Uses 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 monotherapy and combination therapy for the treatment of cancer. Also provided are pharmaceutical compositions comprising such IL-2, IL-4, and / or IL-13 cytokine fusions.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 317,876, filed March 8, 2022, U.S. Provisional Application No. 63 / 328,689, filed April 7, 2022, U.S. Provisional Application No. 63 / 375,132, filed September 9, 2022, U.S. Provisional Application No. 63 / 375,299, filed September 12, 2022, and U.S. Provisional Application No. 63 / 381,072, filed October 26, 2022, the disclosures of which are incorporated herein by reference in their entireties. [Background technology]

[0002] Interleukin-2 (IL-2) is a pluripotent cytokine produced primarily by activated CD4+ T cells and plays a key role in mediating normal immune responses. IL-2 promotes the proliferation and expansion of activated T lymphocytes, enhances B cell growth, and activates monocytes and natural killer cells. These activities have led to the testing of IL-2 and its use as an approved cancer treatment (aldesleukin, Proleukin®). In eukaryotic cells, human IL-2 is synthesized as a 153-amino acid precursor polypeptide, from which 20 amino acids are removed to generate the mature, secreted IL-2 (Taniguchi 1983). Recombinant human IL-2 has been produced in E. coli (Rosenberg 1984), insect cells (Smith 1985), and mammalian COS cells (Taniguchi 1983).

[0003] 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 since been shown to have a wide range of actions. 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)).

[0004] IL-2 functions by interacting with three distinct 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 to be identified was IL-2Rα, a 55 kD polypeptide (p55) that appears upon T cell activation, and the receptor was originally called Tac (T activation) antigen. IL-2Rα interacts with IL-2 approximately 10 -8 K of M dIL-2 binds to the IL-2 receptor IL-2α, also known as the "high-affinity" IL-2 receptor. Binding of IL-2 to cells expressing only IL-2Rα does not result in any detectable biological response. In most cases, IL-2 acts through three distinct receptors: IL-2Rα, IL-2Rβ, and IL-2Rγ. Most cells, such as resting T cells, do not respond to IL-2 because they express only IL-2Rβ and IL-2Rγ, which have low affinity for IL-2. Upon stimulation, resting T cells express the relatively high-affinity IL-2 receptor IL-2Rα. When IL-2 binds to IL-2Rα, this receptor sequentially binds to IL-2Rβ and IL-2Rγ, resulting in T cell activation. An IL-2 "superkine" was previously developed that exhibited enhanced activity due to its enhanced binding affinity for IL-2Rβ (Levin et al., Nature 484:529 (2012)).

[0005] Despite the wealth of knowledge regarding IL-2, including IL-2 superagonists, there remains a need in the art for bispecific IL-2 cytokine fusions. The present invention fulfills this need and provides an IL-2 superagonist or IL-2 agonist as a fusion with another protein. In some embodiments, the IL-2 mutein portion of the bispecific fusion contains the substitutions L80F, R81D, L85V, I86V, and I92F, numbered relative to wild-type IL-2. Summary of the Invention

[0006] IL-2 exerts a wide range of effects on the immune system, playing an important role in regulating both immune activation and homeostasis. Bispecific IL-2 cytokine fusions have been described as immune system stimulators and are used both as monotherapy and in combination with anti-PD-1 antibodies or other immune checkpoint inhibitors and / or therapeutic agents for the treatment of cancer.

[0007] 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 Tables 3, 8, 9, or 10.

[0008] 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 Tables 2, 3, 4, 8, or 10.

[0009] In some embodiments, the present invention provides bifunctional molecules comprising (i) an IL-13-based amino acid sequence of Table 8 and (ii) an amino acid sequence of any one of Tables 2, 3, 4, 9, or 10.

[0010] In some embodiments, the present invention provides bifunctional molecules comprising (i) an IL-7, IL-12, IL-15, IL-18, or IL-33-based amino acid sequence of Table 10 and (ii) an amino acid sequence of one of Tables 2, 3, 4, 8, or 9.

[0011] In some embodiments, the present invention provides bifunctional molecules comprising the 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 in Table 2.

[0012] In some embodiments, the present invention provides bifunctional molecules comprising the amino acid sequence of SEQ ID NO: 395, 484, 501, 502, 503, 504, 505, 506, 507, or 508 and an IL-7, IL-12, IL-15, or IL-18, IL-33-based amino acid sequence in Table 10.

[0013] 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 in any one of Tables 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, or 39.

[0014] In some embodiments, the present invention provides a method for the production of a polypeptide comprising the amino acid sequence of SEQ ID NO: 395, 484, 501, 502, 503, 504, 505, 506, 507, or 508, and a polypeptide comprising the amino acid sequence 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: 11 (H9-F42A / Y45A / E62A, H9-FYEAAA), SEQ ID NO: 12 (H9-F42A / Y45A / E62A, H9-FYEAAA), SEQ ID NO: 13 (H9-F42A / Y45A / E62A, H9-FYEAAA), SEQ ID NO: 14 (H9-F42A / Y45A / E62A, H9-FYEAAA), SEQ ID NO: 15 (H9-F42A / Y45A / E62A, H9-FYEAAA), SEQ ID NO: 16 (H9-F42A / Y45A / E62A, H9-FYEAAA), SEQ ID NO: 17 (H9-F42A / Y45A / E62A, H9-FYEAAA), SEQ ID NO: 18 (H9-F42A / Y45A / E62A, H9-FYEAAA), SEQ ID NO: 19 (H9-F42A / Y45A / and an amino acid sequence selected from the group consisting of 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, 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).

[0015] In some embodiments, the bifunctional molecule comprises the following substitutions compared to wild-type IL-13: L10H, E15R, R86T, D87G, T88R, and R108K.

[0016] 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.

[0017] In some embodiments, the bifunctional molecule further comprises an R39 polymorphism and / or a Q111 polymorphism.

[0018] In some embodiments, the bifunctional molecule comprises the following substitutions compared to wild-type IL-2: L80F, R81D, L85V, I86V, I92F.

[0019] In some embodiments, the bifunctional molecule further comprises the following substitutions compared to wild-type IL-2: F42A and E62A.

[0020] In some embodiments, the bifunctional molecule further comprises the following substitution compared to wild-type IL-2: C125S.

[0021] In some embodiments, the bifunctional molecule comprises the following substitutions compared to wild-type IL-4: R121K, Y124F, S125R.

[0022] In some embodiments, the bifunctional molecule comprises the following substitutions compared to wild-type IL-4: K117R, T118V, R121Q, D122S, Y124W, S125F, S128G, S129A.

[0023] In some embodiments, the invention provides bifunctional molecules comprising one or more amino acid sequences of any one of Tables 2, 3, 4, 8, 9, or 10, and comprising one or more cytokine binding moieties of Tables 2, 3, 4, 8, 9, or 10.

[0024] In some embodiments, the invention provides bifunctional molecules comprising one or more amino acid sequences of any one of Tables 5, 6, 7, 11, 12, 13, 15, or 39, and comprising one or more cytokine binding moieties of Tables 5, 6, 7, 11, 12, 13, 15, or 39.

[0025] In some embodiments, the present invention provides a method for the preparation of a medicament comprising the steps of SEQ ID NOs: 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, 9, 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, 1 78, 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 9, 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 32, 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, 39 ...9, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 4 2, 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.

[0026] In some embodiments, the bifunctional molecule further comprises an Fc domain, albumin, an anti-PD1 antibody, or an anti-CD3 antibody.

[0027] In some embodiments, the bifunctional molecule is selected from the group consisting of mPD1 IgG-MDNA132 L39 / 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 (1:1 KIH), mPD1 IgG-MDNA109FEAAS125(KiH), huPD1 IgG-MDNA109FEAAS125(KiH), mPD1 IgG-MDNA413R39 / Q111, huPD1 IgG-MDNA413 R39 / Q111, MDNA413R39 / Q111-Fc (1:1 KIH), huPD1 IgG-MDNA109FEAAC125(KiH), mPD1 IgG-MDNA109FEAAC125(KiH), manti-PD1-MDNA132.15 (1:1 KIH), hu anti-PD1-MDNA109FEAA-T3A-C125S (1:1 KIH), huPD1-MDNA109FEAA(KiH), mPD1-MDNA109FEAA(KiH), MDNA109FEAA-Fc-MDNA132.15 (2:1:1 KIH), MDNA132.15-Fc-MDNA413 (1:1:2 KIH), huPD1-MDNA109FEAA(KiH)*, mPD1-MDNA109FEAA(KiH)*, anti-mPD1-MDNA109(KiH), or anti-huPD1-MDNA109(KIH).

[0028] In some embodiments, the bifunctional molecule comprises SEQ ID NO: 395 (MDNA132.15).

[0029] In some embodiments, the bifunctional molecule comprises SEQ ID NO: 484 (MDNA132R.15).

[0030] In some embodiments, the bifunctional molecule comprises SEQ ID NO: 501 (MDNA132-Q111).

[0031] In some embodiments, the bifunctional molecule comprises SEQ ID NO: 502 (MDNA132-R111).

[0032] In some embodiments, the bifunctional molecule comprises SEQ ID NO: 503 (cpMDNA132.15-Q111).

[0033] In some embodiments, the bifunctional molecule comprises SEQ ID NO: 504 (cpMDNA132.15-R111).

[0034] In some embodiments, the bifunctional molecule comprises SEQ ID NO: 505 (cpMDNA132.15-Q111-PE).

[0035] In some embodiments, the bifunctional molecule comprises SEQ ID NO: 506 (cpMDNA132.15-R111-PE).

[0036] In some embodiments, the bifunctional molecule comprises SEQ ID NO: 507 (MDNA132.15-Q111-PE).

[0037] In some embodiments, the bifunctional molecule comprises SEQ ID NO: 508 (MDNA132.15-R111-PE).

[0038] In some embodiments, the bifunctional molecule comprises an IL-2-based sequence that has increased binding affinity for CD122 (IL-2Rβ) compared to wild-type human IL-2.

[0039] In some embodiments, the bifunctional molecule comprises an IL-2-based sequence that has increased binding ability to IL-2Rβ compared to wild-type human IL-2.

[0040] In some embodiments, the bifunctional molecule comprises an IL-2-based sequence that has reduced and / or no IL2Rα binding.

[0041] In some embodiments, the IL-2 based sequence further comprises the following amino acid substitutions: F42A and / or E62A (numbering is based on wild-type human IL-2 of SEQ ID NO: 2).

[0042] In some embodiments, the bifunctional molecules exhibit reduced binding affinity for CD25 (IL-2Rα), induce proliferation of immune cells (including CD8 T cells and NK cells), and / or induce activation of effector immune cells (including CD8 T cells and NK cells).

[0043] In some embodiments, the bifunctional molecule comprises an IL-2-based sequence that has reduced binding affinity for CD25 compared to wild-type human IL-2.

[0044] In some embodiments, the bifunctional molecule induces limited and / or no activity with respect to the proliferation and / or activation of immunosuppressive regulatory T cells (Tregs).

[0045] In some embodiments, the bifunctional molecule binds to IL-2R and PD1 on a target cell.

[0046] In some embodiments, the bifunctional molecule comprises a cytokine binding moiety and an anti-PD1 antibody; i) induces activation of tumor-infiltrating CD8+ T cells; ii) Prevents exhaustion of tumor-infiltrating CD8+ T cells, as in i).

[0047] In some embodiments, the cytokine binding moiety and the anti-PD1 antibody are covalently linked.

[0048] 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.

[0049] In some embodiments, the bifunctional molecule induces a decrease in expression of the inhibitory PD1 receptor and / or induces a decrease in expression of TIM3 in CD8+ T cells compared to untreated cells and / or cells treated with a non-covalently bound cytokine-binding moiety and an anti-PD1 antibody.

[0050] In some embodiments, the bifunctional molecule induces increased expression of granzymes in tumor-infiltrating CD8+ T cells compared to untreated cells and / or cells treated with a non-covalently bound cytokine-binding moiety and an anti-PD1 antibody.

[0051] In some embodiments, the bifunctional molecule binds to IL-2R and CD3 on a target cell.

[0052] In some embodiments, the bifunctional molecule comprises a cytokine binding moiety and an anti-CD3 antibody.

[0053] In some embodiments, the bifunctional molecule comprises an IL-13-based sequence with increased binding affinity for IL-13Rα1 and decreased binding affinity for IL-13Rα2.

[0054] In some embodiments, the bifunctional molecule has at least a 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 increase in binding affinity to IL-13Rα1 and at least a 30-fold, 40-fold, 50-fold, or 60-fold or more decrease in binding affinity to IL-13Rα2.

[0055] In some embodiments, the bifunctional molecule inhibits pSTAT6 signaling by at least 20%, at least 30%, at least 40%, or at least 50%.

[0056] In some embodiments, the bifunctional molecule inhibits IL-13-induced TF-1 proliferation by at least 20%, at least 30%, at least 40%, or at least 50%.

[0057] In some embodiments, the bifunctional molecule inhibits IL-4 and / or IL-13-induced M2 polarization of macrophages by at least 20%, at least 30%, at least 40%, or at least 50%.

[0058] In some embodiments, the bifunctional molecule comprises an IL-4-based sequence that exhibits at least a 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, or 120-fold increase in specific binding to type I or type II IL-4R compared to native IL-4.

[0059] 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: a. SEQ ID NOs: 271, 272, and 273; b. SEQ ID NOs: 274, 275, and 276; c. SEQ ID NOs: 283, 284, and 285, and d. SEQ ID NOs: 365, 366, and 367.

[0060] In some embodiments, the bifunctional molecule is covalently linked to an antibody selected from the group consisting of dupilumab, nivolumab (OPDIVO®), BMS-936558, MDX-1106, ONO-4538, AMP224, CT-011, and MK-3475 (pembrolizumab or KEYTRUDA®), cemiplimab (REGN2810), SHR-1210 (CTR20160175 and CTR20170090), SHR-1210 (CTR20170299 and CTR20170322), JS-001 (CTR20160274), IBI308 (CTR20160735), and / or BGB-A317 (CTR20160872).

[0061] In some embodiments, the bifunctional molecule is selected from the group consisting of 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), agonist antibodies targeting immune stimulatory proteins (including anti-CD40 mAbs such as CP-870,893, lucatumumab, dacetuzumab), anti-CD137 mAb (anti-4-1BB antibody) (BMS-663513, uremulab (anti-4-1BB antibody)), and anti-PD-L1 mAbs (such as PD-L1 mAb). BB antibodies, such as, for example, see U.S. Pat. Nos. 7,288,638 and 8,962,804 (incorporated by reference in their entireties), lirilumab (anti-KIRmAB, IPH2102 / BMS-986015, blocks NK cell inhibitory receptors), and PF-05082566 (utomilumab, see, for example, U.S. Pat. Nos. 8,821,867, 8,337,850, and 9,468,678, and International Publication No. WO2012 / 032433 (incorporated by reference in their entireties) ), anti-OX40 mAbs (see, e.g., WO2006 / 029879 or WO2010 / 096418, which are incorporated by reference in their entireties), anti-GITR mAbs (such as TRX518 (see, e.g., U.S. Pat. No. 7,812,135, which is incorporated by reference in its entirety)), anti-CD27 mAbs (such as varlilumab CDX-1127 (see, e.g., WO2016 / 145085, and U.S. Patent Application Publication Nos. 2011 / 0274685 and 2012 / 0213771), (see, e.g., WO 2013 / 006490 or U.S. Patent Publication No. 2016 / 0257758, which are incorporated by reference in their entireties), anti-ICOS mAb (such as MEDI-570, JTX-2011, and anti-TIM-3 antibodies (see, e.g., WO 2013 / 006490 or U.S. Patent Publication No. 2016 / 0257758, which are incorporated by reference in their entireties), Herceptin, anti-EGFR, anti-VEGF, anti-TIGIT, anti-LAG3, anti-CD8, anti-CD47, anti-SIRS alpha, and / or anti-CD112R.

[0062] In some embodiments, the present invention provides a composition comprising any one or more amino acid sequences of SEQ ID NOs: 395, 484, 501, 502, 503, 504, 505, 506, 507, or 508.

[0063] In some embodiments, the present invention provides a composition comprising the amino acid sequence of SEQ ID NO: 395 (MDNA132.15).

[0064] In some embodiments, the invention provides a composition comprising the amino acid sequence of SEQ ID NO: 484 (MDNA132R.15).

[0065] In some embodiments, the present invention provides a composition comprising the amino acid sequence of SEQ ID NO: 501 (MDNA132-Q111).

[0066] In some embodiments, the invention provides a composition comprising the amino acid sequence of SEQ ID NO: 502 (MDNA132-R111).

[0067] In some embodiments, the present invention provides a composition comprising the amino acid sequence of SEQ ID NO: 503 (cpMDNA132.15-Q111).

[0068] In some embodiments, the invention provides a composition comprising the amino acid sequence of SEQ ID NO: 504 (cpMDNA132.15-R111).

[0069] In some embodiments, the invention provides a composition comprising the amino acid sequence of SEQ ID NO: 505 (cpMDNA132.15-Q111-PE).

[0070] In some embodiments, the invention provides a composition comprising the amino acid sequence of SEQ ID NO: 506 (cpMDNA132.15-R111-PE).

[0071] In some embodiments, the present invention provides a composition comprising the amino acid sequence of SEQ ID NO: 507 (MDNA132.15-Q111-PE).

[0072] In some embodiments, the invention provides a composition comprising the amino acid sequence of SEQ ID NO: 508 (MDNA132.15-R111-PE).

[0073] In some embodiments, the present invention provides nucleic acids encoding the bifunctional molecules or compositions described herein.

[0074] In some embodiments, the invention provides vectors comprising the nucleic acids described herein.

[0075] 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 described herein.

[0076] In some embodiments, the present invention provides a method of treating cancer in a subject in need thereof, comprising administering a nucleic acid encoding a bifunctional molecule or composition described herein.

[0077] In some embodiments, the present invention provides methods of treating cancer in a subject in need thereof, comprising administering a vector comprising a nucleic acid encoding a bifunctional molecule or composition described herein.

[0078] In some embodiments, the cancer is a solid tumor.

[0079] 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, lymphatic cancer, gastrointestinal cancer, prostate cancer, ovarian cancer, breast cancer, basal-like breast tumor, endometrial cancer, multiple myeloma, melanoma, lymphoma, lung cancer (including small cell lung cancer), kidney cancer, gastric cancer, brain tumor, and central nervous system tumor.

[0080] In some embodiments, the cancer is colon cancer.

[0081] In some embodiments, the present invention provides methods of treating a viral disease in a subject in need thereof, comprising administering a vector comprising a nucleic acid encoding a bifunctional molecule or composition described herein.

[0082] In some embodiments, the viral disease is human papillomavirus (HPV) and / or hepatitis (eg, hepatitis A, hepatitis B, hepatitis C, and / or hepatitis D).

[0083] 1. A method of treating cancer comprising administering a combination therapy, (i) a therapeutic antibody; and (ii) a bifunctional molecule or composition described herein, optionally wherein the bifunctional molecule comprises an IL-2, IL-4, or IL-13-based sequence described herein; and A method comprising:

[0084] In some embodiments, the therapeutic antibody is an anti-PD-1 antibody or inhibitor, or an anti-PD-L1 antibody or inhibitor.

[0085] In some embodiments, the therapeutic antibody is selected from the group consisting of dupilumab, nivolumab (OPDIVO®), BMS-936558, MDX-1106, ONO-4538, AMP224, CT-011, and MK-3475 (pembrolizumab or KEYTRUDA®), cemiplimab (REGN2810), SHR-1210 (CTR20160175 and CTR20170090), SHR-1210 (CTR20170299 and CTR20170322), and IFN-γ-γ (IFN-γ-γ). ), JS-001 (CTR20160274), IBI308 (CTR20160735), and / or BGB-A317 (CTR20160872), anti-CTLA4 mAb (ipilimumab, tremelimumab, etc.), anti-PD-L1 antagonist antibodies (BMS-936559 / MDX-1105, MEDI4736, RG-7446 / MPDL3280A, etc.), anti-LAG-3 (IMP-321, etc.), agonist antibodies targeting immune stimulatory proteins (CP-870, 89 3, lucatumumab, including anti-CD40 mAbs such as dacetuzumab), anti-CD137 mAb (anti-4-1-BB antibody) (BMS-663513 urelumab (anti-4-1BB antibody, see, e.g., U.S. Pat. Nos. 7,288,638 and 8,962,804, which are incorporated by reference in their entireties)), lirilumab (anti-KIRmAB, IPH2102 / BMS-986015, blocks NK cell inhibitory receptors), and PF-05082566 (anti-KIRmAB). Mirumab (see, e.g., U.S. Pat. Nos. 8,821,867, 8,337,850, and 9,468,678, and International Publication No. WO2012 / 032433, which are incorporated by reference in their entireties), anti-OX40 mAb (see, e.g., WO2006 / 029879 or WO2010 / 096418, which are incorporated by reference in their entireties), anti-GITR mAb (TRX518 (see, e.g., U.S. Pat. Nos. 7,812,135, which is incorporated by reference in its entirety), anti-CD27 mAb (varlilumab CDX-1127 (see, e.g., WO2016 / 145085, and U.S. Patent Application Publication Nos. 2011 / 0274685 and 2012 / 0213771, which are incorporated by reference in their entireties), anti-ICOS mAb (e.g., MEDI-5 70, JTX-2011, and anti-TIM-3 antibodies (see, e.g., WO2013 / 006490 or U.S. Patent Publication No. 2016 / 0257758, which are incorporated by reference in their entireties), Herceptin, anti-EGFR, anti-VEGF, anti-TIGIT, anti-LAG3, anti-CD8, anti-CD47, anti-SIRS alpha, and / or anti-CD112R.

[0086] In some embodiments, the anti-PD-1 antibody or inhibitor is selected from the group consisting of nivolumab (OPDIVO®), BMS-936558, MDX-1106, ONO-4538, AMP224, CT-011, and MK-3475 (pembrolizumab or KEYTRUDA®), cemiplimab (REGN2810), SHR-1210 (CTR20160175 and CTR20170090), SHR-1210 (CTR20170299 and CTR20170322), JS-001 (CTR20160274), IBI308 (CTR20160735), BGB-A317 (CTR20160872), and a PD-1 antibody listed in Table 38.

[0087] In some embodiments, the anti-PD-L1 antibody or inhibitor is selected from the group consisting of atezolizumab, avelumab, and durvalumab.

[0088] 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, lymphatic cancer, gastrointestinal cancer, prostate cancer, ovarian cancer, breast cancer, basal-like breast tumor, endometrial cancer, multiple myeloma, melanoma, lymphoma, lung cancer (including small cell lung cancer), renal cancer, gastric cancer, and brain cancer.

[0089] In some embodiments, the cancer is colon cancer.

[0090] In embodiments, the present disclosure provides a pharmaceutical composition comprising a bifunctional molecule described herein and a pharmaceutically acceptable carrier.

[0091] A pharmaceutical composition comprising an anti-PD-1 antibody or inhibitor, a bifunctional molecule or composition described herein, and a pharmaceutically acceptable carrier, optionally wherein the anti-PD-1 antibody or inhibitor and the bifunctional molecule are covalently attached to the pharmaceutical composition.

[0092] A pharmaceutical composition comprising an anti-PD-L1 antibody or inhibitor, a bifunctional molecule or composition described herein, and a pharmaceutically acceptable carrier, optionally wherein the anti-PD-L1 antibody or inhibitor and the bifunctional molecule are covalently attached to the pharmaceutical composition.

[0093] A pharmaceutical composition comprising an anti-CD3 antibody or inhibitor, a bifunctional molecule or composition described herein, and a pharmaceutically acceptable carrier, optionally wherein the anti-CD3 antibody or inhibitor and the bifunctional molecule are covalently linked.

[0094] A pharmaceutical composition comprising a therapeutic antibody or inhibitor, a bifunctional molecule or composition described herein, and a pharmaceutically acceptable carrier, optionally wherein the therapeutic antibody or inhibitor and the bifunctional molecule are covalently linked.

[0095] In some embodiments, the present invention provides a use according to any of the preceding method paragraphs, comprising administering a bifunctional molecule or composition described herein to treat cancer in a subject in need thereof. [Brief explanation of the drawings]

