA109 family of "IL-2 super factors" has been engineered to improve PK characteristics and enhance selectivity, thereby further improving the therapeutic window.

[0534] In some embodiments, MDNA11 is a "beta-only" superkine with a uniquely enhanced affinity for CD122. In some embodiments, MDNA11 preferentially stimulates immune effector cells. In some embodiments, MDNA11 demonstrates monotherapy anti-tumor efficacy and combination effects with anti-PD1 in the MC38 tumor model. In some embodiments, MDNA11, together with anti-CTLA4, induces tumor clearance, protects against re-attack, and promotes antigen-specific CD8 T cells. In some embodiments, MDNA11 induces immune effector cells but not T cells in NHPs. reg persistent and continuous proliferation and expansion.

[0535] In some embodiments, antibody-targeted superkines (STAbs) enhance accumulation in tumors. In some embodiments, STAbs overcome checkpoint resistance and "cold" tumors.

[0536] In some embodiments, IL-4 and IL-13 receptors play a role in cancer. In some embodiments, MDNA55 is an enhanced super factor with a payload targeting type 2 IL-4R expressed on tumor cells and tumor microenvironment (MDSC and TAM). In some embodiments, MDNA413 is a super antagonist that blocks IL-4 and IL-13 signaling via type 2 IL-4R to inhibit MDSC and TAM. In some embodiments, MDNA 132 is a super factor that selectively targets decoy IL-13Rα2 overexpressed on solid tumors. In some embodiments, MDNA132 is an engineered version of human IL-13 targeting tumor-specific antigens. In some embodiments, MDNA132 plays a role in locating T cell adaptors and checkpoint inhibitors to tumors.

[0537] In some embodiments, MDNA413 is an engineered version of human IL- 13 that exhibits antagonist activity. In some embodiments, Fc-MDNA413 inhibits IL-4 and IL-13 induced signaling and function.

[0538] In some embodiments, the dual specific cytokine (DUCK Cancer) is MDNA109FEAA-Fc-MDNA413 and has the following structure: Figure 70 The mechanism of action shown in .

[0539] Biological products that achieve selective alteration of IL-13 activity are of interest for many therapeutic purposes, including the treatment of certain cancers by engineering T cell specificity. The present invention addresses this problem.

[0540] Provided are methods and compositions for enhancing anti-tumor immune effector cells (such as T cells, NK cells, etc.) with targeted compositions, including but not limited to chimeric antigen receptors (CARs);T cell antigen coupling agents (TACs);Antibody-coupled T cell receptors (ACTRs);With bispecific T cell exchange agents (BiTEs), wherein IL-13 or IL-4 super factors provide target-specific ligands.In additional embodiments, immune effector cells express IL-2, IL-4 or IL-13 mutant protein bifunctional molecules.

[0541] Provided are immune cell targeting or expression constructs comprising IL-2, IL-4 or IL-13 mutant protein bifunctional molecule sequences, which may include any IL-2, IL-4 or IL-13 mutant protein bifunctional molecule sequences as described herein. Super factors can be used to target immune cells to cells expressing at least one receptor, such as tumor cells. In some embodiments, the IL-2, IL-4 or IL-13 mutant protein bifunctional molecule comprises any IL-2, IL-4 or IL-13 mutant protein bifunctional molecule or variant disclosed herein. In some embodiments, the IL-2 mutant protein sequence is 90% identical to any one of SEQ ID NO: 2 or SEQ ID NO: 6 to SEQ ID NO: 10 or SEQ ID NO: 16. In some embodiments, the IL-2 mutein comprises any one of 5-1 SEQ ID NO:5, 5-2 SEQ ID NO:6, 6-6 SEQ ID NO:7, A2 SEQ ID NO:8, B1 SEQ ID NO:9, B11 SEQ ID NO:10, C5 SEQ ID NO:11, D10 SEQ ID NO:12, E10 SEQ ID NO:13, G8 SEQ ID NO:14, H4 SEQ ID NO:15, and H9 SEQ ID NO:16. In some embodiments, the substitutions in the IL-2 mutein comprise L80F, R81D, L85V, I86V, and I92F, numbered according to wild-type human IL-2 of SEQ ID NO:2. In some embodiments, the bifunctional molecule comprises a sequence that is 90% identical to any one of: SEQ ID NO: NO:1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,3 5, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124,125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158、159、160、161、162、163、164、165、166、167、168、169、170、171、172、173、174、175、176、177、178、179、180、181、182、183、184、185、186、187、188、189、190、191、192、193、194、195、196、197、198、199、200、201、202、203、204、205、206、207、208、209、210、211、212、213、214、215、216、217、218、219、220、221、222、223、224、225、226、227、228、229、230、231、232、233、234、235、236、237、238、239、240、241、242、243、244、245、246、247、248、249、250、251、252、253、254、255、256、257、258、259、260、261、262、263、264、265、266、267、268、269、270、271、272、273、274、275、276、277、278、279、280、281、282、283、284、285、286、287、288、289、290、291、292、293、294、295、296、297、298、299、300、301、302、303、304、305、306、307、308、309、310、311、312、313、314、315、316、317、318、319、320、321、322、323、324、325、326、327、328、329、330、331、332、333、334、335、336、337、338、339、340、341、342、343、344、345、346、347、348、349、350、351、352、353、354、355、356、357、358、359、360、361、362、363、364、365、366、367、368、369、370、371、372、373、374、375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 4 39, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 4554, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472 72, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 501, 502, 503, 504, 505, 506, 507, and / or 508.

[0542] The length of the IL-2, IL-4 or IL-13 mutant protein component of the bifunctional molecule construct can be at least about 50 amino acids, at least about 75, at least about 100, at least about 110, at least about 115 amino acids in length, to the full length of the wild-type protein at the transmembrane domain, i.e., a length of about 116 amino acids. For example, the super factor or mutant protein can be fused to the hinge domain, transmembrane domain or signaling domain of the CAR. Exemplary polypeptide sequences are provided.

[0543] Included as IL-2, IL-4, or IL-13 superkines or muteins are amino acid and nucleic acid coding sequences that are 90%, 95%, 98%, or 99% identical to these sequences, including those sequences but further comprising additional nucleotides at the 3' or 5' end, for example, any number of additional nucleotides or codons, such as 3, 6, 9, 12 or more nucleotides, or longer sequences of up to about 12, 20, 50, or 100 additional nucleotides, as well as any sequence that, due to the degeneracy of the genetic code, encodes the same amino acid sequence as these nucleic acids. In particular, sequences that have been codon-optimized (CO) for expression by a desired host are considered part of the present invention. In some embodiments, the amino acid sequences are 90% identical. In some embodiments, the amino acid sequences are 95% identical. In some embodiments, the amino acid sequences are 98% identical. In some embodiments, the amino acid sequences are 99% identical. In some embodiments, the polypeptides are linked to immune cell targeting or expression constructs comprising IL-2, IL-4, or IL-13 muteins. In some embodiments, the immune cell targeting or expression construct comprising an IL-2, IL-4, or IL-13 mutant protein comprises one or more signaling domains derived from CD3-ζ, CD28, DAP10, OX-40, ICOS, and CD137. In some embodiments, the immune cell targeting or expression construct comprising an IL-2, IL-4, or IL-13 mutant protein comprises one or more signaling domains derived from CD3-ζ. In some embodiments, the immune cell targeting or expression construct comprising an IL-2, IL-4, or IL-13 mutant protein comprises one or more signaling domains derived from CD28. In some embodiments, the immune cell targeting or expression construct comprising an IL-2, IL-4, or IL-13 mutant protein comprises one or more signaling domains derived from DAP10. In some embodiments, the immune cell targeting or expression construct comprising an IL-2, IL-4, or IL-13 mutant protein comprises one or more signaling domains derived from OX-40. In some embodiments, the immune cell targeting or expression construct comprising an IL-2, IL-4, or IL-13 mutant protein comprises one or more signaling domains derived from CD 137. In some embodiments, the immune cell targeting or expression construct comprising an IL-2, IL-4, or IL-13 mutant protein comprises an IL-2 variant / IL-2 superkine, an IL-4 variant / IL-4 superkine, or an IL-13 variant / IL-13 superkine, including those provided herein.In some embodiments, the immune cell targeting or expression construct comprising an IL-2, IL-4, or IL-13 mutant protein comprises an IL-2 variant / IL-2 superkine, an IL-4 variant / IL-4 superkine, or an IL-13 variant / IL-13 superkine, including those provided in SEQ ID NO: 1 to SEQ ID NO: 495 or SEQ ID NO: 501 to SEQ ID NO: 508.

[0544] 1. NK cells

[0545] In some embodiments, immune cells are natural killer (NK) cells. NK cells recognize infected or transformed cells through a variety of cell surface receptors (including NKG2D, CD16) and natural cytotoxicity receptors (NCR) (such as NKp44, NKp46 and NKp30). These receptors activate signal transduction adapter proteins (such as DAP10, DAP12 and CD3ζ), which contain immune tyrosine activation motifs (ITAMs), which promote the release of cytolytic granules containing perforin and granzymes, and mediate the production and release of cytokines and chemokines (such as IFN-γ and TNF-α). Importantly, the cytotoxicity mediated by NK cells does not depend on the presentation of self HLA. Therefore, since NK cells can be used in allogeneic environments and can provide ready-made cell products, NK cells have important clinical significance as cell-based cancer therapy.

[0546] Since natural killer cells do not require HLA matching, they can replace T cells for adoptive immunotherapy, so they can be used as allogeneic effector cells. Clinical trials of adoptively transferred allogeneic NK cells show that these cells can survive for weeks to months in patients. In addition, compared with hematological malignancies (especially acute myeloid leukemia) that are generally more sensitive to NK cells, CAR expression in NK cells enables these cells to more effectively kill solid tumors that are generally tolerant to NK cell-mediated activity. CARs that can be used for NK cell targeting include, for example, first-generation CAR constructs, which contain CD3 ζ as the only signaling domain. Second-generation and third-generation CARs can also be used for NK cells. In some embodiments, the extracellular domain of NKG2D (NK cell activation receptor) is directly connected to CD3 ζ.

[0547] NK cells for modification include cell lines or peripheral blood NK cells, and if a large number of cells are needed, these cell lines or peripheral blood NK cells can be separated from donors by simple blood sampling or by single blood component extraction. Activated PB-NK cells express more extensive activation receptors, such as CD16, NKp44 and NKp46 and KIR, which play an important role in NK cell licensing. In addition, PB-NK cells can be provided without irradiating cells, so there is in vivo amplification ability. Another source of NK cells suitable for CAR expression is NK cells derived from human pluripotent stem cells (i.e., induced pluripotent stem cells (iPSC) or human embryonic stem cells (hESC)). These NK cells show a phenotype similar to PB-NK cells, and hESC / iPSC-NK cells can be grown on a clinical scale.

[0548] 2. Chimeric Antigen Receptor (CAR)

[0549] In addition to the super factor sequence, CAR also contains a signaling domain for CD3ζ and a signaling domain for one or more costimulatory receptors, which also promote the recycling, survival and / or amplification of immune cells expressing the CAR. The signaling domain of the costimulatory receptor is the intracellular portion of each receptor protein that produces an activation signal in the cell. Examples are amino acids 180-220 of a natural CD28 molecule and amino acids 214-255 of a natural 4-1BB molecule.

[0550] The example of suitable hinge and membrane spanning region that super factor is connected to signal transduction zone can include but not limited to immunoglobulin, human CD8a and the constant (Fc) region of artificial joint, and they are used to remove the targeting part from cell surface, to improve the approach and combination with target cell.The example of suitable membrane spanning domain includes the membrane spanning domain of leukocyte CD marker, preferably the membrane spanning domain of CD4 or CD28.The example of intracellular receptor signal transduction domain includes T cell antigen receptor complex, preferably the ζ chain of CD3, but can use any membrane spanning domain that is enough to anchor CAR in the membrane.Technicians know that many membrane spanning regions and the structural elements (such as lipophilic amino acid regions) that produce membrane spanning domain in many membrane proteins, therefore any simple sequence can be substituted. Suitable for improving the function of CAR expressing cells and the T cell costimulatory signal transduction receptor of activity include but not limited to CD28, CD137 and OX-40.

[0551] Signal transduction via CD28 is required for the production and proliferation of IL2, IL-4 or IL-13, but does not play a major role in the maintenance of T cell function and activity. CD137 (a member of the tumor necrosis factor receptor family expressed after CD28 activation) and OX-40 are involved in driving the long-term survival of T cells and the accumulation of T cells. The ligands of these receptors are usually expressed on professional antigen presenting cells (such as dendritic cells and activated macrophages), but not on tumor cells. Compared with the expression of CAR containing only CD3ζ signaling domain, CAR with CD28 and / or 4-1BB signaling domain has a significant effect on the expression of CD4 + Expression in T cells enhances the activity and antitumor efficacy of these cells, and these constructs can be referred to as second- or third-generation CARs.

[0552] Included as target CAR constructs are tandem CARs, for example, see Hegde et al. (2016) J. Clin. Invest 126 (8): 3036-3052, which are specifically incorporated by reference. In such constructs, a binding portion for a tumor-specific antigen is combined in series with an IL-13 super factor. The binding portion can be, for example, a scFv specific for a tumor cell antigen, including but not limited to HER-2, EGFR, CD20, etc., as known in the art.