[0096] [Figure 1A] Examples of sequences for IgG1, IgG2, IgG3 and IgG4 are shown. [Figure 1B] Examples of sequences for IgG1, IgG2, IgG3 and IgG4 are shown. [Figure 2] The sequence of an exemplary H9-Fc fusion is shown. [Figure 3] Exemplary oncolytic viruses. [Figure 4] Modulation of the TME with IL-2 / IL-13 bispecific superkines immunologically targets "cold tumors," which do not respond to checkpoint inhibitors due to the tumor-induced TME: 1) low counts of CD8+ cells and NK cells, high counts of Tregs, and 2) high numbers of immunosuppressive myeloid cells (i.e., TAMs and MDSCs). [Figure 5A] 1 shows bispecific sequence information for various construct embodiments. [Figure 5B] 1 shows bispecific sequence information for various construct embodiments. [Figure 5C] 1 shows bispecific sequence information for various construct embodiments. [Figure 5D] 1 shows bispecific sequence information for various construct embodiments. [Figure 5E] 1 shows bispecific sequence information for various construct embodiments. [Figure 5F] 1 shows bispecific sequence information for various construct embodiments. [Figure 5G] 1 shows bispecific sequence information for various construct embodiments. [Figure 5H]1 shows bispecific sequence information for various construct embodiments. [Figure 5I] 1 shows bispecific sequence information for various construct embodiments. [Figure 5J] 1 shows bispecific sequence information for various construct embodiments. [Figure 5K] 1 shows bispecific sequence information for various construct embodiments. [Figure 5L] 1 shows bispecific sequence information for various construct embodiments. [Figure 5M] 1 shows bispecific sequence information for various construct embodiments. [Figure 5N] 1 shows bispecific sequence information for various construct embodiments. [Figure 5O] 1 shows bispecific sequence information for various construct embodiments. [Figure 5P] 1 shows bispecific sequence information for various construct embodiments. [Figure 5Q] 1 shows bispecific sequence information for various construct embodiments. [Figure 5R] 1 shows bispecific sequence information for various construct embodiments. [Figure 5S] 1 shows bispecific sequence information for various construct embodiments. [Figure 5T] 1 shows bispecific sequence information for various construct embodiments. [Figure 6] The role of IL-4 receptor and IL-13 receptor in cancer. [Figure 7] Mechanism of action of the bispecific cytokine (DUCK Cancer) MDNA109FEAA-Fc-MDNA413. [Figure 8] SPR analysis of the binding of mouse anti-PD1-MDNA109FEAAS125 to human IL-2R alpha (CD25) and human IL-2R beta (CD122). [Figure 9]Figure 1 shows the results of SPR analysis of the binding of mouse anti-PD1-MDNA109FEAAS125 to human PD-1 and mouse PD-1. [Figure 10A] Figure 1 shows the results of SPR analysis of the binding of human anti-PD1-MDNA109FEAAS125 to human PD-1 and mouse PD-1. [Figure 10B] SPR analysis of the binding of IL2-Fc, mouse anti-PD1-MDNA109FEAAC125, and human anti-PD1-MDNA109FEAAC125 to human IL-2R alpha (CD25) and human IL-2R beta (CD122). [Figure 11A] These are the results of a PD-1 reporter assay. [Figure 11B] These are the results of a PD-1 reporter assay. [Figure 11C] These are the results of a PD-1 reporter assay. [Figure 12A] This shows the results of a pSTAT5 phosphorylation reporter assay. [Figure 12B] This shows the results of a pSTAT5 phosphorylation reporter assay. [Figure 12C] This shows the results of a pSTAT5 phosphorylation reporter assay. [Figure 12D] This shows the results of a pSTAT5 phosphorylation reporter assay. [Figure 12E] This shows the results of a pSTAT5 phosphorylation reporter assay. [Figure 13] 1 shows the results of a Jurkat IL2Rβγ bioassay. [Figure 14] FIG. 1 is a schematic diagram of the study design for the IT treatment trial in the CT26 model. [Figure 15] Figure 1 shows tumor growth inhibition in a CT26 colon cancer model by IT treatment. [Figure 16] Results of tumor growth inhibition in a CT26 colon cancer model IP treatment study. [Figure 17] Survival curves for intraperitoneal (IP) treatment of CT26 colon cancer with murine anti-PD1-MDNA109FEAAC125. [Figure 18] FIG. 1 is a schematic diagram of the study design for IP treatment studies in the B16F10 model. [Figure 19] 1 shows tumor growth inhibition in a B16F10 melanoma model tumor growth inhibition test. [Figure 20] 1 is a survival curve for a B16F10 melanoma cell line treatment trial. [Figure 21] These are the results of a CTLL-2 assay. [Figure 22] 1 is a study summary of the in vivo CT26 colon tumor efficacy study. [Figure 23] Results of in vivo efficacy studies in CT26 colon tumors. [Figure 24] Results of in vivo efficacy studies in CT26 colon tumors. [Figure 25A] SPR analysis of IL-13Rα1 and IL-13Rα2 binding by Fc-MDNA413R39 / Q111 (1:2). [Figure 25B] SPR analysis of mouse and cynomolgus monkey IL-13Rα1 binding by Fc-MDNA413R39 / Q111 (1:2). [Figure 26] HEK Blue IL-4 competition assay of MDNA413R39 / Q111-FcKIH, MDNA109FEAAC125-Fc-MDNA413R39 / Q111 (2:1:2), Fc-MDNA413R39 / Q111 (1:2), and MDNA413R39 / Q111-Fc-MDNA132L39 / Q111KIH. [Figure 27] HEK Blue IL-13 competition assay of MDNA413R39 / Q111-FcKIH, MDNA109FEAAC125-Fc-MDNA413R39 / Q111, Fc-MDNA413R39 / Q111 (1:2), and MDNA413R39 / Q111-Fc-MDNA132L39 / Q111KIH. [Figure 28] Fc-MDNA413R39 / Q111 (1:2) showed dose-dependent inhibition of TF-1 proliferation at both the EC50 (upper panel) and EC80 (lower panel) of rhIL-13. [Figure 29] Phenotypic analysis of IL-4-treated macrophages in the presence of Fc-MDNA413R39 / Q111 (1:2). The dotted line represents the IL-4 only control. The dashed line represents the M0 macrophage control. [Figure 30] Phenotypic analysis of IL-13-treated macrophages in the presence of Fc-MDNA413R39 / Q111 (1:2). The dotted line represents the IL-13 only control. The dashed line represents the M0 macrophage control. [Figure 31A] Growth inhibition of various tumor types by Fc-MDNA413R39 / Q111 (1:2). [Figure 31B] Growth inhibition of various tumor types by Fc-MDNA413R39 / Q111 (1:2). [Figure 31C] Growth inhibition of various tumor types by Fc-MDNA413R39 / Q111 (1:2). [Figure 31D] Growth inhibition of various tumor types by Fc-MDNA413R39 / Q111 (1:2). [Figure 31E] Growth inhibition of various tumor types by Fc-MDNA413R39 / Q111 (1:2). [Figure 32] Inhibition of tumor growth in a B16F10 melanoma model by Fc-MDNA413R39 / Q111 (1:2), MDNA19 / MDNA109FEAAC125-Fc, the combination of Fc-MDNA413R39 / Q111 (1:2) + MDNA19 / MDNA109FEAAC125-Fc, or the combination of Fc-MDNA413R39 / Q111 (1:2) + MDNA109FEAAC125-Fc-MDNA413R39 / Q111 (2:1:2). [Figure 33] A to B show the inhibition of B16F10 melanoma tumor growth by Fc-MDNA413R39 / Q111(1:2), the combination of Fc-MDNA413R39 / Q111(1:2) + MDNA19 / MDNA109FEAAC125-Fc, the combination of Fc-MDNA413R39 / Q111(1:2) + anti-PD-1 antibody, MDNA19 / MDNA109FEAAC125-Fc, and anti-PD-1 antibody. [Figure 34] HEK Blue IL-2 assay results. [Figure 35] These are the results of the Jurkat IL-2 bioassay. [Figure 36] PK profile of mouse anti-PD1-MDNA109FEAAC125 upon IP (intraperitoneal) administration. [Figure 37] A to C are PD profiles of mouse anti-PD1-MDNA109FEAAC125 upon IP (intraperitoneal) administration. [Figure 38] These are the results of the E0771 tumor growth inhibition test. [Figure 39] 1 shows the body weight of animals during the MTD study using Fc-MDNA413R39 / Q111 (1:2). [Figure 40] PK profile of Fc-MDNA413R39 / Q111 (1:2) after the first and second doses indicated for MTD testing. [Figure 41] Figure 1 shows tumor growth inhibition of B16F10 melanoma (A) and CT26 colon carcinoma (B) by Fc-MDNA413R39 / Q111 (1:2). [Figure 42A] 1 shows the results of the SPR test described in Example 5. [Figure 42B] 1 shows the results of the SPR test described in Example 5. [Figure 42C] 1 shows the results of the SPR test described in Example 5. [Figure 43] AB are the results of the IL13Ra1 and IL13Ra2 binding assay described in Example 5. [Figure 44A] 1 shows the results of the CD3 epitope and CD3 epsilon / delta binding assays described in Example 5. [Figure 44B] 1 shows the results of the CD3 epitope and CD3 epsilon / delta binding assays described in Example 5. [Figure 44C] 1 shows the results of the CD3 epitope and CD3 epsilon / delta binding assays described in Example 5. [Figure 45]1A to 1C are the results of the IL2Rα and IL2Rβ binding assay described in Example 5. [Figure 46] 1 shows the results of the in vivo imaging analysis described in Example 5. [Figure 47A] 1 shows the results of the PTNG binding affinity analysis described in Example 5. [Figure 47B] 1 shows the results of the PTNG binding affinity analysis described in Example 5. [Figure 47C] 1 shows the results of the PTNG binding affinity analysis described in Example 5. [Figure 48A] 1 shows the results of the MDNA132BiSKIT binding affinity analysis described in Example 5. [Figure 48B] 1 shows the results of the MDNA132BiSKIT binding affinity analysis described in Example 5. [Figure 48C] 1 shows the results of the MDNA132BiSKIT binding affinity analysis described in Example 5. [Figure 49] 1 shows the results of the Fc-MDNA132L39 / Q111 (1:1 KIH) binding analysis described in Example 5. [Figure 50] 1 shows the results of the mouse anti-CD3-MDNA132L39 / Q111 (1:1 KIH) binding analysis described in Example 5. [Figure 51] 1 shows the results of the receptor internalization assay described in Example 5. [Figure 52] 1 shows the results of the Jurkat IL2Rβγ bioassay described in Example 5. [Figure 53] 1 shows the results of the PD1 reporter assay described in Example 5. [Figure 54] Schematic diagram of MDNA11. The IL-2 moiety contains mutations that enhance affinity for CD122 and inhibit binding to CD25, and the albumin moiety extends in vivo half-life and promotes tumor accumulation. [Figure 55]Body weights of BALB / c mice treated with MDNA11 at the SUD or fixed dose schedule. Each line corresponds to an individual mouse: SUD (Groups #1-#4) and fixed dose (Group #5). Arrows indicate MDNA11 administration at the indicated dose. [Figure 56] Body weights of MDNA11 mice treated with MDNA11 by subcutaneous injection. (A) Average body weight of the treatment group. Individual body weights of mice in each treatment group are shown in (B)–(F). Dotted vertical lines (on days 1 and 8 of the study) indicate administration of MDNA11. [Figure 57] Survival curves of mice treated with MDNA11 by subcutaneous injection. Kaplan-Meier plots show overall survival for each group. Mice surviving at the end of the study were censored on day 15. N=3 per group. [Figure 58] CD122 binding of MDNA11 and MDNA19. Sensorgrams of MDNA11 (top) and MDNA19 (bottom) showing binding to human CD122. Unlabeled (left) and Vivo Tag800-labeled constructs (right) show very similar binding profiles to CD122. [Figure 59] CD25 binding of MDNA11, MDNA19, and rhIL-2. Sensorgrams of MDNA11 (top), MDNA19 (center), and rhIL-2 binding to human CD25. Unlabeled (left) and VivoTag800-labeled (right) MDNA11 and MDNA19 show no binding to CD25. [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 panels, the rightmost mouse was a PBS-treated control. (B) Ex vivo IVIS imaging of CT26 tumors in mice treated with VivoTag800-MDNA19 (left) or VivoTag800-MDNA11 (right), harvested 144 hours post-administration (end of study). White arrows indicate tumors in MDNA19-treated mice. Black arrows indicate tumors in PBS-treated control mice. [Figure 61] The proposed mechanism of action of MDNA223 (anti-PD1-MDNA109FEAA) is that MDNA109FEAA binds to the intermediate affinity receptor (CD122) via cis-activation, selectively stimulating effector T cells, while anti-PD1 inhibits co-inhibitory PD1 on the same cells. [Figure 62] Survival curves for all groups indicated for subcutaneous MTD testing in BALB / c mice. [Figure 63] Animal weights at the stepwise MTD dosing of MDNA223. All animals were treated with 0.5 mg / kg in week 1 and 1 mg / kg in week 2. Animals were treated in weeks 3 and 4 as indicated in the figure legends. All animals were treated with 8 mg / kg in week 5. Data are presented as mean + SEM. [Figure 64] (a) Mean tumor measurements for groups 1-6 treated with the indicated treatments in the tumor growth inhibition study (IP treatment) in the B16F10 melanoma model. The downward black arrow indicates the dosing schedule (once weekly for 3 weeks). Data are presented as mean + SEM. (b) Tumor growth inhibition rates for the indicated groups on day 14 of the study, (c) survival curves for the indicated groups in the study, and (d) survival rates at the end of the study. [Figure 65](a) Mean tumor measurements for groups 1-5 treated as indicated in the tumor growth inhibition study (IP treatment) in the E0771 breast model. The downward black arrow indicates the dosing schedule (once weekly for 2 weeks). Data are presented as mean + SEM. (b) Tumor growth inhibition rates for the treatment groups on day 15 of the study, (c) survival curves for the treatment groups in the study, and (d) survival rates at the end of the study. [Figure 66] (a) Tumor measurements for all groups in the CT26 colon cancer tumor growth inhibition study in the CT26 colon model. Data are presented as mean SEM. For animals euthanized early or found dead, tumor volumes were measured to determine group means. (b) Tumor growth inhibition rates for the groups on day 15 of the study; (c) survival curves for the groups in the study; and (d) survival rates at the end of the study. [Figure 67] PK ELISA analysis: Time versus concentration data are presented on the y-axis as log10. Error bars represent the standard error of the mean of technical replicates. Zero values ​​were replaced with a value of 1.0 ng / mL to allow plotting on a logarithmic scale. (a) SQ route of administration, (b) IV route of administration, (c) IP route of administration. [Figure 68] Flow cytometry analysis of CD4, CD8, NK, and Treg proliferation marker Ki67: Samples were collected on the indicated days after treatment with MDNA223 (IV, IP, or SQ), and cells were isolated and stained for analytical markers. Data are presented as mean + SEM. [Figure 69] Flow cytometry analysis of absolute CD4, CD8, NK, and Treg counts as a ratio to CD45+ cells: Samples were taken on the indicated days after treatment with MDNA223 (IV, IP, or SQ), and cells were isolated and stained with analytical markers. Data are presented as mean + SEM. [Figure 70]Flow cytometry analysis for TIL analysis in B16F10 tumors. Tumors were harvested 7 days after the indicated dose and processed for flow cytometry. Data are presented as mean + SEM. (a) Percentage of intratumoral CD45 cells; (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; (e) Intratumoral Treg cells per gram of tumor. [Figure 71] Flow cytometry analysis for TIL analysis in B16F10 tumors. Tumors were harvested 7 days after the indicated dose and processed for flow cytometry. Data are presented as the mean + SEM of the ratio of CD8+ T cells to Treg cells. [Figure 72] Flow cytometry analysis of TILs in B16F10 tumors. Tumors were harvested 7 days after the indicated dose and processed for flow cytometry. Data are presented as the mean + SEM of (a) CD8+PD1+ T cell population, (b) CD8+Tim3-GrzB+ T cell population, and (c) CD8+Tim3+GrzB- T cell population. [Figure 73] (a) Mean tumor growth plot for groups indicated for the E0771 tumor growth inhibition study. Data are presented as mean + SEM. (b) Survival curves for indicated treatments. [Figure 74] Principle of the IL13RSPR assay using mouse and cynomolgus monkey receptors. [Figure 75] Figure 1 shows a plot of Fc-MDNA413(R39) plasma concentrations after each dose. Mean Fc-MDNA413 concentrations are presented on a logarithmic scale for each group and time point after dosing. Bars represent the standard error of the mean. Values ​​below the limit of quantitation were assigned a value of 1 ng / mL to allow plotting on a logarithmic scale. [Figure 76] Composite graph of Fc-MDNA413(R39) drug exposure. Mean Fc-MDNA413 concentrations are presented on a logarithmic scale for each group and time point. Bars represent the standard error of the mean. Values ​​below the limit of quantitation were assigned a value of 1 ng / mL to allow plotting on a logarithmic scale. [Figure 77] Body weight of CT26 colon carcinoma animals: Animals were treated as indicated and body weights were measured twice weekly throughout the study. Data are presented as mean and standard deviation. [Figure 78] CT26 colon cancer model. (a) Tumor growth curves for the vehicle and Fc-MDNA413 groups. Data points represent the mean for each group. Error bars indicate the standard error of each data point. For animals euthanized early or found dead, tumor volumes were measured to determine group means. Downward arrows indicate the day of administration. (b) Percent tumor growth inhibition on days 18 and 21 of the study. (c) Survival curves for the indicated groups. [Figure 79] Animal weight 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. [Figure 80] B16F10 melanoma model. (a) Tumor measurements for the treatment groups. Data points represent the mean for each group. Error bars indicate the standard error of each data point. For animals euthanized early or found dead, tumor volumes were measured to determine the group mean. (b) Percent tumor growth inhibition on study day 22 (Experiment #1). [Figure 81] B16F10 melanoma model: survival curves for the indicated groups (Experiment No. 1). The table shows the survival rate for each group at the end of the study. [Figure 82] B16F10 melanoma model (Experiment #2). Tumor measurements for the indicated groups. Data points represent the mean for each group. Error bars indicate the standard error of each data point. For animals euthanized early or found dead, tumor volumes were measured to determine group means. (a) Fc-MDNA413 in combination with MDNA19; (b) Fc-MDNA413 in combination with anti-PD1; (c) Percent tumor growth inhibition on day 15 of the study. [Figure 83]B16F10 melanoma model (Experiment No. 2). Figures show survival curves for animals treated in the study: (a) Fc-MDNA413 in combination with MDNA19, (b) Fc-MDNA413 in combination with anti-PD1, and (c) the table shows the survival rate for each group at the end of the study. [Figure 84] Principle of IL-13R SPR assay to examine binding affinity. [Figure 85] As shown, representative sensorgrams of various constructs showing binding affinity to human IL-13Rα1 and IL-13Rα2. [Figure 86] Flow cytometry data showing the efficiency of IL-13Rα2 transduction in EMT6 cells. A375 cells constitutively express IL-13Rα2 and were therefore used as a positive control for flow cytometry analysis. [Figure 87] Flow cytometry analysis of the expression of IL-13 decoy receptor in EMT6 and EMT6 / IL-13Rα2 tumors in BALB / c mice. [Figure 88] Receptor internalization data are MFI data plotted against time. Cells were treated with ligand as indicated and surface ligand binding was examined at different time points using FITC-conjugated anti-Fc antibody. [Figure 89] Principle of the IL13RSPR assay using the human IL-13 receptor. The left panel shows the principle used to test Fc fusions, while the right panel shows the principle used to test antibody fusions. [Figure 90] Assay principle: The active construct Fc-MDNA132.15 was captured directly (right panel) or via an anti-human IgG (Fc) antibody (left panel) on a CM5 chip. The analytes were either the human IL-13 receptor (left panel) or the human Fc receptor (right panel). [Figure 91] Representative sensorgrams of various constructs showing binding affinity to human IL13Rα1 and IL13Rα2. [Figure 92]Representative sensorgrams of various constructs showing binding affinity to human CD25 and CD122. [Figure 93] Representative sensorgrams of various constructs showing binding affinity to mouse PD1. [Figure 94] Representative sensorgrams show the binding affinity of active Fc-MDNA132.15 to human IL-13Rα1 (left) and human IL-13Rα2 (right). [Figure 95] Representative sensorgrams show the binding affinity of the active Fc-MDNA132.15 to human CD32b / c (left) and human CD16a (right). [Figure 96] A graph of hIL-13 dose response in the surrogate antagonist assay was generated. OD650nm was plotted as a function of hIL-13 concentration on a semi-logarithmic graph. The four-parameter logistic curve fit is presented as a solid line. Error bars represent the standard error of the mean of replicate wells. [Figure 97] 1 is a representative sensorgram of unlabeled and labeled Fc-MDNA132.15 showing binding affinity to mouse IL13Rα2. [Figure 98] In vivo imaging data are shown at the indicated time points. In all panels, the two rightmost mice were not treated with Fc-MDNA132.15 and served as controls. EMT6 and EMT6 / IL13Ra2 tumors are located in the left and right flanks, respectively. [Figure 99] In vivo imaging data are shown at the indicated time points. In all panels, the two rightmost mice were not treated with Fc-MDNA132.15 and served as controls. A549 and U87 tumors are located in the left and right flanks, respectively. [Figure 100]Fc-MDNA413 exhibits tumor growth similar to vehicle control, and MDNA19 exhibits modest tumor growth inhibition in the TRAMP-C1 prostate tumor model. However, the combination of Fc-MDNA413 and MDNA19 exhibits superior tumor growth inhibition compared to either agent alone. [Figure 101] Figure 1 shows dose-response graphs for A375 and U87 cells. Viability was normalized, with 0% defined as the minimum mean and 100% defined as the maximum mean for each data set. Viability was then plotted as a function of construct concentration (pM). The mean viability of positive control wells is represented by the dotted line on the bar graph. [Figure 102] Figure 1 shows a graph of the dose response in EMT6-IL13Rα2 and EMT6 wild-type cells. Viability was normalized, with 0% defined as the minimum mean and 100% defined as the maximum mean for each EMT6-IL13Rα2 data set. Viability was then plotted as a function of construct concentration (pM). For EMT6 wild-type cells, the mean viability at each concentration is plotted as a bar graph, with positive control wells presented as a dotted line. [Figure 103] Figure 1 shows dose-response graphs for A375 and U87 cells. Viability was normalized, with 0% defined as the minimum mean and 100% defined as the maximum mean for each data set. Viability was then plotted as a function of construct concentration (pM). The mean viability of positive control wells is represented by the dotted line on the bar graph. [Figure 104] Figure 1 shows a graph of the dose response in EMT6-IL13Rα2 and EMT6 wild-type cells. Viability was normalized, with 0% defined as the minimum mean and 100% defined as the maximum mean for each EMT6-IL13Rα2 data set. Viability was then plotted as a function of construct concentration (pM). For EMT6 wild-type cells, the mean viability at each concentration is plotted as a bar graph, with positive control wells presented as a dotted line. DETAILED DESCRIPTION OF THE INVENTION

[0097] In order that the present disclosure may be more readily understood, certain terms and phrases are defined below and throughout the specification.

[0098] definition All references cited herein are incorporated by reference in their entirety as if fully set forth. Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by those skilled 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) can provide those skilled in the art with a general guide to many of the terms used in this disclosure. Where appropriate, procedures involving the use of commercially available kits and reagents are generally performed according to manufacturer-defined protocols and / or parameters unless otherwise noted.

[0099] As used herein, "IL-2" refers to wild-type IL-2, whether native or recombinant. Mature human IL-2 occurs as a 133 amino acid sequence (excluding a signal peptide consisting of an additional 20 N-terminal amino acids) as described in Fujita, et al., PNAS USA, 80, 7437-7441 (1983). The amino acid sequence of human IL-2 (SEQ ID NO: 1, full length) can be found in GenBank under the accession locator NP_000577.2. The amino acid sequence of mature human IL-2 is shown in SEQ ID NO: 2 (human wild-type mature; numbering of substitution positions is based on this sequence). The amino acid sequence of mouse (Mus musculus) IL-2 can be found in GenBank under the accession locator SEQ ID NO: 3. The amino acid sequence of mature mouse IL-2 is shown in SEQ ID NO: 4. SEQ ID NO: 1 MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT SEQ ID NO: 2 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT SEQ ID NO: 3 MYSMQLASCVTLTLVLLVNSAPTSSSTSSSTAEAQQQQQQQQQQQHLEQLLMDLQELLSRMENYRNLKLPRMLTFKFYLPKQATELKDLQCLEDELGPLRHVLDLTQSKSFQLEDAENFISNIRVTVVKLKGSDNTFECQFDDESATVVDFLRRWIAFCQSIISTSPQ SEQ ID NO:4 APTSSSTSSSTAEAQQQQQQQQQQQHLEQLLMDLQELLSRMENYRNLKLPRMMLTFKFYLPKQATELKDLQCLEDELGPLRHVLDLTQSKSFQLEDAENFISNIRVTVVKLKGSDNTFECQFDDESATVVDFLRRWIAFCQSIISTSPQ

[0100] As used herein, "IL-2 mutein" refers to an IL-2 polypeptide in which a specific substitution has been made in the interleukin-2 protein. The IL-2 mutein is characterized by amino acid insertions, deletions, substitutions, and modifications at one or more sites in the native IL-2 polypeptide chain or at other residues in the native IL-2 polypeptide chain. According to the present disclosure, any of these insertions, deletions, substitutions, and modifications results in an IL-2 mutein that retains IL-2Rβ binding activity. Exemplary muteins can include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid substitutions.

[0101] Muteins also contain conservative modifications and substitutions at other positions in IL-2 (i.e., positions that have minimal effect on the secondary or tertiary structure of the mutein). Such conservative substitutions include those described by Dayhoff in *The Atlas of Protein Sequence and Structure* 5 (1978) and by Argos in *EMBO J., 8:779-785 (1989). For example, amino acids belonging to one of the following groups are conservatively altered: Group I (ala, pro, gly, gln, asn, ser, thr), Group II (cys, ser, tyr, thr), Group III (val, ile, leu, met, ala, phe), Group IV (lys, arg, his), Group V (phe, tyr, trp, his), and Group VI (asp, glu).

[0102] "Numbered relative to IL-2" means identifying a given amino acid by reference to the position where that amino acid is normally found in the mature sequence of wild-type IL-2, e.g., R81 refers to arginine, the 81st amino acid found in SEQ ID NO:2; L80 refers to leucine, the 80th amino acid found in SEQ ID NO:2; L85 refers to leucine, the 85th amino acid found in SEQ ID NO:2; I86 refers to isoleucine, the 86th amino acid found in SEQ ID NO:2; I92 refers to isoleucine, the 92nd amino acid found in SEQ ID NO:2; F42 refers to phenylalanine, the 42nd amino acid found in SEQ ID NO:2; and K43 refers to lysine, the 43rd amino acid found in SEQ ID NO:2.

[0103] As used herein, the abbreviations for the genetically encoded L-enantiomer amino acids used in the methods of the disclosure are conventional and are as shown in Table 1 below. TIFF2025509333000001.tif173170

[0104] "Hydrophilic amino acid" refers to an amino acid that has a hydrophobicity of less than 0 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).

[0105] The term "cell type having IL-2Rαβγ receptor" refers to cells known to have this type of receptor, i.e., T cells, activated T cells, B cells, activated monocytes, and activated NK cells. The term "cell type having IL-2Rαβγ receptor" refers to cells known to have that type of receptor, i.e., B cells, resting monocytes, and resting NK cells.

[0106] The term "identity," as used herein in reference to polypeptide or DNA sequences, refers to the identity of subunit sequences between two molecules. When a subunit position in both molecules is occupied by the same monomeric subunit (i.e., the same amino acid residue or the same nucleotide), the molecules are identical at that position. The similarity between two amino acid sequences or two nucleotide sequences is a linear function of the number of identical positions. Generally, sequences are aligned to maximize correspondence. Where necessary, identity can be calculated using published techniques and widely available computer programs such as the GCS program package (Devereux et al., Nucleic Acids Res. 12:387, 1984), BLASTP, BLASTN, and 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 from the Genetics Computer Group at the University of Wisconsin Biotechnology Center (1710 University Avenue, Madison, Wis. 53705) using its default parameters.

[0107] The terms "polypeptide," "protein," or "peptide" refer to any chain of amino acid residues, regardless of its length or post-translational modification (eg, glycosylation or phosphorylation).

[0108] When a variant IL-2 polypeptide of the disclosure is "substantially pure," the polypeptide can be at least about 60% by weight (dry weight) of the polypeptide of interest, e.g., a polypeptide comprising the amino acid sequence of the variant IL-2. For example, the polypeptide can be at least about 75%, 80%, 85%, 90%, 95%, or 99% by weight of the polypeptide of interest. Purity can be measured by any appropriate standard method, such as column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

[0109] An "agonist" is a compound that interacts with a target to increase or promote increased activation of that target.

[0110] A "partial agonist" is a compound that interacts with the same target as an agonist, but where increasing doses of the partial agonist do not produce as great a biochemical and / or physiological effect as an agonist.

[0111] "Superagonists" (also called "superkines") are a class of agonists that can produce a maximal response that is greater than the endogenous agonist for the target receptor, i.e., greater than 100% effective.

[0112] "Operably linked" is intended to mean that the nucleotide sequence of interest (i.e., the sequence encoding the IL-2 mutein) is linked to a regulatory sequence(s) in a manner that allows for expression of the nucleotide sequence of interest (e.g., in an in vitro transcription / translation system or within 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, e.g., Goeddel (1990) in Gene Expression Technology: Methods in Enzymology 185 (Academic Press, San Diego, Calif.). Regulatory sequences include sequences that direct constitutive expression of a nucleotide sequence in many types of host cell and sequences that direct expression of a nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be apparent to those skilled in the art that the design of the expression vector can depend on factors such as the choice of host cell to be transformed, the level of expression of the desired protein, etc. The expression constructs of the present invention can be introduced into host cells to thereby produce the human IL-2 muteins disclosed herein, or to produce biologically active variants thereof.

[0113] The terms "host cell" and "recombinant host cell" are used interchangeably herein. It is 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 successive generations, either due to mutation 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.

[0114] As used herein, the terms "transformation" and "transfection" refer to various art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into host cells, including calcium phosphate co-precipitation, calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, particle gun or electroporation.

[0115] 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., that are compatible with pharmaceutical administration. Supplementary active compounds (e.g., antibiotics) can also be incorporated into the compositions of the present invention.

[0116] 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.

[0117] As used herein, the terms "cancer" (or "cancerous"), "hyperproliferative," "tumor," and / or "neoplasia" refer to cells capable of autonomous growth (i.e., an abnormal state or condition characterized by rapidly proliferating cell growth). Hyperproliferative and neoplastic disease states can be classified as pathological (i.e., characterizing or constituting a disease state) or non-pathological (i.e., deviating from normal but not associated with a disease state). These terms are intended to include any type of cancerous growth, oncogenic process, metastatic tissue, or malignantly transformed cell, tissue, or organ, regardless of invasive histopathological type or stage. "Pathological hyperproliferative" cells are found in disease states characterized by malignant tumor growth. An example of non-pathological hyperproliferative cells is 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 lung, breast, thyroid, lymph glands and lymphatic tissue, reproductive system, gastrointestinal tract, and genitourinary tract, and adenocarcinomas, which are generally considered to include malignancies such as most colon cancers, renal cell carcinoma, prostate cancer and / or testicular cancer, non-small cell carcinoma of the lung, cancer of the small intestine, and cancer of the esophagus. Cancer may generally 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, lymphatic cancer, gastrointestinal cancer, prostate cancer, ovarian cancer, breast cancer, basal cell breast tumors, endometrial cancer, multiple myeloma, melanoma, lymphoma, lung cancer (including small cell lung cancer), kidney cancer, stomach cancer, brain tumors, and central nervous system tumors. Central nervous system tumors include glioma, glioblastoma, glioblastoma multiforme (GBM), refractory glioblastoma multiforme (rGBM), recurrent glioblastoma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, meningioma, neuroblastoma, retinoblastoma, medulloblastoma, adult pituitary adenoma, O6-methylguanine methyltransferase (MGMT) positive and negative central nervous system tumors, and furin positive central nervous system tumors.

[0118] The term "carcinoma" is art-recognized and refers to malignant tumors of epithelial or endocrine tissue, including respiratory, gastrointestinal, genitourinary, testicular, breast, prostate, endocrine, and melanoma. "Adenocarcinoma" refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures.

[0119] As used herein, the term "hematopoietic neoplastic disorder" refers to a disease involving hyperplastic / neoplastic cells of hematopoietic origin, e.g., a disease arising from the myeloid, lymphoid, or erythroid lineages, or their precursor cells. Preferably, the disease arises from a poorly differentiated acute leukemia (e.g., erythroblastic leukemia and acute megakaryoblastic leukemia). Additional 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 include, but are not limited to, acute lymphoblastic leukemia (ALL) (including B-lineage ALL and T-lineage ALL), chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), hairy cell leukemia (HLL), and Waldenstrom's macroglobulinemia (WM). Additional forms of malignant lymphoma include, but are not limited to, non-Hodgkin's lymphoma and its subtypes, peripheral T-cell lymphoma, adult T-cell leukemia / lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), large granular lymphocytic leukemia (LGF), Hodgkin's disease, and Reed-Sternberg disease.

[0120] As used herein, the terms "treatment," "treating," and the like refer to obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, 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 adverse effects caused by the disease. "Treatment," as used herein, encompasses any treatment of a disease in a mammal, particularly a human, and includes (a) preventing the disease from occurring in a subject predisposed to or at risk of the disease but not yet diagnosed with the disease; (b) inhibiting the disease, i.e., preventing its development; and (c) alleviating the disease, i.e., reversing the disease. A therapeutically effective amount can be an amount that reduces tumor number, tumor size, and / or increases survival rates.

[0121] 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 apes and humans), sport mammals (e.g., horses), livestock mammals (e.g., sheep, goats, etc.), pet mammals (dogs, cats, etc.), and rodents (e.g., mice, rats, etc.).

[0122] The terms "pharmaceutically acceptable" and "physiologically acceptable" refer to a biologically acceptable formulation, gas, liquid, or solid, or mixture thereof, suitable for one or more routes of administration, in vivo delivery, or contact. A "pharmaceutically acceptable" or "physiologically acceptable" composition is a substance that is not biologically or otherwise undesirable; e.g., the substance can be administered to a subject without causing significant undesirable biological effects. That is, such a pharmaceutical composition can be used, for example, when administering an IL-2 mutein to a subject. In particular, an IL-2 mutein containing the following substitutions is administered to a subject with cancer in combination with an anti-PD-1 antibody. In some embodiments, the administered IL-2 mutein further comprises a substitution at position F42A. In some embodiments, the administered IL-2 mutein further comprises a substitution at position K43N.

[0123] The phrase "unit dosage form," as used herein, refers to physically discrete units suitable for unitary administration to a subject to be treated, each unit containing a predetermined amount, optionally with a pharmaceutical carrier (excipient, diluent, vehicle, or filler), that, 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 tumor number. In some embodiments, the therapeutic effect is a reduction in tumor size. In some embodiments, the therapeutic effect is an increase in survival rate.

[0124] In some embodiments, unit dosage forms include liquid compositions or freeze-dried or lyophilized compositions, and may be contained, for example, in ampoules and vials, to which, for example, a sterile liquid carrier can be added prior to in vivo administration or delivery. Individual unit dosage forms may be included in multi-dose kits or containers. The IL-2 muteins in combination with anti-PD-1 antibodies, and pharmaceutical compositions thereof, may be packaged in single or multiple unit dosage forms for ease of administration and uniformity of dosage.

[0125] A "therapeutically effective amount" will fall in a relatively broad range that can be determined through experimentation and / or clinical trials. For example, in vivo injection, e.g., direct injection into the subject's tissue or vascular system (e.g., liver tissue or vein). Other effective dosages can be readily determined by one of ordinary skill in the art through routine testing to determine dose-response curves.

[0126] An "effective amount" or "sufficient amount" refers to an amount, in one or more doses, that alone or in combination with one or more other compositions (therapeutic agents such as drugs), treatments, protocols or therapeutic regimens (including, for example, vaccine regimens), results in a detectable response of any duration (long or short term), an expected or desired outcome or benefit in a subject, to any measurable or detectable degree, or of any duration (e.g., minutes, hours, days, months or years, or a cure), of any time period (long or short term).

[0127] An "effective amount" or "sufficient amount" for treatment (e.g., treatment to ameliorate or produce a therapeutic effect or improvement) is typically effective to measurably produce a response to one, more than one, or all of the adverse symptoms, outcomes, or complications of the disease, e.g., one or more adverse symptoms, disorders, illnesses, pathologies, or complications caused by or associated with the disease. However, decreasing, reducing, inhibiting, suppressing, limiting, or controlling the progression or worsening of the disease is also a sufficient result. 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 tumors. In some embodiments, an effective amount is an amount sufficient to increase survival rates.

[0128] "Prevention" and grammatical variations thereof refer to methods in which contact, administration, or in vivo delivery to a subject occurs prior to the onset of disease. Administration or in vivo delivery to a subject can occur prior to the onset 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 described methods and uses, even if the disease is not yet apparent in the subject. Thus, such subjects include those who screen positive for producing an insufficient or missing functional gene product (protein), or an abnormal, partially functional, or non-functional gene product (protein) that leads to disease, even if the disease symptoms are not yet apparent in the subject, and those who screen positive for an abnormal or defective (mutant) gene product (protein) that leads to disease.

[0129] I. Detailed Description Described herein are bispecific IL-2 cytokine fusions (also referred to herein as bifunctional molecules) comprising an IL-2 mutein fused to a second cytokine. Described herein are IL-2 muteins comprising the substitutions L80F, R81D, L85V, I86V, and I92F that have improved binding to the IL-2Rβ receptor and can be included in the bispecific IL-2 cytokine fusions. Described herein are also IL-2 muteins for use in monotherapy and in combination with anti-PD-1 antibodies. In some embodiments, the IL-2 mutein comprising L80F, R81D, L85V, I86V, and I92F (numbered relative to wild-type human IL-2 (SEQ ID NO: 2, wild-type hIL-2)) is designated H9. Such IL-2 muteins find use in combination with anti-PD-1 antibodies, for example, for the treatment of cancer. Also provided are nucleic acids encoding such IL-2 muteins, methods for making such IL-2 muteins, pharmaceutical compositions comprising such IL-2 muteins, and therapeutic methods using such IL-2 muteins.

[0130] A. IL-2 Muteins for Use in Bispecific IL-2 Cytokine Fusions The substituted amino acid residue(s) can be (but are not necessarily) conservative substitutions, which typically include substitutions within the following groups: glycine, alanine, valine, isoleucine, leucine, aspartic acid, glutamic acid, asparagine, glutamine, serine, threonine, lysine, arginine, and phenylalanine, tyrosine. These mutations can be at amino acid residues that interface with IL-2Rβ and / or IL-2Rγ.

[0131] More specifically, mutations (either conservative or non-conservative substitutions by addition(s) or deletion(s)) 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. Exemplary IL-2 mutein sequences are SEQ ID NO:5 for 5-1, SEQ ID NO:6 for 5-2, SEQ ID NO:7 for 6-6, SEQ ID NO:8 for A2, SEQ ID NO:9 for B1, SEQ ID NO:10 for B11, SEQ ID NO:11 for C5, SEQ ID NO:12 for D10, SEQ ID NO:13 for E10, SEQ ID NO:14 for G8, SEQ ID NO:15 for H4, and SEQ ID NO:16 for H9.

[0132] In some embodiments, the substitutions in the IL-2 mutein include L80F, R81D, L85V, I86V, and I92F (numbered relative to wild-type human IL-2 of SEQ ID NO:2). In some embodiments, the IL-2 mutein further includes the substitution F42A, where the numbering is relative to wild-type human IL-2 of SEQ ID NO:2. In some embodiments, the IL-2 mutein further includes the substitution Y45A, where the numbering is relative to wild-type human IL-2 of SEQ ID NO:2. In some embodiments, the IL-2 mutein further includes the substitution E62A, where the numbering is relative 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 (numbered relative to wild-type human IL-2 of SEQ ID NO:2). In some embodiments, substitutions in an IL-2 mutein include F42A, Y45A, L80F, R81D, L85V, I86V, and I92F (numbered relative to wild-type human IL-2 of SEQ ID NO: 2). In some embodiments, substitutions in an IL-2 mutein include F42A, E62A, L80F, R81D, L85V, I86V, and I92F (numbered relative to wild-type human IL-2 of SEQ ID NO: 2). In some embodiments, substitutions in an IL-2 mutein include F42A, Y45A, E62A, L80F, R81D, L85V, I86V, and I92F (numbered relative to wild-type human IL-2 of SEQ ID NO: 2). In some embodiments, substitutions in an IL-2 mutein include E62A, L80F, R81D, L85V, I86V, and I92F (numbered relative to wild-type human IL-2 of SEQ ID NO: 2). In some embodiments, substitutions in an IL-2 mutein include Y45A, E62A, L80F, R81D, L85V, I86V, and I92F (numbered relative to wild-type human IL-2 of SEQ ID NO: 2). In some embodiments, substitutions in an IL-2 mutein include Y45A and E62A (numbered relative to wild-type human IL-2 of SEQ ID NO: 2).

[0133] In some embodiments, substitutions in an IL-2 mutein that increase and / or enhance binding to IL-2Rβ include L80F, R81D, L85V, I86V, and I92F (numbered relative to wild-type human IL-2 of SEQ ID NO: 2). In some embodiments, an IL-2 mutein for use in the invention comprises L80F, R81D, L85V, I86V, and I92F and has increased binding to IL-2Rβ. In some embodiments, an IL-2 mutein for use in the invention further comprises a substitution at position F42A. In some embodiments, an IL-2 mutein for use in the invention further comprises a substitution at position K43N. In some embodiments, the mutein comprises the substitutions L80F, R81D, L85V, I86V, and I92F, as well as one or more substitutions selected from the group consisting of F42A, Y45A, and E62A (all relative to wild-type human IL-2 (SEQ ID NO: 2)).

[0134] 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.

[0135] In some embodiments, the IL-2 muteins of the invention, which have increased binding affinity to IL-2Rβ compared to wild-type human IL-2, comprise the amino acid substitutions L80F, R81D, L85V, I86V, and I92F. [ka]

[0136] In some embodiments, the IL-2 mutein is an IL-2Rβ / IL-2Rγ cIn some embodiments, the IL-2 muteins of the invention have an enhanced ability to stimulate one or more signal transduction pathways that depend on heterodimerization of STAT5. In some embodiments, the IL-2 muteins of the invention have an enhanced ability to stimulate STAT5 phosphorylation in IL-2Rβ+ cells compared to wild-type human IL-2. In some embodiments, the IL-2 muteins stimulate 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%, or 95% or greater than the level at which wild-type IL-2 stimulates STAT5 phosphorylation in IL-2Rβ+ cells. In some embodiments, the IL-2 mutein stimulates 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%, or 195% or greater compared to the level at which wild-type IL-2 stimulates STAT5 phosphorylation in IL-2Rβ+ cells. In some embodiments, the IL-2Rβ+ cells are T cells. In certain embodiments, the T cells are CD8+ T cells. In some embodiments, the CD8+ T cells are freshly isolated CD8+ T cells. In another embodiment, the CD8+ T cells are activated CD8+ T cells. In another embodiment, the IL-2Rβ+ cells are natural killer (NK) cells. In some embodiments, the IL-2 mutein comprises the following substitutions relative to wild-type human IL-2 (SEQ ID NO: 2): L80F, R81D, L85V, I86V, and I92F.

[0137] In some embodiments, the mutein has an enhanced ability to stimulate ERK1 / ERK2 signaling in IL-2Rβ+ cells compared to wild-type human IL-2. In some embodiments, the IL-2 mutein 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%, or 95% or greater of the level at which wild-type IL-2 stimulates pERK1 / ERK2 signaling in IL-2Rβ+ cells. In some embodiments, the IL-2 mutein stimulates phosphorylation of pERK1 / ERK2 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%, or 195% or greater compared to the level at which wild-type IL-2 stimulates phosphorylation of pERK1 / ERK2 in IL-2Rβ+ cells. In some embodiments, the IL-2Rβ+ cells are T cells. In certain embodiments, the T cells are CD8+ T cells. In some embodiments, the CD8+ T cells are freshly isolated CD8+ T cells. In another embodiment, the CD8+ T cells are activated CD8+ T cells. In another embodiment, the IL-2Rβ+ cells are natural killer (NK) cells. In some embodiments, the IL-2 mutein contains the following substitutions relative to wild-type human IL-2 (SEQ ID NO: 2): L80F, R81D, L85V, I86V, and I92F.

[0138] 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, phosphorylation of STAT5 and ERK1 / 2 can be measured using antibodies specific for the phosphorylated forms of these molecules in combination with flow cytometry analysis as described herein. In some embodiments, the mutein has an enhanced ability to stimulate PI3 kinase signaling in IL-2Rβ+ cells compared to wild-type human IL-2. In some embodiments, the IL-2 mutein stimulates PI3 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% or 95% or less of the level at which wild-type IL-2 stimulates PI3 kinase signaling in IL-2Rβ+ cells. In some embodiments, the IL-2 mutein stimulates PI3 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%, or 195% or greater compared to the level at which wild-type IL-2 stimulates phosphorylation of PI3 kinase signaling in IL-2Rβ+ cells. In some embodiments, the IL-2Rβ+ cells are T cells. In certain embodiments, the T cells are CD8+ T cells. In some embodiments, the CD8+ T cells are activated CD8+ T cells. In another embodiment, the IL-2Rβ+ cells are natural killer (NK) cells. In some embodiments, the IL-2 mutein contains the following substitutions compared to wild-type human IL-2 (SEQ ID NO: 2): L80F, R81D, L85V, I86V, and I92F. PI3 kinase signaling can be measured using any suitable method known in the art. For example, PI3 kinase signaling can be measured using an antibody specific for phosphorylated S6 ribosomal protein in conjunction with flow cytometry analysis as described herein.

[0139] In some embodiments, the IL-2 mutein is a stimulator of IL-2STAT5 and / or IL-15STAT5 phosphorylation in CD8+ T cells. In some embodiments, the mutein is a promoter of IL-2-induced and / or IL-15-induced proliferation of CD8+ T cells. In some embodiments, the mutein is a stimulator of IL-2-dependent, TCR-induced cell proliferation. In some embodiments, the IL-2 mutein contains the following substitutions compared to wild-type human IL-2 (SEQ ID NO: 2): L80F, R81D, L85V, I86V, and I92F.

[0140] IL-2 promotes the differentiation of Th1, Th9, and Treg T cells and inhibits Th17 differentiation. Thus, without being bound by any particular theory of action, it is believed that IL-2 muteins that function as IL-2 superagonists can promote the differentiation of Th1, Th9, and / or Treg cells or inhibit the differentiation of Th17 cells. In some embodiments, the IL-2 mutein is a promoter of IL-2-dependent Th1, Th9, and / or Treg differentiation. In some embodiments, the mutein is an inhibitor of Th17 differentiation. In some embodiments, the IL-2 mutein contains the following substitutions compared to wild-type human IL-2 (SEQ ID NO: 2): L80F, R81D, L85V, I86V, and I92F.

[0141] In some embodiments, the IL-2 mutein has reduced signaling to CD25 and / or signals independently of CD25 (e.g., reduced or lost binding to CD25) compared to wild-type human IL-2. In some embodiments, reduced signaling and / or independence of signaling with respect to CD25 allows preferential activation of effector T cells while limiting stimulation of Tregs. In some embodiments, reduced signaling and / or independence of signaling with respect to CD25 allows reduced toxicity. In some embodiments, the mutein comprises one or more substitutions selected from the group consisting of L80F, R81D, L85V, I86V, and I92F, and F42A, Y45A, and E62A, all relative to wild-type human IL-2 (SEQ ID NO: 2).

[0142] In some embodiments, the IL-2 mutein is capable of increasing and / or restoring responsiveness to anergic NK cells. In some embodiments, the IL-2 mutein is capable of increasing and / or restoring responsiveness to anergic NK cells in a tumor microenvironment. In some embodiments, the IL-2 mutein comprises the following substitutions relative to wild-type human IL-2 (SEQ ID NO: 2): L80F, R81D, L85V, I86V, and I92F.

[0143] In some embodiments, the mutein is an inhibitor of IL-2-dependent natural killer (NK) cell activation. IL-2-induced NK cell activation can be measured by any suitable method known in the art, for example, by measuring IL-2-induced CD69 expression and / or cytotoxicity, as described herein.

[0144] In some embodiments, increased binding affinity to IL-2Rβ refers to any binding affinity to IL-2Rβ that is greater than the binding affinity of wild-type human IL-2 to IL-2Rβ, ie, the binding affinity is increased by 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, or 240-fold or more compared to the binding affinity of wild-type human IL-2 to IL-2Rβ.

[0145] In some embodiments, increased binding affinity to IL-2Rβ means that the binding affinity to IL-2Rβ is greater than the binding affinity of wild-type human IL-2 to IL-2Rβ, and in some embodiments, the binding affinity is increased by 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, or 240-fold or more compared to the binding affinity of wild-type human IL-2 to IL-2Rβ.

[0146] In some embodiments, an IL-2 mutein of the invention that has increased binding affinity to IL-2Rβ compared to wild-type human IL-2 also has reduced binding to CD25 and comprises the amino acid substitutions F42A, L80F, R81D, L85V, I86V, and I92F. In some embodiments, the reduction in binding affinity is about 220-fold, i.e., the Kd is reduced from about 6.6 nM for wild-type human IL-2 to about 1.4 μM for the mutein comprising F42A, L80F, R81D, L85V, I86V, and I92F. In some embodiments, the IL-2 mutein has the amino acid sequence: [ka]

[0147] In some embodiments, the IL-2 muteins of the invention that have increased binding affinity to IL-2Rβ compared to wild-type human IL-2 also have reduced binding to CD25 and contain the following amino acid substitutions: K43N, L80F, R81D, L85V, I86V, and I92F. In some embodiments, the reduced binding affinity is due to glycosylation at position 43 and the K43N substitution. In IL-2 muteins containing the K43N, L80F, R81D, L85V, I86V, and I92F amino acid substitutions, binding to CD25 is reduced and / or abolished by substituting asparagine for lysine (K43N). In some embodiments, the IL-2 mutein has the following amino acid sequence: [ka]

[0148] In some embodiments, reduced binding affinity to CD25 means that any binding affinity to CD25 is lower than the binding affinity of wild-type human IL-2, ie, the binding affinity to CD25 is 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 less than the binding affinity of wild-type human IL-2 to CD25.

[0149] In some embodiments, an IL-2 mutein of the invention, which has increased binding affinity to IL-2Rβ and reduced binding affinity to CD25 compared to wild-type human IL-2, comprises the following amino acid substitutions: F42A, Y45AL80F, R81D, L85V, I86V, and I92F. In some embodiments, the IL-2 mutein has the following amino acid sequence: [ka]

[0150] In some embodiments, an IL-2 mutein of the invention, which has increased binding affinity to IL-2Rβ and reduced binding affinity to CD25 compared to wild-type human IL-2, comprises the following amino acid substitutions: F42A, E62A, L80F, R81D, L85V, I86V, and I92F. In some embodiments, the IL-2 mutein has the following amino acid sequence: [ka]

[0151] In some embodiments, an IL-2 mutein of the invention, which has increased binding affinity to IL-2Rβ and reduced binding affinity to CD25 compared to wild-type human IL-2, comprises the following 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: [ka]

[0152] In some embodiments, the sequence of the IL-2 mutein is 90% identical to any one of SEQ ID NO:2, SEQ ID NO:6-10, or SEQ ID NO:16. In some embodiments, the sequence of the IL-2 mutein is 95% identical to any one of SEQ ID NO:2 or SEQ ID NO:6-10. In some embodiments, the sequence of the IL-2 mutein is 98% identical to any one of SEQ ID NO:2 or SEQ ID NO:6-10. In some embodiments, the sequence of the IL-2 mutein is 99% identical to any one of SEQ ID NO:2 or SEQ ID NO:6-10.

[0153] Further exemplary IL-2 sequences are shown in the table below. TIFF2025509333000008.tif232170TIFF2025509333000009.tif227170TIFF2025509333000010.tif180170

[0154] B. IL-2 Mutein Fusion Protein IL-2 muteins can be prepared as fusion polypeptides, i.e., chimeric polypeptides (including, for example, bispecific IL-2 cytokine fusions), comprising an IL-2 mutein of the invention and a heterologous polypeptide (i.e., a polypeptide that is not IL-2 or a variant thereof) (see, e.g., U.S. Pat. No. 6,451,308). Exemplary heterologous polypeptides can extend the circulating half-life of the chimeric polypeptide in vivo, thereby further enhancing the properties of the mutant IL-2 polypeptides of the invention. In various embodiments, the polypeptide that extends circulating half-life can be serum albumin (e.g., human serum albumin), PEG, a PEG derivative, or the Fc region of an IgG subclass antibody (lacking the heavy chain variable region of IgG). Exemplary Fc regions may contain mutations that inhibit complement fixation and Fc receptor binding, or may be lytic, i.e., capable of lysing cells by either binding complement or another mechanism such as antibody-dependent complement lysis (ADCC, U.S. Patent No. 08 / 355,502, filed December 12, 1994).

[0155] The "Fc region" can be a naturally occurring polypeptide or a synthetic polypeptide homologous to the IgG C-terminal domain produced by digesting IgG with papain. IgGFc has a molecular weight of approximately 50 kDa. A mutant IL-2 polypeptide can comprise the entire Fc region, or a smaller Fc region portion that retains the ability to extend the circulating half-life of a chimeric polypeptide of which it is a part. In addition, the full-length Fc region or a fragmented Fc region can 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) comprises an IgG1, IgG2, IgG3, or IgG4 Fc region (see, e.g., the sequences in Figures 2A-2B). In some embodiments, the Fc region comprises the following substitution: N297A.

[0156] In some embodiments, the IL-2 mutein is linked directly or indirectly to a heterologous fusion polypeptide.

[0157] In some embodiments, the IL-2 mutein is linked directly to the Fc region. In some embodiments, the IL-2 mutein is linked to the Fc region via a linker peptide, such as GGGGS. In some embodiments, the linker is (GGGGS)n, where 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 GGGSGGGGSGGGGGS (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 comprises one or more protease cleavage sites (e.g., is a protease-cleavable linker). The linker may further comprise one or more protease cleavage sites or may be susceptible to cleavage by oxidation and / or reduction. In one example, a peptide linker may be used that is susceptible to cleavage by enzymes of the complement system, urokinase, tissue plasminogen activator, trypsin, plasmin, caspase, kallikrein, cathepsin, legumain, MMP, thrombin, urokinase-type plasminogen activator (uPA), matriptase, or another enzyme with proteolytic activity. According to another example, the linker may include a disulfide bond (e.g., a disulfide bond at a cysteine ​​molecule).By way of another example, the linker may comprise a protease-cleavable Val-Cit (VC) linker, Phe-Arg linker, Val-Lys linker, Val-Ala linker, Val-Arg linker, Val-Leu-Lys linker, Gly-Phe-Leu-Gly linker, Ala-Phe-Lys linker, pol-L-lysine linker, β-Ala-Leu-Ala-Leu linker, Arg-Arg-Ala-Leu-Ala-Leu linker, peptidomimetic linker, legumain-cleavable Ala-Ala-Asn tripeptide linker, peptide linkers cleaved by cathepsin B and other lysosomal proteases (such as Gly-Phe-Leu-Gly and Ala-Leu-Ala-Leu), caspase 3 DEVD sequence, or a self-immolative linker. For example, the linkers disclosed in Poreba, M, FEBS J. 287(10):1936-1969 (2020) (incorporated herein by reference) are contemplated by the present disclosure. Many tumors naturally release high levels of glutathione (a reducing agent), which can reduce disulfide bonds and subsequently release the cargo moiety at the delivery site. In some embodiments, the linker is a protease-cleavable linker that can be cleaved by matrix metalloproteinases (MMPs). MMPs are overexpressed in situ in tumors, and cleavable linkers in this context are contemplated by the present disclosure. For example, the linkers disclosed in Hsu, EJ, et al., Nat. Commun. 12(2768):1-13 (2021) (incorporated herein by reference) are contemplated by the present disclosure. 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.

[0158] Fc regions can be "lytic" or "nonlytic," but are typically nonlytic. Nonlytic 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 IgGFc contains a Leu residue at position 235 of IgGFc. That is, the Fc receptor binding site can be disrupted by mutating or deleting Leu235. For example, substituting Leu235 with Glu inhibits the ability of the Fc region to bind to the high-affinity Fc receptor. The function of the mouse C'1q binding site can be disrupted by mutating or deleting residues Glu318, Lys320, and Lys322 of IgG. For example, substituting Glu318, Lys320, and Lys322 with Ala residues prevents IgG1Fc from inducing antibody-dependent complement lysis. In contrast, the soluble IgGFc region has a high-affinity Fc receptor binding site and a C'1q binding site. The high-affinity Fc receptor binding site contains a Leu residue at position 235 of IgGFc, and the C'1q binding site contains residues Glu318, Lys320, and Lys322 of IgG1. Soluble IgGFc contains wild-type residues or conservative amino acid substitutions at these sites. Soluble IgGFc can target cells for antibody-dependent cellular cytotoxicity or complement-induced cytolysis (CDC). Mutations suitable for human IgG are also known (see, e.g., Morrison et al., The Immunologist 2:119-124, 1994 and Brekke et al., The Immunologist 2:125, 1994).

[0159] In another embodiment, a chimeric polypeptide can comprise an IL-2 mutein of the present invention and a polypeptide that functions as an antigenic tag, such as a FLAG sequence. The FLAG sequence is recognized by a highly specific biotinylated anti-FLAG antibody, as described herein (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.

[0160] In another embodiment, the chimeric polypeptide comprises a mutant IL-2 polypeptide and a heterologous polypeptide (such as an agglutinin subunit called Aga2p) that functions to enhance expression of the mutant IL-2 polypeptide or to induce cellular localization of the mutant IL-2 polypeptide (see, e.g., Boder and Wittrup, Nature Biotechnol. 15:553-7, 1997).

[0161] In another embodiment, a chimeric polypeptide can be produced comprising a mutant IL-2 and an antibody or its antigen-binding portion. The antibody or antigen-binding portion of the chimeric protein can function as a targeting moiety. For example, the antibody or antigen-binding portion 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.

[0162] In some embodiments, the chimeric polypeptides comprise a fusion to an antibody or antigen-binding portion thereof that inhibits the interaction of the PD-1 receptor with its ligand PD-L1 and / or an antibody to a component of the PD-1 / PD-L1 signaling pathway. Antibodies known in the art that bind to PD-1 and inhibit the interaction of PD-1 with 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 may find use in the present invention include, for example, 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 (cemiplimab, Regeneron), human monoclonal antibody MDX-1106 (Bristol-Myers Squibb), and / or human monoclonal antibody PD-1107 (Bristol-Myers Squibb). Suitable antibodies include, but are not limited to, PD-L1 (Squibb), and / or the humanized anti-PD-1 IgG4 antibody PDR001 (Novartis). In some embodiments, the PD-1 antibody is obtained from clone: ​​RMP1-14 (rat IgG) (BioXcell catalog number BP0146). Other suitable antibodies include the anti-PD-1 antibodies disclosed in U.S. Pat. No. 8,008,449, incorporated herein by reference. In some embodiments, the antibody or antigen-binding portion thereof specifically binds to PD-L1 and inhibits the interaction between PD-L1 and PD-1, thereby enhancing immune activity.Any antibody known in the art that binds to PD-L1, inhibits the interaction between PD-1 and PD-L1, and stimulates an anti-tumor immune response is suitable for use in the chimeric polypeptides disclosed herein. For example, antibodies that target PD-L1 and are in clinical trials include BMS-936559 (Bristol-Myers Squibb) and MPDL3280A (Genetech). Other suitable antibodies that target PD-L1 are disclosed in U.S. Patent No. 7,943,743, which is incorporated herein by reference. One of skill in the art will appreciate that any antibody that binds to PD-1 or PD-L1, inhibits 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 to an anti-PD-1 antibody. In some embodiments, the chimeric polypeptide comprises a fusion to an anti-PD-L1 antibody.

[0163] In some embodiments, the chimeric polypeptide comprises a fusion to an antibody or antigen-binding portion thereof that targets CTLA-4 and inhibits the interaction of CTLA-4 with CD80 and CD86. Exemplary antibodies that target CTLA-4 include ipilimumab (MDX-010, MDX-101, Bristol-Myers Squibb), which has been approved by the FDA, and tremelimumab (ticilimumab, CP-675, 206, Pfizer), which is currently undergoing human trials. Other suitable antibodies that target CTLA-4 are disclosed in WO2012 / 120125, U.S. Patent Nos. 6,984,720 and 6,682,7368, and U.S. Patent Application Publication Nos. 2002 / 0039581, 2002 / 0086014, and 2005 / 0201994, which are incorporated herein by reference. Those skilled in the art will appreciate that any antibody that binds to CTLA-4 and inhibits the interaction of CTLA-4 with CD80 and CD86 while stimulating an anti-tumor immune response is suitable for use in the chimeric polypeptides disclosed herein. In some embodiments, the chimeric polypeptides comprise a fusion to an anti-CTLA-4 antibody.

[0164] In some embodiments, the chimeric polypeptide comprises a fusion to an antibody or antigen-binding portion thereof that targets LAG-3 and inhibits the interaction of LAG-3 with MHC class II molecules. An exemplary antibody that targets LAG-3 is IMP321 (Immutep), which is currently undergoing human trials. Other suitable antibodies that target LAG-3 are disclosed in U.S. Patent Application Publication No. 2011 / 0150892 (incorporated herein by reference). One of skill in the art will appreciate that any antibody that binds to LAG-3, inhibits the interaction of LAG-3 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 to an anti-LAG-3 antibody.