[0553] In various embodiments, the antigen binding domain binds to an antigen on a target cell (e.g., a cancer cell). The antigen binding domain can bind to an antigen, such as, but not limited to, a tumor target antigen. In some cases, the antigen binding domain binds to one or more antigens. Exemplary antigen binding domains can bind to antigens including, but not limited to, D19; CD123; CD22; CD30; CD171; CS-1 (also known as CD2 subgroup 1, CRACC, SLAMF7, CD319, and 19A24); C-type lectin-like molecule 1 (CLL-1 or CLECL1); CD33; epidermal growth factor receptor variant III (EGFRvIII); ganglioside G2 (GD2); ganglioside GD3; TNF receptor family member B cell maturation (BCMA); Tn antigen ((Tn Ag) or (GalNAcαSer / Thr)); prostate-specific membrane antigen (PSMA); receptor tyrosine kinase-like orphan receptor 1 (ROR1); Fms-like tyrosine kinase 3 (FLT3); tumor-associated glycoprotein 72 (TAG72); CD38; CD44v6; carcinoembryonic antigen (CEA); epithelial cell adhesion molecule (EPCAM); B7H3 (CD276); KIT (CD117); interleukin-13 receptor subunit alpha-2 (IL-13Rα2 or CD213A2); mesothelin; interleukin-11 receptor alpha (IL-11Ra); prostate stem cell antigen (PSCA); protease serine 21 (testosterone or PRSS21); vascular endothelial growth factor receptor 2 (VEGFR2); Lewis (Y) antigen; CD24 Platelet-derived growth factor receptor beta (PDGFR-β); stage-specific embryonic antigen 4 (SSEA-4); CD20; folate receptor alpha; receptor tyrosine-protein kinase ERBB2 (Her2 / neu); mucin 1, cell surface-associated (MUC1); epidermal growth factor receptor (EGFR); neural cell adhesion molecule (NCAM); prostatic enzyme; prostatic acid phosphatase (PAP); elongation factor 2, mutant (ELF2M); ephrin B2; fibroblast activation protein alpha (FAP); insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX); proteasome (prosome, macropain) subunit, beta type, 9 (LMP2); glycoprotein 100 (gp 100); oncogene fusion protein consisting of the breakpoint cluster region (BCR) and Abelson murine leukemia virus oncogene homolog 1 (Abl) (bcr-abl); tyrosinase; ephrin type A receptor 2 (EphA2); fucosyl GM1; sialyl Lewis adhesion molecule (sLe);Ganglioside GM3 (aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer); transglutaminase 5 (TGS5); high molecular weight melanoma-associated antigen (HMWMAA); o-acetyl GD2 ganglioside (OAcGD2); folate receptor beta; tumor endothelial marker 1 (TEM1 / CD248); tumor endothelial marker 7-related (TEM7R); claudin 6 (CLDN6); thyroid stimulating hormone receptor (TSHR); G protein-coupled receptor type C family 5, member D (GPRC5D) Chromosome X open reading frame 61 (CXORF61); CD97; CD179a; anaplastic lymphoma kinase (ALK); polysialic acid; placenta-specific 1 (PLAC1); globoH glycoceramide hexasaccharide moiety (GloboH); mammary differentiation antigen (NY-BR-1); urokinase 2 (UPK2); hepatitis A virus cellular receptor 1 (HAVCR1); adrenergic receptor β3 (ADRB3); pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex, locus K 9 (LY6K); olfactory receptor 51E2 (OR51E2); TCR gamma alternate reading frame protein (TARP); Wilms tumor protein (WT1); cancer / testis antigen 1 (NY-ESO-1); cancer / testis antigen 2 (LAGE-1a); melanoma-associated antigen 1 (MAGE-A1); ETS translocation variant gene 6 located on chromosome 12p (ETV6-AML); sperm protein 17 (SPA17); X antigen family, member 1A (XAGE1); angiopoietin-binding cell surface receptor 2 (Tie 2); melanoma cancer testis antigen-1 (MAD-CT-1); melanoma cancer testis antigen-2 (MAD-CT-2); Fos-related antigen 1; tumor protein p53 (p53); p53 mutants; prostein; survivin; telomerase; prostate cancer tumor antigen 1 (PCTA-1 or galectin 8), melanoma antigen recognized by T cells 1 (MelanA or MART 1); rat sarcoma (Ras) mutant; human telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoint; melanoma inhibitor of apoptosis (ML-IAP); ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-acetylglucosamine transferase V (NA17); paired box protein Pax-3 (PAX3); androgen receptor; cyclin B1; v-myc avian myelocytic neoplasia viral oncogene neuroblastoma-derived homolog (MYCN); Ras homolog family member C (RhoC); tyrosinase-related protein 2 (TRP-2);Cytochrome P450 1B1 (CYP1B1); CCCTC-binding factor (zinc finger protein)-like (BORIS or brother of imprinted site regulator factor), squamous cell carcinoma antigen recognized by T cells 3 (SART3); paired box protein Pax-5 (PAX5); pre-acrosomal protein-binding protein sp32 (OY-TES1); lymphocyte-specific protein tyrosine kinase (LCK); kinase anchoring protein 4 (AKAP-4); synovial sarcoma, breakpoint X 2 (SSX2); receptor for advanced glycation end products (RAGE-1); renal ubiquitin 1 (RU1); renal ubiquitin 2 (RU2); leguminase; human papillomavirus E6 (HPV E6); human papillomavirus E7 (HPV E7); intestinal carboxylesterase; mutant heat shock protein 70-2 (mut hsp70-2); CD79a; CD79b; CD72; leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment of the IgA receptor (FCAR or CD89); leukocyte immunoglobulin-like receptor subfamily A, member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12, member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); mucin-like hormone receptor-like 2 with an EGF-like module (EMR2); lymphocyte antigen 75 (LY75); glypican 3 (GPC3); Fc receptor-like 5 (FCRL5); and immunoglobulin lambda-like polypeptide 1 (IGLL1).

[0554] In some embodiments, antigen binding domains include monoclonal antibodies, polyclonal antibodies, synthetic antibodies, human antibodies, humanized antibodies, non-human antibodies, nanobodies, single chain variable fragments (scFv), F (ab ') 2, Fab ', Fab, Fv and the like. Antigen binding domains can be connected to the transmembrane domain of CAR. In some embodiments, the nucleic acid encoding the antigen binding domains is operably connected to the nucleic acid encoding the transmembrane domain of CAR.

[0555] In some embodiments, the membrane-spanning domain can be derived from a membrane-bound protein or a transmembrane protein.In certain embodiments, compared with the wild-type amino acid sequence of the membrane-spanning domain of a membrane-bound protein or a transmembrane protein, the membrane-spanning domain includes one or more, for example, 1, 2, 3, 4, 5, 6, 7, 8 or more amino acid modifications (for example, substitutions, insertions and deletions). Non-limiting examples of the membrane-spanning domain of CAR include at least α, β or ζ chains of T cell receptors, CD28, CD3ε (CD3ξ), CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154 or one or more membrane-spanning regions of erythropoietin receptor. In some embodiments, the membrane-spanning domain includes human immunoglobulin (Ig) hinge region, such as IgG4Fc hinge. In other embodiments, the transmembrane domain is a recombinant or synthetic domain comprising hydrophobic amino acid residues (e.g., leucine and valine). In some cases, the transmembrane domain comprises phenylalanine, tryptophan, and valine at one or both ends of the domain.

[0556] The transmembrane domain connects the antigen binding domain to the intracellular signaling domain of the CAR. In some embodiments, the nucleic acid encoding the antigen binding domain is operably linked to the nucleic acid encoding the transmembrane domain, which is operably linked to the nucleic acid encoding the intracellular signaling domain.

[0557] In some embodiments, the intracellular signaling domain of CAR includes a signal activation domain or a signal transduction domain. Therefore, the intracellular signaling domain includes any part of the intracellular signaling domain of the protein that is sufficient to transduce or transmit a signal (e.g., an activation signal) or mediate a cellular response in the cell. Non-limiting examples include TCR, CD2, CD3ζ, CD3γ, CD3δ, CD3ε, CD7, CD27, CD86, common FcRγ, FcRβ, CD79a, CD79b, FcγRIIa, DAP10, DAP12, T cell receptor (TCR), CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGH T, NKG2C, B7-H3, ligands specifically binding to CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD127, CD160, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, IT GAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRTAM, Ly9( In some embodiments, the intracellular signaling domain comprises a co-stimulatory molecule (such as an intracellular domain of a co-stimulatory molecule (such as CD3, CD27, CD28, CD127, ICOS, 4-1BB (CD137), PD-1, T cell receptor (TCR), any derivative thereof or any variant thereof).In some embodiments, the intracellular signaling domain of the CAR is selected from the group consisting of: MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), activating NK cell receptors, BTLA, Toll ligand receptors, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD11a / CD18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS (CD2 78), GITR, BAFFR, LIGHT, HVEM(LIGHTR), KIRDS2, SLAMF7, NKp80(KLRF1), NKp44, NKp30, NKp46, CD19, CD4, C D8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d , ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, and ligands that specifically bind to CD83.

[0558] 3.ICE

[0559] Targeted therapies targeting IL13Rα2 have been developed, including bacterial toxins conjugated to IL13, nanoparticles, oncolytic viruses, and immunotherapies using monoclonal antibodies, IL13Rα2-pulsed dendritic cells, and chimeric antigen receptors targeting IL13Rα2 (see Kahlon et al. (2004) Cancer Research. 64(24):9160–9166; Kong et al. (2012) Clinical Cancer Research. 18(21):5949–5960; Thaci et al. (2014) Neuro-Oncology; and clinical trials NCT02208362, NCT00730613, and NCT01082926). In some normal situations, these targeted therapies can be used to deliver bifunctional molecules of IL-2, IL-4, or IL-13 mutant proteins to tumors.

[0560] ICE (Il-13 directed cell engager), including bispecific T cell engagers (BiTEs), is a fusion protein comprising an IL-13 superkine fused to an antibody variable region that specifically binds to CD3. In some embodiments, the antibody variable region is in a single-chain variable fragment (scFv). The superkine can be fused to the variable region via a linker. An Fc region is optionally provided. ICE molecules are capable of interacting with and engaging T cells, NK cells, NKT cells, dendritic cells (DCs), and / or macrophages.

[0561] Bispecific T cell adaptors (BiTEs) are bispecific antibodies that can bind to the TCR of T cells and target tumor cells through two modules: a ligand targeting cancer; and a CD3 binding domain, a CD28 binding domain, a CD137 binding domain, an OX-40 binding domain, an ICOS binding domain, or a DAP10 binding domain. T cells are bridged to the scFv domain of the tumor. In some embodiments, the bispecific fusion protein comprising a super factor may also be referred to as a bifunctional super factor (BiSKIT) as an immunotherapy. In some embodiments, novel interleukin super agonists, partial agonists, and super antagonists are designed using directed evolution. In some embodiments, long-acting IL-2, IL-4, and IL-13 super factors are further designed using a rational approach, including bifunctional molecules as described herein, without masking functional activity.

[0562] 4.TAC

[0563] The TAC construct comprises an IL-2, IL-4, or IL-13 superkine fused to a ligand that binds a protein associated with a TCR complex; fused to a T cell receptor signaling domain polypeptide. The domains may be separated by a linker. The protein associated with the TCR complex may be CD3. The ligand that binds a protein associated with the TCR complex may be a single-chain antibody. The ligand that binds a protein associated with the TCR complex may be UCHT1 or a variant thereof. The T cell receptor signaling domain polypeptide may comprise a cytoplasmic domain and a transmembrane domain. The cytoplasmic domain may be a CD4 cytoplasmic domain, and the transmembrane domain may be a CD4 transmembrane domain.

[0564] 5. ACTR

[0565] ACTR is a hybrid approach of CAR and established monoclonal antibody oncology therapeutics. ACTR consists of a typical CAR construct that can bind to the heavy chain of an antibody through a high-affinity variant of the Fc receptor CD16. The super factor is fused to a portion recognized by the CAR, which may include but is not limited to the Fc region of an antibody with high affinity for CD16.

[0566] Immune cell targeting or expression construct encoding sequence can be produced by any means known in the art, including recombinant DNA technology. Nucleic acids encoding several regions of chimeric receptors can be prepared by standard molecular cloning techniques known in the art (genomic library screening, PCR, primer-assisted connection, site-directed mutagenesis, etc.), and these nucleic acids are assembled into complete encoding sequences, which is convenient. The resulting coding region can be inserted into an expression vector, and used to transform a suitable expression host cell line, such as allogeneic or autologous T lymphocytes, allogeneic or autologous NK cells (including primary culture, cell line, iPSC derived cells, etc.) colony. These methods can be used for cells in vitro (for example, in a cell-free system), in culture, such as in vitro or in vitro cells. For example, cells expressing IL-2, IL-4 or IL-13 super factor CAR can be cultured in culture medium and amplified in vitro.

[0567] Any non-IL-2 superkine (including any IL-4 or IL-13 containing molecule as described herein) immune cell targeting or expression construct can also be used to specifically direct immune cells to target specific tumor cells. By introducing a superkine immune cell targeting or expression construct into T cells, anti-tumor effector cells (e.g., CD4 + or CD8 +Effector T cells) are redirected to recognize these tumor cells, and the construct comprises one or more signaling domains derived from CD3-ζ, CD28, DAP10, OX-40, ICOS, and CD137. In some embodiments, the cells may also comprise a transgene capable of expressing an IL-2 mutant protein as described herein. IL-2, IL-4, or IL-13 superkine immune cell targeting or expression constructs can specifically guide immune cells to target cells expressing a binding partner of the construct, including tumor cells. By introducing an IL-2 superkine immune cell targeting or expression construct into T cells, anti-tumor effector cells (e.g., CD4 + or CD8 + The constructs comprise one or more signaling domains derived from CD3-ζ, CD28, DAP10, OX-40, ICOS, and CD137.