[0165] In some embodiments, the chimeric polypeptide comprises a fusion to an antibody or antigen-binding portion thereof that targets TIGIT and inhibits the interaction of TIGIT with CD155 (PVR) and / or CD112 (PVRL2, nectin-2). One skilled in the art will appreciate that any antibody that binds to TIGIT and inhibits the interaction of TIGIT with CD155 (PVR) and / or CD112 (PVRL2, nectin-2) and stimulates an anti-tumor immune response or immunostimulatory response that results in systemic anti-tumor activity is suitable for use in the chimeric polypeptides disclosed herein. In some embodiments, the chimeric polypeptide comprises a fusion to an anti-TIGIT antibody.

[0166] In some embodiments, the chimeric polypeptide comprises a fusion to an antibody, or antigen-binding portion thereof, that targets CD112R and inhibits the interaction of CD112R (also known as PVRIG) with CD112 and / or PVRL2 / Nectin-2. One of skill in the art will appreciate that any antibody that binds to CD112R and inhibits the interaction of CD112R with CD112 and / or PVRL2 / Nectin-2, while stimulating an anti-tumor immune response or immunostimulatory response that confers systemic anti-tumor activity, is suitable for use in the chimeric polypeptides disclosed herein. In some embodiments, the chimeric polypeptide comprises a fusion to an anti-CD112R antibody.

[0167] In some embodiments, the chimeric polypeptide comprises a fusion to an antibody or antigen-binding portion thereof targeting B7-H3 or B7-H4. The B7 family does not have a single clearly defined receptor, 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 Publication No. 2013 / 0149236 (incorporated herein by reference). Those skilled in the art 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 to an anti-B7-H3 antibody or an anti-B7-H4 antibody.

[0168] In some embodiments, the chimeric polypeptide comprises a fusion to an antibody or antigen-binding portion thereof that targets TIM-3 and inhibits the interaction between TIM-3 and galectin-9. Suitable antibodies that target TIM-3 are disclosed in U.S. Patent Application Publication No. 2013 / 0022623, which is incorporated herein by reference. One of skill in the art will appreciate that any antibody that binds to TIM-3, inhibits the interaction between TIM-3 and 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 to an anti-TIM-3 antibody.

[0169] In some embodiments, the chimeric polypeptide comprises a fusion to an antibody, or antigen-binding portion thereof, that targets 4-1BB / CD137 and inhibits the interaction of 4-1BB / CD137 with CD137L. One of skill in the art will appreciate that any antibody that binds to 4-1BB / CD137 and inhibits the interaction of 4-1BB / CD137 with CD137L or another ligand, while stimulating an anti-tumor immune response or immunostimulatory response that results in systemic anti-tumor activity, is suitable for use in the chimeric polypeptides disclosed herein. In some embodiments, the chimeric polypeptide comprises a fusion to an anti-4-1BB / CD137 antibody.

[0170] In some embodiments, the chimeric polypeptide comprises a fusion to an antibody or antigen-binding portion thereof that targets GITR and inhibits the interaction of GITR with its ligand. One skilled in the art will appreciate that any antibody that binds to GITR and inhibits the interaction of GITR with GITRL or another ligand, while stimulating an anti-tumor immune response or immunostimulatory response that results in systemic anti-tumor activity, is suitable for use in the chimeric polypeptides disclosed herein. In some embodiments, the chimeric polypeptide comprises a fusion to an anti-GITR antibody.

[0171] In some embodiments, the chimeric polypeptide comprises a fusion to an antibody or antigen-binding portion thereof that targets OX40 and inhibits the interaction of OX40 with its ligand. One of skill in the art will understand that any antibody that binds to OX40 and inhibits the interaction of OX40 with OX40L or another ligand, while stimulating an anti-tumor immune response or immunostimulatory response that provides systemic anti-tumor activity, is suitable for use in the chimeric polypeptides disclosed herein. In some embodiments, the chimeric polypeptide comprises a fusion to an anti-OX40 antibody.

[0172] In some embodiments, the chimeric polypeptide comprises a fusion to an antibody or antigen-binding portion thereof that targets CD40 and inhibits the interaction of CD40 with its ligand. One of skill in the art will appreciate that any antibody that binds to CD40, inhibits the interaction of CD40 with its ligand, and stimulates an anti-tumor immune response or immunostimulatory response that results in systemic anti-tumor activity is suitable for use in the chimeric polypeptides disclosed herein. In some embodiments, the chimeric polypeptide comprises a fusion to an anti-CD40 antibody.

[0173] In some embodiments, the chimeric polypeptide comprises a fusion to an antibody or antigen-binding portion thereof that targets ICOS and inhibits the interaction of ICOS with its ligand. One skilled in the art will appreciate that any antibody that binds to ICOS, inhibits the interaction of ICOS with its ligand, and stimulates an anti-tumor immune response or immunostimulatory response that results in systemic anti-tumor activity is suitable for use in the chimeric polypeptides disclosed herein. In some embodiments, the chimeric polypeptide comprises a fusion to an anti-ICOS antibody.

[0174] In some embodiments, the chimeric polypeptide comprises a fusion to an antibody or antigen-binding portion thereof that targets CD28 and inhibits the interaction of CD28 with its ligand. One of skill in the art will appreciate that any antibody that binds to CD28, inhibits the interaction of CD28 with its ligand, and stimulates an anti-tumor immune response or immunostimulatory response that results in systemic anti-tumor activity is suitable for use in the chimeric polypeptides disclosed herein. In some embodiments, the chimeric polypeptide comprises a fusion to an anti-CD28 antibody. In some embodiments, the chimeric polypeptide comprises a fusion to an anti-CD3 antibody, including a T cell engager anti-CD3 antibody.

[0175] In some embodiments, the chimeric polypeptide comprises a fusion to an antibody or antigen-binding portion thereof that targets IFNα and inhibits the interaction of IFNα with its ligand. One of skill in the art will appreciate that any antibody that binds to IFNα and inhibits the interaction of IFNα with its ligand, while stimulating an anti-tumor immune response or immunostimulatory response that results in systemic anti-tumor activity, is suitable for use in the chimeric polypeptides disclosed herein. In some embodiments, the chimeric polypeptide comprises a fusion to an anti-IFNα antibody.

[0176] In some embodiments, the chimeric polypeptide comprises a tumor antigen or a fusion to a polypeptide that targets a tumor antigen. Generally, tumor antigens allow tumor cells to be distinguished from their normal cellular counterparts and can 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 aberrantly expressed cellular proteins, tumor antigens produced by oncogenic viruses, carcinoembryonic antigens, altered cell surface glycolipids and glycoproteins, and / or cell type-specific differentiation antigens. Such tumor antigens can include melanoma antigens, cancer-testis antigens, epithelial tumor antigens, cell cycle regulatory proteins, prostate-specific antigens (including, for example, prostate cancer antigens such as those disclosed in U.S. Pat. No. 5,538,866), and lymphomas (U.S. Pat. Nos. 4,816,249, 5,068,177, and 5,227,159). Examples of tumor antigens include HMW mucin to which 2G3 and 369F10 bind, c-erbB-2-related tumor antigen (a glycoprotein of approximately 42 kD or 55 kD), antigens of approximately 40 kD, 60 kD, 100 kD, and 200 kD to which 113F1 binds, 9-O-acetyl GD3, p97, alpha-fetoprotein (AFP) (e.g., in the case of germ cell tumors and / or hepatocellular carcinoma), carcinoembryonic antigen (CEA) (e.g., in the case of intestinal cancer, but may also be in the case of lung cancer or breast cancer), CA-125 (e.g., in the case of ovarian cancer), and MUC-1 (e.g., in the case of breast cancer). These include, but are not limited to, tumor antigens (e.g., in the case of breast cancer), epithelial tumor antigens (ETAs) (e.g., in the case of malignant melanoma), melanoma-associated antigens (MAGEs) (e.g., in the case of malignant melanoma), cancer / testis antigen 1 (CTAG1B), melanoma-associated antigen 1 (MAGEA1), aberrant Ras products, aberrant p53 products, overexpression of cyclins (including, for example, cyclin B1), fibronectin mutations, post-translational changes in MUC1 glycoprotein, and secreted tumor antigens (including, for example, gangliosides).

[0177] Other fusions can include fusions with pro-apoptotic payloads. Exemplary sequences are shown in the table below. In some embodiments, an IL-2 mutein as described herein is fused to a pro-apoptotic payload, e.g., a BAD, BAX, BAK, BIK, and / or BID sequence. In some embodiments, the pro-apoptotic payload is a peptide comprising a Bcl-2 domain and / or a subsequence of a BAD, BAX, BAK, BIK, and / or BID sequence. Exemplary pro-apoptotic fusions are shown in Table 3 below. In certain embodiments, an IL-2 antagonist can be fused to a pro-apoptotic payload for the treatment of cancer. An "antagonist" is a compound that blocks the action of an agonist, for example, by blocking, reducing, inhibiting, or neutralizing the activity of the agonist. An "antagonist" can also block, inhibit, or reduce the constitutive activity of a target, e.g., a target receptor, even in the absence of a specified agonist. Typically, the cancer treatment methods of the present invention use IL-2 muteins that have agonist or superagonist activity relative to wild-type IL-2; however, when such antagonists are fused to a pro-apoptotic payload, IL-2 muteins with antagonist properties can be used. In some embodiments, the IL-2 antagonist contains the following amino acid substitutions relative to wild-type IL-2 of SEQ ID NO:2: L18R, Q22E, Q126T, and S130R. In some embodiments, the IL-2 antagonist comprises the following amino acid substitutions relative to wild-type IL-2 of SEQ ID NO: 2: L18R, Q22E, L80F, R81D, L85V, I86V, and Q126T. In some embodiments, the IL-2 antagonist comprises the following amino acid substitutions relative to wild-type IL-2 of SEQ ID NO: 2: L18R, Q22E, L80F, R81D, L85V, I86V, Q126T, and S130R. Exemplary antagonists that can be fused to pro-apoptotic payloads such as those set forth above are shown in Table 4 below. TIFF2025509333000012.tif228170TIFF2025509333000013.tif239170TIFF2025509333000014.tif110170

[0178] Other fusions may include fusions with anti-apoptotic payloads used to prolong activation of CD8 cells, NK cells, and anergic NK cells, exemplary sequences of which are shown in the table below. Such sustained activation of T cells may prove beneficial in therapeutic methods for cancer therapy. TIFF2025509333000015.tif203170TIFF2025509333000016.tif239170

[0179] Other exemplary IL-2 fusions include those listed in the table below. TIFF2025509333000017.tif96170TIFF2025509333000018.tif233170TIFF20255093 33000019.tif203170TIFF2025509333000020.tif149170TIFF2025509333000021.ti f192170TIFF2025509333000022.tif132170TIFF2025509333000023.tif132170TIFF 2025509333000024.tif132170TIFF2025509333000025.tif132170TIFF20255093330 00026.tif132170TIFF2025509333000027.tif132170TIFF2025509333000028.tif13 7170TIFF2025509333000029.tif137170TIFF2025509333000030.tif132170TIFF202 5509333000031.tif132170TIFF2025509333000032.tif137170TIFF20255093330000 33.tif142170TIFF2025509333000034.tif132170TIFF2025509333000035.tif239170

[0180] In some embodiments, the IL-2 mutein-Fc fusion comprises one of the following sequences: TIFF2025509333000036.tif218170TIFF2025509333000037.tif79170

[0181] In some embodiments, the IL-2 mutein sequence is 90% identical to any one of SEQ ID NO:12-15 and / or SEQ ID NO:20-80 (e.g., any of the IL-2 sequences presented herein). In some embodiments, the IL-2 mutein sequence is 95% identical to any one of SEQ ID NO:12-15 and / or SEQ ID NO:20-80 (e.g., any of the IL-2 sequences presented herein). In some embodiments, the IL-2 mutein sequence is 98% identical to any one of SEQ ID NO:12-15 and / or SEQ ID NO:20-80 (e.g., any of the IL-2 sequences presented herein). In some embodiments, the IL-2 mutein sequence is 99% identical to any one of SEQ ID NO:12-15 and / or SEQ ID NO:20-80 (e.g., any of the IL-2 sequences presented herein). C. IL-4, IL-7, IL-13, IL-12, IL-15, IL-18, or IL-33 for use in bispecific IL-2 cytokine fusions

[0182] In some embodiments, an IL-2 mutein can be fused to an IL-4 mutein as described herein. In some embodiments, an IL-2 mutein can be fused to an IL-13 mutein as described herein. In some embodiments, an IL-2, IL-4, or IL-13 mutein can be fused to IL-7. In some embodiments, an IL-2, IL-4, or IL-13 mutein can be fused to IL-10. In some embodiments, an IL-2, IL-4, or IL-13 mutein can be fused to IL-12. In some embodiments, an IL-2, IL-4, or IL-13 mutein can be fused to IL-15. In some embodiments, an IL-2, IL-4, or IL-13 mutein can be fused to IL-18. In some embodiments, an IL-2, IL-4, or IL-13 mutein can be fused to IL-33. In some embodiments, such fusions function to specifically target cancer cells and / or cancer stem cells, reducing or inhibiting the growth of cancer stem cells, as well as targeting immunosuppressive cells in the tumor microenvironment (TME).

[0183] Any IL-13 sequence or variant thereof can be used in a fusion with an IL-2 mutein as described herein. In some embodiments, the IL-2 mutein comprises any one of SEQ ID NO:5 for 5-1, SEQ ID NO:6 for 5-2, SEQ ID NO:7 for 6-6, SEQ ID NO:8 for A2, SEQ ID NO:9 for B1, SEQ ID NO:10 for B11, SEQ ID NO:11 for C5, SEQ ID NO:12 for D10, SEQ ID NO:13 for E10, SEQ ID NO:14 for G8, SEQ ID NO:15 for H4, and SEQ ID NO:16 for H9. Exemplary IL-13 polypeptide sequences are set forth in SEQ ID NOs:81-128 and in the table below. In some embodiments, the IL-13 polypeptide sequence is as set forth in any one of SEQ ID NOs:81-128. 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 polypeptide sequence of IL-13 is SEQ ID NO: 84. In some embodiments, the polypeptide sequence of IL-13 is SEQ ID NO: 85. In some embodiments, the polypeptide sequence of IL-13 is SEQ ID NO: 86. In some embodiments, the polypeptide sequence of IL-13 is SEQ ID NO: 87. In some embodiments, the polypeptide sequence of IL-13 is SEQ ID NO: 88. In some embodiments, the polypeptide sequence of IL-13 is SEQ ID NO: 89. In some embodiments, the polypeptide sequence of IL-13 is SEQ ID NO: 90. In some embodiments, the polypeptide sequence of IL-13 is SEQ ID NO: 91. In some embodiments, the polypeptide sequence of IL-13 is SEQ ID NO: 92. In some embodiments, the polypeptide sequence of IL-13 is SEQ ID NO: 93. In some embodiments, the polypeptide sequence of IL-13 is SEQ ID NO: 94. In some embodiments, the polypeptide sequence is SEQ ID NO: 95. In some embodiments, the polypeptide sequence of IL-13 is SEQ ID NO: 96. In some embodiments, the polypeptide sequence of IL-13 is SEQ ID NO: 97.In some embodiments, the polypeptide sequence of IL-13 is SEQ ID NO: 98. In some embodiments, the polypeptide sequence of IL-13 is SEQ ID NO: 99. In some embodiments, the polypeptide sequence is SEQ ID NO: 100. In some embodiments, the polypeptide sequence of IL-13 is SEQ ID NO: 101. In some embodiments, the polypeptide sequence of IL-13 is SEQ ID NO: 102. In some embodiments, the polypeptide sequence of IL-13 is SEQ ID NO: 103. In some embodiments, the polypeptide sequence of IL-13 is SEQ ID NO: 104. In some embodiments, the polypeptide sequence of IL-13 is SEQ ID NO: 105. In some embodiments, the polypeptide sequence of IL-13 is SEQ ID NO: 106. In some embodiments, the polypeptide sequence of IL-13 is SEQ ID NO: 107. In some embodiments, the polypeptide sequence of IL-13 is SEQ ID NO: 108. In some embodiments, the polypeptide sequence of IL-13 is SEQ ID NO: 109. In some embodiments, the polypeptide sequence of IL-13 is SEQ ID NO: 110. In some embodiments, the polypeptide sequence is SEQ ID NO: 111. In some embodiments, the polypeptide sequence of IL-13 is SEQ ID NO: 112. In some embodiments, the polypeptide sequence of IL-13 is SEQ ID NO: 113. In some embodiments, the polypeptide sequence of IL-13 is SEQ ID NO: 114. In some embodiments, the polypeptide sequence of IL-13 is SEQ ID NO: 115. In some embodiments, the polypeptide sequence of IL-13 is SEQ ID NO: 116. In some embodiments, the polypeptide sequence of IL-13 is SEQ ID NO: 117. In some embodiments, the polypeptide sequence of IL-13 is SEQ ID NO: 118. In some embodiments, the polypeptide sequence of IL-13 is SEQ ID NO: 119. In some embodiments, the polypeptide sequence of IL-13 is SEQ ID NO: 120. In some embodiments, the polypeptide sequence of IL-13 is SEQ ID NO: 121. In some embodiments, the polypeptide sequence of IL-13 is SEQ ID NO: 122.In some embodiments, the polypeptide sequence of IL-13 is SEQ ID NO: 123. In some embodiments, the polypeptide sequence of IL-13 is SEQ ID NO: 124. In some embodiments, the polypeptide sequence of IL-13 is SEQ ID NO: 125. In some embodiments, the polypeptide sequence of IL-13 is SEQ ID NO: 126. In some embodiments, the polypeptide sequence of IL-13 is SEQ ID NO: 127. In some embodiments, the polypeptide sequence of IL-13 is SEQ ID NO: 128. IL-13. In some embodiments, the polypeptide sequence of IL-13 is 90% identical to any one of SEQ ID NOs: 81-128. In some embodiments, the polypeptide sequence of IL-13 is 95% identical to any one of SEQ ID NOs: 81-128. In some embodiments, the polypeptide sequence of IL-13 is 98% identical to any one of SEQ ID NOs: 81-128. In some embodiments, the polypeptide sequence of IL-13 is 99% identical to any one of SEQ ID NOs: 81-128.

[0184] In some embodiments, any one of SEQ ID NOs: 81 through 128 is linked to IL-2 or an IL-2 mutein as described herein. In some embodiments, SEQ ID NO: 81 is linked to an IL-2 or an IL-2 mutein as described herein. In some embodiments, SEQ ID NO: 82 is linked to an IL-2 or an IL-2 mutein as described herein. In some embodiments, SEQ ID NO: 83 is linked to an IL-2 or an IL-2 mutein as described herein. In some embodiments, SEQ ID NO: 84 is linked to an IL-2 or IL-2 mutein as described herein. In some embodiments, SEQ ID NO: 85 is linked to an IL-2 or IL-2 mutein as described herein. In some embodiments, SEQ ID NO: 86 is linked to an IL-2 or IL-2 mutein as described herein. In some embodiments, SEQ ID NO: 87 is linked to an IL-2 or IL-2 mutein as described herein. In some embodiments, SEQ ID NO: 88 is linked to an IL-2 or IL-2 mutein as described herein. In some embodiments, SEQ ID NO:89 is linked to IL-2 or an IL-2 mutein as described herein. In some embodiments, SEQ ID NO:90 is linked to an IL-2 or an IL-2 mutein as described herein. In some embodiments, SEQ ID NO:91 is linked to an IL-2 or an IL-2 mutein as described herein. In some embodiments, SEQ ID NO:92 is linked to an IL-2 or an IL-2 mutein as described herein. In some embodiments, SEQ ID NO:93 is linked to an IL-2 or an IL-2 mutein as described herein. In some embodiments, SEQ ID NO:94 is linked to an IL-2 or an IL-2 mutein as described herein. In some embodiments, SEQ ID NO:94 is linked to an IL-2 or IL-2 mutein as described herein.In some embodiments, SEQ ID NO:96 is linked to IL-2 or an IL-2 mutein as described herein. In some embodiments, SEQ ID NO:97 is linked to an IL-2 or an IL-2 mutein as described herein. In some embodiments, SEQ ID NO:98 is linked to an IL-2 or an IL-2 mutein as described herein. In some embodiments, SEQ ID NO:99 is linked to an IL-2 or an IL-2 mutein as described herein. In some embodiments, SEQ ID NO:100 is linked to an IL-2 or IL-2 mutein as described herein. In some embodiments, SEQ ID NO:101 is linked to an IL-2 or IL-2 mutein as described herein. In some embodiments, SEQ ID NO:102 is linked to an IL-2 or IL-2 mutein as described herein. In some embodiments, SEQ ID NO:103 is linked to an IL-2 or IL-2 mutein as described herein. In some embodiments, SEQ ID NO:104 is linked to an IL-2 or IL-2 mutein as described herein. In some embodiments, SEQ ID NO: 105 is linked to IL-2 or an IL-2 mutein as described herein. In some embodiments, SEQ ID NO: 106 is linked to an IL-2 or an IL-2 mutein as described herein. In some embodiments, SEQ ID NO: 107 is linked to an IL-2 or an IL-2 mutein as described herein. In some embodiments, SEQ ID NO: 108 is linked to an IL-2 or an IL-2 mutein as described herein. In some embodiments, SEQ ID NO: 109 is linked to an IL-2 or an IL-2 mutein as described herein. In some embodiments, SEQ ID NO: 110 is linked to an IL-2 or an IL-2 mutein as described herein. In some embodiments, SEQ ID NO: 111 is linked to an IL-2 or IL-2 mutein as described herein.In some embodiments, SEQ ID NO: 112 is linked to IL-2 or an IL-2 mutein as described herein. In some embodiments, SEQ ID NO: 113 is linked to an IL-2 or an IL-2 mutein as described herein. In some embodiments, SEQ ID NO: 114 is linked to an IL-2 or an IL-2 mutein as described herein. In some embodiments, SEQ ID NO: 115 is linked to an IL-2 or IL-2 mutein as described herein. In some embodiments, SEQ ID NO: 116 is linked to an IL-2 or IL-2 mutein as described herein. In some embodiments, SEQ ID NO: 117 is linked to an IL-2 or IL-2 mutein as described herein. In some embodiments, SEQ ID NO: 118 is linked to an IL-2 or IL-2 mutein as described herein. In some embodiments, SEQ ID NO: 119 is linked to an IL-2 or IL-2 mutein 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 IL-2 or an IL-2 mutein as described herein. In some embodiments, SEQ ID NO: 122 is linked to IL-2 or an IL-2 mutein as described herein. In some embodiments, SEQ ID NO: 123 is linked to IL-2 or an IL-2 mutein as described herein. In some embodiments, SEQ ID NO: 124 is linked to IL-2 or an IL-2 mutein as described herein. In some embodiments, SEQ ID NO: 125 is linked to IL-2 or an IL-2 mutein as described herein. In some embodiments, SEQ ID NO: 126 is linked to IL-2 or an IL-2 mutein as described herein. In some embodiments, SEQ ID NO: 127 is linked to an IL-2 or IL-2 mutein as described herein.In some embodiments, SEQ ID NO: 128 is linked to IL-2 or an IL-2 mutein as described herein. In some embodiments, the IL-2 mutein comprises any one of SEQ ID NO: 5 for 5-1, SEQ ID NO: 6 for 5-2, SEQ ID NO: 7 for 6-6, SEQ ID NO: 8 for A2, SEQ ID NO: 9 for B1, SEQ ID NO: 10 for B11, SEQ ID NO: 11 for C5, SEQ ID NO: 12 for D10, SEQ ID NO: 13 for E10, SEQ ID NO: 14 for G8, SEQ ID NO: 15 for H4, and SEQ ID NO: 16 for H9.

[0185] In some embodiments, the IL-13 peptides of the present invention include: (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; and (10) K89. (13) K105T, K105A, K105R, K105E, (14) F107L, F107I, F107V, F107M, (15) R108K, R108T, R108M, and (16) E15R amino acid substitutions, which alter affinity for one or both of IL-13Rα1 and IL-13Rα2. In another embodiment, the altered residues are at 2 or more, 3 or more, 4 or more, 5 or more, and up to 14 amino acids in the combined set of contact residues defined above. As described in International Publication No. WO2013 / 112871, the disclosure of which is incorporated herein by reference in its entirety. In some embodiments, the amino acid substitutions include, but are not limited to, those shown in Figure 4.

[0186] 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 one of the specific changes listed. 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 comprises 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 of these modifications is linked to IL-2 or an IL-2 mutein as described herein. In some embodiments, the amino acid substitutions include, but are not limited to, those shown in Figure 4. In some embodiments, the IL-2 mutein comprises any one of SEQ ID NO:5 for 5-1, SEQ ID NO:6 for 5-2, SEQ ID NO:7 for 6-6, SEQ ID NO:8 for A2, SEQ ID NO:9 for B1, SEQ ID NO:10 for B11, SEQ ID NO:11 for C5, SEQ ID NO:12 for D10, SEQ ID NO:13 for E10, SEQ ID NO:14 for G8, SEQ ID NO:15 for H4, and SEQ ID NO:16 for H9.

[0187] Specific sets of alterations that result in increased selectivity for binding to IL-13Rα2 over IL-13Rα1 compared to the native IL-13 sequence may include, but are not limited to, the sets below. [L10D, R11I, V18I, R86K, D87K, k89R, R108K] (e.g., C2, e.g., SEQ ID NO: 109) [L10A, R86T, D87G, T88K, K89R, L101N, K104R, K105A, R108K] (e.g., C3, e.g., SEQ ID NO: 110) [L10V, K89R, L101N, K105E, R108T] (e.g., C4, e.g., SEQ ID NO: 111 or SEQ ID NO: 93) [R11S, I14M, T88S, L101N, K105A, R108K] (e.g., C7, e.g., SEQ ID NO: 112 or SEQ ID NO: 94) [L10H, R11L, V18I, R86K, D87E, K89R, L101N, K105T, R108K] (C9, e.g., SEQ ID NO: 113) [L10H, R86T, D87G, T88R, R108K] (C11, e.g., SEQ ID NO: 98 or SEQ ID NO: 115) [L10H, E15R, R86T, D87G, T88R, R108K] (MDNA132+E15R, e.g., SEQ ID NO: 395) [L10A, V18F, R86K, D87K, K89R, L101I, K104R, R108K] (D7, e.g., SEQ ID NO: 117) ·[L10T / D, R11I, V18I, R86K, D87K / G, T88S, K89R, L101Y, K104R, K105T, R108K] ·[L10A / V, R86T, D87G, T88K, K89R, L101N, K104R, K105A / E, R108K / T]

[0188] 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 includes L10H, E15R, R86T, D87G, T88R, and R108K (MDNA132+E15R, e.g., SEQ ID NO: 395). In some embodiments, the set of modifications includes L10A, V18F, R86K, D87K, K89R, L101I, K104R, and R108K (D7, e.g., SEQ ID NO: 117). In some embodiments, the set of modifications includes 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, dR108T. 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, a polypeptide comprising one or more modifications is linked to IL-2 or an IL-2 mutein as described herein. In some embodiments, the amino acid substitutions include, but are not limited to, those shown in Figure 4. In some embodiments, the IL-2 mutein comprises any one of SEQ ID NO:5 for 5-1, SEQ ID NO:6 for 5-2, SEQ ID NO:7 for 6-6, SEQ ID NO:8 for A2, SEQ ID NO:9 for B1, SEQ ID NO:10 for B11, SEQ ID NO:11 for C5, SEQ ID NO:12 for D10, SEQ ID NO:13 for E10, SEQ ID NO:14 for G8, SEQ ID NO:15 for H4, and SEQ ID NO:16 for H9.

[0189] Specific sets of alterations that result in increased selectivity for binding to IL-13Rα1 over IL-13Rα2 compared to the native IL-13 sequence may include, but are not limited to, the sets below. ·[L10V, V18I, D87S, D88S, L101F, K104R, K105T] ·[R11S, V18I, R86K, D87G, T88S, K89M, L101Y, K104R, K105T] ·[L10V, V18I, D87S, T88S, L101F, K104R, K105T] ·[L10V / I, D87S, T88S, K89R, L101H / F, K104R, K105T] ·[L10I, V18I, R86T, D87G, T88S, K89R, L101Y / H, K104R, K105A] ·[L10V, V18I, D87S, T88S, L101F, K104R, K105T] ·[V18I, R86T, D87G, T88S, L101Y, K104R, K105A] ·[R11I, V18I, R86K, D87G, T88S, L101H, K104R, K105A, F107M] These substitutions are optionally combined with the substitutions [E12A / G / S, R65D / E]. [L10V, V18I, D87S, T88S, L101F, K104R, K105T, and R39 polymorphisms] [L10V, V18I, D87S, T88S, L101F, K104R, K105T, and Q111 polymorphisms] [L10V, V18I, D87S, T88S, L101F, K104R, K105T, and R39 and Q111 polymorphisms] [E15R] ·[L10V, V18I, D87S, D88S, L101F, K104R, K105T, E15R] ·[R11S, V18I, R86K, D87G, T88S, K89M, L101Y, K104R, K105T, E15R] ·[L10V, V18I, D87S, T88S, L101F, K104R, K105T, E15R] ·[L10V / I, D87S, T88S, K89R, L101H / F, K104R, K105T, E15R] ·[L10I, V18I, R86T, D87G, T88S, K89R, L101Y / H, K104R, K105A, E15R] ·[L10V, V18I, D87S, T88S, L101F, K104R, K105T, E15R] ·[V18I, R86T, D87G, T88S, L101Y, K104R, K105A, E15R] ·[R11I, V18I, R86K, D87G, T88S, L101H, K104R, K105A, F107M, E15R] These substitutions are optionally combined with the substitutions [E12A / G / S, R65D / E] and / or E15R]. [L10V, E15R, V18I, D87S, T88S, L101F, K104R, K105T, and R39 polymorphisms] [L10V, E15R, V18I, D87S, T88S, L101F, K104R, K105T, and Q111 polymorphisms] [L10V, E15R, V18I, D87S, T88S, L101F, K104R, K105T, and R39 and Q111 polymorphisms]

[0190] In some embodiments, the set of modifications comprises L10V, V18I, D87S, D88S, L101F, K104R, and K105T. In some embodiments, the set of modifications comprises R11S, V18I, R86K, D87G, T88S, K89M, L101Y, K104R, and K105T. In some embodiments, the set of modifications comprises L10V, V18I, D87S, T88S, L101F, K104R, and K105T. In some embodiments, the set of modifications comprises L10V, V18I, D87S, T88S, L101F, K104R, K105T, and an R39 polymorphism. In some embodiments, the set of modifications comprises the L10V, V18I, D87S, T88S, L101F, K104R, K105T, and Q111 polymorphisms. In some embodiments, the set of modifications comprises the L10V, V18I, D87S, T88S, L101F, K104R, K105T, and Q111 polymorphisms. In some embodiments, the set of modifications comprises the L10V, V18I, D87S, T88S, L101F, K104R, K105T, R39 polymorphism, and Q111 polymorphism. In some embodiments, the set of modifications comprises the L10V / I, D87S, T88S, K89R, L101H / F, K104R, and K105T. In some embodiments, the set of modifications comprises L10I, V18I, R86T, D87G, T88S, K89R, L101Y / H, K104R, and K105A. In some embodiments, the set of modifications comprises L10V, V18I, D87S, T88S, L101F, K104R, and K105T. In some embodiments, the set of modifications comprises V18I, R86T, D87G, T88S, L101Y, K104R, and K105A. In some embodiments, the set of modifications 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 set of modifications comprises L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12S, and R65D / E. In some embodiments, the set of modifications comprises L10V / I, D87S, T88S, K89R, L101H / F, K104R, K105T, E12S, and R65D / E. In some embodiments, the set of modifications comprises L10I, V18I, R86T, D87G, T88S, K89R, L101Y / H, K104R, K105A, E12S, and R65D / E. In some embodiments, the set of modifications 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 set of modifications comprises R11I, V18I, R86K, D87G, T88S, L101H, K104R, K105A, F107M, E12S, and R65D / E. In some embodiments, the set of modifications comprises L10V, V18I, D87S, D88S, L101F, K104R, K105T, E12A, and R65D. 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 R65D. In some embodiments, the set of modifications comprises L10V / I, D87S, T88S, K89R, L101H / F, K104R, K105T, E12A, and R65D. In some embodiments, the set of modifications comprises L10I, V18I, R86T, D87G, T88S, K89R, L101Y / H, K104R, K105A, E12A, and R65D. In some embodiments, the set of modifications comprises L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12A, and R65D. In some embodiments, the set of modifications comprises V18I, R86T, D87G, T88S, L101Y, K104R, K105A, E12A, and R65D. In some embodiments, the set of modifications comprises R11I, V18I, R86K, D87G, T88S, L101H, K104R, K105A, F107M, E12A, and R65D. In some embodiments, the set of modifications comprises L10V, V18I, D87S, D88S, L101F, K104R, K105T, E12G, and R65D. In some embodiments, the set of modifications comprises R11S, V18I, R86K, D87G, T88S, K89M, L101Y, K104R, K105T, E12G, and R65D. In some embodiments, the set of modifications 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 set of modifications comprises L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12G, and R65D. In some embodiments, the set of modifications comprises V18I, R86T, D87G, T88S, L101Y, K104R, K105A, E12G, and R65D. In some embodiments, the set of modifications comprises R11I, V18I, R86K, D87G, T88S, L101H, K104R, K105A, F107M, E12G, and R65D. In some embodiments, the set of modifications comprises L10V, V18I, D87S, D88S, L101F, K104R, K105T, E12S, and R65D. In some embodiments, the set of modifications comprises R11S, V18I, R86K, D87G, T88S, K89M, L101Y, K104R, K105T, E12S, and R65D. In some embodiments, the set of modifications comprises L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12S, and R65D.In some embodiments, the set of modifications comprises L10V / I, D87S, T88S, K89R, L101H / F, K104R, K105T, E12S, and R65D. In some embodiments, the set of modifications comprises L10I, V18I, R86T, D87G, T88S, K89R, L101Y / H, K104R, K105A, E12S, and R65D. In some embodiments, the set of modifications comprises L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12S, and R65D. In some embodiments, the set of modifications comprises V18I, R86T, D87G, T88S, L101Y, K104R, K105A, E12S, and R65D. In some embodiments, the set of modifications comprises R11I, V18I, R86K, D87G, T88S, L101H, K104R, K105A, F107M, E12S, and R65D. In some embodiments, the set of modifications 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 set of modifications comprises L10V / I, D87S, T88S, K89R, L101H / F, K104R, K105T, E12A, and R65E. In some embodiments, the set of modifications comprises L10I, V18I, R86T, D87G, T88S, K89R, L101Y / H, K104R, K105A, 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 set of modifications comprises V18I, R86T, D87G, T88S, L101Y, K104R, K105A, E12A, and R65E. In some embodiments, the set of modifications comprises R11I, V18I, R86K, D87G, T88S, L101H, K104R, K105A, F107M, E12A, and R65E.In some embodiments, the set of modifications comprises L10V, V18I, D87S, D88S, L101F, K104R, K105T, E12G, and R65E. In some embodiments, the set of modifications comprises R11S, V18I, R86K, D87G, T88S, K89M, L101Y, K104R, K105T, E12G, and R65E. In some embodiments, the set of modifications comprises L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12A / G / S, and R65E. In some embodiments, the set of modifications comprises L10V / I, D87S, T88S, K89R, L101H / F, K104R, K105T, E12G, and R65E. In some embodiments, the set of modifications comprises L10I, V18I, R86T, D87G, T88S, K89R, L101Y / H, K104R, K105A, E12G, and R65E. In some embodiments, the set of modifications comprises L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12G, and R65E. In some embodiments, the set of modifications 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 set of modifications comprises L10I, V18I, R86T, D87G, T88S, K89R, L101Y / H, K104R, K105A, E12S, 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 V18I, R86T, D87G, T88S, L101Y, K104R, K105A, E12S, and R65E. In some embodiments, the set of modifications comprises R11I, V18I, R86K, D87G, T88S, L101H, K104R, K105A, F107M, E12S, and R65E. In some embodiments, the set of modifications includes 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 includes 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 IL-2 or an IL-2 mutein as described herein. In some embodiments, the amino acid substitutions include, but are not limited to, those shown in Figure 3. In some embodiments, the IL-2 mutein comprises any one of SEQ ID NO:5 for 5-1, SEQ ID NO:6 for 5-2, SEQ ID NO:7 for 6-6, SEQ ID NO:8 for A2, SEQ ID NO:9 for B1, SEQ ID NO:10 for B11, SEQ ID NO:11 for C5, SEQ ID NO:12 for D10, SEQ ID NO:13 for E10, SEQ ID NO:14 for G8, SEQ ID NO:15 for H4, and SEQ ID NO:16 for H9.

[0191] A table of IL-13 sequences is shown below. TIFF2025509333000038.tif240170TIFF2025509333000039.tif239170TIFF2025509333000040.tif226170TIFF2025509333000041.tif239170TIFF2025509333000042.tif217170TIFF2025509333000043.tif239170TIFF2025509333000044.tif156170

[0192] Fusions with IL-2 muteins or variants can use any IL-4 sequence or variant thereof, including those described herein. In some embodiments, the IL-2 mutein comprises any one of SEQ ID NO:5 for 5-1, SEQ ID NO:6 for 5-2, SEQ ID NO:7 for 6-6, SEQ ID NO:8 for A2, SEQ ID NO:9 for B1, SEQ ID NO:10 for B11, SEQ ID NO:11 for C5, SEQ ID NO:12 for D10, SEQ ID NO:13 for E10, SEQ ID NO:14 for G8, SEQ ID NO:15 for H4, and SEQ ID NO:16 for H9. Exemplary polypeptide sequences are set forth in SEQ ID NOs:130-135, including any of the sequences set forth herein. In some embodiments, the IL-4 polypeptide sequence is set forth in any one of SEQ ID NOs:130-135. 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 polypeptide sequence of IL-4 is SEQ ID NO: 133. In some embodiments, the polypeptide sequence of IL-4 is SEQ ID NO: 134. In some embodiments, the polypeptide sequence of IL-4 is SEQ ID NO: 135. In some embodiments, the polypeptide sequence of IL-4 is 98% identical to any one of SEQ ID NOs: 130-135. In some embodiments, the polypeptide sequence of IL-4 is 99% identical to any one of SEQ ID NOs: 130-135. In some embodiments, any one of SEQ ID NOs: 130-135 is linked to IL-2 or an IL-2 mutein as described herein. In some embodiments, SEQ ID NO: 130 is linked to an IL-2 or an IL-2 mutein as described herein. In some embodiments, SEQ ID NO: 131 is linked to an IL-2 or an IL-2 mutein as described herein. In some embodiments, SEQ ID NO: 132 is linked to an IL-2 or an IL-2 mutein as described herein.In some embodiments, SEQ ID NO: 133 is linked to IL-2 or an IL-2 mutein as described herein. In some embodiments, SEQ ID NO: 134 is linked to IL-2 or an IL-2 mutein as described herein. In some embodiments, SEQ ID NO: 135 is linked to IL-2 or an IL-2 mutein as described herein. In some embodiments, the IL-2 mutein comprises any one of SEQ ID NO: 5 for 5-1, SEQ ID NO: 6 for 5-2, SEQ ID NO: 7 for 6-6, SEQ ID NO: 8 for A2, SEQ ID NO: 9 for B1, SEQ ID NO: 10 for B11, SEQ ID NO: 11 for C5, SEQ ID NO: 12 for D10, SEQ ID NO: 13 for E10, SEQ ID NO: 14 for G8, SEQ ID NO: 15 for H4, and SEQ ID NO: 16 for H9. 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.

[0193] A table of IL-4 sequences is shown below. TIFF2025509333000045.tif239170TIFF2025509333000046.tif115170

[0194] In some embodiments, the IL-2 mutein can be fused to sequences of IL-7, IL-12, IL-15, IL-18, and / or IL-33. In some embodiments, such fusions function to specifically target the fusion construct to NK cells and / or CD8+ cells. In some embodiments, the IL-2 mutein comprises any one of SEQ ID NO:5 for 5-1, SEQ ID NO:6 for 5-2, SEQ ID NO:7 for 6-6, SEQ ID NO:8 for A2, SEQ ID NO:9 for B1, SEQ ID NO:10 for B11, SEQ ID NO:11 for C5, SEQ ID NO:12 for D10, SEQ ID NO:13 for E10, SEQ ID NO:14 for G8, SEQ ID NO:15 for H4, and SEQ ID NO:16 for H9. In some embodiments, SEQ ID NO:136 is linked to IL-2 or an IL-2 mutein as described herein. In some embodiments, SEQ ID NO:137 is linked to IL-2 or an IL-2 mutein as described herein. In some embodiments, SEQ ID NO: 138 is linked to IL-2 or an IL-2 mutein as described herein. In some embodiments, SEQ ID NO: 139 is linked to IL-2 or an IL-2 mutein as described herein. In some embodiments, SEQ ID NO: 140 is linked to IL-2 or an IL-2 mutein as described herein. In some embodiments, SEQ ID NO: 141 is linked to IL-2 or an IL-2 mutein as described herein. In some embodiments, the IL-2 mutein can be fused to the sequences of IL-7, IL-12, IL-15, IL-18, and / or IL-33, as shown in the table below (SEQ ID NOs: 136-141). TIFF2025509333000047.tif223170TIFF2025509333000048.tif67170

[0195] Exemplary IL-2 mutein sequences include any one of SEQ ID NO:5 for 5-1, SEQ ID NO:6 for 5-2, SEQ ID NO:7 for 6-6, SEQ ID NO:8 for A2, SEQ ID NO:9 for B1, SEQ ID NO:10 for B11, SEQ ID NO:11 for C5, SEQ ID NO:12 for D10, SEQ ID NO:13 for E10, SEQ ID NO:14 for G8, SEQ ID NO:15 for H4, and SEQ ID NO:16 for H9.

[0196] In some embodiments, the cytokine-cytokine fusion is one of the fusions included in the table below. TIFF2025509333000049.tif239170TIFF2025509333000050.tif235170TIFF2025509333000051.tif239170

[0197] In some embodiments, the cytokine-cytokine fusion is one of the fusions included in the table below (see Table 12 and Figure 54 of WO2021258213, which is incorporated by reference in its entirety). TIFF2025509333000052.tif240170TIFF2025509333000053.tif178170TIFF2025509333000054.tif232170TIFF2025509333 000055.tif85170TIFF2025509333000056.tif179170TIFF2025509333000057.tif166170TIFF2025509333000058.tif193170 TIFF2025509333000059.tif162170TIFF2025509333000060.tif162170TIFF2025509333000061.tif222170TIFF20255093330 00062.tif239170TIFF2025509333000063.tif241170TIFF2025509333000064.tif221170TIFF2025509333000065.tif135170 TIFF2025509333000066.tif210170TIFF2025509333000067.tif240170TIFF2025509333000068.tif210170TIFF2025509333000069.tif193170TIFF2025509333000070.tif240170TIFF2025509333000071.tif240170TIFF2025509333000072.tif215170TIFF2025509333000073.tif224170TIFF2025509333000074.tif240170TIFF2025509333000075.tif240170TIFF2025509333000076.tif236170TIFF2025509333000077.tif240170TIFF2025509333000078.tif172170TIFF2025509333000079.tif172170TIFF2025509333000080.tif172170TIFF2025509333000081.tif172170TIFF2025509333000082.tif177170TIFF2025509333000083.tif177170TIFF2025509333000084.tif211170TIFF2025509333000085.tif240170TIFF2025509333000086.tif240170TIFF2025509333000087.tif228170TIFF2025509333000088.tif188170TIFF2025509333000089.tif226170TIFF2025509333000090.tif238170TIFF2025509333000091.tif237170TIFF2025509333000092.tif240170TIFF2025509333000093.tif211170TIFF2025509333000094.tif240170TIFF2025509333000095.tif240170TIFF2025509333000096.tif214170TIFF2025509333000097.tif222170TIFF2025509333000098.tif222170TIFF2025509333000099.tif226170TIFF2025509333000100.tif240170TIFF2025509333000101.tif218170TIFF2025 509333000102.tif134170TIFF2025509333000103.tif165170TIFF2025509333000104.tif167170TIFF2025509333000105.tif13 3170TIFF2025509333000106.tif126170TIFF2025509333000107.tif182170TIFF2025509333000108.tif177170TIFF202550933 3000109.tif220170TIFF2025509333000110.tif240170TIFF2025509333000111.tif233170TIFF2025509333000112.tif240170.

[0198] D. Recombinant Expression of IL-2, IL-4, or IL-13 Mutein Bifunctional Molecules, Expression Vectors, and Host Cells In various embodiments, the polypeptides used in practicing the invention are synthetic or produced by expressing recombinant nucleic acid molecules. Where the polypeptide is chimeric (e.g., a fusion protein comprising at least a mutant IL-2 polypeptide and a heterologous polypeptide (including bispecific IL-2 cytokine fusions)), the polypeptide can be encoded by a hybrid nucleic acid molecule comprising one sequence encoding all or a portion of an IL-2, IL-4, or IL-13 mutein bifunctional molecule and a second sequence encoding all or a portion of a heterologous polypeptide. For example, the IL-2, IL-4, or IL-13 mutein bifunctional molecules of the invention described herein may be fused to a hexahistidine tag to facilitate purification of bacterially expressed proteins or a hemagglutinin tag to facilitate purification of eukaryotically expressed proteins.

[0199] Methods for constructing DNA sequences encoding IL-2, IL-4, or IL-13 mutein bifunctional molecules and expressing these sequences in an appropriately transformed host include, but are not limited to, the use of PCR-mediated mutagenesis. Mutations consisting of deletions or additions of amino acid residues to the IL-2 polypeptide can also be made using standard recombinant techniques. For deletions or additions, the nucleic acid molecule encoding IL-2 is optionally digested with an appropriate restriction enzyme. The resulting fragments can be expressed directly or further manipulated, for example, by ligating the fragment to a second fragment. Ligation can be facilitated if the two ends of the nucleic acid molecule contain overlapping complementary nucleotides, although blunt-ended fragments can also be ligated. Nucleic acids generated by PCR can also be used to generate a variety of mutant sequences.

[0200] The complete amino acid sequence can be used to construct a reverse-translated gene. DNA oligomers containing nucleotide sequences encoding IL-2, IL-4, or IL-13 mutein bifunctional molecules can be synthesized. For example, several small oligonucleotides encoding portions of the desired polypeptide can be synthesized and then ligated. Individual oligonucleotides typically contain 5' or 3' overhangs for complementary assembly.

[0201] In addition to producing mutant polypeptides by expressing nucleic acid molecules altered by molecular biological recombinant techniques, the IL-2, IL-4, or IL-13 mutein bifunctional molecules of the present invention can be chemically synthesized, which can be produced by conventional methods by those skilled in the art.

[0202] Once assembled (by synthesis, site-directed mutagenesis, or another method), 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 appropriate expression control sequences for expression of the IL-2, IL-4, or IL-13 mutein bifunctional molecule in the desired host to be transformed. Proper assembly can be confirmed by nucleotide sequencing, restriction enzyme mapping, and expression of a biologically active polypeptide in a suitable host. As is well known in the art, to obtain high levels of expression of a transfected gene in a host, the gene must be operably linked to transcriptional and translational expression control sequences that are functional in the selected expression host.

[0203] The DNA sequence encoding the IL-2, IL-4, or IL-13 mutein bifunctional molecule, whether 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 recognized by the cell selected for expression of the IL-2, IL-4, or IL-13 mutein bifunctional molecule. The signal sequence can be a prokaryotic sequence, a eukaryotic sequence, or a combination of the two. It can also be the native IL-2 signal sequence. The inclusion of a signal sequence depends on whether it is desired to secrete the IL-2, IL-4, or IL-13 mutein bifunctional molecule from the recombinant cell in which it is produced. If the selected cell is a prokaryotic cell, it is generally preferred that the DNA sequence not encode a signal sequence. If the selected cell is eukaryotic, it is generally preferred that a signal sequence be encoded, most preferably the wild-type IL-2 signal sequence.

[0204] E. Oncolytic viruses targeting parts In some examples, oncolytic viruses can be targeted using the bispecific IL-2 cytokine fusions and / or IL-2, IL-4, or IL-13 mutein bifunctional molecules described herein (see, e.g., Allen et al., Mol. Ther. 16:1556-64, 2008). In some examples, oncolytic viruses are armed against tumors or the TME with IL-2, IL-4, or IL-13 mutein bifunctional molecules. Many viruses can be used as oncolytic viruses, including adenoviruses, self-replicating alphaviruses, and oncolytic vaccinia viruses (see, e.g., WO2013038066, incorporated herein by reference in its entirety, particularly FIG. 17). Other oncolytic viruses can include Seneca Valley virus, Newcastle disease virus (also known as Newcastle virus), Maraba virus, vesicular stomatitis virus (VSV), herpesviruses (including HSV-1), measles virus, poliovirus, reovirus, coxsackievirus, lentivirus, morbillivirus, influenza virus, Sindbis virus, myxoma virus, and / or retrovirus (see, e.g., 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) (both of which are incorporated by reference in their entireties). In some embodiments, oncolytic viruses include, but are not limited to, adenoviruses, self-replicating alphaviruses, vaccinia viruses, Seneca Valley viruses, Newcastle disease viruses, Maraba viruses, vesicular stomatitis viruses (VSV), herpes viruses (including HSV-1 and HSV-2), measles viruses, polioviruses, reoviruses, coxsackie viruses, lentiviruses, morbilliviruses, influenza viruses, Sindbis viruses, myxoma viruses, and / or retroviruses.The IL-2 superkine of the present invention (H9 and IL-2 variants as described herein) can also be used to induce T cells / OVs into the TME. IL-2 variants (such as H9) can boost effector T cells and NK cells, while suppressing Treg activity. Other oncolytic viruses include oncoVex / T-VEC, which involves intratumoral injection of a conditionally replicating herpes simplex virus (which preferentially infects cancer cells). Viruses engineered to express GM-CSF can also replicate inside cancer cells, causing their lysis and, in the process, releasing new viruses and a series of tumor antigens and secreting GM-CSF. Such oncolytic virus vaccines enhance DC function within the tumor microenvironment to stimulate antitumor immune responses. These oncolytic viruses can be used to target or deliver the IL-2, IL-4, or IL-13 muteins described herein, including the bifunctional molecules described herein, to tumors. These oncolytic viruses can be used to target or deliver the IL-2, IL-4, or IL-13 mutein bifunctional molecules described herein to tumors. In some embodiments, the IL-2, IL-4, or IL-13 mutein bifunctional molecule is any IL-2, IL-4, or IL-13 mutein bifunctional molecule or variant disclosed herein. In some embodiments, the sequence of the IL-2 mutein is 90% identical to any one of SEQ ID NO:2, SEQ ID NO:6-SEQ ID NO:10, or SEQ ID NO:16. In some embodiments, the IL-2 mutein or bifunctional molecule comprises any one of SEQ ID NO:5 for 5-1, SEQ ID NO:6 for 5-2, SEQ ID NO:7 for 6-6, SEQ ID NO:8 for A2, SEQ ID NO:9 for B1, SEQ ID NO:10 for B11, SEQ ID NO:11 for C5, SEQ ID NO:12 for D10, SEQ ID NO:13 for E10, SEQ ID NO:14 for G8, SEQ ID NO:15 for H4, and SEQ ID NO:16 for H9. In some embodiments, substitutions in the IL-2 mutein or bifunctional molecule include L80F, R81D, L85V, I86V, and I92F (numbered relative to wild-type human IL-2 of SEQ ID NO:2).In some embodiments, the oncolytic virus comprises a transgene capable of expressing an IL-2 mutein or bifunctional molecule as described herein. In some embodiments, the oncolytic virus comprises a transgene capable of expressing an IL-2 mutein or bifunctional molecule comprising the amino acid substitutions L80F, R81D, L85V, I86V, and I92F (numbered relative to wild-type human IL-2 of SEQ ID NO: 2). In some embodiments, the oncolytic virus comprises a nucleic acid encoding an IL-2 mutein or bifunctional molecule comprising the amino acid substitutions L80F, R81D, L85V, I86V, and I92F (numbered relative to wild-type human IL-2 of SEQ ID NO: 2). In some embodiments, the oncolytic virus comprises a transgene that is expressed as a therapeutic payload. In some embodiments, the therapeutic payload is IL-2 as described herein. In some embodiments, the therapeutic payload is an IL-2 mutein or bifunctional molecule comprising the following amino acid substitutions, L80F, R81D, L85V, I86V, and I92F, numbered relative to wild-type human IL-2 of SEQ ID NO: 2. In some embodiments, an oncolytic virus can be used to target or deliver a bifunctional molecule comprising (i) an IL-2-based amino acid sequence of Table 2 and (ii) an amino acid sequence of any one of Tables 3, 4, 8, 9, or 10. In some embodiments, an oncolytic virus can be used to target or deliver a bifunctional molecule comprising (i) an IL-4-based amino acid sequence of Table 4 or 9 and (ii) an amino acid sequence of any one of Tables 2, 3, 8, or 10. In some embodiments, an oncolytic virus can be used to target or deliver 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 Tables 2, 3, 4, 9, or 10.In some embodiments, oncolytic viruses can be used to target or deliver bifunctional molecules comprising (i) an IL-7, IL-12, IL-15, or IL-18, IL-33-based amino acid sequence of Table 10 and (ii) an amino acid sequence of one of Tables 2, 3, 4, 8, or 9. In some embodiments, oncolytic viruses can be used to target or deliver bifunctional molecules 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. In some embodiments, the oncolytic virus comprises an oncolytic virus having an amino acid sequence of SEQ ID NO: 395, 484, 501, 502, 503, 504, 505, 506, 507, or 508 and an amino acid sequence 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, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, S and an amino acid sequence selected from the group consisting of 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, 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, the oncolytic virus 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, and optionally, the bifunctional molecule further comprises an R39 polymorphism and / or a Q111 polymorphism.In some embodiments, oncolytic viruses can be used to target or deliver bifunctional molecules that include the following substitutions relative to wild-type IL-13: L10V, E12A, V18I, R65D, D87S, T88S, L101F, K104R, and K105T, and optionally, the bifunctional molecule further includes an R39 polymorphism and / or a Q111 polymorphism. In some embodiments, oncolytic viruses can be used to target or deliver bifunctional molecules that include the following substitutions relative to wild-type IL-2: L80F, R81D, L85V, I86V, I92F, and optionally, the bifunctional molecule further includes the following substitutions relative to wild-type IL-2: F42A and E62A, and / or optionally, the bifunctional molecule further includes the following substitution relative to wild-type IL-2: C125S. In some embodiments, oncolytic viruses can be used to target or deliver bifunctional molecules that comprise the following substitutions relative to wild-type IL-4: R121K, Y124F, S125R. In some embodiments, oncolytic viruses can be used to target or deliver bifunctional molecules that comprise the following substitutions relative 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 that comprise one or more amino acid sequences of any one of Tables 2, 3, 4, 8, 9, or 10 and that include one or more cytokine-binding moieties of Tables 2, 3, 4, 8, 9, or 10. In some embodiments, the oncolytic virus comprises one or more amino acid sequences of any one of Tables 5, 6, 7, 11, 12, 13, 15, or 39 and can be used to target or deliver bifunctional molecules that comprise one or more cytokine binding moieties of Tables 5, 6, 7, 11, 12, 13, 15, or 39. In some embodiments, the oncolytic virus is selected from the group consisting of SEQ ID NOs: 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 4, 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, 2 08, 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 9, 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 2, 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, ​​3 63, 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 Bifunctional molecules comprising any one or more of the amino acid sequences of 56, 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 can be used to target or deliver the

[0205] In some embodiments, the oncolytic virus is an oncolytic vaccinia virus. In some embodiments, the oncolytic vaccinia virus vector is characterized in that the viral particle is an intracellular mature virus (IMV), an intracellular enveloped virus (IEV), a cell-associated enveloped virus (CEV), or an extracellular enveloped virus (EEV) type particle. In some embodiments, the oncolytic vaccinia virus particle is an EEV or IMV type type. In some embodiments, the oncolytic vaccinia virus particle is an EEV type particle.

[0206] Generally, the construction of recombinant oncolytic vaccinia viruses, cells, and pharmaceutical compositions containing vectors that replicate preferentially in tumor cells and express at least one transgene (e.g., an IL-2, IL-4, or IL-13 mutein bifunctional molecule as described herein) promotes antitumor effects and apoptosis induction, and modulates the host immune response in a subject. According to the present invention, oncolytic adenoviruses and oncolytic vaccinia viruses can be combined with IL-2 expression or targeting moieties, as described herein, to target the oncolytic vaccinia virus or oncolytic adenovirus and / or express IL-2, IL-4, or IL-13 mutein bifunctional molecule. Oncolysis releases tumor antigens and provides costimulatory danger signals. However, efficacy can be further improved by arming the virus. For example, CD40 ligand (CD40L, CD154) is known to induce apoptosis in tumor cells and also triggers several immune mechanisms. One of these is the T helper type 1 (Thl) response, which activates cytotoxic T cells and reduces immunosuppression. The present invention provides oncolytic viruses that express the IL-2, IL-4, or IL-13 mutein bifunctional molecules of the present invention. In some embodiments, the present invention provides oncolytic viruses that are targeted (e.g., "armed") by a targeting moiety of the present invention.