[0568] For example, IL-2, IL-4 or IL-13 super factor immune cell targeting or expression constructs are infected or transfected into human immune cells using non-viral plasmid vectors and electroporation methods as known in the art; viral vectors and infection methods, etc. CARs containing costimulatory signaling domains can enhance the duration and / or retention of anti-tumor activity in a manner that can significantly improve the clinical efficacy of adoptive therapy regimens. CD4 + and CD8 + T cell effector functions as well as NK cell functions can be triggered via these receptors, and therefore these cell types are contemplated for use in the present invention. + T cells can be used to lyse target cells and produce IL-2, IL-4, or IL-13 in the presence of target cells, among other functions. + and / or CD8 + Expression of appropriate co-stimulatory CARs in T cells is used to provide the most effective cell population for adoptive immunotherapy, which is thus composed of one or both of professional helper T cells and killer T cells that exhibit enhanced and / or long-term viability and anti-tumor activity. In some embodiments, the IL-2, IL-4 or IL-13 superkine immune cell targeting or expression construct comprises an IL-2 variant / IL-2 superkine, an IL-4 variant / IL-4 superkine or an IL-13 variant / IL-13 superkine, including Figure 2In some embodiments, the IL-2, IL-4, or IL-13 superkine immune cell targeting or expression construct comprises an IL-2 variant / IL-2 superkine, an IL-4 variant / IL-4 superkine, or an IL-13 variant / IL-13 superkine, including any of those provided herein.

[0569] For various purposes, the polypeptides of the present invention may be further modified, for example, by linking to a variety of other oligopeptides or proteins. For example, post-translational modifications, such as by prenylation, acetylation, amidation, carboxylation, glycosylation, pegylation, etc. Such modifications may also include glycosylation modifications, such as by modifying the glycosylation form of the polypeptide during its synthesis and processing or in another processing step; for example, those modifications performed by exposing the polypeptide to enzymes that affect glycosylation, such as mammalian glycosylases or deglycosylases.

[0570] Methods well known to those skilled in the art can be used to construct T cell targeting construct expression vectors containing coding sequences and appropriate transcription / translation control signals. These methods include, for example, in vitro recombinant DNA technology, synthetic techniques, and in vivo recombination / genetic recombination. Alternatively, RNA capable of encoding the target polypeptide can be chemically synthesized. Those skilled in the art can easily utilize well-known codon usage tables and synthetic methods to provide suitable coding sequences for any polypeptide of the present invention. Nucleic acids can be isolated and obtained in high purity. Typically, the nucleic acids obtained (whether DNA or RNA) will be substantially free of other naturally occurring nucleic acid sequences, with a purity of typically at least about 50%, typically at least about 90%, and are typically "recombinant," for example, having one or more nucleotides on the flanks that are typically not associated with them on naturally occurring chromosomes. The nucleic acids of the present invention can be provided as linear molecules or in circular molecules and can be provided in self-replicating molecules (vectors) or in molecules without replication sequences. The expression of the nucleic acids can be regulated by themselves or by other regulatory sequences known in the art. The nucleic acids of the present invention can be introduced into suitable host cells using a variety of techniques available in the art.

[0571] According to the present invention, immune cell targeting or expression construct vectors and immune cell targeting or expression construct modified cells can be provided in pharmaceutical compositions suitable for therapeutic use (e.g., suitable for human treatment). In some embodiments, the pharmaceutical compositions of the present invention comprise one or more therapeutic entities of the present invention, or pharmaceutically acceptable salts, esters, or solvates thereof. In some other embodiments, the pharmaceutical compositions of the present invention comprise one or more therapeutic entities of the present invention in combination with another therapeutic agent (e.g., another anti-tumor agent).

[0572] The therapeutic entities of the present invention are typically administered in the form of a pharmaceutical composition comprising an active therapeutic agent and another pharmaceutically acceptable excipient. Such formulations may comprise one or more non-toxic pharmaceutically acceptable carriers, diluents, excipients and / or adjuvants. The preferred form depends on the intended mode of administration and therapeutic application. Depending on the desired formulation, the composition may also comprise a pharmaceutically acceptable, non-toxic carrier or diluent, which is defined as a vehicle commonly used to formulate a pharmaceutical composition for administration to animals or humans. The diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, physiological phosphate-buffered saline, Ringer's solution, dextrose solution, and Hank's solution. In addition, the pharmaceutical composition or formulation may also comprise other carriers, adjuvants, or non-toxic, non-therapeutic, non-immunogenic stabilizers, etc.

[0573] In yet other embodiments, the pharmaceutical compositions of the present invention may also contain large, slowly metabolized macromolecules such as proteins, polysaccharides such as chitosan, polylactic acid, polyglycolic acid and copolymers (such as latex functionalized Sepharose TM , agarose, cellulose, etc.), polymeric amino acids, amino acid copolymers, and lipid aggregates (such as oil droplets or liposomes).

[0574] The maximum tolerated dose (MTD) of CAR immune cells can be determined during clinical trial development, for example, up to about 10 4 T cells / kg body weight, up to about 10 5 cells / kg body weight, up to about 10 6 cells / kg body weight, up to approximately 5x10 6 cells / kg body weight, up to about 10 7 cells / kg body weight, up to approximately 5x10 7 cells / kg body weight, or higher, as determined empirically. In some embodiments, the maximum tolerated dose (MTD) of CAR immune cells is up to about 10 4 T cells / kg body weight. In some embodiments, the maximum tolerated dose (MTD) of CAR immune cells is up to about 10 5 T cells / kg body weight. In some embodiments, the maximum tolerated dose (MTD) of CAR immune cells is up to about 10 6 T cells / kg body weight. In some embodiments, the maximum tolerated dose (MTD) of CAR immune cells is up to about 10 7 T cells / kg body weight. In some embodiments, the maximum tolerated dose (MTD) of CAR immune cells is up to about 5x10 6T cells / kg body weight. In some embodiments, the maximum tolerated dose (MTD) of CAR immune cells is up to about 5x10 7 T cells / kg body weight.

[0575] The toxicity of the cells described herein can be determined by standard pharmaceutical procedures in cell culture or experimental animals, for example by measuring the LD 50 (dose lethal to 50% of the population) or LD 100 (a dose that is lethal to 100% of the population). The dose ratio between toxicity and therapeutic effect is the therapeutic index. The data obtained from these cell culture assays and animal studies can be used to formulate a dosage range that is non-toxic for human use. The dosages described herein are preferably within a circulating concentration range that includes an effective dose with little or no toxicity. The dosage can vary within this range depending on the dosage form employed and the route of administration used. The individual physician can choose the exact formulation, route of administration, and dosage based on the patient's condition.

[0576] After the dose escalation phase, patients in the expansion group are treated with immune cells at the MTD. Exemplary treatment regimens employ administration once every two weeks, once a month, or once every three to six months. The therapeutic entities of the present invention are typically administered on multiple occasions. The intervals between single doses can be weekly, monthly, or annual. Intervals can also be irregular, as indicated by measuring blood levels of the therapeutic entity in the patient.

[0577] In preventive applications, for example, to maintain remission in a patient, a relatively low dose may be administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment throughout their lives. In other therapeutic applications, a relatively high dose at relatively short intervals is sometimes necessary until the progression of the disease is reduced or terminated, preferably until the patient shows partial or complete improvement in the symptoms of the disease. Thereafter, a preventive regimen may be administered to the patient.

[0578] Examples of additional therapeutic agents that can be co-administered and / or co-formulated with immune cell targeting or expression constructs include: antiproliferative or cytoreductive therapies, which are used therapeutically to eliminate tumor cells and other unwanted cells in the host, and include the use of therapies such as delivery of ionizing radiation and administration of chemotherapeutic agents. Chemotherapeutic agents are well known in the art and are used in conventional doses and regimens, or in reduced doses or regimens, including, for example, topoisomerase inhibitors such as anthracyclines, including compounds daunorubicin, doxorubicin (doxorubicin), epirubicin, idarubicin, doxorubicin, MEN 10755, etc. Other topoisomerase inhibitors include podophyllotoxin analogs etoposide and teniposide, as well as anthracenediones, mitoxantrone, and amsacrine. Other antiproliferative agents interfere with microtubule assembly, such as the vinca alkaloid family. Examples of vinca alkaloids include vinblastine, vincristine; vinorelbine; vindesine; vindoline; vincamine, etc. DNA damaging agents include nucleotide analogs and alkylating agents. Alkylating agents include nitrogen mustards such as dichloromethyldiethylamine, cyclophosphamide, and melphalan (L-phenylalanine nitrogen mustard); and nitrosoureas such as carmustine (BCNU), lomustine (CCNU), semustine (methyl-CCNU), streptozotocin, and chlorozotocin. Nucleotide analogs include pyrimidines such as cytarabine (CYTOSAR-U), cytosine arabinoside, fluorouracil (5-FU), and floxuridine (FudR); purines such as thioguanine (6-thioguanine), mercaptopurine (6-MP), pentostatin, and fluorouracil (5-FU); and folic acid analogs such as methotrexate, 10-propargyl-5,8-dideazafolate (PDDF, CB3717), 5,8-dideazatetrahydrofolate (DDATHF), and folinic acid. Other chemotherapeutic agents of interest include metal complexes, such as cisplatin (cis-DDP), carboplatin, oxaliplatin, etc.; ureas, such as hydroxyurea; and hydrazines, such as N-methylhydrazine.

[0579] For example, ionizing radiation (IR) is used to treat about 60% of cancer patients by depositing energy that damages or destroys cells in the treated area, and for the purposes of the present invention, can be delivered in conventional doses and regimens or in reduced doses. Radiation damage to cells is nonspecific and has complex effects on DNA. The efficacy of the treatment depends on the damage to cancer cells being greater than that to normal cells. Radiation therapy can be used to treat every type of cancer. Some types of radiation therapy involve photons, such as X-rays or gamma rays. Another technique for delivering radiation to cancer cells is internal radiation therapy, which places radioactive implants directly into the tumor or body cavity, thereby concentrating the radiation dose to a very small area. Suitable doses of ionizing radiation can range from at least about 2 Gy to no more than about 10 Gy, typically about 5 Gy. Suitable doses of ultraviolet radiation can range from at least about 5 J / m 2 to no more than about 50 J / m 2 In the range of 10 J / m 2 The sample may be collected at least about 4 hours and no more than about 72 hours after the UV radiation, typically around about 4 hours.

[0580] Treatment can also be combined with immunomodulators, including agents that stimulate immune co-stimulatory molecules (e.g., CD40, OX40, etc.); and / or (iii) agents that antagonize immunosuppressive molecules (e.g., CTLA-4, PD-1, PD-L1, etc.). Active agents are administered over a period of time to produce an additive or synergistic effect on the depletion of cancer cells in the host. Methods of administration include, but are not limited to, systemic administration, intratumoral administration, and the like.

[0581] In some embodiments, an individual cancer is selected for treatment with a combination therapy because the cancer is a type of cancer that responds to checkpoint inhibitors, such as PD-1 antagonists, PD-L1 antagonists, CTLA4 antagonists, TIM-3 antagonists, BTLA antagonists, VISTA antagonists, LAG3 antagonists, etc. In some embodiments, such immunomodulators are CTLA-4, PD1, or PDL1 antagonists, such as avelumab, nivolumab, pembrolizumab, ipilimumab, etc. In some such embodiments, the cancer is, but is not limited to, sarcoma, carcinoma, head and neck cancer, glioblastoma, bladder cancer, oral cancer, mesothelioma, pancreatic cancer, liver cancer, colorectal cancer, lung cancer, skin cancer, lymphoma, gastrointestinal cancer, prostate cancer, ovarian cancer, breast cancer, basaloid breast tumors, endometrial cancer, multiple myeloma, melanoma, lymphoma, lung cancer (including small cell lung cancer), kidney cancer, gastric cancer, brain cancer, and CNS tumors. CNS tumors include glioma, glioblastoma, glioblastoma multiforme (GBM), refractory glioblastoma multiforme (rGBM), recurrent glioblastoma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma (menangioma), meningioma, neuroblastoma, retinoblastoma, medulloblastoma, adult pituitary adenoma, O6-methylguanine methyltransferase (MGMT) positive or negative CNS tumors and furin (furin) positive CNS tumors. In some such embodiments, cancer is the type with high neoantigen or mutagenic burden (see Vogelstein et al. (2013) Science 339 (6127): 1546-1558, which is specifically incorporated herein by reference).

[0582] In some embodiments, an individual cancer is selected for treatment with a combination therapy of the invention because the cancer is a type of cancer that responds to an immune response agonist, such as a CD28 agonist, an OX40 agonist; a GITR agonist, a CD137 agonist, a CD27 agonist, an HVEM agonist, an anti-CTLA4 antagonist, an anti-PD-L1 agonist / antagonist, a LAG-3 antagonist, a CD40 agonist, a 4-1BB agonist, a KIR agonist, an ICOS agonist, an EGFR antagonist, a VEGF antagonist, a TIGIT antagonist, and / or a CD112R antagonist.