[0207] In some embodiments, the oncolytic virus is a modified vaccinia virus vector, viral particle, host cell, pharmaceutical composition, and kit comprising a vaccinia virus genome, wherein the thymidine kinase gene has been inactivated by either a substitution in the thymidine kinase (TK) gene and / or a deletion resulting in the loss of at least one nucleotide in the open reading frame (thereby resulting in a partially deleted thymidine kinase gene), wherein the vaccinia growth factor gene has been deleted, and wherein 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 mutein bifunctional molecule capable of expressing as described herein). In another aspect, the modified vaccinia virus vector, viral particle, pharmaceutical composition, or kit can be used in cancer therapy to elicit an immune response in a subject, in a method of inhibiting the growth of malignant cells in a mammal, in cancer therapy or prophylaxis, to detect the presence of the modified vaccinia virus in a subject, and, optionally, as an in situ cancer vaccine in combination with an adenovirus. In some embodiments, the invention provides methods for producing a modified vaccinia virus comprising a vaccinia virus genome, wherein the thymidine kinase (TK) gene has been inactivated by a substitution in the TK gene and / or a deletion resulting in the loss of at least one nucleotide in the open reading frame (thereby resulting in a partially deleted thymidine kinase gene), a vaccinia growth factor gene has been 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 sustaining the production of vaccinia virus particles and harboring the modified vaccinia vector; culturing the producer cell under conditions suitable for virus replication and production; and recovering the virus particles.

[0208] In some embodiments, the present invention provides methods of administering an oncolytic virus that is "armed" with or includes a nucleic acid encoding an IL-2, IL-4, or IL-13 mutein bifunctional molecule as described herein, where the IL-2, IL-4, or IL-13 mutein bifunctional molecule is expressed at the location of a tumor or expressed throughout the subject's body. In some embodiments, the present invention also provides methods of administering an oncolytic virus that is "armed" or targeted by an IL-2, IL-4, or IL-13 mutein bifunctional molecule as described herein. The route of administration will, of course, vary with the location and nature of the tumor and may include, for example, intradermal, transdermal, parenteral, intravenous, intramuscular, intranasal, subcutaneous, local (e.g., near the tumor, particularly the vasculature or vasculature adjacent to the tumor), percutaneous, intratracheal, intraperitoneal, intraarterial, intravesical, intratumoral, inhalation, perfusion, lavage, and oral administration. The composition is formulated with reference to the particular route of administration.

[0209] 1. Oncolytic vaccinia virus Vaccinia virus is a member of the Orthopoxvirus genus in the Poxviridae family. Vaccinia virus has a large double-stranded DNA genome (approximately 200 kb, approximately 200 genes), and a complex morphogenetic pathway results in the production of infectious virions with unique morphology from each infected cell. The virus particle contains a lipid membrane(s) surrounding a core. The viral core contains viral structural proteins, a compacted viral DNA genome, and transcriptase. Vaccinia virus dimensions are approximately 360 × 270 × 250 nm and weigh approximately 5–10 fg. Genes are compactly packed with little non-coding DNA, and open reading frames (ORFs) lack introns. There are three classes of genes: early, middle, and late. Early genes (approximately 100 genes, immediate early, and delayed early) encode proteins primarily involved in immune regulation and viral DNA replication. The intermediate genes encode regulatory proteins required for expression of the late genes (e.g., transcription factors), and the late genes encode proteins required to make viral particles and enzymes that are packaged into new virions to initiate the next round of infection. Vaccinia virus replicates within the cytoplasm of the cell.

[0210] Various vaccinia virus strains have been identified (e.g., Copenhagen strain, Modified Vaccinia Ankara (MVA) strain, Lister strain, Tian Tan strain, Wyeth strain (New York City Board of Health strain), Western Reserve (WR) strain). The genome of WR vaccinia has been sequenced (accession number AY243312). In some embodiments, the oncolytic vaccinia virus is a Copenhagen strain, Modified Vaccinia Ankara (MVA) strain, Lister strain, Tian Tan strain, Wyeth strain, or Western Reserve (WR) strain vaccinia virus.

[0211] Different forms of virus particles have different roles in the viral life cycle. Several forms of virus particles exist: intracellular mature virus (IMV), intracellular enveloped virus (IEV), cell-associated enveloped virus (CEV), and extracellular enveloped virus (EEV). EEV particles have an outer membrane derived from the trans-Golgi network. This outer membrane has two important roles: a) to protect the internal IMV from immune attack, and b) to mediate virus attachment to the cell surface.

[0212] CEV and EEV are surrounded by host-derived membranes, which helps the virus evade host antibodies and complement. IMV and EEV particles have several differences in biological properties and play different roles in the viral life cycle. EEV and IMV bind to different (unknown) receptors (1) and enter cells by different mechanisms. EEV particles enter cells by endocytosis, a pH-sensitive process. After internalization, the outer membrane of EEV ruptures within acidified endosomes, exposing the IMV, which fuses with the endosomal membrane and releases the viral core into the cytoplasm. On the other hand, IMV enters cells by fusion of the viral membrane with the cellular membrane, a pH-independent process. In addition, CEV induces the formation of an actin tail from the cell surface, which guides the virion toward nearby uninfected cells.

[0213] Furthermore, EEV is resistant to neutralization by antibodies (NAb) and complement toxicities, whereas IMV is not. Therefore, EEV mediates widespread spread in vitro and in vivo. The comet inhibition test is one method for measuring EEV-specific antibodies. Even if free EEV cannot be neutralized by EEV NAb, EEV NAb blocks the release of EEV from infected cells, resulting in the disappearance of the comet-like pattern. EEV has a higher specific infectivity than IMV particles (which have a low particle / pfu ratio), making it an attractive candidate for therapeutic use. However, the EEV outer membrane is a very fragile structure, and EEV particles must be handled carefully, making it difficult to obtain EEV particles in the quantities required for therapeutic applications. The EEV outer membrane ruptures at low pH (approximately 6). Once the EEV outer membrane ruptures, the inner enveloped virus particles retain full infectivity as IMV.

[0214] Some host cell-derived proteins colocalize with EEV preparations but not with IEVs, and the amount of cellular proteins varies depending on the host cell line and virus strain. For example, WR EEV contains more cellular proteins than the IHD-J strain of VV. Host cell-derived proteins can modify the biological functions of EEV particles. For example, when the host membrane protein CD55 is introduced onto the surface of EEV, the surface becomes resistant to complement toxicity. In the present invention, we have shown that human A549 cell-derived proteins on the surface of EEV particles can target the virus to human cancer cells. A similar phenomenon has been demonstrated in studies with human immunodeficiency virus type 1, where the host-derived ICAM-1 glycoprotein increased viral infectivity. The IEV membrane contains at least nine proteins, two of which are absent in CEV / EEV. The F12L and A36R proteins are involved in transport of IEV to the cell surface; these proteins are left behind and do not become part of CEV / EEV (9, 11). Seven proteins, F13L, A33R, A34R, A56R, B5R, E2, and (K2L), are common to (IEV), CEV, and EEV. In the Western Reserve strain of vaccinia virus, typically up to 1% of virus particles are EEV, which are released into the culture supernatant before oncolysis of producer cells. The International Health Department (IHD)-J strain of vaccinia releases 50-fold more EEV particles. However, IHD has not been studied for use in human cancer therapy. The IHD-W phenotype was primarily due to a point mutation in the A34R lectin-like protein. Furthermore, deletion of A34R increases the number of released EEV. EEV particles can first be detected on the cell surface (as CEV) 6 hours after infection and in the supernatant 5 hours later (IHD-J strain). Infection at a low multiplicity of infection (MOI) results in a higher proportion of EEV than infection with a higher viral load. The balance between CEV and EEV is influenced by the host cell and the virus strain.

[0215] Vaccinia was used to eradicate smallpox and has since been used as an expression vector for foreign genes and as a live recombinant vaccine against infectious diseases and cancer. Vaccinia virus is the most widely used poxvirus in humans, and therefore there is ample safety data for its use in humans. In global smallpox vaccination programs, hundreds of thousands of people have been safely vaccinated with modified vaccinia virus strains, with only rare serious adverse events reported. These include systemic vaccinia (dissemination of vaccinia throughout the body), erythema multiforme (toxic / allergic reaction), eczema vaccinatum (widespread infection of the skin), progressive vaccinia (tissue destruction), and postvaccinial encephalitis.

[0216] In early clinical trials between the 1960s and 1990s, a total of 44 melanoma patients were treated with wild-type vaccinia virus, resulting in a 50% overall objective response rate of the inoculated tumor. Some beneficial immunological responses were also observed (36). Wild-type vaccinia virus has also been used to treat bladder cancer, lung cancer, kidney cancer, and myeloma with only minor adverse events. JX-594 (an oncolytic vaccinia virus, Wyeth strain, encoding GM-CSF) has been successfully evaluated in three phase 1 trials, and preliminary results from a randomized phase 2 trial have been presented at scientific meetings.

[0217] Vaccinia virus has several characteristics that make it an interesting virus for cancer gene therapy. Vaccinia virus has a natural tropism for cancer cells, and deleting some of its viral genes can significantly enhance selectivity. The present invention relates to the use of double-deleted vaccinia viruses (vvdd) in which two viral genes, viral thymidine kinase (TK) and vaccinia growth factor (VGF), have been at least partially deleted. The TK and VGF genes are required for viral replication in normal cells but are dispensable in cancer cells. Partial deletions of TK can be engineered in the TK region to confer activity.

[0218] TK-deleted vaccinia viruses depend on cellular nucleotide pools present in dividing cells for DNA synthesis and replication. In some embodiments, TK deletion significantly limits viral replication in quiescent cells, resulting in efficient viral replication only in actively dividing cells (e.g., cancer cells). VGF is secreted from infected cells and exerts a paracrine priming effect on surrounding cells by acting as a mitogen. Replication of VGF-deleted vaccinia viruses is significantly attenuated in quiescent (non-cancer) cells. The effects of TK and VGF deletion have been shown to be synergistic.

[0219] 2. Oncolytic adenovirus Generally, adenoviruses are 36 kb linear, double-stranded DNA viruses (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), AAV9_hu14, avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV. "Primate AAV" refers to an AAV that can infect primates, "non-primate AAV" refers to an AAV that can infect non-primate mammals, "bovine AAV" refers to an AAV that can infect bovine mammals, and so on.

[0220] Upon infection of host cells with adenovirus, adenoviral DNA is maintained episomally, thereby reducing the potential for genotoxicity associated with integrated vectors. Adenoviruses are also structurally stable, and no genome rearrangements have been detected, even after extensive amplification. Adenoviruses can infect virtually all epithelial cells, regardless of their cell cycle stage (see, e.g., US20060147420, incorporated herein by reference in its entirety). Furthermore, the adenoviral E1a and E4 regions are essential for efficient productive infection of human cells. The E1a gene is the first viral gene transcribed during productive infection, and its transcription does not depend on the action of any other viral gene products. However, expression of the E1a gene is required for transcription of the remaining early viral genes. In addition to regulating E1a gene expression, the E1a promoter also integrates signals for viral genome packaging and sites required for the initiation of viral DNA replication. See Schmid, S.I., and Hearing, P. in Current Topics in Microbiology and Immunology, vol. 199: pages 67-80 (1995).

[0221] In some embodiments, the oncolytic virus is an oncolytic adenovirus.It has been established that natural viruses can be engineered to produce oncolytic effects 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)).In the case of adenovirus, specific deletions in the adenovirus genome can reduce the virus's ability to replicate in normal resting cells, while maintaining its ability to replicate in tumor cells.One such conditionally replicating adenovirus, Δ24, has been described by Fueyo et al. (2000); see also US Patent Application Publication No. 20030138405 (each of which is incorporated herein by reference). The Δ24 adenovirus is derived from adenovirus type 5 (Ad-5) and contains a 24 base pair deletion in the CR2 portion of the E1A gene. See, e.g., WO2001036650A2 (incorporated herein by reference in its entirety).

[0222] Oncolytic adenoviruses include conditionally replicating adenoviruses (CRADs), such as Δ24, which possess several properties that make them candidates for use as biotherapeutics. One such property is the ability to replicate in permissive cells or tissues, amplifying the original input of the oncolytic virus and facilitating the spread of the agent to neighboring tumor cells, resulting in a direct antitumor effect.

[0223] In some embodiments, the oncolytic component of Δ24 is expressed via a transgene expression approach to produce armed Δ24. Armed Δ24 adenoviruses can be used to provide or enhance bystander effects within tumors and / or to detect / image or enhance oncolytic adenovirus in patients or tumor-associated tissues and / or tumor-associated cells. In some embodiments, combining oncolytic adenoviruses with various transgene strategies (e.g., expression of IL-2, IL-4, or IL-13 mutein bifunctional molecules) can improve therapeutic potential, including potential for various refractory tumors, as well as improve imaging capabilities. In certain embodiments, oncolytic adenoviruses can be administered with replication-deficient adenoviruses, other oncolytic viruses, replication-competent adenoviruses, and / or wild-type adenoviruses. Each virus can be administered simultaneously with, before, or after the other adenovirus.

[0224] In some embodiments, the E1a adenoviral vector comprises a basal adenoviral E1a promoter, including the CAAT box, TATA box, and start site for transcription initiation, replaced with a tumor-specific basal promoter, preferably E2F-responsive, more preferably the human E2F-1 promoter. Thus, the virus is suppressed in cells lacking or in which molecules that activate transcription from E2F-responsive promoters are nonfunctional. This is the case for normal, non-dividing, i.e., quiescent cells. Because the transcription factor E2F is bound to pRb, i.e., the retinoblastoma protein, E2F is unavailable to bind to and activate E2F-responsive promoters. In contrast, cells containing free E2F should be capable of E2F-driven transcription. An example of such a cell is a neoplastic cell lacking pRb function and capable of productive viral infection. In some embodiments, the E1a adenoviral vector is targeted using an IL-2 moiety, as described herein.

[0225] Retention of the enhancer sequence, packaging signal, and DNA replication initiation site in the E1a promoter allows adenoviral infection to proceed to wild-type levels in neoplastic cells lacking pRb function. Essentially, the modified E1a promoter confers tumor-specific transcriptional activation, resulting in sufficient tumor-specific killing and enhanced safety in normal cells.

[0226] In some embodiments, E1a adenoviral vectors are prepared by replacing the endogenous E1a promoter with an E2F-responsive promoter, while leaving intact the element upstream of nucleotide 375 in the adenovirus type 5 genome. The nucleotide numbering is as described in Schmid, S.I., and Hearing, P., Current Topics in Microbiology and Immunology, vol. 199: pages 67-80 (1995). This element contains all seven A repeat motifs identified for viral genome packaging. The sequence from nucleotide 375 to nucleotide 536 is deleted using the BsaAI or BsrBI restriction initiation site, while retaining the 23 base pairs upstream of the translation initiation codon of the E1A protein. 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 may be isolated as described in the Examples.

[0227] The E4 region is involved in many of the events that occur late in adenovirus infection and is required for efficient viral DNA replication, late mRNA accumulation and protein synthesis, splicing, and cessation of host cell protein synthesis. Adenoviruses lacking most of the E4 transcription unit are severely replication-deficient and generally require growth in E4-complementing cell lines to achieve 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 essential for mediating maximal transcriptional activity have been characterized in this promoter. In addition to these sequences, the E4 promoter region contains regulatory sequences necessary for viral DNA replication. A description of the E4 promoter and the location of these regulatory sequences can be found in Figures 2 and 3 of U.S. Pat. No. 7,001,596, incorporated herein by reference in its entirety.

[0228] In some embodiments, the adenoviral vector has the E4 basal promoter replaced with a promoter that has been shown to exhibit tumor specificity, preferably an E2F-responsive promoter, more preferably the human E2F-1 promoter.The reason why the E2F-responsive promoter is preferred for driving E4 expression is the same as that discussed above for the E1a adenoviral vector in which the E1a promoter is replaced with an E2F-responsive promoter. The tumor suppressor function of pRb correlates with its ability to repress E2F-responsive promoters, such as the E2F-1 promoter (Adams, P.D., and W.G. Kaelin, Jr. 1995, Cancer Biol. 6:99-108; Sellers, W.R., and W.G. Kaelin. 1996, published erratum appears in Biochim Biophys Acta 1996 Dec. 9;1288(3), E-1, Biochim Biophys Acta. 1288:M1-5; Sellers, W.R., J.W. Lodgers, and W.G. Kaelin, Jr. 1995, Proc Natl Acad Sci USA. 92:11544-8). The human E2F-1 promoter has been extensively characterized and shown to respond to the pRb signaling pathway, including pRb / p107, E2F-1 / -2 / -3 and G1 cyclin / cdk complexes, and 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, JAM McNeil, JB Lawrence, and WG Kaelin, Jr. 1996, Gene. 173:163-9). This regulation is largely, if not entirely, due to the presence of multiple E2F sites within the E2F-1 promoter.Thus, viruses with this modification (or these modifications) are expected to be attenuated in normal cells containing an intact (wild-type) pRb pathway, but to exhibit a normal infection / replication profile in cells lacking the inhibitory function of pRb.To maintain the normal infection / replication profile of this mutant virus, we retained the inverted terminal repeats (ITRs) at the distal end of the E4 promoter. The ITRs contain all the regulatory elements required for viral DNA replication (Hatfield, L. and P. Hearing. 1993, J. Virol. 67:3931-9; Rawlins, D.R., P.J. Rosenfeld, R.J. Wides, M.D. Challberg, and T.J. Kelly, Jr. 1984, Cell. 37:309-19; Rosenfeld, P.J., E.A.O. Neill, R.J. Wides, and T.J. Kelly. 1987, Mol. Cell. Biol. 7:875-86; Wides, R.J., M.D. Challberg, D.R. Rawlins, and T.J. Kelly. 1987, Mol. Cell. Biol. 7:864-74), which facilitates the achievement of wild-type levels of virus in tumor cells infected with this virus that lack the pRb pathway.

[0229] In some embodiments, the E4 promoter is located near the right end of the viral genome and controls transcription of multiple open reading frames (ORFs) (Freyer, G.A., Y. Katoh, and R.J. Roberts. 1984, Nucleic Acids Res. 12:3503-19; Tigges, M.A., and H.J. 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. LeMoullec, U. Pettersson, and M. Perricaudet. 1984, J. Virol. 51:822-31). Many regulatory elements that mediate transcriptional activity in this promoter have been characterized (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, PA 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 contains elements involved in viral DNA replication (Hatfield, L., and P. Hearing. 1993, J. Virol. 67:3931-9; Rawlins, D.R., P.J. Rosenfeld, R.J. Wides, M.D. Challberg, and T.J. Kelly, Jr. 1984, Cell. 37:309-19; Rosenfeld, P.J., E.A.O. Neill, R.J. Wides, and T.J. Kelly. 1987, Mol. Cell. Biol. 7:875-86; Wides, R.J., M.D. Challberg, D.R. Rawlins, and T.J. Kelly. 1987, Mol. Cell. Biol. 7:864-74). A description of the E4 promoter and the location of these regulatory sequences can be seen in Figures 1 and 2. See also Jones, C., and KALee. Mol Cell Biol. 11:4297-305 (1991). Taking these considerations into account, we designed the E4 promoter shuttle by creating two novel restriction enzyme 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 removes nucleotides 35,581 to 35,817. This effectively eliminates bases −208 to +29 relative to the E4 transcription start site, including all sequences known to have the greatest effect on E4 transcription. Notably, this sequence contains the two inverted repeats of the E4F binding site, which have been shown to have the greatest 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 essential for viral DNA replication, are retained.

[0230] In some embodiments, the E2F-responsive promoter is the human E2F-1 promoter. Key regulatory elements in the E2F-1 promoter that mediate response to the pRb pathway have been mapped both in vitro and in vivo (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 incorporated SpeI and XhoI sites at those base pairs, creating the same sites present in the E4 promoter shuttle and allowing direct replacement of the E4 promoter with the E2F-1 promoter. F. Nucleic Acid Molecules Encoding Variant IL-2, IL-4, and / or IL-13

[0231] In some embodiments, the IL-2, IL-4, or IL-13 muteins of the present invention can be obtained by expression of nucleic acid molecules, either alone or as part of a bifunctional molecule (including a bispecific IL-2 cytokine fusion) as described above. Just as IL-2, IL-4, or IL-13 mutein bifunctional molecules can be described in terms of their identity to a wild-type IL-2 polypeptide, nucleic acid molecules encoding IL-2 muteins will necessarily have a certain identity to nucleic acid molecules encoding wild-type IL-2. For example, nucleic acid molecules encoding IL-2 muteins of the present invention 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).

[0232] In some embodiments, the IL-2 muteins of the present invention can be obtained by expression of nucleic acid molecules, alone or as part of chimeric polypeptides (including bispecific IL-4 cytokine fusions) as described above. Just as IL-4 muteins can be described in terms of their identity to wild-type IL-4 polypeptides, nucleic acid molecules encoding IL-4 muteins will necessarily have a certain identity to nucleic acid molecules encoding wild-type IL-2. For example, nucleic acid molecules encoding IL-4 muteins of the present invention 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 nucleic acids encoding wild-type IL-4 (e.g., SEQ ID NO: 129).

[0233] In some embodiments, the IL-13 muteins of the present invention can be obtained by expression of a nucleic acid molecule, alone or as part of a chimeric polypeptide (including a bispecific IL-13 cytokine fusion) as described above. As IL-13 muteins can be described in terms of their identity to a wild-type IL-13 polypeptide, a nucleic acid molecule encoding an IL-13 mutein will necessarily have a certain identity to a nucleic acid molecule encoding wild-type IL-13. For example, a nucleic acid molecule encoding an IL-13 mutein of the present invention 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).

[0234] The provided nucleic acid molecules can include naturally occurring sequences or sequences that differ from sequences found in nature but, due to the degeneracy of the genetic code, encode the same polypeptide. 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 the nucleotides within these types of nucleic acids. In addition, the nucleic acid molecules can be double-stranded or single-stranded (i.e., either the sense or antisense strand).

[0235] The nucleic acid molecule is not limited to a sequence encoding a polypeptide, but can also include some or all of the non-coding sequences upstream or downstream from the coding sequence (e.g., the coding sequence for IL-2, IL-4, or IL-13). Those skilled in the art of molecular biology are familiar with conventional procedures for isolating nucleic acid molecules. These molecules can be prepared, for example, by treating genomic DNA with restriction enzymes or by performing the polymerase chain reaction (PCR). If the nucleic acid molecule is ribonucleic acid (RNA), the molecule can be prepared, for example, by in vitro transcription.

[0236] Exemplary isolated nucleic acid molecules of the present disclosure can include fragments not found in nature, etc. That is, the present disclosure includes recombinant molecules 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 its natural chromosomal location).

[0237] As described above, the IL-2, IL-4, or IL-13 mutein bifunctional molecules of the present invention may be present as part of a chimeric polypeptide. In addition to, or instead of, the heterologous polypeptides described above, the nucleic acid molecules of the present invention may contain sequences encoding a "marker" or "reporter." Examples of marker or reporter genes include β-lactamase, chloramphenicol acetyltransferase (CAT), adenosine deaminase (ADA), aminoglycoside phosphotransferase (neo), and the like. 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 recognize additional useful reagents, for example, additional sequences that can serve the function of a marker or reporter.

[0238] The nucleic acid molecules of the present invention can be obtained by introducing mutations into DNA encoding IL-2 obtained from any living cell (e.g., a mammalian cell). That is, the nucleic acids of the present invention (and the polypeptides they encode) can be from a mouse, rat, guinea pig, cow, sheep, horse, pig, rabbit, monkey, baboon, dog, or cat. In one embodiment, the nucleic acid molecule is a human molecule.

[0239] G. Chimeric Antigen Receptor (CAR) Targeted immunotherapy has emerged as a promising area of ​​research in the treatment of malignant tumors and has attracted significant interest in recent years. Indeed, engineered or genetically modified T cells targeting CD19 malignant cells have been reported to cure lymphoma patients. This has led to increased interest in 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 mutein bifunctional molecules described herein to tumors, or even to express systemic IL-2, IL-4, or IL-13 mutein bifunctional molecules. In some embodiments, the IL-2, IL-4, or IL-13 mutein bifunctional molecule is any IL-2, IL-4, or IL-13 mutein bifunctional molecule or variant disclosed herein. In some embodiments, the sequence of the IL-2 mutein is 90% identical to any one of SEQ ID NO:2 or SEQ ID NO:6-10 or SEQ ID NO:16. In some embodiments, the IL-2 mutein comprises any one of SEQ ID NO:5 for 5-1, SEQ ID NO:6 for 5-2, SEQ ID NO:7 for 6-6, SEQ ID NO:8 for A2, SEQ ID NO:9 for B1, SEQ ID NO:10 for B11, SEQ ID NO:11 for C5, SEQ ID NO:12 for D10, SEQ ID NO:13 for E10, SEQ ID NO:14 for G8, SEQ ID NO:15 for H4, and SEQ ID NO:16 for H9. In some embodiments, substitutions in the IL-2 mutein include L80F, R81D, L85V, I86V, and I92F (numbered relative to wild-type human IL-2 of SEQ ID NO:2). In some embodiments, the bifunctional molecule is selected from the group consisting of SEQ ID NOs: 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, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381 1, 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, 48 24, 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, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467 7, 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.

[0240] Genetic engineering of autologous or allogeneic T cells, NK cells, macrophages, gamma delta T cells, or NKT cells to specifically target specific tumor antigens provides a strategy for circumventing the ineffective induction of cytotoxic immune responses by most tumor cells. In some embodiments, these genetically engineered T cells or NK cells can be used to target the IL-2, IL-4, or IL-13 mutein bifunctional molecules described herein to tumors, e.g., so that the IL-2, IL-4, or IL-13 mutein bifunctional molecules are expressed at the tumor site. These techniques are based on the genetic modification of human immune cells, which may be harvested from patients or donors by leukapheresis. Specific cells, usually T cells, are purified and engineered to express receptors that target cancer antigens of interest. Engineering may involve transduction via retroviruses, lentiviruses, transposons, mRNA electroporation, or the like. Immune cells may be expanded to a desired number and introduced into a patient, where the engineered cells can specifically kill cancer cells through cell-mediated cytotoxicity (cytotoxic T cells) and / or induce an immune response against cancer cells through immune recognition of tumors, release of cytokines, and recruitment of immune cells.

[0241] For example, the application of chimeric antigen receptors (CARs) in immunogene therapy for malignant tumors is a promising approach. In these CARs, an antibody or ligand-binding domain is fused to the zeta signaling chain of the T cell receptor. The resulting CAR immune cells are redirected with their newly acquired specificity to attack tumors expressing the surface antigen or receptor recognized by the genetically engineered T cell receptor, and the CAR immune cells then act as a cellular therapy that attacks tumors through a highly regulated normal host immune response. These cells circulate freely throughout the cerebral and systemic circulation, reducing the need for colocalization and bioavailability.

[0242] Several generations of CAR immune cells have been developed. CARs are created by fusing a tumor-specific scFv antibody or other extracellular ligand-binding domain to either the CD3ζ signaling domain associated with a TCR or another intracellular signaling domain derived from a costimulatory protein receptor. This architecture allows CARs to possess the tumor specificity of a B cell antigen receptor and activate T cells through the T cell antigen receptor, independent of MHC binding. First-generation CARs contained a single intracellular signaling domain, typically along with a CD3ζ signaling domain, to enable TCR signaling. Second-generation CARs have two intracellular signaling domains: a costimulatory domain containing either a CD28 signaling domain or a 4-1BB signaling domain, combined with a CD3ζ signaling domain. This configuration allows antigen recognition by the scFv region of the CAR to activate and proliferate T cells. Third-generation CARs have two costimulatory domains and a CD3ζ signaling domain. The first costimulatory domain is either a CD28 domain or a 4-1BB domain, and the second costimulatory domain is either a CD28 domain, a 4-1BB domain, or an OX40 domain. In fourth-generation "armored CAR T cells," second-generation CARs are combined with various additional genes, including cytokines, and costimulatory ligands to enhance the tumoricidal activity of CAR T cells. See, e.g., 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 entire contents of which are incorporated herein by reference.

[0243] An alternative approach to targeting T cells includes T cell antigen couplers, such as those described in International Publication No. WO 2015 / 117229, entitled "Trifunctional T cell antigen coupler and methods and uses thereof," specifically incorporated herein by reference. T cell antigen coupler systems contain three linked domains: a target-specific polypeptide ligand, a ligand that binds to a protein associated with the TCR complex, e.g., an scFv that binds to CD3 (TCR, T cell receptor), to stimulate T cell activation, and a T cell receptor signaling domain, e.g., the CD4 transmembrane intracellular domain, which amplifies T cell activation. TACs are engineered to work with the intrinsic molecular machinery of T cells by stimulating T cell activation through the TCR.