[0583] In some embodiments, the immunomodulator is OX40, CD137, or a GITR agonist such as tremelimumab. In some such embodiments, the cancer is, but is not limited to, melanoma or small cell lung cancer. In some such embodiments, the cancer is of a type with a high neoantigen or mutagenic burden.

[0584] In some embodiments, the combination therapy includes an antibody known in the art that binds to PD-1 and disrupts the interaction between PD-1 and its ligand PD-L1 and stimulates an anti-tumor immune response. In some embodiments, the antibody, or antigen-binding portion thereof, specifically binds to PD-1. For example, antibodies that target PD-1 and can be used in the present invention include, for example, but are not limited to, nivolumab (BMS-936558, Bristol-Myers Squibb), pembrolizumab (ramborizumab, MK03475 or MK-3475, Merck), humanized anti-PD-1 antibody JS001 (Shanghai Junshi), monoclonal anti-PD-1 antibody TSR-042 (Tesaro, Inc.), pidilizumab (anti-PD-1 mAb CT-011, Medivation), anti-PD-1 monoclonal antibody BGB-A317 (BeiGene) and / or anti-PD-1 antibody SHR-1210 (Shanghai Hengrui), human monoclonal antibody REGN2810 (Regeneron), human monoclonal antibody MDX-1106 (Bristol-Myers Squibb) and / or humanized anti-PD-1 IgG4 antibody PDR001 (Novartis). In some embodiments, the PD-1 antibody is from the following clone: ​​RMP1-14 (rat IgG)-BioXcell cat#BP0146. Other suitable antibodies include the anti-PD-1 antibodies disclosed in U.S. Patent No. 8,008,449, which is incorporated herein by reference. In some embodiments, the antibody or its antigen-binding portion specifically binds to PD-L1 and inhibits its interaction with PD-1, thereby enhancing immune activity. Any antibody known in the art that binds to PD-L1 and disrupts the interaction between PD-1 and PD-L1, and stimulates an anti-tumor immune response, is suitable for use in the combination therapy disclosed herein. For example, antibodies targeting PD-L1 and in clinical trials include BMS-936559 (Bristol-Myers Squibb) and MPDL3280A (Genetech). Other suitable antibodies targeting PD-L1 are disclosed in U.S. Patent No. 7,943,743, which is incorporated herein by reference. One of ordinary skill will understand that any antibody that binds to PD-1 or PD-L1, disrupts the PD-1 / PD-L1 interaction, and stimulates an anti-tumor immune response is suitable for use in a combination therapy approach.

[0585] In some embodiments, the combination therapy includes antibodies known in the art that bind to CTLA-4 and disrupt its interaction with CD80 and CD86. Exemplary antibodies targeting CTLA-4 include FDA-approved ipilimumab (MDX-010, MDX-101, Bristol-Myers Squibb) and tremelimumab (tesitumumab, CP-675, 206, Pfizer) currently undergoing human trials. Other suitable antibodies targeting CTLA-4 are disclosed in WO 2012 / 120125, U.S. Patent Nos. 6,984720 and 6,682,7368, and U.S. Patent Application Nos. 2002 / 0039581, 2002 / 0086014, and 2005 / 0201994, which are incorporated herein by reference. Those of ordinary skill will appreciate that any antibody that binds to CTLA-4, disrupts its interaction with CD80 and CD86, and stimulates an anti-tumor immune response is suitable for use in a combination therapy. In some embodiments, the combination therapy includes an antibody known in the art that binds to LAG-3 and disrupts its interaction with MHC class II molecules. An exemplary antibody targeting LAG-3 is IMP321 (Immutep), which is currently undergoing human trials. Other suitable antibodies targeting LAG-3 are disclosed in U.S. Patent Application No. 2011 / 0150892, which is incorporated herein by reference. Those of ordinary skill will appreciate that any antibody that binds to LAG-3, disrupts its interaction with MHC class II molecules, and stimulates an anti-tumor immune response is suitable for use in a combination therapy.

[0586] In some embodiments, the combination therapy includes an antibody known in the art that binds to TIM-3 and disrupts its interaction with galectin-9. Suitable antibodies targeting TIM-3 are disclosed in U.S. Patent Application No. 2013 / 0022623, which is incorporated herein by reference. A person of ordinary skill will appreciate that any antibody that binds to TIM-3, disrupts its interaction with galectin-9, and stimulates an anti-tumor immune response is suitable for use in a combination therapy.

[0587] In some embodiments, the combination therapy includes antibodies known in the art that bind to 4-1BB / CD137 and disrupt its interaction with CD137L. One of ordinary skill will understand that any antibody that binds to 4-1BB / CD137, disrupts its interaction with CD137L or another ligand, and stimulates an overall anti-tumor immune response or immunostimulatory response that produces anti-tumor activity is suitable for use in a combination therapy.

[0588] In some embodiments, the combination therapy includes an antibody known in the art that binds to GITR and disrupts its interaction with its ligand. One of ordinary skill will understand that any antibody that binds to GITR, disrupts its interaction with GITRL or another ligand, and stimulates an anti-tumor immune response or immunostimulatory response that generally produces anti-tumor activity is suitable for use in a combination therapy approach.

[0589] In some embodiments, the combination therapy includes an antibody known in the art that binds to OX40 and disrupts its interaction with its ligand. One of ordinary skill will understand that any antibody that binds to OX40, disrupts its interaction with OX40L or another ligand, and stimulates an anti-tumor immune response or immunostimulatory response that generally produces anti-tumor activity is suitable for use in a combination therapy approach.

[0590] In some embodiments, the combination therapy includes an antibody known in the art that binds to CD40 and disrupts its interaction with its ligand. One of ordinary skill will understand that any antibody that binds to CD40, disrupts its interaction with its ligand, and stimulates an anti-tumor immune response or immunostimulatory response that generally produces anti-tumor activity is suitable for use in a combination therapy approach.

[0591] In some embodiments, the combination therapy includes an antibody known in the art that binds to ICOS and disrupts its interaction with its ligand. One of ordinary skill will understand that any antibody that binds to ICOS, disrupts its interaction with its ligand, and stimulates an anti-tumor immune response or immunostimulatory response that generally produces anti-tumor activity is suitable for use in a combination therapy method.

[0592] In some embodiments, the combination therapy includes an antibody known in the art that binds to CD28 and disrupts its interaction with its ligand. One of ordinary skill will understand that any antibody that binds to CD28, disrupts its interaction with its ligand, and stimulates an anti-tumor immune response or immunostimulatory response that generally produces anti-tumor activity is suitable for use in a combination therapy method.

[0593] In some embodiments, the combination therapy includes an antibody known in the art that binds to IFNα and disrupts its interaction with its ligand. One of ordinary skill will appreciate that any antibody that binds to IFNα, disrupts its interaction with its ligand, and stimulates an anti-tumor immune response or immunostimulatory response that produces an overall anti-tumor activity is suitable for use in a combination therapy.

[0594] "Anticancer therapy" is a compound, composition or treatment (e.g., surgery) that prevents or delays the growth and / or metastasis of cancer cells. Such anticancer therapies include, but are not limited to, surgery (e.g., removal of all or part of a tumor), chemotherapy, radiation, gene therapy, hormone manipulation, immunotherapy (e.g., therapeutic antibodies and cancer vaccines), and antisense or RNAi oligonucleotide therapy. Examples of available chemotherapeutic drugs include, but are not limited to, hydroxyurea, busulfan, cisplatin, carboplatin, chlorambucil, melphalan, cyclophosphamide, ifosfamide, danorubicin, doxorubicin, epirubicin, mitoxantrone, vincristine, vinblastine, Navelbine.RTM. (vinorelbine), etoposide, teniposide, paclitaxel, docetaxel, gemcitabine, cytosine, arabinoside, bleomycin, neostatin, suramin, taxol, mitomycin C, Avastin, Herceptin.RTM., fluorouracil and temozolomide, etc. These compounds are also suitable for use with standard combination therapies employing two or more chemotherapeutic agents.It will be understood that anti-cancer therapies encompass novel compounds or treatments developed in the future.

[0595] The above-mentioned pharmaceutical compositions and / or preparations contain one or more therapeutic entities in an amount effective to achieve the intended purpose. Therefore, the term "therapeutically effective dose" refers to the amount of a therapeutic entity that improves cancer symptoms. The determination of the therapeutically effective dose of a compound is entirely within the capabilities of those skilled in the art. For example, the therapeutically effective dose can first be estimated in a cell culture assay or an animal model (such as those described herein). Animal models can also be used to determine appropriate concentration ranges and routes of administration. Standard methods known to those of ordinary skill in the art can then be used to determine useful doses and routes in other animals (including humans).

[0596] Kits comprising the compositions of the present invention and instructions for use are also within the scope of the present invention. The kits may also contain at least one additional agent, such as a chemotherapeutic drug, an anti-tumor antibody, or the like. The kits typically include a label indicating the intended use of the kit contents. The term label includes any written or recorded material on or provided with the kit or otherwise accompanying the kit.

[0597] Now that the present invention has been fully described, it will be apparent to those skilled in the art that various changes and modifications may be made without departing from the spirit or scope of the invention. In some embodiments, the kit comprises an IL-2 super factor immune cell targeting or expression construct comprising an IL-2 variant / IL-2 super factor as described herein. In some embodiments, the kit comprises an IL-2 super factor immune cell targeting or expression construct comprising an IL-2 variant / IL-2 super factor, including those provided herein. In some embodiments, the IL-2 super factor immune cell targeting or expression construct comprises an IL-2 variant / IL-2 super factor, including those provided herein.

[0598] 6. Exemplary Immune Cell Targeting or Expression Construct Embodiments

[0599] An immune cell targeting or expression construct comprising: an interleukin-2 receptor beta (IL-2Rβ) binding protein, wherein the binding protein has an equilibrium dissociation constant for IL-2Rβ that is less than the equilibrium dissociation constant for wild-type human IL-2 (hIL-2); linked to the immune cell targeting or expression construct. In some embodiments, the bispecific IL-2 cytokine fusion is also an interleukin-2 receptor beta (IL-2Rβ) binding protein.

[0600] In some embodiments, the immune cell targeting or expression construct exhibits a cytotoxic effect on T cells, such as CD8+ T cells or CD4+ T cells.

[0601] In some embodiments, the construct is a chimeric antigen receptor (CAR) and wherein the IL-2 superkines is fused to a transmembrane domain; linked to an intracellular signaling region.

[0602] In some embodiments, the intracellular signaling region comprises a CD3 signaling domain.

[0603] In some embodiments, the intracellular signaling region comprises one or more of a CD28 signaling domain, a CD137 signaling domain, an OX-40 signaling domain, an ICOS signaling domain, a DAP10 signaling domain.

[0604] In some embodiments, the construct is a T cell antigen coupling (TAC) in which the IL-2 superkines is fused to a ligand that binds a protein associated with the TCR complex; fused to a T cell receptor signaling domain polypeptide.

[0605] In some embodiments, the protein associated with the TCR complex is CD3.

[0606] In some embodiments, the T cell receptor signaling domain polypeptide comprises a CD4 cytoplasmic domain and a CD4 transmembrane domain.

[0607] In some embodiments, the construct is an antibody-coupled T cell receptor (ACTR) comprising a chimeric antigen receptor component that binds with high affinity to an IL-2, IL-4, or IL-13 mutein superkine.

[0608] In some embodiments, the CAR component comprises CD16, and the IL-2, IL-4, or IL-13 mutein superkine is fused to an Fc sequence.

[0609] In some embodiments, the construct is an ICE comprising an IL-2 superkine fused to the variable region of an antibody that binds to a component of the T cell receptor. In some embodiments, the construct is an ICE comprising an IL-2, IL-4, or IL-13 mutein or bifunctional molecule as described herein fused to the variable region of an antibody that binds to a component of the T cell receptor.

[0610] In some embodiments, the BiTE component of the T cell receptor is CD3.

[0611] In some embodiments, the IL-2Rβ binding protein comprises the following amino acid substitutions, numbered according to wild-type hIL-2: L80F, R81D, L85V, I86V, and I92F.

[0612] In some embodiments, a nucleic acid encoding an IL-2, IL-4, or IL-13 mutant protein as described herein is provided. In some embodiments, a vector comprising the nucleic acid is provided.

[0613] In some embodiments, a T cell comprising a construct according to any of the above is provided. In some embodiments, a NK cell comprising a construct according to any of the above is provided. In some embodiments, the T cell is a CD4 + In some embodiments, the T cells are CD8 + T cells.

[0614] Also provided are isolated populations of the aforementioned immune cells and pharmaceutical preparations comprising the aforementioned immune cell populations.

[0615] H. Expression of mutant IL-2, IL-4, or IL-13 gene products

[0616] The nucleic acid molecules described above can be contained in vectors that are capable of directing expression of these nucleic acid molecules in, for example, cells that have been transduced with the vectors. Thus, in addition to the subject IL-2, IL-4, or IL-13 muteins and / or bispecific IL-2, IL-4, or IL-13 cytokine fusions, expression vectors comprising nucleic acid molecules encoding the subject IL-2, IL-4, or IL-13 muteins and cells transfected with these vectors are also preferred embodiments.

[0617] Of course, it should be understood that not all vectors and expression control sequences will express the DNA sequence described herein equally well. Not all hosts can function equally well with the same expression system. However, those skilled in the art can select between these vectors, expression control sequences and hosts without having to perform too many experiments. For example, when selecting a vector, the host must be considered because the vector must be replicated in the host. The copy number of the vector, the ability to control the copy number, and the expression of any other protein (such as an antibiotic marker) encoded by the vector should also be considered. For example, usable vectors include those that allow the DNA encoding IL-2, IL-4 or IL-13 mutant proteins or bifunctional molecules to be amplified in copy number. Such amplifiable vector is well known in the art. These include, for example, vectors capable of amplification by DHFR amplification (see, e.g., Kaufman, U.S. Pat. No. 4,470,461, Kaufman and Sharp, "Construction of a Modular Dihydrafolate Reductase cDNA Gene: Analysis of Signals Utilized for Efficient Expression", Mol. Cell. Biol., 2, pp. 1304-1319 (1982)) or glutamine synthetase ("GS") amplification (see, e.g., U.S. Pat. No. 5,122,464 and European Published Application 338,841).

[0618] In some embodiments, human IL-2, IL-4 or IL-13 mutant proteins or bifunctional molecules disclosed herein will be expressed from a vector, preferably from an expression vector. The vector can be used for autonomous replication in a host cell, or can be integrated into the genome of the host cell after being introduced into the host cell, thereby replicating together with the host genome (e.g., non-episomal mammalian vectors). The expression vector can guide the expression of the coding sequence operably connected thereto. Generally speaking, expression vectors useful in recombinant DNA technology are typically in the form of plasmids (vectors). However, other forms of expression vectors are also included, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses).

[0619] Exemplary recombinant expression vectors can include one or more regulatory sequences, selected based on the host cell to be used for expression, operably linked to the nucleic acid sequence to be expressed.

[0620] Expression constructs or vectors can be designed to express IL-2, IL-4 or IL-13 muteins or variants thereof or bifunctional molecules in prokaryotic or eukaryotic host cells.

[0621] Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. Suitable methods for transforming or transfecting host cells can be found in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Plainview, NY) and other standard molecular biology laboratory manuals.

[0622] Protein expression in prokaryotes is most commonly carried out in Escherichia coli using vectors containing constitutive or inducible promoters. Strategies for maximizing expression of recombinant proteins in Escherichia coli are described in, for example, Gottesman (1990), Gene Expression Technology: Methods in Enzymology 185 (Academic Press, San Diego, Calif.), pp. 119-128 and Wada et al. (1992) Nucleic Acids Res. 20: 2111-2118. Methods for growing, harvesting, disrupting, or extracting IL-2 muteins or variants thereof from cells are generally described in, for example, U.S. Pat. Nos. 4,604,377, 4,738,927, 4,656,132, 4,569,790, 4,748,234, 4,530,787, 4,572,798, 4,748,234, and 4,931,543, which are incorporated herein by reference in their entireties.

[0623] In some embodiments, recombinant IL-2, IL-4 or IL-13 muteins or biologically active variants or bifunctional molecules thereof can also be produced in eukaryotic organisms such as yeast or human cells. Suitable eukaryotic host cells include insect cells (examples of baculovirus vectors that can be used to express proteins in cultured insect cells (e.g., Sf9 cells) include the pAc series (Smith et al. (1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170:31-39)), yeast cells (examples of vectors for expression in Saccharomyces cerevisiae include pYepSec1 (Baldari et al. (1987) EMBO J. 6:229-234), pMFa (Kurjan and Herskowitz (1982) Cell 30:933-943), pJRY88 (Schultz et al. (1987) Gene 54:113-123), pYES2 (Invitrogen Corporation, San Diego), and pYES3 (Invitrogen Corporation, San Diego). Diego, Calif.) and pPicZ (Invitrogen Corporation, San Diego, Calif.); or mammalian cells (mammalian expression vectors include pCDM8 (Seed (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6:187:195)). Suitable mammalian cells include Chinese hamster ovary cells (CHO) or COS cells. In mammalian cells, the control functions of the expression vector are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma virus, adenovirus 2, cytomegalovirus, and simian virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells, see Chapters 16 and 17 of Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Plainview, NY). See Goeddel (1990), Gene Expression Technology: Methods in Enzymology 185 (Academic Press, San Diego, Calif.).

[0624] The sequences encoding the human IL-2, IL-4, or IL-13 muteins or bifunctional molecules of the present disclosure can be optimized for expression in a target host cell. The GC content of the sequence can be adjusted to the average level of a given cellular host, as calculated with reference to known genes expressed in the host cell. Methods for codon optimization are well known in the art. Codons within the coding sequence of the IL-2, IL-4, or IL-13 mutein or bifunctional molecule can be optimized to enhance expression in the host cell such that about 1%, about 5%, about 10%, about 25%, about 50%, about 75%, or up to 100% of the codons within the coding sequence are optimized for expression in a particular host cell.

[0625] Suitable vectors for use include T7-based vectors for bacteria (see, e.g., Rosenberg et al., Gene 56:125, 1987), the pMSXND expression vector for mammalian cells (Lee and Nathans, J. Biol. Chem. 263:3521, 1988), and baculovirus-derived vectors for insect cells (e.g., the expression vector pBacPAK9 from Clontech, Palo Alto, Calif.).

[0626] In some embodiments, the nucleic acid insert encoding a subject IL-2, IL-4 or IL-13 mutein or bifunctional molecule in such vectors can be operably linked to a promoter selected based on, for example, the cell type in which expression is sought.

[0627] In selecting an expression control sequence, a variety of factors should also be considered. These factors include, for example, the relative strength of the sequence, its controllability, and its compatibility with the actual DNA sequence encoding the subject IL-2, IL-4, or IL-13 mutein or bifunctional molecule, particularly with respect to potential secondary structures. The choice of host should take into account their compatibility with the selected vector, the toxicity of the product encoded by the DNA sequence of the present invention, their secretory characteristics, their ability to correctly fold polypeptides, their fermentation or culture requirements, and the ease of purification of the product encoded by the DNA sequence.

[0628] Within these parameters, one skilled in the art can select various vector / expression control sequence / host combinations that will express the desired DNA sequence either in fermentation or in large-scale animal culture, for example using CHO cells or COS 7 cells.

[0629] In some embodiments, the choice of expression control sequences and expression vectors will depend on the choice of host. A variety of expression host / vector combinations can be used. Expression vectors useful for eukaryotic hosts include, for example, vectors with expression control sequences from SV40, bovine papilloma virus, adenovirus, and cytomegalovirus. Expression vectors useful for bacterial hosts include known bacterial plasmids, such as plasmids from Escherichia coli, including col E1, pCRI, pER32z, pMB9, and their derivatives; plasmids with a wider host range such as RP4; phage DNA, such as many derivatives of lambda phage, such as NM989, and other DNA phages such as M13 and filamentous single-stranded DNA phages. Expression vectors useful for yeast cells include 2μ plasmids and their derivatives. Vectors useful for insect cells include pVL 941 and pFastBac TM 1 (Gibco BRL, Gaithersburg, Md.). Cate et al., "Isolation Of The Bovine And Human Genes For Mullerian Inhibiting Substance And Expression Of The Human Gene In Animal Cells", Cell, 45, pp. 685-98 (1986).

[0630] In addition, any of a variety of expression control sequences can be used in these vectors. Such useful expression control sequences include expression control sequences associated with the structural genes of the aforementioned expression vectors. Examples of useful expression control sequences include, for example, early and late promoters of SV40 or adenovirus, the lac system, the trp system, the TAC or TRC system, the major operator and promoter region of lambda phage, such as the PL, the control region of the fd coat protein, the promoter of 3-phosphoglycerate kinase or other glycolytic enzymes, the promoter of acid phosphatase such as PhoA, the promoter of the yeast a mating system, the polyhedron promoter of baculovirus, and other sequences known to control gene expression in prokaryotic or eukaryotic cells or their viruses, and various combinations thereof.

[0631] The T7 promoter can be used in bacteria, the polyhedrin promoter can be used in insect cells, and the cytomegalovirus or metallothionein promoters can be used in mammalian cells. In addition, with respect to higher eukaryotes, tissue-specific and cell-type-specific promoters are widely available. These promoters are named for their ability to direct the expression of nucleic acid molecules in a given tissue or cell type in vivo. The skilled artisan is familiar with many promoters and other regulatory elements that can be used to direct nucleic acid expression.

[0632] In addition to the sequence that contributes to the transcription of the inserted nucleic acid molecule, the vector can also include genes for replication origin and other encoding selective markers. For example, the neomycin resistance (neor) gene confers G418 resistance to the cell expressing it, and therefore allows phenotypic selection of transfected cells. Those skilled in the art can easily determine whether a given regulatory element or selective marker is suitable for use in a specific experimental environment.

[0633] Viral vectors useful in the present invention include, for example, retroviral, adenoviral and adeno-associated viral vectors, herpes virus, simian virus 40 (SV40), and bovine papilloma virus vectors (see, e.g., Gluzman (ed.), Eukaryotic Viral Vectors, CSH Laboratory Press, Cold Spring Harbor, NY).

[0634] Prokaryotic or eukaryotic cells that contain and express nucleic acid molecules encoding the subject IL-2 muteins disclosed herein are also a feature of the present invention. The cells of the present invention are transfected cells, i.e., cells into which a nucleic acid molecule (e.g., a nucleic acid molecule encoding an IL-2, IL-4, or IL-13 mutein or bifunctional molecule) has been introduced by recombinant DNA technology. Progeny of such cells are also considered within the scope of the present invention.

[0635] The exact components of the expression system are not important. For example, IL-2 mutant proteins can be produced in prokaryotic hosts such as bacterial Escherichia coli or in eukaryotic hosts such as insect cells (for example, Sf21 cells) or mammalian cells (for example, CHO, HEK293, COS cells, NIH 3T3 cells or HeLa cells). These cells can be obtained from many sources, including the American Type Culture Collection (Manassas, Va.). When selecting an expression system, it is important whether the components are compatible with each other. A technician or a person of ordinary skill can make such a decision. In addition, if guidance is needed when selecting an expression system, the technician can consult Ausubel et al. (Current Protocols in Molecular Biology, John Wiley and Sons, New York, NY, 1993) and Pouwels et al. (Cloning Vectors: A Laboratory Manual, 1985 Supplement 1987).

[0636] The expressed polypeptide can be purified from the expression system using conventional biochemical procedures and used, for example, as a therapeutic agent as described herein.

[0637] In some embodiments, depending on the host organism used to produce the mutant protein, the IL-2, IL-4 or IL-13 mutant protein or bifunctional molecule obtained will be glycosylated or non-glycosylated. If bacteria are selected as the host, the IL-2, IL-4 or IL-13 mutant protein or bifunctional molecule produced will be non-glycosylated. On the other hand, eukaryotic cells will glycosylate the IL-2, IL-4 or IL-13 mutant protein or bifunctional molecule, but may be glycosylated in a different manner than native IL-2. The IL-2, IL-4 or IL-13 mutant protein or bifunctional molecule produced by the transformed host can be purified according to any suitable method. Various methods are known for purifying IL-2, IL-4 or IL-13 mutant proteins or bifunctional molecules. See, e.g., Current Protocols in Protein Science, Volume 2. Editors: John E. Coligan, Ben M. Dunn, Hidde L. Ploehg, David W. Speicher, Paul T. Wingfield, Unit 6.5 (Copyright 1997, John Wiley and Sons, Inc.). IL-2, IL-4, or IL-13 muteins or bifunctional molecules can be isolated from inclusion bodies produced in E. coli or from conditioned medium of mammalian or yeast cultures producing a given mutein using cation exchange, gel filtration, and / or reversed-phase liquid chromatography.

[0638] Another exemplary method for constructing a DNA sequence encoding an IL-2, IL-4 or IL-13 mutant protein or bifunctional molecule is by chemical synthesis. This includes direct peptide synthesis by chemical means from a protein sequence encoding an IL-2 mutant protein exhibiting the described properties. This method can incorporate natural and unnatural amino acids at positions that affect the interaction of the IL-2, IL-4 or IL-13 mutant protein or bifunctional molecule with its corresponding receptor. Alternatively, a gene encoding the desired IL-2, IL-4 or IL-13 mutant protein or bifunctional molecule can be synthesized by chemical means using an oligonucleotide synthesizer. Such oligonucleotides are designed based on the amino acid sequence of the desired IL-2, IL-4 or IL-13 mutant protein or bifunctional molecule, and preferably those codons that are favorable in the host cell in which the recombinant mutant protein will be produced are selected. In this regard, it is well known that the genetic code is degenerate, that is, an amino acid can be encoded by more than one codon. For example, Phe (F) is encoded by two codons, TIC or TTT, Tyr (Y) is encoded by TAC or TAT, and his (H) is encoded by CAC or CAT. Trp (W) is encoded by a single codon, TGG. Thus, it will be appreciated that for a given DNA sequence encoding a specific IL-2 mutant protein, there will be many degenerate DNA sequences encoding the IL-2 mutant protein. For example, it will be appreciated that, in addition to the preferred DNA sequence of mutant protein H9, there will be many degenerate DNA sequences encoding the IL-2, IL-4, or IL-13 mutant proteins or bifunctional molecules shown. These degenerate DNA sequences are considered to be within the scope of the present disclosure. Therefore, in the context of the present invention, "degenerate variants thereof" means all DNA sequences encoding a specific mutant protein and thus enabling the expression of that specific mutant protein.

[0639] The biological activity of the IL-2, IL-4 or IL-13 muteins or bifunctional molecules can be determined by any suitable method known in the art. Such assays include PHA-blast proliferation and NK cell proliferation.