[0244] Antibodies coupled to T cell receptors are another approach to targeting T cells. ACTR is a hybrid approach to oncology medicine that combines CARs and established monoclonal antibodies. 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. ACTR-T cells can target tumors by binding to ligands targeted to specific cancer antigens. T cell activation is driven by the CAR module.

[0245] In some embodiments, platforms that use directed evolution in conjunction with yeast display result in tunable superkines. In some embodiments, such platforms generate extensive libraries of IL-2, IL-4, and IL-13 superkines with unique properties. In some embodiments, MDNA109 is an engineered version of human IL-2 with enhanced agonist activity. In some embodiments, the MDNA109 family of "IL-2 superkines" has been engineered to have improved PK properties and enhanced selectivity to further improve the therapeutic window.

[0246] In some embodiments, MDNA11 is a "beta-only" superkine with uniquely enhanced affinity for CD122. In some embodiments, MDNA11 preferentially stimulates immune effector cells. In some embodiments, MDNA11 shows anti-tumor efficacy in monotherapy and in combination with anti-PD1 in the MC38 tumor model. In some embodiments, MDNA11 in conjunction with anti-CTLA4 induces tumor regression, protects against rechallenge, and promotes antigen-specific CD8 T cells. In some embodiments, MDNA11 induces tolerance and sustained proliferation and expansion of immune effector cells in NHPs, but not T regs So we will not induce you.

[0247] In some embodiments, Superkine Targeted with Antibody (STAb) enhances accumulation in tumors. In some embodiments, STAb overcomes checkpoint resistance and "cold" tumors.

[0248] In some embodiments, IL-4 receptors and IL-13 receptors play important roles in cancer. In some embodiments, MDNA55 is an enhanced superkine with a potent payload that targets type 2 IL-4R expressed on tumor cells and the tumor microenvironment (MDSCs and TAMs). In some embodiments, MDNA413 is a superantagonist that blocks IL-4 and IL-13 signaling through type 2 IL-4R, suppressing MDSCs and TAMs. In some embodiments, MDNA132 is a superkine that selectively targets decoy IL-13Rα2, which is overexpressed on solid tumors. In some embodiments, MDNA132 is an engineered version of human IL-13 that targets tumor-specific antigens. In some embodiments, MDNA132 plays a role in localizing T cell engagers and checkpoint inhibitors to tumors.

[0249] 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.

[0250] In some embodiments, the bispecific cytokine (DUCK Cancer) is MDNA109FEAA-Fc-MDNA413, which has a mechanism of action as shown in Figure 70 of WO2021258213, which is incorporated by reference herein in its entirety.

[0251] Biologics that selectively alter the activity of IL-13 are of interest for many therapeutic purposes, including the treatment of certain cancers by manipulating T cell specificity. The present invention addresses this problem.

[0252] Methods and compositions are provided for enhancing anti-tumor immune effector cells, e.g., T cells, NK cells, etc., by targeting compositions including, but not limited to, chimeric antigen receptors (CARs), T cell antigen couplers (TACs), antibody-binding T cell receptors (ACTRs), and bispecific T cell engagers (BiTEs), where the IL-13 superkine or IL-4 superkine provides the target-specific ligand. In further embodiments, the immune effector cells express IL-2, IL-4, or IL-13 mutein bifunctional molecules.

[0253] Immune cell targeting or expression constructs comprising an IL-2, IL-4, or IL-13 mutein bifunctional molecule sequence are provided, and the constructs can include any IL-2, IL-4, or IL-13 mutein bifunctional molecule sequence as described herein. Superkines are useful for targeting immune cells to cells expressing at least one receptor, such as tumor cells. In some embodiments, the IL-2, IL-4, or IL-13 mutein bifunctional molecule comprises any IL-2, IL-4, or IL-13 mutein bifunctional molecule or variant disclosed herein. In some embodiments, the sequence of the IL-2 mutein is 90% identical to any one of SEQ ID NO:2, SEQ ID NO:6-SEQ ID NO:10, or SEQ ID NO:16. In some embodiments, the IL-2 mutein comprises any one of SEQ ID NO: 5 for 5-1, SEQ ID NO: 6 for 5-2, SEQ ID NO: 7 for 6-6, SEQ ID NO: 8 for A2, SEQ ID NO: 9 for B1, SEQ ID NO: 10 for B11, SEQ ID NO: 11 for C5, SEQ ID NO: 12 for D10, SEQ ID NO: 13 for E10, SEQ ID NO: 14 for G8, SEQ ID NO: 15 for H4, and SEQ ID NO: 16 for H9. In some embodiments, substitutions in the IL-2 mutein comprise L80F, R81D, L85V, I86V, and I92F (numbered relative to wild-type human IL-2 of SEQ ID NO: 2). In some embodiments, the bifunctional molecule is selected from the group consisting of SEQ ID NOs: 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, 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, 03, 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.

[0254] The IL-2, IL-4, or IL-13 mutein component of the bifunctional molecular construct can be at least about 50 amino acids in length, at least about 75 amino acids in length, at least about 100 amino acids in length, at least about 110 amino acids in length, at least about 115 amino acids in length, up to the full length of the wild-type protein in the transmembrane domain, i.e., about 116 amino acids in length. For example, the superkine or mutein can be fused to the hinge domain, transmembrane domain, or signaling domain of a CAR. Exemplary polypeptide sequences are shown.

[0255] Included as IL-2, IL-4, or IL-13 superkines or muteins are amino acid sequences and nucleic acid coding sequences that are 90%, 95%, 98%, or 99% identical to these sequences, longer sequences that include these sequences but also include additional nucleotides at the 3' or 5' end, e.g., any number of additional nucleotides or codons (e.g., 3, 6, 9, or 12 or more nucleotides, or up to about 12, 20, 50, or 100 additional nucleotides), as well as any sequences that, due to the degeneracy of the genetic code, encode 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 contemplated as part of the 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 polypeptide is linked to an IL-2, IL-4, or IL-13 mutein comprising immune cell targeting construct or expression construct. In some embodiments, the IL-2, IL-4, or IL-13 mutein comprising immune cell targeting construct or expression construct comprises one or more signaling domains derived from CD3-zeta, CD28, DAP10, OX-40, ICOS, and CD137. In some embodiments, the IL-2, IL-4, or IL-13 mutein comprising immune cell targeting construct or expression construct, or expression, comprises one or more signaling domains derived from CD3-zeta. In some embodiments, the IL-2, IL-4, or IL-13 mutein comprising immune cell targeting construct or expression construct comprises one or more signaling domains derived from CD28. In some embodiments, the IL-2, IL-4, or IL-13 mutein comprising immune cell targeting constructs or expression constructs comprise one or more signaling domains derived from DAP10.In some embodiments, the IL-2, IL-4, or IL-13 mutein comprising immune cell targeting constructs or expression constructs comprise one or more signaling domains derived from OX-40. In some embodiments, the IL-2, IL-4, or IL-13 mutein comprising immune cell targeting constructs or expression constructs comprise one or more signaling domains derived from CD137. In some embodiments, the IL-2, IL-4, or IL-13 mutein comprising immune cell targeting constructs or expression constructs comprise an IL-2 variant / IL-2 superkine, an IL-4 variant / IL-4 superkine, or an IL-13 variant / IL-13 superkine, including those set forth herein. In some embodiments, the IL-2, IL-4, or IL-13 mutein containing immune cell targeting construct 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 those set forth in SEQ ID NOs: 1-495 or 501-508.

[0256] 1.NK cells In some embodiments, the immune cells are natural killer (NK) cells. NK cells recognize infected or transformed cells through multiple cell surface receptors, including NKG2D, CD16, and natural killer cell cytotoxicity receptors (NCRs) (such as NKp44, NKp46, and NKp30). These receptors activate signaling adapter proteins, such as DAP10, DAP12, and CD3ζ, which contain immunoreceptor tyrosine-based activation motifs (ITAMs), which initiate the release of cytotoxic granules, including perforin and granzymes, and mediate the production and release of cytokines and chemokines, such as IFN-γ and TNF-α. Importantly, NK cell-mediated cytotoxicity is independent of autologous HLA presentation. Therefore, NK cells remain of great clinical interest as cell-based cancer therapies due to their potential for use in allogeneic settings and the potential for off-the-shelf cell products.

[0257] Natural killer cells offer an alternative to the use of T cells in adoptive immunotherapy because natural killer cells can be used as allogeneic effector cells without the need for HLA matching. Clinical trials of adoptively transferred allogeneic NK cells have shown that these cells can survive for weeks to months in patients. In addition, expressing CARs on NK cells enables these cells to kill solid tumors, which are often resistant to NK cell-mediated activity, more effectively than hematologic malignancies (particularly acute myeloid leukemia), which are typically more sensitive to NK cells. CARs useful for targeting NK cells include, for example, first-generation CAR constructs containing CD3ζ as the sole signaling domain. Second- and third-generation CARs are also useful in NK cells. In some embodiments, the extracellular domain of NKG2D (an NK cell-activating receptor) is directly linked to CD3ζ.

[0258] NK cells for modification include cell lines or peripheral blood NK cells, which can be isolated from donors through simple blood draws or, if more cells are needed, by apheresis. Activated PB-NK cells express a wide range of activating receptors, such as CD16, NKp44, and NKp46, as well as KIR (which play an important role in NK cell licensing). In addition, PB-NK cells have the ability to expand in vivo, allowing them to be administered without irradiation. Another source of NK cells suitable for expressing CARs are NK cells derived from human pluripotent stem cells (both induced pluripotent stem cells (iPSCs) or human embryonic stem cells (hESCs)). These NK cells exhibit a phenotype similar to that of PB-NK cells, and hESC / iPSC-NK cells can be grown on a clinical scale.

[0259] 2. Chimeric antigen receptor (CAR) In addition to the superkine sequence, CARs contain the signaling domain of CD3ζ and one or more costimulatory receptors that further promote the recycling, survival, and / or expansion of CAR-expressing immune cells. The costimulatory receptor signaling domain is the intracellular portion of each receptor protein that generates an activation signal in the cell. Examples are amino acids 180-220 of the native CD28 molecule and amino acids 214-255 of the native 4-1BB molecule.

[0260] Examples of suitable hinge and transmembrane regions linking the superkine to the signaling domain may include, but are not limited to, the constant (Fc) region of immunoglobulin, human CD8a, and artificial linkers that serve to move the targeting moiety away from the cell surface for improved access and binding to the target cell. Examples of suitable transmembrane domains include the transmembrane domains of leukocyte CD markers, preferably the transmembrane domains of CD4 or CD28. Examples of intracellular receptor signaling domains include the zeta chain of the T cell antigen receptor complex, preferably CD3, although any transmembrane region sufficient to anchor the CAR within the membrane can be used. Those skilled in the art are aware of numerous transmembrane regions and structural elements (such as lipophilic amino acid regions) that make up transmembrane domains in numerous membrane proteins, and therefore any convenient sequence can be substituted. Examples of T cell costimulatory signaling receptors suitable for improving the function and activity of CAR-expressing cells include, but are not limited to, CD28, CD137, and OX-40.

[0261] Signaling through CD28 is necessary for the production and proliferation of IL2, IL-4, or IL-13, but does not play a central role in sustaining T cell function and activity. CD137 (a tumor necrosis factor receptor family member expressed after CD28 activation) and OX-40 are involved in promoting long-term T cell survival and T cell accumulation. The ligands for these receptors are typically expressed on professional antigen-presenting cells such as dendritic cells and activated macrophages, but not on tumor cells. CD4 + Expression of CARs incorporating the CD28 and / or 4-1BB signaling domains in T cells enhances the activity and antitumor efficacy of these cells compared to cells expressing CARs containing only the CD3ζ signaling domain (these constructs are sometimes referred to as second- or third-generation CARs).

[0262] CAR constructs of interest include tandem CARs, see, e.g., Hegde et al. (2016) J. Clin. Invest 126(8):3036-3052 (specifically incorporated herein by reference). In such constructs, a binding moiety for a tumor-specific antigen is combined in tandem with the IL-13 superkine. The binding moiety can be, for example, an scFv specific for a tumor cell antigen, including, but not limited to, HER-2, EGFR, CD20, etc., as known in the art.

[0263] 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 include D19, CD123, CD22, CD30, CD171, CS-1 (also known as CD2 subset 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 (testisin 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), prostase, prostatic acid phosphatase (PAP), elongation factor 2 mutated (ELF2M), ephrin B2, fibroblast activation protein alpha (FAP), insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX), proteasome (prosome, macropenetrance) subunit beta type 9 (LMP2), glycoprotein 100 (gp100),Oncogene fusion protein consisting of breakpoint cluster region (BCR) and Abelson murine leukemia viral 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 β, tumor Endothelial marker 1 (TEM1 / CD248), tumor endothelial marker 7-related (TEM7R), claudin 6 (CLDN6), thyroid-stimulating hormone receptor (TSHR), G protein-coupled receptor class C group 5 member D (GPRC5D), X chromosome open reading frame 61 (CXORF61), CD97, CD179a, anaplastic lymphoma kinase (ALK), polysialic acid, placenta-specific 1 (PLAC1), hexasaccharide moiety of globoH glycoceramide (GloboH), mammary differentiation antigen (NY-BR-1), uroplakin 2 (UPK2), and hepatitis A viral cell receptor 1 (HAVCR1), adrenergic receptor beta 3 (ADRB3), pannexin 3 (PANX3), G protein-coupled receptor 20 (GPR20), lymphocyte antigen 6 complex locus K9 (LY6K), olfactory receptor 51E2 (OR51E2), TCR gamma alternative 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 (ETV) located on chromosome 12p 6-AML), sperm protein 17 (SPA17), X antigen family member 1A (XAGE1), angiopoietin-binding cell surface receptor 2 (Tie2), 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 mutant, prostein, survivin, telomerase, prostate cancer tumor antigen-1 (PCTA-1 or galectin 8), melanoma antigen recognized by T cells 1 (MelanA or MART1), rat sarcoma (Ras) mutant,Human telomerase reverse transcriptase (hTERT), sarcoma translocation breakpoints, melanoma inhibitor of apoptosis (ML-IAP), ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene), N-acetylglucosaminyl-transferase V (NA17), paired box protein Pax-3 (PAX3), androgen receptor, cyclin B1, v-myc avian myelocytoma viral oncogene neuroblastoma-derived homolog (MYCN), Ras homolog family member C (RhoC), tyrosinase-related protein 2 (TRP-2), cytochrome P4501B1 (CYP1B1), CCCTC-binding factor (zinc finger protein)-like (BORIS, i.e., Brother of the Regulator of Imprinted Proteins). Sites), squamous cell carcinoma antigen recognized by T cells 3 (SART3), paired box protein Pax-5 (PAX5), proacrosin-binding protein sp32 (OY-TES1), lymphocyte-specific protein tyrosine kinase (LCK), A-kinase anchor protein 4 (AKAP-4), synovial sarcoma X breakpoint 2 (SSX2), receptor for advanced glycation end products (RAGE-1), renal ubiquitous 1 (RU1), renal ubiquitous 2 (RU2), legumain, human papillomavirus E6 (HPV E6), human papillomavirus E7 (HPV E7), intestinal carboxylesterase, heat shock protein 70-2 mutant (mutated) hsp70-2), CD79a, CD79b, CD72, leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), Fc fragment of 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), EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2), lymphocyte antigen 75 (LY75), glypican-3 (GPC3), Fc receptor-like 5 (FCRL5), and immunoglobulin lambda-like polypeptide 1 (IGLL1).

[0264] In some embodiments, the antigen binding domain comprises a monoclonal antibody, a polyclonal antibody, a synthetic antibody, a human antibody, a humanized antibody, a non-human antibody, a nanobody, a single chain variable fragment (scFv), F(ab')2, Fab', Fab, Fv, etc. The antigen binding domain can be linked to a transmembrane domain of a CAR. In some embodiments, a nucleic acid encoding the antigen binding domain is operably linked to a nucleic acid encoding the transmembrane domain of a CAR.

[0265] In some embodiments, the transmembrane domain can be derived from a membrane-associated protein or a transmembrane protein. In certain embodiments, the transmembrane domain comprises one or more, e.g., 1, 2, 3, 4, 5, 6, 7, or 8 or more amino acid modifications (e.g., substitutions, insertions, and deletions) compared to the wild-type amino acid sequence of the transmembrane domain of the membrane-associated protein or transmembrane protein. Non-limiting examples of transmembrane domains of CARs include at least the transmembrane region(s) of the α, β, or ζ chain of the T cell receptor, CD28, CD3 empsilon (CD3ξ), CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or the erythropoietin receptor. In some embodiments, the transmembrane domain comprises a human immunoglobulin (Ig) hinge region, e.g., an IgG4 Fc hinge. In another embodiment, the transmembrane domain is a recombinant or synthetic domain that includes hydrophobic amino acid residues (e.g., leucine and valine). Optionally, the transmembrane domain includes phenylalanine, tryptophan, and valine at one or both ends of the domain.

[0266] The transmembrane domain links 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 a nucleic acid encoding the transmembrane domain, which is operably linked to the nucleic acid encoding the intracellular signaling domain.

[0267] In some embodiments, the intracellular signaling domain of the CAR comprises a signal activation domain or signal transduction domain. Thus, the intracellular signaling domain includes any portion of the intracellular signaling domain of a protein that is sufficient to transmit or deliver a signal, e.g., an activation signal, or mediate a cellular response within a 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, LIGHT , NKG2C, B7-H3, ligands that specifically bind 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, ITGAD , 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(CD229), CD 160 (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, NKp44, NKp30, NKp46, NKG2D, and derivatives, variants, or fragments of any of these.In certain embodiments, the intracellular signaling domain comprises the intracellular domain of a costimulatory molecule such as CD3, CD27, CD28, CD127, ICOS, 4-1BB (CD137), PD-1, a T cell receptor (TCR), any derivative or any variant thereof. In some embodiments, the intracellular signaling domain of the CAR comprises the intracellular domain of an MHC class I molecule, a TNF receptor protein, an immunoglobulin-like protein, a cytokine receptor, an integrin, a signaling lymphocyte activation molecule (SLAM protein), an activating NK cell receptor, BTLA, a Toll ligand receptor, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD5, ICAM-1, LFA-1 (CD11a / CD6), or a CAR-like protein. 18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLR F1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, V LA-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), CEACAM 1, 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 a ligand that specifically binds to CD83.

[0268] 3.ICE Targeted therapies have been developed against IL13Rα2, including IL13-conjugated bacterial toxins, nanoparticles, oncolytic viruses, and immunotherapy using monoclonal antibodies, IL13Rα2-pulsed dendritic cells, and IL13Rα2-targeted chimeric antigen receptors (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 embodiments, these targeted therapies can be used to deliver IL-2, IL-4, or IL-13 mutein bifunctional molecules to tumors.

[0269] ICE (IL-13-mediated cell engager) comprises a bispecific T cell engager (BiTE), 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 a single-chain variable fragment (scFv). The superkine may be fused to the variable region via a linker. An Fc region is optionally provided. ICE molecules can interact with and bind to T cells, NK cells, NKT cells, dendritic cells (DCs), and / or macrophage cells.

[0270] Bispecific T cell engagers (BiTEs) are bispecific antibodies that can bind the TCR of a T cell to a target tumor cell through two modules: a cancer-targeting ligand and an scFv domain of CD3-, CD28-, CD137-, OX-40-, ICOS-, or DAP10-binding domain that bridges T cells to tumors. In some embodiments, bispecific fusion proteins containing superkines are sometimes referred to as Bi-functional SuperKines as ImmunoTherapies (BiSKIT). In some embodiments, novel interleukin superagonists, partial agonists, and superantagonists are designed using directed evolution. In some embodiments, rational approaches are used to further engineer long-acting IL-2, IL-4, and IL-13 superkines, including the bifunctional molecules described herein, without blocking functional activity.

[0271] 4.TAC The TAC construct comprises an IL-2 superkine, IL-4 superkine, or IL-13 superkine fused to a ligand that binds to a protein associated with the TCR complex, the ligand being fused to a T cell receptor signaling domain polypeptide. These domains may be separated by a linker. The protein associated with the TCR complex may be CD3. The ligand that binds to a protein associated with the TCR complex may be a single-chain antibody. The ligand that binds to 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 the cytoplasmic domain of CD4, and the transmembrane domain is the transmembrane domain of CD4.

[0272] 5.ACTR ACTR is a hybrid approach to CAR and established monoclonal antibody-based oncology medicine. 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. A superkine is fused to a moiety recognized by the CAR, which may include, but is not limited to, the Fc region of an antibody with high affinity for CD16.

[0273] Immune cell targeting constructs or expression constructs encoding sequences can be produced by any means known in the art, including recombinant DNA techniques. Nucleic acids encoding some of the nucleic acid regions of the chimeric receptor can be conveniently prepared and assembled into complete coding sequences by standard molecular cloning techniques known in the art (e.g., screening of genomic libraries, PCR, primer ligation, site-directed mutagenesis, etc.). The resulting coding regions can be inserted into expression vectors and used to transform appropriate expression host cell lines, such as populations of allogeneic or autologous T lymphocytes, allogeneic or autologous NK cells (including primary cultures, cell lines, iPSC-derived cells, etc.). This method can be used with cells in vitro (e.g., in a cell-free system) or in culture, e.g., in vitro or ex vivo. For example, cells expressing IL-2, IL-4, or IL-13 superkine CARs can be cultured and expanded in vitro in a culture medium.

[0274] Any non-IL-2 superkine immune cell targeting or expression construct, including any IL-4 or IL-13, including any of the molecules described herein, can also be used to specifically induce immune cells to target specific tumor cells. Anti-tumor effector cells, e.g., CD4, can be targeted by introducing into T cells superkine immune cell targeting or expression constructs containing one or more signaling domains from CD3-zeta, CD28, DAP10, OX-40, ICOS, and CD137. + or CD8 + Effector T cells are generated and redirected to recognize tumor cells as described above. In some embodiments, the cells can further comprise a transgene capable of expressing an IL-2 mutein as described herein. IL-2, IL-4, or IL-13 superkine immune cell targeting or expression construct can specifically induce immune cells to target the construct to cells expressing the binding partner, including tumor cells. Anti-tumor effector cells, e.g., CD4, can be targeted by introducing into T cells an IL-2 superkine immune cell targeting or expression construct containing one or more signaling domains from CD3-zeta, CD28, DAP10, OX-40, ICOS, and CD137. + or CD8 + Effector T cells are generated and redirected to recognize tumor cells as described above.

[0275] The IL-2, IL-4, or IL-13 superkine immune cell targeting or expression construct is infected or transfected into human immune cells using, for example, non-viral plasmid vectors and electroporation methods, viral vectors and infection methods, etc., as known in the art. 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 protocols. CD4 +and CD8 + T cell effector functions, as well as NK cell functions, can be elicited via these receptors, and so these types of cells are contemplated for use with the present invention. Among other functions of these cells are CD8+ cells expressing the IL-2, IL-4, or IL-13 superkine CARs of the present invention. + T cells may be used to lyse target cells and produce IL-2, IL-4, or IL-13 in the presence of target cells. + T cells and CD8 + Expression of an appropriate costimulatory CAR on either or both T cells provides the most effective cell population for adoptive immunotherapy, i.e., a population of professional helper and / or killer T cells with enhanced and / or long-lasting viability and anti-tumor activity. In some embodiments, the IL-2, IL-4, or IL-13 superkine containing immune cell targeting construct 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 those shown in FIG. 2 and in the Tables herein. In some embodiments, the IL-2, IL-4, or IL-13 superkine immune cell targeting construct 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 shown herein.

[0276] The polypeptides of the invention can be further modified for a variety of purposes, e.g., conjugated to a wide variety of other oligopeptides or proteins. For example, they can be post-translationally modified by, e.g., prenylation, acetylation, amidation, carboxylation, glycosylation, pegylation, etc. Such modifications can include altered glycosylation, e.g., by altering the glycosylation pattern of a polypeptide during polypeptide synthesis and processing, or in further processing steps, e.g., by exposing the polypeptide to enzymes that affect glycosylation, such as mammalian glycosylating or deglycosylating enzymes.

[0277] Methods well known to those of skill in the art can be used to construct T cell targeting construct expression vectors containing a coding sequence and appropriate transcriptional / translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo recombination / genetic recombination. Alternatively, RNA capable of encoding a polypeptide of interest can be chemically synthesized. Those skilled in the art can readily use well-known codon usage tables and synthetic methods to generate suitable coding sequences for any of the polypeptides of the invention. The nucleic acid may be isolated and obtained with sufficient purity. Typically, the nucleic acid, either DNA or RNA, will be obtained substantially free of other naturally occurring nucleic acid sequences, generally at least about 50% pure, usually at least about 90%, and is typically "recombinant," e.g., flanked by one or more nucleotides not normally associated in the natural chromosome. The nucleic acids of the invention can be provided as linear or circular molecules, and can be provided within autonomously replicating molecules (vectors) or molecules that do not contain replication sequences. Expression of the nucleic acid can be regulated by the nucleic acid itself or by other regulatory sequences known in the art. Nucleic acids of the invention can be introduced into suitable host cells using a variety of techniques available in the art.

[0278] According to the present invention, immune cell targeting construct vectors or expression construct vectors, and immune cell targeting construct or expression construct-modified cells, can be provided in pharmaceutical compositions suitable for therapeutic use, e.g., human treatment. In some embodiments, pharmaceutical compositions of the present invention comprise one or more therapeutic agents of the present invention, or pharmaceutically acceptable salts, esters, or solvates thereof. In some embodiments, pharmaceutical compositions of the present invention comprise one or more therapeutic agents of the present invention in combination with another therapeutic agent, e.g., another anti-tumor agent.

[0279] Therapeutic agents of the present invention are often administered as pharmaceutical compositions containing an active therapeutic agent and another pharmaceutically acceptable excipient. Such formulations can include one or more non-toxic pharmaceutically acceptable carriers, diluents, excipients, and / or adjuvants. The preferred form depends on the intended method of administration and therapeutic application. Depending on the desired formulation, the composition can also include a non-toxic pharmaceutically acceptable carrier or diluent, which are defined as vehicles commonly used to formulate pharmaceutical compositions 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 include other carriers, adjuvants, or non-toxic, non-therapeutic, non-immunogenic stabilizers.

[0280] In yet another embodiment, the pharmaceutical compositions of the present invention can also include large, slowly metabolized macromolecules such as proteins, polysaccharides (such as chitosan), polylactic acids, polyglycolic acids, copolymers (such as latex-functionalized Sepharose™, agarose, cellulose), polymeric amino acids, amino acid copolymers, and lipid aggregates (such as oil droplets or liposomes).

[0281] The maximum tolerated dose (MTD) of CAR immune cells may be determined during clinical trial development and, when empirically determined, may be, for example, up to about 10 per kg of body weight. 4T cells, up to about 10 per kg of body weight 5 cells, up to about 10 per kg of body weight 6 cells, up to about 5 × 10 per kg of body weight 6 cells, up to about 10 per kg of body weight 7 cells, up to about 5 × 10 per kg of body weight 7 In some embodiments, the maximum tolerated dose (MTD) of CAR immune cells is up to about 10 per kg of body weight. 4 In some embodiments, the maximum tolerated dose (MTD) of CAR immune cells is up to about 10 per kg of body weight. 5 In some embodiments, the maximum tolerated dose (MTD) of CAR immune cells is up to about 10 per kg of body weight. 6 In some embodiments, the maximum tolerated dose (MTD) of CAR immune cells is up to about 10 per kg of body weight. 7 In some embodiments, the maximum tolerated dose (MTD) of CAR immune cells is up to about 5 x 10 per kg of body weight. 6 In some embodiments, the maximum tolerated dose (MTD) of CAR immune cells is up to about 5 x 10 per kg of body weight. 7 T cells.

[0282] Toxicity of the cells described herein can be determined by standard pharmaceutical procedures in cell culture or experimental animals, e.g., LD 50 (median lethal dose of a population) or LD 100 The therapeutic index can be determined by determining the lethal dose (total population lethal dose). The dose ratio between toxic and therapeutic effects is the therapeutic index. Data obtained from these cell culture assays and animal studies can be used to formulate a dosage range that is non-toxic for use in humans. The dosages described herein preferably fall within a range of circulating concentrations that include the 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 exact formulation, route of administration, and dosage can be selected by the individual physician in view of the patient's condition.

[0283] Patients in the expansion cohort are treated with immune cells at the MTD after the dose escalation phase. Exemplary treatment regimens involve administration once every two weeks, once a month, or once every three to six months. The therapeutic agents of the present invention are typically administered multiple times. The interval between each administration can be one week, one month, or one year. The interval can also be irregular, such as determined by measuring the blood level of the therapeutic agent in the patient.