[0640] I. Anti-PD-1 Antibodies and Combinations

[0641] Anti-PD-1 antibodies for use and / or fusion with any IL-2, IL-4, or IL-13 mutein bifunctional molecules according to the present invention and the methods described herein include, but are not limited to, nivolumab, BMS-936558, MDX-1106, ONO-4538, AMP224, CT-011, and MK-3475 (pembrolizumab), seplizumab (REGN2810), SHR-1210 (CTR2016), and MK-3475 (pembrolizumab). 0175 and CTR20170090), SHR-1210 (CTR20170299 and CTR20170322), JS-001 (CTR20160274), IBI308 (CTR20160735), BGB-A317 (CTR20160872) and / or PD-1 antibodies as listed in U.S. Patent Publication No. 2017 / 0081409 (see, e.g., Table 38). There are two approved anti-PD-1 antibodies, pembrolizumab ( MK-3475-033) and nivolumab ( CheckMate 078) and more antibodies under development, which can be used in the combinations described herein. Exemplary anti-PD-1 antibody sequences are shown in Figure 5, and any of these sequences can be used with the combination methods of IL-2 mutant proteins as described herein. In some embodiments, the IL-2 mutant protein used in combination with the anti-PD-1 antibody is a fusion mutant protein as described herein.

[0642] Table 38: PD-1 Antibody Sequences from U.S. Patent Publication No. 2017 / 0081409

[0643]

[0644]

[0645]

[0646]

[0647]

[0648]

[0649]

[0650]

[0651]

[0652]

[0653]

[0654]

[0655] In some embodiments, the IL-2 mutant protein portion of a bispecific IL-2 cytokine fusion comprising the substitutions L80F, R81D, L85V, I86V, and I92F numbered according to human wild-type IL-2 (SEQ ID NO: 2) is used in combination with an anti-PD-1 antibody or inhibitor. In some embodiments, the IL-2 mutant protein comprising the substitutions L80F, R81D, L85V, I86V, and I92F numbered according to human wild-type IL-2 (SEQ ID NO: 2) is used in combination with nivolumab. In some embodiments, the IL-2 mutant protein comprising the substitutions L80F, R81D, L85V, I86V, and I92F numbered according to human wild-type IL-2 (SEQ ID NO: 2) is used in combination with pembrolizumab. In some embodiments, an IL-2 mutant protein comprising the substitutions L80F, R81D, L85V, I86V, and I92F, numbered according to human wild-type IL-2 (SEQ ID NO: 2), is used in combination with seplizumab. In some embodiments, an IL-2 mutant protein comprising the substitutions L80F, R81D, L85V, I86V, and I92F, numbered according to human wild-type IL-2 (SEQ ID NO: 2), is used in combination with BMS-936558. In some embodiments, an IL-2 mutant protein comprising the substitutions L80F, R81D, L85V, I86V, and I92F, numbered according to human wild-type IL-2 (SEQ ID NO: 2), is used in combination with MDX-1106. In some embodiments, an IL-2 mutant protein comprising the substitutions L80F, R81D, L85V, I86V, and I92F, numbered according to human wild-type IL-2 (SEQ ID NO: 2), is used in combination with ONO-4538. In some embodiments, an IL-2 mutant protein comprising the substitutions L80F, R81D, L85V, I86V, and I92F, numbered according to human wild-type IL-2 (SEQ ID NO: 2), is used in combination with AMP224. In some embodiments, an IL-2 mutant protein comprising the substitutions L80F, R81D, L85V, I86V, and I92F, numbered according to human wild-type IL-2 (SEQ ID NO: 2), is used in combination with CT-011. In some embodiments, an IL-2 mutant protein comprising substitutions L80F, R81D, L85V, I86V, and I92F, numbered according to human wild-type IL-2 (SEQ ID NO: 2), is used in combination with MK-3475. In some embodiments, the IL-2 mutant protein further comprises an F42A substitution, wherein the numbering is according to wild-type human IL-2 of SEQ ID NO: 2. In some embodiments, the IL-2 mutant protein further comprises a Y45A substitution, wherein the numbering is according to wild-type human IL-2 of SEQ ID NO: 2.In some embodiments, the IL-2 mutein further comprises an E62A substitution, wherein the numbering is according to wild-type human IL-2 of SEQ ID NO: 2.

[0656] In some embodiments, an IL-2 mutein comprising a bispecific IL-2 cytokine fusion comprising substitutions L80F, R81D, L85V, I86V, and I92F numbered according to human wild-type IL-2 (SEQ ID NO: 2) is used in combination with any of the reference antibodies. In some embodiments, the IL-2 mutein further comprises an F42A substitution, wherein the numbering is according to wild-type human IL-2 of SEQ ID NO: 2. In some embodiments, the IL-2 mutein further comprises a Y45A substitution, wherein the numbering is according to wild-type human IL-2 of SEQ ID NO: 2. In some embodiments, the IL-2 mutein further comprises an E62A substitution, wherein the numbering is according to wild-type human IL-2 of SEQ ID NO: 2. In some embodiments, the IL-2 mutein further comprises an E62A substitution, wherein the numbering is according to wild-type human IL-2 of SEQ ID NO: 2. In some embodiments, the IL-2 mutein is any IL-2 mutein or variant disclosed herein. In some embodiments, the IL-2 mutant protein sequence is 90% identical to any one of SEQ ID NO: 2 or SEQ ID NO: 6 to SEQ ID NO: 10 or SEQ ID NO: 16. In some embodiments, the IL-2 mutant protein comprises any one of 5-1 SEQ ID NO: 5, 5-2 SEQ ID NO: 6, 6-6 SEQ ID NO: 7, A2 SEQ ID NO: 8, B1 SEQ ID NO: 9, B11 SEQ ID NO: 10, C5 SEQ ID NO: 11, D10 SEQ ID NO: 12, E10 SEQ ID NO: 13, G8 SEQ ID NO: 14, H4 SEQ ID NO: 15, and H9 SEQ ID NO: 16. In some embodiments, the IL-2 mutant protein used in combination with an anti-PD-1 antibody is a fusion mutant protein as described herein. In some embodiments, the IL-2 mutant protein used in combination with an anti-PD-1 antibody is a fusion mutant protein as described herein.

[0657] In some embodiments, the IL-2 cytokine fusion comprises a sequence selected from the group consisting of:

[0658] a. SEQ ID nos. 271, 272, and 273;

[0659] b. SEQ ID n0:274, 275 and 276;

[0660] c. SEQ ID nos. 283, 284, and 285; and

[0661] d. SEQ ID nO:365, 366 and 367.

[0662] In some embodiments, the IL-2 cytokine fusion comprises the sequences of SEQ ID NOs: 271, 272, and 273.

[0663] In some embodiments, the IL-2 cytokine fusion comprises the sequences of SEQ ID NOs: 274, 275, and 276.

[0664] In some embodiments, the IL-2 cytokine fusion comprises the sequences of SEQ ID NOs: 283, 284, and 285.

[0665] In some embodiments, the IL-2 cytokine fusion comprises the sequences of SEQ ID NOs: 365, 366, and 367.

[0666] mPD1 IgG-MDNA109FEAA S125 (KiH)*

[0667] Mutations: F42A, E62A, L80F, R81D, L85V, I86V, I92F, C125S

[0668] Gene 1:

[0669]

[0670] Gene 2:

[0671]

[0672] Gene 3:

[0673]

[0674] huPD1 IgG-MDNA109FEAA S125 (KiH)*

[0675] Mutations: F42A, E62A, L80F, R81D, L85V, I86V, I92F, C125S

[0676] Gene 1:

[0677]

[0678]

[0679] Gene 2:

[0680]

[0681] Gene 3:

[0682]

[0683] mPD1 IgG-MDNA413 R39 / Q111 *

[0684] Mutations: L10V, E12A, L39R, V18I, R65D, D87S, T88S, L101F, K104R, K105T

[0685] Gene 1:

[0686]

[0687]

[0688] Gene 3:

[0689]

[0690] huPD1 IgG-MDNA413 R39 / Q111 *

[0691] Mutations: L10V, E12A, L39R, V18I, R65D, D87S, T88S, L101F, K104R, K105T

[0692] Gene 1:

[0693]

[0694] Gene 3:

[0695]

[0696]

[0697] *Partial sequence

[0698] MDNA413 R39 / Q111 -Fc(1:1KIH)

[0699] L10V, E12A, V18I, L39R, R65D, D87S, T88S, L101F, K104R, K105T

[0700] Gene 1:

[0701]

[0702] Gene 2:

[0703]

[0704] huPD1 IgG-MDNA109FEAA C125 (KiH)*

[0705] Mutations: F42A, E62A, L80F, R81D, L85V, I86V, I92F

[0706] Gene 1:

[0707]

[0708] Gene 2:

[0709]

[0710] Gene 3:

[0711]

[0712]

[0713] *Partial sequence

[0714] mPD1 IgG-MDNA109FEAA C125 (KiH)* (*Partial sequence) Gene 1:

[0715]

[0716]

[0717] Gene 2:

[0718]

[0719] Gene 3:

[0720]

[0721] J. Anti-PD-L1 Antibodies and Combinations

[0722] In some embodiments, any of the IL-2, IL-4, or IL-13 mutein bifunctional molecules described herein can be used in combination and / or fused with an anti-PD-L1 antibody. There are three approved anti-PD-L1 antibodies, namely, atezolizumab ( MPDL3280A), Avelumab ( MSB001071 8C) and durvalumab (MEDI4736) and other anti-PD-L1 antibodies under development. There are many anti-PD-L1 antibodies available, and more are under development that can be used in combination with IL-2 mutant proteins as described herein. In some embodiments, the PD-L1 antibody is an antibody described in U.S. Patent Publication No. 2017 / 0281764 and International Patent Publication No. WO 2013 / 079174 (avelumab) and No. WO 2010 / 077634 (or U.S. Patent Application No. 20160222117 or U.S. Patent No. 8,217,149; atezolizumab). In some embodiments, the PD-L1 antibody comprises a heavy chain sequence of SEQ ID NO: 34 and a light chain sequence of SEQ ID NO: 36 (from US2017 / 281764). In some embodiments, the PD-L1 antibody is atezolizumab ( MPDL3280A; iMpower110). In some embodiments, the PD-L1 antibody is avelumab ( MSB001071 8C). In some embodiments, the PD-L1 antibody is durvalumab (MEDI4736). In some embodiments, the PD-L1 antibody includes, for example, atezolizumab (iMpower133), BMS-936559 / MDX-1105 and / or RG-7446 / MPDL3280A and / or YW243.55.S70, and any of the exemplary anti-PD-L1 antibodies provided herein. In some embodiments, an IL-2 mutant protein comprising substitutions L80F, R81D, L85V, I86V, and I92F numbered according to human wild-type IL-2 (SEQ ID NO: 2) is used in combination with any of the reference antibodies. In some embodiments, the IL-2 mutant protein further comprises an F42A substitution, wherein the numbering is according to wild-type human IL-2 of SEQ ID NO: 2. In some embodiments, the IL-2 mutein further comprises a Y45A substitution, wherein the numbering is according to wild-type human IL-2 of SEQ ID NO: 2. In some embodiments, the IL-2 mutein further comprises an E62A substitution, wherein the numbering is according to wild-type human IL-2 of SEQ ID NO: 2. In some embodiments, the IL-2 mutein further comprises an E62A substitution, wherein the numbering is according to wild-type human IL-2 of SEQ ID NO: 2. In some embodiments, the IL-2 mutein is any IL-2 mutein or variant disclosed herein. In some embodiments, the IL-2 mutein sequence is 90% identical to any one of SEQ ID NO: 2 or SEQ ID NO: 6 to SEQ ID NO: 10 or SEQ ID NO: 16. In some embodiments, the IL-2 mutant protein comprises any one of 5-1 SEQ ID NO: 5, 5-2 SEQ ID NO: 6, 6-6 SEQ ID NO: 7, A2 SEQ ID NO: 8, B1 SEQ ID NO: 9, B11 SEQ ID NO: 10, C5 SEQ ID NO: 11, D10 SEQ ID NO: 12, E10 SEQ ID NO: 13, G8 SEQ ID NO: 14, H4 SEQ ID NO: 15, and H9 SEQ ID NO: 16. In some embodiments, the IL-2 mutant protein used in combination with and / or fused to an anti-PD-L1 antibody is a fusion mutant protein as described herein. In some embodiments, the IL-2 / IL-13 mutant protein used in combination with an anti-PD-L1 antibody is a fusion mutant protein as described herein.

[0723] K. Other immunotherapy combinations

[0724] Other antibodies and / or immunotherapies used and / or fused according to the methods of the invention include, but are not limited to, anti-CTLA4 mAbs, such as ipilimumab, tremelimumab; anti-PD-L1 antagonist antibodies, such as BMS-936559 / MDX-1105, MEDI4736, RG-7446 / MPDL3280A; anti-LAG-3, such as IMP-321; agonistic antibodies targeting immunostimulatory proteins, including anti-CD40 mAbs, such as CP-870,893, rukatumomab, dacitumomab; anti-CD137 mAbs (anti-4-1-BB antibodies), such as BMS-663513; urelumab (anti-4-1BB antibody; see, e.g., U.S. Patent Nos. 7,288,638 and 8,962,804, which are incorporated herein by reference in their entireties); lirelumab (anti-KIR mAbs; IPH2102 / BMS-986015; blocking NK cell inhibitory receptors) and PF-05082566 (utamilumab; see, e.g., U.S. Pat. Nos. 8,821,867, 8,337,850, and 9,468,678, and International Patent Application Publication No. WO 2012 / 032433, herein incorporated by reference in their entireties); anti-OX40 mAbs (see, e.g., WO 2006 / 029879 or WO 2010 / 096418, herein incorporated by reference in their entireties); anti-GITR mAbs, such as TRX518 (see, e.g., U.S. Pat. No. 7,812,135, herein incorporated by reference in its entirety); anti-CD27 mAbs, such as valerumab CDX-1127 (see, e.g., WO 2016 / 145085 and U.S. Patent Publication Nos. US2011 / 0274685 and US2012 / 0213771, which are incorporated herein by reference in their entireties); anti-ICOS mAbs (e.g., MEDI-570, JTX-2011; and anti-TIM-3 antibodies (see, e.g., WO 2013 / 006490 or U.S. Patent Publication No. US2016 / 0257758, which are incorporated herein by reference in their entireties). In some embodiments, an IL-2 mutein comprising the substitutions L80F, R81D, L85V, I86V, and I92F, numbered according to human wild-type IL-2 (SEQ ID NO: 2), is used in combination with any of the reference antibodies. In some embodiments, the IL-2 mutein further comprises an F42A substitution, wherein the numbering is according to SEQ ID NO: In some embodiments, the IL-2 mutein further comprises a Y45A substitution, wherein the numbering is according to wild-type human IL-2 of SEQ ID NO: 2.In some embodiments, the IL-2 mutein further comprises an E62A substitution, wherein the numbering is according to wild-type human IL-2 of SEQ ID NO: 2. In some embodiments, the IL-2 mutein further comprises an E62A substitution, wherein the numbering is according to wild-type human IL-2 of SEQ ID NO: 2. In some embodiments, the IL-2 mutein is any IL-2 mutein or variant disclosed herein. In some embodiments, the IL-2 mutein sequence is 90% identical to any one of SEQ ID NO: 2 or SEQ ID NO: 6 to SEQ ID NO: 10 or SEQ ID NO: 16. In some embodiments, the IL-2 mutein comprises any one of 5-1 SEQ ID NO: 5, 5-2 SEQ ID NO: 6, 6-6 SEQ ID NO: 7, A2 SEQ ID NO: 8, B1 SEQ ID NO: 9, B11 SEQ ID NO: 10, C5 SEQ ID NO: 11, D10 SEQ ID NO: 12, E10 SEQ ID NO: 13, G8 SEQ ID NO: 14, H4 SEQ ID NO: 15, and H9 SEQ ID NO: 16. In some embodiments, the IL-2 / IL-13 mutein used in combination with an antibody is a fusion mutein with an antibody as described herein.

[0725] Other antibodies can also include monoclonal antibodies directed against solid tumors, as well as sarcomas, carcinomas, head and neck cancers, glioblastomas, bladder cancer, oral cancer, mesothelioma, pancreatic cancer, liver cancer, colorectal cancer, lung cancer, skin cancer, lymphomas, gastrointestinal cancer, prostate cancer, ovarian cancer, breast cancer, basaloid breast tumors, endometrial cancer, multiple myeloma, melanoma, lymphoma, lung cancer (including small cell lung cancer), kidney cancer, gastric cancer, brain cancer, and CNS tumors (see generally www.clinicaltrials.gov). CNS tumors include gliomas, glioblastomas, glioblastoma multiforme (GBM), refractory glioblastoma multiforme (rGBM), recurrent glioblastomas, astrocytomas, medulloblastomas, craniopharyngiomas, ependymomas, pinealomas, hemangioblastomas, acoustic neuromas, oligodendrogliomas, meningiomas, meningiomas, neuroblastomas, retinoblastomas, medulloblastomas, adult pituitary adenomas, O6-methylguanine methyltransferase (MGMT) positive or negative CNS tumors and furin positive CNS tumors. In some embodiments, IL-2 mutant proteins comprising substitutions L80F, R81D, L85V, I86V and I92F numbered according to human wild-type IL-2 (SEQ ID NO: 2) are used in combination with any one of the reference antibodies. In...

Claims

1. A bifunctional molecule comprising (i) an IL-2-based amino acid sequence of Table 2 or Table 4, and (ii) an amino acid sequence of any one of Table 3, Table 8, Table 9 or Table 10.

2. A bifunctional molecule comprising (i) an IL-4-based amino acid sequence of Table 9, and (ii) an amino acid sequence of any one of Table 2, Table 3, Table 4, Table 8 or Table 10.

3. A bifunctional molecule comprising (i) an IL-13-based amino acid sequence of Table 8, and (ii) an amino acid sequence of any one of Table 2, Table 3, Table 4, Table 9 or Table 10.

4. A bifunctional molecule comprising (i) an amino acid sequence based on IL-7, IL-12, IL-15, IL-18 or IL-33 of Table 10, and (ii) an amino acid sequence of any one of Table 2, Table 3, Table 4, Table 8 or Table 9.

5. A bifunctional molecule comprising an amino acid sequence of SEQ ID NO: 395, 484, 501, 502, 503, 504, 505, 506, 507 or 508, and an IL-2-based amino acid sequence of Table 2.

6. A bifunctional molecule comprising an amino acid sequence of SEQ ID NO: 395, 484, 501, 502, 503, 504, 505, 506, 507 or 508, and an amino acid sequence based on IL-7, IL-12, IL-15 or IL-18, IL-33 in Table 10.

7. A bifunctional molecule comprising the amino acid sequence of SEQ ID NO: 395, 484, 501, 502, 503, 504, 505, 506, 507 or 508, and the amino acid sequence of any one of Table 2, Table 3, Table 4, Table 5, Table 6, Table 7, Table 8, Table 9, Table 10, Table 11, Table 12, Table 13, Table 15 or Table 39.

8. A bifunctional molecule comprising an amino acid sequence of SEQ ID NO:395, 484, 501, 502, 503, 504, 505, 506, 507 or 508, and an amino acid sequence selected from the group consisting of SEQ ID NO:6 (H9-F42A), SEQ ID NO:7 (H9-K43N), SEQ ID NO:8 (H9-F42A / Y45A; H9-FYAA), SEQ ID NO:9 (H9-F42A / E62A; H9-FEAA), SEQ ID NO:10; H9-F42A / Y45A / E62A; H9-FYEAAA), SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28 NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31 (MDNA109 or H9), SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35 and SEQ ID NO: 146 (F42A, E62A, L80F, R81D, L85V, I86V, I92F and C125S).

9. The bifunctional molecule of any one of claims 1 to 7, wherein compared to wild-type IL-13, the bifunctional molecule comprises the following substitutions: L10H, E15R, R86T, D87G, T88R and R108K.

10. The bifunctional molecule of any one of claims 1 to 7, wherein the bifunctional molecule comprises the following substitutions compared to wild-type IL-13: L10V, E12A, V18I, R65D, D87S, T88S, L101F, K104R and K105T.

11. The bifunctional molecule of claim 9 or 10, wherein the bifunctional molecule further comprises R39 polymorphism and / or Q111 polymorphism.

12. The bifunctional molecule of any one of claims 1 to 11, wherein compared to wild-type IL-2, the bifunctional molecule comprises the following substitutions: L80F, R81D, L85V, I86V, I92F.

13. The bifunctional molecule of any one of claims 1 to 12, wherein compared to wild-type IL-2, the bifunctional molecule further comprises the following substitutions: F42A and E62A.

14. The bifunctional molecule of any one of claims 1 to 13, wherein compared to wild-type IL-2, the bifunctional molecule further comprises the following substitution: C125S.

15. The bifunctional molecule of any one of claims 1 to 14, wherein compared to wild-type IL-4, the bifunctional molecule comprises the following substitutions: R121K, Y124F, S125R.

16. The bifunctional molecule of any one of claims 1 to 15, wherein compared to wild-type IL-4, the bifunctional molecule comprises the following substitutions: K117R, T118V, R121Q, D122S, Y124W, S125F, S128G, S129A.

17. A bifunctional molecule comprising one or more amino acid sequences of any one of Table 2, Table 3, Table 4, Table 8, Table 9 or Table 10, including one or more cytokine binding portions of Table 2, Table 3, Table 4, Table 8, Table 9 or Table 10.

18. A bifunctional molecule comprising one or more amino acid sequences of any one of Table 5, Table 6, Table 7, Table 11, Table 12, Table 13, Table 15 or Table 39, including one or more cytokine binding portions of Table 5, Table 6, Table 7, Table 11, Table 12, Table 13, Table 15 or Table 39.

19. A bifunctional molecule comprising any one or more of the following amino acid sequences: SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 ,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42 ,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,6 2, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 8 2, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146 6, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 2 06, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265,266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325 5, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 3 85, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444 , 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 501, 502, 503, 504, 505, 506, 507, and / or 508. , 20. The bifunctional molecule of any one of claims 1 to 19, further comprising an Fc domain, albumin, an anti-PD1 antibody or an anti-CD3 antibody.

21. The bifunctional molecule of any one of claims 1 to 20, wherein the bifunctional molecule is mPD1 IgG-MDNA132 L39 / Q111 (KiH), huPD1 IgG-MDNA132 L39 / Q111 (KiH), mPD1 IgG-MDNA109FEAAS125 (Ki H), huPD1 IgG-MDNA109FEAAS125 (KiH), mPD1 IgG-MDNA413R39 / Q111, huPD1 IgG-MDNA413 R39 / Q111, MDNA413R39 / Q111-Fc (1:1 KIH), mPD1 IgG-MDNA109FEAAS125 (KiH), huPD1 IgG-MDNA109FEAAS125 (KiH), mPD1 IgG-MDNA413R39 / Q111, huPD1 IgG-MDNA413 R39 / Q111, MDNA413R39 / Q111-Fc(1:1KIH), huPD1 IgG-MDNA109FEAAC125(KiH), mPD1 IgG-MDNA109FEAA C125(KiH), m anti-PD1-MDNA132.15(1:1KIH), hu anti-PD1-MDNA 109FEAA-T3A-C125S(1:1KIH), huPD1-MDNA109FEAA(KiH), mPD1-MDNA109FEAA(KiH), MDN A109FEAA-Fc-MDNA132.15(2:1:1KIH), MDNA132.15-Fc-MDNA413(1:1:2KIH), huPD1-MDNA 109FEAA (KiH)*, mPD1-MDNA109FEAA(KiH)*, anti-mPD1-MDNA109(KIH) or anti-huPD1-MDNA109(KIH).

22. The bifunctional molecule of any one of claims 1-20, wherein the bifunctional molecule comprises SEQ ID NO: 395 (MDNA132.15).

23. The bifunctional molecule of any one of claims 1-20, wherein the bifunctional molecule comprises SEQ ID NO: 484 (MDNA132R.15).

24. The bifunctional molecule of any one of claims 1 to 20, wherein the bifunctional molecule comprises SEQ ID NO: 501 (MDNA132-Q111).

25. The bifunctional molecule of any one of claims 1-20, wherein the bifunctional molecule comprises SEQ ID NO: 502 (MDNA132-R111).

26. The bifunctional molecule of any one of claims 1-20, wherein the bifunctional molecule comprises SEQ ID NO: 503 (cpMDNA132.15-Q111).

27. The bifunctional molecule of any one of claims 1-20, wherein the bifunctional molecule comprises SEQ ID NO: 504 (cpMDNA132.15-R111).

28. The bifunctional molecule of any one of claims 1-20, wherein the bifunctional molecule comprises SEQ ID NO: 505 (cpMDNA132.15-Q111-PE).

29. The bifunctional molecule of any one of claims 1 to 20, wherein the bifunctional molecule comprises SEQ ID NO: 506 (cpMDNA132.15-R111-PE).

30. The bifunctional molecule of any one of claims 1-20, wherein the bifunctional molecule comprises SEQ ID NO: 507 (MDNA132.15-Q111-PE).

31. The bifunctional molecule of any one of claims 1-20, wherein the bifunctional molecule comprises SEQ ID NO: 508 (MDNA132.15-R111-PE).

32. The bifunctional molecule of any one of claims 1 to 31, wherein the bifunctional molecule comprises an IL-2 based sequence that exhibits increased binding affinity to CD122 (IL-2Rβ) compared to wild-type human IL-2.

33. The bifunctional molecule of any one of claims 1-31, wherein the bifunctional molecule comprises an IL-2 based sequence that exhibits increased binding ability to IL-2Rβ compared to wild-type human IL-2.

34. The bifunctional molecule of any one of claims 1-33, wherein the bifunctional molecule comprises an IL-2 based sequence that exhibits abolished IL2Rα binding and / or no IL2Rα binding.

35. The bifunctional molecule of claim 34, wherein the IL-2 based sequence further comprises the following amino acid substitutions: F42A and / or E62A, wherein the numbering is according to wild-type human IL-2 of SEQ ID NO:

2.

36. The bifunctional molecule of claim 35, wherein the bifunctional molecule exhibits reduced binding affinity to CD25 (IL-2Rα), induces expansion of immune cells (including CD8 T cells and NK cells), and / or induces activation of effector immune cells (including CD8 T cells and NK cells).

37. The bifunctional molecule of claim 36, wherein the bifunctional molecule comprises an IL-2 based sequence that exhibits reduced binding affinity to CD25 compared to wild-type human IL-2.

38. The bifunctional molecule of claim 37, wherein the bifunctional molecule induces limited activity and / or no activity in the expansion and / or activation of immunosuppressive regulatory T cells (Tregs).

39. The bifunctional molecule of any one of claims 1-38, wherein the bifunctional molecule binds to IL-2R and PD1 on a target cell.

40. The bifunctional molecule of claim 39, wherein the bifunctional molecule comprises a cytokine binding moiety and an anti-PD1 antibody, and: i) induce activation of tumor-infiltrating CD8+ T cells; and ii) preventing the depletion of the same tumor-infiltrating CD8+ T cells as in i).

41. The bifunctional molecule of claim 40, wherein the cytokine binding moiety and the anti-PD1 antibody are covalently linked.

42. The bifunctional molecule of any one of claims 40 or 41, wherein tumor-infiltrating CD8+ T cells are analyzed for expression of one or more of the following markers: inhibitory PD1 receptor, TIM3 and / or cytotoxic granzyme B.

43. The bifunctional molecule of any one of claims 40-42, wherein the bifunctional molecule induces a decrease in expression of the inhibitory PD1 receptor and / or induces a decrease in TIM3 expression in CD8+ T cells compared to untreated cells and / or cells treated with the cytokine binding portion and the anti-PD1 antibody that are not covalently linked.

44. The bifunctional molecule of any one of claims 40-43, wherein the bifunctional molecule induces an increase in granzyme expression in tumor-infiltrating CD8+ T cells compared to untreated cells and / or cells treated with the cytokine binding portion and the anti-PD1 antibody that are not covalently linked.

45. The bifunctional molecule of any one of claims 1-44, wherein the bifunctional molecule binds to IL-2R and CD3 on a target cell.

46. ​​The bifunctional molecule of claim 45, wherein the bifunctional molecule comprises a cytokine binding moiety and an anti-CD3 antibody.

47. The bifunctional molecule of any one of claims 1 to 46, wherein the bifunctional molecule comprises an IL-13 based sequence that exhibits increased binding affinity to IL-13Rα1 and decreased binding affinity to IL-13Rα2.

48. The bifunctional molecule of claim 47, wherein the increased binding affinity for IL-13Rα1 is at least 5-fold, 8-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 110-fold, 120-fold or more, and wherein the decreased binding affinity for IL-13Rα2 is at least 30-fold, 40-fold, 50-fold or 60-fold or more.

49. The bifunctional molecule of claim 47 or 48, wherein the bifunctional molecule blocks pSTAT6 signaling by at least 20%, at least 30%, at least 40% or at least 50%.

50. The bifunctional molecule of any one of claims 47-49, wherein the bifunctional molecule blocks IL-13-induced TF-1 proliferation by at least 20%, at least 30%, at least 40% or at least 50%.

51. The bifunctional molecule of any one of claims 47-50, wherein the bifunctional molecule blocks IL-4 and / or IL-13-induced M2 macrophage polarization by at least 20%, at least 30%, at least 40% or at least 50%.

52. The bifunctional molecule of any one of claims 1-51, wherein the bifunctional molecule comprises an IL-4-based sequence that exhibits at least 5-fold, 8-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 110-fold, 120-fold increased specific binding to type I or type II IL-4R compared to native IL-4.

53. The bifunctional molecule of any one of claims 1 to 52, wherein the bifunctional molecule comprises an IL-2 mutein or an IL-2 based sequence comprising a sequence selected from the group consisting of: a. SEQ ID NO: 271, 272 and 273; b. SEQ ID NO: 274, 275 and 276; c. SEQ ID NO: 283, 284 and 285; and d. SEQ ID NO:365, 366 and 367.

54. The bifunctional molecule of any one of claims 1-53, wherein the bifunctional molecule is covalently linked to an antibody selected from the group consisting of: dupilumab, nivolumab BMS-936558, MDX-1106, ONO-4538, AMP224, CT-011, and MK-3475 (pembrolizumab or ), seplizumab (REGN2810), SHR-1210 (CTR20160175 and CTR20170090), SHR-1210 (CTR20170299 and CTR20170322), JS-001 (CTR20160274), IBI308 (CTR20160735) and / or BGB-A317 (CTR20160872).

55. The bifunctional molecule of any one of claims 1-53, wherein the bifunctional molecule is covalently linked to an antibody selected from the group consisting of: anti-CTLA4 mAb, such as ipilimumab, tremelimumab; anti-PD-L1 antagonist antibodies, such as BMS-936559 / MDX-1105, MEDI4736, RG-7446 / MPDL3280A; anti-LAG-3, such as IMP-321; agonistic antibodies targeting immunostimulatory proteins, including anti-CD40 mAbs, such as CP-870,893, rukatumomab, dacitumomab; anti-CD137 mAbs (anti-4-1-BB antibodies), such as BMS-663513; Urelumab (anti-4-1BB antibody; see, e.g., U.S. Pat. Nos. 7,288,638 and 8,962,804, which are incorporated herein by reference in their entirety); Lirelumab (anti-KIR mAbs; IPH2102 / BMS-986015; blocking NK cell inhibitory receptors) and PF-05082566 (utomilumab; see, e.g., U.S. Pat. Nos. 8,821,867, 8,337,850, and 9,468,678 and International Patent Application Publication No. WO 2012 / 032433, which are incorporated herein by reference in their entireties); anti-OX40 mAbs (see, e.g., WO 2006 / 029879 or WO 2010 / 096418, which are incorporated herein by reference in their entireties); anti-GITR mAbs, such as TRX518 (see, e.g., U.S. Pat. No. 7,812,135, which are incorporated herein by reference in their entireties); anti-CD27 mAbs, such as valirumab CDX-1127 (see, e.g., WO 2016 / 145085 and U.S. Pat. No. 2011 / 0274685 and US2012 / 0213771, which are incorporated herein by reference in their entirety); anti-ICOS mAb (e.g., MEDI-570, JTX-2011; and anti-TIM-3 antibodies (see, e.g., WO 2013 / 006490 or U.S. Patent Publication No. US2016 / 0257758, which are incorporated herein by reference in their entirety), Herceptin, anti-EGFR, anti-VEGF, anti-TIGIT, anti-LAG3, anti-CD8, anti-CD47, anti-sirs-α and / or anti-CD112R.

56. A composition comprising any one or more amino acid sequences of SEQ ID NO:395, 484, 501, 502, 503, 504, 505, 506, 507 or 508.

57. A nucleic acid encoding the bifunctional molecule or composition of any one of claims 1-56.

58. A vector comprising the nucleic acid of claim 57.

59. A method of treating cancer in a subject in need thereof, the method comprising administering the bifunctional molecule or composition of any one of claims 1-56.

60. A method of treating cancer in a subject in need thereof, the method comprising administering a nucleic acid encoding the bifunctional molecule or composition of any one of claims 1-56.

61. A method of treating cancer in a subject in need thereof, the method comprising administering a vector comprising a nucleic acid encoding the bifunctional molecule or composition of any one of claims 1-56.

62. The method of any one of claims 59-61, wherein the cancer is a solid tumor.

63. The method of any one of claims 59-62, wherein the cancer is selected from the group consisting of sarcoma, carcinoma, head and neck cancer, glioblastoma, bladder cancer, oral cancer, mesothelioma, pancreatic cancer, liver cancer, colorectal cancer, lung cancer, skin cancer, lymphoma, gastrointestinal cancer, prostate cancer, ovarian cancer, breast cancer, basaloid breast tumors, endometrial cancer, multiple myeloma, melanoma, lymphoma, lung cancer (including small cell lung cancer), kidney cancer, gastric cancer, brain cancer, and CNS tumors.

64. The method of claim 63, wherein the cancer is colon cancer.

65. A method of treating a viral disease in a subject in need thereof, the method comprising administering a vector comprising a nucleic acid encoding the bifunctional molecule or composition of any one of claims 1-56.

66. The method of claim 65, wherein the viral disease is human papillomavirus (HPV) and / or hepatitis, such as hepatitis A, hepatitis B, hepatitis C and / or hepatitis D.

67. A method of treating cancer comprising administering a combination therapy include: i) therapeutic antibodies, and ii) The bifunctional molecule or composition of any one of claims 1-56, optionally wherein the bifunctional molecule comprises an IL-2, IL-4 or IL-13 based sequence of any one of claims 1-56.

68. The method of claim 67, wherein the therapeutic antibody is an anti-PD-1 antibody or inhibitor, or an anti-PD-L1 antibody or inhibitor.

69. The method of claim 67 or 68, wherein the therapeutic antibody or inhibitor is selected from the group consisting of: dupilumab, nivolumab BMS-936558, MDX-1106, ONO-4538, AMP224, CT-011, and MK-3475 (pembrolizumab or ), seplizumab (REGN2810), SHR-1210 (CTR20160175 and CTR20170090), SHR-1210 (CTR20170299 and CTR20170322), JS-001 (CTR20160274), IBI308 (CTR20160735), and / or BGB-A317 (CTR20160872); anti-CTLA4 mAbs, such as ipilimumab, tremelimumab; anti-PD-L1 antagonist antibodies, such as BMS-936559 / MDX-1105, MEDI4736, RG-7446 / MPDL3280A; anti-LAG-3, such as IMP-321; agonistic antibodies targeting immunostimulatory proteins, including anti-CD40 mAbs, such as CP-870,893, rucamumab, dacitumomab; anti-CD137 mAbs (anti-4-1-BB antibodies), such as BMS-663513, urelumab (anti-4-1BB antibody; see, e.g., U.S. Pat. Nos. 7,288,638 and 8,962,804, which are incorporated herein by reference in their entirety), lirelumab (anti-KIR mAbs; IPH2102 / BMS-986015; blocking NK cell inhibitory receptors) and PF-05082566 (utamilumab; see, e.g., U.S. Pat. Nos. 8,821,867, 8,337,850, and 9,468,678 and International Patent Application Publication No. WO 2012 / 032433, which are incorporated herein by reference in their entireties); anti-OX40 mAbs (see, e.g., WO 2006 / 029879 or WO 2010 / 096418, which are incorporated herein by reference in their entireties); anti-GITR mAbs, such as TRX518 (see, e.g., U.S. Pat. No. 7,812,135, which are incorporated herein by reference in their entireties); anti-CD27 mAbs, such as valirumab CDX-1127 (see, e.g., WO 2016 / 145085 and U.S. Patent Publication Nos. US2011 / 0274685 and US 2012 / 0213771, which are incorporated herein by reference in their entirety); anti-ICOS mAb (e.g., MEDI-570, JTX-2011; and anti-TIM-3 antibodies (see, e.g., WO 2013 / 006490 or U.S. Patent Publication No. US2016 / 0257758, which are incorporated herein by reference in their entirety), Herceptin, anti-EGFR, anti-VEGF, anti-TIGIT, anti-LAG3, atezolizumab, avelumab, durvalumab and / or anti-CD112R.

70. The method of claim 68 or 69, wherein the anti-PD-1 antibody or inhibitor is selected from the group consisting of: nivolumab BMS-936558, MDX-1106, ONO-4538, AMP224, CT-011, and MK-3475 (pembrolizumab or ), seplizumab (REGN2810), SHR-1210 (CTR20160175 and CTR20170090), SHR-1210 (CTR20170299 and CTR20170322), JS-001 (CTR20160274), IBI308 (CTR20160735), BGB-A317 (CTR20160872), and PD-1 antibodies as listed in Table 38.

71. The method of claim 68 or 69, wherein the anti-PD-L1 antibody or inhibitor is selected from the group consisting of atezolizumab, avelumab, and durvalumab.

72. The method of any one of claims 67-71, wherein the cancer is selected from the group consisting of sarcoma, carcinoma, head and neck cancer, glioblastoma, bladder cancer, oral cancer, mesothelioma, pancreatic cancer, liver cancer, colorectal cancer, lung cancer, skin cancer, lymphoma, gastrointestinal cancer, prostate cancer, ovarian cancer, breast cancer, basaloid breast tumors, endometrial cancer, multiple myeloma, melanoma, lymphoma, lung cancer (including small cell lung cancer), kidney cancer, stomach cancer, and brain cancer.

73. The method of any one of claims 67-72, wherein the cancer is colon cancer.

74. A pharmaceutical composition comprising the bifunctional molecule of any one of claims 1-56 and a pharmaceutically acceptable carrier.

75. A pharmaceutical composition comprising an anti-PD-1 antibody or inhibitor, a bifunctional molecule or composition according to any one of claims 1-56, and a pharmaceutically acceptable carrier, wherein optionally the anti-PD-1 antibody or inhibitor and the bifunctional molecule are covalently linked.

76. A pharmaceutical composition comprising an anti-PD-L1 antibody or inhibitor, a bifunctional molecule or composition according to any one of claims 1-56, and a pharmaceutically acceptable carrier, wherein optionally the anti-PD-L1 antibody or inhibitor and the bifunctional molecule are covalently linked.

77. A pharmaceutical composition comprising an anti-CD3 antibody or inhibitor, a bifunctional molecule or composition according to any one of claims 1-56, and a pharmaceutically acceptable carrier, wherein optionally the anti-CD3 antibody or inhibitor and the bifunctional molecule are covalently linked.

78. A pharmaceutical composition comprising the therapeutic antibody or inhibitor of claim 61, the bifunctional molecule or composition of any one of claims 1-56, and a pharmaceutically acceptable carrier, wherein optionally the therapeutic antibody or inhibitor and the bifunctional molecule are covalently linked.

79. Use according to any of the preceding method claims comprising administering the bifunctional molecule or composition of any of claims 1-56 for treating cancer in a subject in need thereof.

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