[0284] For example, in prophylactic use to maintain remission in a patient, a relatively low dosage may be administered at relatively infrequent intervals over a long period of time. Some patients continue treatment for the rest of their lives. Other therapeutic uses may require a relatively high dosage at relatively short intervals until the progression of the disease is reduced or stopped, preferably until the patient experiences partial or complete improvement in the symptoms of the disease. The patient can then be administered a prophylactic regimen.

[0285] Examples of additional therapeutic agents that can be co-administered and / or co-formulated with immune cell targeting constructs or expression constructs include antiproliferative or cytoreductive therapies, including the use of therapies such as the delivery of ionizing radiation and the administration of chemotherapeutic agents, used in therapy to eliminate tumor cells and other unwanted cells in a host. Chemotherapeutic agents are well known in the art, including topoisomerase inhibitors such as anthracyclines (including compounds such as daunorubicin, adriamycin (doxorubicin), epirubicin, idarubicin, annamycin, and MEN10755), and are used at conventional or reduced doses and regimens. Other topoisomerase inhibitors include the podophyllotoxin analogs etoposide and teniposide, as well as anthracenedione, mitoxantrone, and amsacrine. Other antiproliferative agents, such as the vinca alkaloid family, interfere with microtubule polymerization. Examples of vinca alkaloids include vinblastine, vincristine, vinorelbine (NAVELBINE), vindesine, vindoline, vincamine, etc. DNA damaging agents include nucleotide analogs, alkylating agents, etc. Alkylating agents include nitrogen mustards such as mechlorethamine, cyclophosphamide, melphalan (L-sarcolysin), etc., and nitrosoureas such as carmustine (BCNU), lomustine (CCNU), semustine (methyl-CCNU), streptozocin, chlorozotocin, etc. 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 folate analogs such as methotrexate, 10-propargyl-5,8-dideazafolate (PDDF, CB3717), 5,8-dideazatetrahydrofolic acid (DDATHF), and leucovorin.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.

[0286] For example, ionizing radiation (IR) is used to treat approximately 60% of cancer patients by depositing energy that damages or destroys cells in the treatment area; for purposes of the present invention, IR may be delivered at conventional doses and regimens or at reduced doses. Radiation-induced cell damage is nonspecific and has complex effects on DNA. The effectiveness of therapy depends on greater cellular damage to cancer cells than to normal cells. Radiation therapy can be used to treat any 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, in which a radioactive implant is placed directly into a tumor or body cavity to concentrate the radiation dose in a small area. Suitable ionizing radiation doses may range from at least about 2 Gy to no more than about 10 Gy, typically about 5 Gy. Suitable ultraviolet radiation doses are at least about 5 J / m 2 ~about 50J / m 2 Range below, typically around 10J / m 2 The sample may be collected within at least about 4 hours to about 72 hours after ultraviolet irradiation, usually around 4 hours after.

[0287] The treatment may be combined with immunomodulatory agents, including (iii) agents that agonize immune costimulatory molecules, such as CD40 and OX40, and / or (iv) agents that antagonize immune inhibitory molecules, such as CTLA-4, PD-1, and PD-L1. The active agents are administered within a time period that provides an additive or synergistic effect on the elimination of cancer cells in the host. Methods of administration include, but are not limited to, systemic administration, intratumoral administration, and the like.

[0288] In some embodiments, an individual cancer is selected for treatment with the combination therapy because it is a type of cancer that responds to a checkpoint inhibitor, e.g., a PD-1 antagonist, a PD-L1 antagonist, a CTLA4 antagonist, a TIM-3 antagonist, a BTLA antagonist, a VISTA antagonist, a LAG3 antagonist, etc. In some embodiments, such an immunomodulatory agent is a CTLA-4 antagonist, a PD1 antagonist, or a PDL1 antagonist, e.g., 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, colon cancer, lung cancer, skin cancer, lymphatic cancer, gastrointestinal cancer, prostate cancer, ovarian cancer, breast cancer, basal-like breast tumor, endometrial cancer, multiple myeloma, melanoma, lymphoma, lung cancer (including small cell lung cancer), kidney cancer, gastric cancer, brain tumor, and central nervous system tumor. Central nervous system tumors include glioma, glioblastoma, glioblastoma multiforme (GBM), refractory glioblastoma multiforme (rGBM), recurrent glioblastoma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, meningioma, neuroblastoma, retinoblastoma, medulloblastoma, adult pituitary adenoma, O6-methylguanine methyltransferase (MGMT) positive or negative central nervous system tumors, and furin-positive central nervous system tumors. In some such embodiments, the cancer is of a type with a high neoantigen load, i.e., a high mutagenic load (see Vogelstein et al. (2013) Science 339(6127):1546-1558, specifically incorporated herein by reference).

[0289] In some embodiments, an individual's cancer is selected for treatment with the combination therapy of the invention because the cancer is a cancer type that responds to an immune response agonist, e.g., 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.

[0290] In some embodiments, the immunomodulatory agent is an OX40, CD137, or 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 load, i.e., a high level of mutagenesis.

[0291] In some embodiments, the combination therapy includes an antibody known in the art that binds to PD-1, inhibits the interaction of PD-1 with 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 may find use in the present invention include, for example, 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 (Regeneron), human monoclonal antibody MDX-1106 (Bristol-Myers Squibb), and / or human monoclonal antibody PD-1 (Bristol-Myers Squibb). Suitable antibodies include, but are not limited to, the PD-1 antibody (Squibb), and / or the humanized anti-PD-1 IgG4 antibody PDR001 (Novartis). In some embodiments, the PD-1 antibody is derived from clone RMP1-14 (rat IgG) (BioXcell catalog number BP0146). Other suitable antibodies include the anti-PD-1 antibodies disclosed in U.S. Patent No. 8,008,449, incorporated herein by reference. In some embodiments, the antibody or antigen-binding portion thereof specifically binds to PD-L1 and inhibits the interaction between PD-L1 and PD-1, thereby enhancing immune activity. Any antibody known in the art that binds to PD-L1 and inhibits the interaction between PD-1 and PD-L1 while stimulating an anti-tumor immune response is suitable for use in the combination therapies disclosed herein.For example, antibodies that target PD-L1 and are in clinical trials include BMS-936559 (Bristol-Myers Squibb) and MPDL3280A (Genetech). Other suitable antibodies that target PD-L1 are disclosed in U.S. Patent No. 7,943,743, which is incorporated herein by reference. One of skill in the art will appreciate that any antibody that binds to PD-1 or PD-L1, inhibits the PD-1 / PD-L1 interaction, and stimulates an anti-tumor immune response is suitable for use in this combination therapy.

[0292] In some embodiments, the combination therapy includes an antibody known in the art that binds to CTLA-4 and inhibits its interaction with CD80 and CD86. Exemplary antibodies targeting CTLA-4 include ipilimumab (MDX-010, MDX-101, Bristol-Myers Squibb), which has been approved by the FDA, and tremelimumab (ticilimumab, CP-675, 206, Pfizer), which is currently undergoing human trials. Other suitable antibodies targeting CTLA-4 are disclosed in WO2012 / 120125, U.S. Patent Nos. 6,984,720 and 6,682,7368, and U.S. Patent Application Publication Nos. 2002 / 0039581, 2002 / 0086014, and 2005 / 0201994, which are incorporated herein by reference. Those skilled in the art will understand that any antibody that binds to CTLA-4, inhibits the interaction of CTLA-4 with CD80 and CD86, and stimulates an anti-tumor immune response is suitable for use in this combination therapy. In some embodiments, the combination therapy includes an antibody known in the art that binds to LAG-3 and inhibits the interaction of LAG-3 with MHC class II molecules. An exemplary antibody that targets LAG-3 is IMP321 (Immutep), which is currently undergoing human trials. Other suitable antibodies that target LAG-3 are disclosed in U.S. Patent Application Publication No. 2011 / 0150892 (incorporated herein by reference). Those skilled in the art will understand that any antibody that binds to LAG-3, inhibits the interaction of LAG-3 with MHC class II molecules, and stimulates an anti-tumor immune response is suitable for use in this combination therapy.

[0293] In some embodiments, the combination therapy includes an antibody known in the art that binds to TIM-3 and inhibits the interaction of TIM-3 with galectin 9. Suitable antibodies that target TIM-3 are disclosed in U.S. Patent Application Publication No. 2013 / 0022623, which is incorporated herein by reference. One of skill in the art will appreciate that any antibody that binds to TIM-3 and inhibits the interaction of TIM-3 with galectin 9 while stimulating an anti-tumor immune response is suitable for use in this combination therapy.

[0294] In some embodiments, the combination therapy includes an antibody known in the art that binds to 4-1BB / CD137 and inhibits the interaction of 4-1BB / CD137 with CD137L. One skilled in the art will appreciate that any antibody that binds to 4-1BB / CD137 and inhibits the interaction of 4-1BB / CD137 with CD137L or another ligand, while stimulating an anti-tumor immune response or immunostimulatory response that results in systemic anti-tumor activity, is suitable for use in this combination therapy.

[0295] In some embodiments, the combination therapy includes an antibody known in the art that binds to GITR and inhibits the interaction of GITR with its ligand. One skilled in the art will appreciate that any antibody that binds to GITR and inhibits the interaction of GITR with GITRL or another ligand, and stimulates an anti-tumor immune response or immunostimulatory response that results in systemic anti-tumor activity, is suitable for use in this combination therapy.

[0296] In some embodiments, the combination therapy includes an antibody known in the art that binds to OX40 and inhibits the interaction of OX40 with its ligand. One of skill in the art will understand that any antibody that binds to OX40 and inhibits the interaction of OX40 with OX40L or another ligand, and stimulates an anti-tumor immune response or immunostimulatory response that provides systemic anti-tumor activity, is suitable for use in this combination therapy.

[0297] In some embodiments, the combination therapy includes an antibody known in the art that binds to CD40 and inhibits the interaction of CD40 with its ligand. One of skill in the art will appreciate that any antibody that binds to CD40 and inhibits the interaction of CD40 with its ligand and stimulates an anti-tumor immune response or immunostimulatory response that results in systemic anti-tumor activity is suitable for use in this combination therapy.

[0298] In some embodiments, the combination therapy includes an antibody known in the art that binds to ICOS and inhibits the interaction of ICOS with its ligand. One skilled in the art will appreciate that any antibody that binds to ICOS and inhibits the interaction of ICOS with its ligand and stimulates an anti-tumor immune response or immunostimulatory response that results in systemic anti-tumor activity is suitable for use in this combination therapy.

[0299] In some embodiments, the combination therapy includes an antibody known in the art that binds to CD28 and inhibits the interaction of CD28 with its ligand. One of skill in the art will appreciate that any antibody that binds to CD28 and inhibits the interaction of CD28 with its ligand and stimulates an anti-tumor immune response or immunostimulatory response that results in systemic anti-tumor activity is suitable for use in this combination therapy.

[0300] In some embodiments, the combination therapy includes an antibody known in the art that binds to IFNα and inhibits the interaction of IFNα with its ligand. One skilled in the art will appreciate that any antibody that binds to IFNα and inhibits the interaction of IFNα with its ligand and stimulates an anti-tumor immune response or immunostimulatory response that results in systemic anti-tumor activity is suitable for use in this combination therapy.

[0301] An "anti-cancer therapy" is a compound, composition, or treatment (e.g., surgery) that prevents or slows the growth and / or metastasis of cancer cells. Such anti-cancer treatments 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 useful chemotherapeutic agents include, but are not limited to, hydroxyurea, busulfan, cisplatin, carboplatin, chlorambucil, melphalan, cyclophosphamide, ifosfamide, daunorubicin, doxorubicin, epirubicin, mitoxantrone, vincristine, vinblastine, Navelbine® (vinorelbine), etoposide, teniposide, paclitaxel, docetaxel, gemcitabine, cytosine, arabinoside, bleomycin, neocarzinostatin, suramin, taxol, mitomycin C, Avastin, Herceptin®, fluorouracil, and temozolomide. The compounds are also suitable for use in standard combination therapy with two or more chemotherapeutic agents. It is understood that anti-cancer treatments include novel compounds or treatments developed in the future.

[0302] The pharmaceutical compositions and / or formulations described above contain one or more therapeutic agents in an amount effective to achieve its intended purpose. That is, the term "therapeutically effective dose" refers to an amount of a therapeutic agent that ameliorates the symptoms of cancer. Determining a therapeutically effective dose of a compound is well within the capabilities of one of ordinary skill in the art. For example, a therapeutically effective dose can be initially estimated using either cell culture assays or animal models such as those described herein. Appropriate concentration ranges and routes of administration can also be determined using animal models. Such information can then be used to determine useful dosages and routes of administration in other animals, including humans, using standard methods known to those skilled in the art.

[0303] A kit comprising the composition of the present invention and instructions for use is also within the scope of the present invention. The kit may further comprise at least one additional reagent, such as a chemotherapeutic agent, an anti-tumor antibody, etc. The kit typically includes a label indicating the intended use of the contents of the kit. The term label includes any written or recorded material provided on or with the kit, or otherwise accompanying the kit.

[0304] While the present invention has now been fully described, it will be apparent to those skilled in the art that various changes and modifications can be made therein without departing from the spirit or scope of the invention. In some embodiments, the kits comprise an IL-2 superkine immune cell targeting construct or expression construct comprising an IL-2 variant / IL-2 superkine as described herein. In some embodiments, the kits comprise an IL-2 superkine immune cell targeting construct or expression construct comprising an IL-2 variant / IL-2 superkine, including those set forth herein. In some embodiments, the IL-2 superkine immune cell targeting construct or expression construct comprises an IL-2 variant / IL-2 superkine, including those set forth herein. 6. Exemplary Immune Cell Targeting or Expression Construct Embodiments

[0305] The immune cell targeting or expression construct comprises an interleukin-2 receptor beta (IL-2Rβ) binding protein, which has an equilibrium dissociation constant for IL-2Rβ that is less than that of 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.

[0306] In some embodiments, the immune cell targeting construct or expression construct exhibits a cytotoxic effect on T cells, e.g., CD8+ T cells or CD4+ T cells.

[0307] In some embodiments, the construct is a chimeric antigen receptor (CAR) in which the IL-2 superkine of the invention is fused to a transmembrane domain linked to an intracellular signaling region.

[0308] In some embodiments, the intracellular signaling region comprises a CD3 signaling domain.

[0309] 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.

[0310] In some embodiments, the construct is a T cell antigen coupler (TAC) in which an IL-2 superkine is fused to a ligand that binds to a protein associated with the TCR complex, and the ligand is fused to a T cell receptor signaling domain polypeptide.

[0311] In some embodiments, the protein associated with the TCR complex is CD3.

[0312] In some embodiments, the T cell receptor signaling domain polypeptide comprises the cytoplasmic domain of CD4 and the transmembrane domain of CD4.

[0313] In some embodiments, the construct is an antibody-binding 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.

[0314] In some embodiments, the CAR component comprises CD16, with an IL-2, IL-4, or IL-13 mutein superkine fused to an Fc sequence.

[0315] In some embodiments, the construct is an ICE comprising an IL-2 superkine fused to the variable region of an antibody that binds a component of the T cell receptor, hi some embodiments, the construct is an ICE comprising an IL-2, IL-4, or IL-13 mutein or bifunctional molecule described herein fused to the variable region of an antibody that binds a component of the T cell receptor.

[0316] In some embodiments, the BiTE component of the T cell receptor is CD3.

[0317] In some embodiments, the IL-2Rβ binding protein comprises the following amino acid substitutions (numbered relative to wild-type hIL-2): L80F, R81D, L85V, I86V, and I92F.

[0318] In some embodiments, nucleic acids encoding the IL-2, IL-4, or IL-13 muteins described herein are provided, and in some embodiments, vectors comprising the nucleic acids are provided.

[0319] In some embodiments, a T cell is provided comprising a construct according to any of the above. In some embodiments, a NK cell is provided comprising a construct according to any of the above. In some embodiments, the T cell is a CD4 + In some embodiments, the T cells are CD8 + T cells.

[0320] Also provided are isolated populations of the above immune cells. Also provided are pharmaceutical formulations comprising the above immune cell populations.

[0321] H. Expression of mutant IL-2, IL-4, or IL-13 gene products The nucleic acid molecules described above can be included within a vector capable of directing expression, for example, in cells transduced with the vector. Thus, preferred embodiments include expression vectors containing nucleic acid molecules encoding the IL-2, IL-4, or IL-13 muteins and / or bispecific IL-2, IL-4, or IL-13 cytokine fusions of the invention, as well as IL-2, IL-4, or IL-13 muteins of the invention, and cells transfected with these vectors.

[0322] It should be understood, of course, that not all vectors and expression control sequences function equally well to express the DNA sequences described herein. Also, not all hosts function equally well in the same expression system. However, one of skill in the art can make a selection from among these vectors, expression control sequences, and hosts without undue experimentation. For example, when selecting a vector, the host must be considered, since the vector must replicate within the host. The vector's copy number, the ability to control that copy number, and the expression of any other proteins encoded by the vector (such as antibiotic markers) must also be considered. For example, vectors that can be used include those that allow DNA encoding IL-2, IL-4, or IL-13 muteins or bifunctional molecules to be amplified in copy number. Such amplifiable vectors are well known in the art. Examples of such vectors include those that can be amplified by DHFR amplification (see, e.g., U.S. Pat. No. 4,470,461 to Kaufman; Kaufman and Sharp, "Construction of a Modular Dihydrafolate Reductase cDNA Gene: Analysis of Signals Utilized for Efficient Expression," Mol. Cell. Biol., 2, pp. 1304-19 (1982)) or glutamine synthetase ("GS") amplification (see, e.g., U.S. Pat. No. 5,122,464 and European Patent Application Publication No. 338,841).

[0323] In some embodiments, the human IL-2, IL-4, or IL-13 muteins or bifunctional molecules of the present disclosure will be expressed from a vector, preferably an expression vector. The vector may be useful for autonomous replication in a host cell, or may be integrated into the genome of the host cell upon introduction into the host cell, thereby replicating along with the host genome (e.g., non-episomal mammalian vectors). Expression vectors are capable of directing the expression of coding sequences operably linked to them. Generally, expression vectors useful in recombinant DNA techniques are often in the form of plasmids (vectors). However, other forms of expression vectors, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), are also included.

[0324] 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.

[0325] The expression construct or expression vector can be designed to express an IL-2, IL-4, or IL-13 mutein or variant or bifunctional molecule thereof in a prokaryotic or eukaryotic host cell.

[0326] 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 (2d ed., Cold Spring Harbor Laboratory Press, Plainview, NY) and other standard molecular biology laboratory manuals.

[0327] Protein expression in prokaryotes is most often carried out in Escherichia coli with vectors containing constitutive or inducible promoters. Strategies for maximizing recombinant protein expression in E. coli can be found, for example, in Gottesman (1990) in 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. Processes for growing, recovering, destroying or extracting IL-2 muteins or variants thereof from cells are substantially as described, for example, in 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, each of which is incorporated by reference in its entirety.

[0328] In some embodiments, recombinant IL-2, IL-4, or IL-13 muteins or biologically active variants or bifunctional molecules thereof may 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 the yeast S. cerenvisiae 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, CA) and the like). Expression vectors include vectors such as pCDM8 (Seed (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6:187:195)), 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 expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, and simian virus 40. Other expression systems suitable for both prokaryotic and eukaryotic cells include Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2 ndSee Chapters 16 and 17 of "Genetic Expression Technology: Methods in Enzymology," ed., Cold Spring Harbor Laboratory Press, Plainview, NY. See Goeddel (1990) in Gene Expression Technology: Methods in Enzymology 185 (Academic Press, San Diego, Calif.).

[0329] 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 the host cells of interest. The GC content of the sequences can be adjusted to the average level for that cellular host, as calculated by reference to known genes expressed in a given host cell. Methods for optimizing codons are well known in the art. Codons in the IL-2, IL-4, or IL-13 mutein or bifunctional molecule coding sequences 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 in the coding sequence are optimized for expression in a particular host cell.

[0330] Vectors suitable for use include T7-based vectors for use in bacteria (see, e.g., Rosenberg et al., Gene 56:125, 1987), the pMSXND expression vector for use in mammalian cells (Lee and Nathans, J. Biol. Chem. 263:3521, 1988), and baculovirus-derived vectors for use in insect cells (e.g., the expression vector pBacPAK9 from Clontech, Palo Alto, Calif.).

[0331] In some embodiments, in such vectors, the nucleic acid insert encoding the IL-2, IL-4, or IL-13 mutein or bifunctional molecule of the invention can be operably linked to a promoter, which is selected, for example, based on the cell type in which expression is desired.

[0332] A variety of factors must also be considered when selecting an expression control sequence, including, for example, the sequence's relative strength, controllability, and compatibility (especially with respect to potential secondary structure) with the actual DNA sequence encoding the IL-2, IL-4, or IL-13 mutein or bifunctional molecule of the present invention. A host should be selected by considering compatibility with the selected vector, toxicity of the product encoded by the DNA sequence of the present invention, the secretion characteristics of the host, ability to properly fold the polypeptide, fermentation or cultivation requirements, and ease of purification of the product encoded by the DNA sequence.

[0333] One skilled in the art can select various vector / expression control sequence / host combinations within these parameters that will express the desired DNA sequence during fermentation or large scale animal culture using, for example, CHO or COS7 cells.

[0334] The choice of expression control sequences and expression vectors will, in some embodiments, depend on the host cell chosen. A wide variety of expression host / expression vector combinations can be used. Useful expression vectors for eukaryotic hosts include, for example, vectors having expression control sequences derived from SV40, bovine papillomavirus, adenovirus, and cytomegalovirus. Useful expression vectors for bacterial hosts include known bacterial plasmids such as E. coli-derived plasmids, including colEl, pCRI, pER32z, pMB9, and their derivatives; broad-host-range plasmids such as RP4; phage DNA, e.g., numerous derivatives of lambda phage, e.g., NM989; and DNA phages such as M13 and filamentous single-stranded DNA phages. Useful expression vectors for yeast cells include the 2μ plasmid and its derivatives. Useful vectors for insect cells include pVL941 and pFastBac™1 (GibcoBRL, 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).

[0335] In addition, any of a wide variety of expression control sequences can be used in these vectors. Such useful expression control sequences include those associated with the structural genes of the expression vectors described above. Examples of useful expression control sequences include, for example, the early and late promoters of SV40 or adenovirus, the lac, trp, TAC, or TRC systems, the major operator and promoter regions of lambda phage, e.g., PL, the coat protein control region of fd, promoters for 3-phosphoglycerate kinase or other glycolytic enzymes, promoters for acid phosphatases such as PhoA, promoters of the yeast a mating system, the polyhedrin promoter of baculovirus, and various combinations thereof, along with other sequences known to control expression of genes in prokaryotic or eukaryotic cells or their viruses.

[0336] The T7 promoter can be used in bacteria, the polyhedrin promoter can be used in insect cells, and the cytomegalovirus promoter or metallothionein promoter can be used in mammalian cells. In addition, in the case of 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 the body. Those skilled in the art are well aware of the numerous promoters and other regulatory elements that can be used to direct the expression of nucleic acids.

[0337] In addition to sequences that facilitate transcription of the inserted nucleic acid molecule, vectors can include an origin of replication and other genes that encode selectable markers, e.g., neomycin resistance (neomycin resistance). r The ) gene confers G418 resistance to expressing cells, thereby allowing for phenotypic selection of transfected cells. One of skill in the art can readily determine whether a given regulatory element or selectable marker is suitable for use in a particular experimental context.

[0338] Viral vectors that can be used in the present invention include, for example, retroviral vectors, adenoviral vectors, adeno-associated vectors, herpes virus vectors, simian virus 40 (SV40) vectors, and bovine papilloma virus vectors (see, e.g., Gluzman (Ed.), Eukaryotic Viral Vectors, CSH Laboratory Press, Cold Spring Harbor, NY).

[0339] Prokaryotic or eukaryotic cells that contain and express a nucleic acid molecule encoding an IL-2 mutein of the invention disclosed herein are also a feature of the invention. The cells of the 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 techniques. The progeny of such cells are also considered within the scope of the invention.

[0340] The exact components of the expression system are not critical. For example, IL-2 muteins can be produced in prokaryotic hosts such as the bacterium E. coli, or eukaryotic hosts such as insect cells (e.g., Sf21 cells) or mammalian cells (e.g., CHO cells, HEK293 cells, COS cells, NIH 3T3 cells, or HeLa cells). These cells are available from many sources, including the American Type Culture Collection (Manassas, Va.). When selecting an expression system, it is important only that the components are compatible with each other. One of skill in the art can make such a judgment. Furthermore, if guidance is needed in selecting an expression system, one of skill in the art 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 Suppl. 1987).

[0341] 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.

[0342] In some embodiments, the resulting IL-2, IL-4, or IL-13 mutein or bifunctional molecule will be glycosylated or deglycosylated, depending on the host organism used to produce the mutein. If a bacterial host is selected, the produced IL-2, IL-4, or IL-13 mutein or bifunctional molecule will be deglycosylated. In eukaryotic cells, on the other hand, the IL-2, IL-4, or IL-13 mutein or bifunctional molecule will be glycosylated, although perhaps not in the same way as native IL-2. The IL-2, IL-4, or IL-13 mutein 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 muteins or bifunctional molecules. See, for example, Current Protocols in Protein Science, Vol. 2. Eds: 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 derived from either mammalian or yeast cultures producing the given mutein using cation exchange chromatography, gel filtration chromatography, and / or reverse-phase liquid chromatography.

[0343] Another exemplary method for constructing a DNA sequence encoding an IL-2, IL-4, or IL-13 mutein or bifunctional molecule is by chemical synthesis. This method involves directly synthesizing a peptide by chemical means of a protein encoding an IL-2 mutein exhibiting the described properties. This method allows for the incorporation of both natural and unnatural amino acids at positions that affect the interaction of the IL-2, IL-4, or IL-13 mutein or bifunctional molecule with its corresponding receptor. Alternatively, genes encoding the desired IL-2, IL-4, or IL-13 mutein 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 mutein or bifunctional molecule, preferably based on the selection of those codons that are preferred in the host cell in which the recombinant mutein will be produced. In this regard, it is well recognized that the genetic code is degenerate, i.e., an amino acid can be coded for 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, for a given DNA sequence encoding a particular IL-2 mutein, it will be apparent that there are numerous degenerate DNA sequences that will encode that IL-2 mutein. For example, in addition to the preferred DNA sequence for mutein H9, it will be apparent that there are numerous degenerate DNA sequences that will encode the indicated IL-2, IL-4, or IL-13 muteins or bifunctional molecules. These degenerate DNA sequences are considered to be within the scope of this disclosure. Thus, in the context of this invention, "degenerate variants thereof" refers to all DNA sequences that encode a particular mutein and thereby provide for the expression of that mutein.

[0344] The biological activity of the IL-2, IL-4, or IL-13 muteins or bifunctional molecules can be assayed by any suitable method known in the art, including PHA blastocyte proliferation and NK cell proliferation.

[0345] I. Anti-PD-1 Antibodies and Combinations Anti-PD-1 antibodies for use and / or fusion with any of the IL-2, IL-4, or IL-13 mutein bifunctional molecules according to the present invention and methods described herein include nivolumab, BMS-936558, MDX-1106, ONO-4538, AMP224, CT-011, and MK-3475 (pembrolizumab), cemiplimab (REGN2810), SHR-1210 (CTR2016017 Anti-PD-1 antibodies include, but are not limited to, PD-1 antibodies such as those listed in U.S. Patent Application Publication No. 2017 / 0081409 (see, e.g., Table 38), including, but not limited to, pembrolizumab (Keytruda®, MK-3475-033) and nivolumab (Opdivo®, CheckMate078), with more in development that can be used in the combination methods described herein. Exemplary anti-PD-1 antibody sequences are shown in Figure 5, any of which can be used in combination with an IL-2 mutein as described herein. In some embodiments, the IL-2 mutein used in combination with an anti-PD-1 antibody is a fusion mutein as described herein. TIFF2025509333000113.tif226170TIFF2025509333000114.tif229170TIFF2025509333000115.tif230170TIFF2025509333000116.tif231170TIFF2025509333000117.tif95170TIFF2025509333000118.tif196170TIFF2025509333000119.tif228170TIFF2025509333000120.tif231170TIFF2025509333000121.tif230170TIFF2025509333000122.tif230170TIFF2025509333000123.tif215170

[0346] In some embodiments, the IL-2 mutein portion of the bispecific IL-2 cytokine fusion contains the substitutions L80F, R81D, L85V, I86V, and I92F (numbered relative to human wild-type IL-2 (SEQ ID NO: 2)) and is used in combination with an anti-PD-1 antibody or inhibitor. In some embodiments, an IL-2 mutein containing the substitutions L80F, R81D, L85V, I86V, and I92F (numbered relative to human wild-type IL-2 (SEQ ID NO: 2)) is used in combination with nivolumab. In some embodiments, an IL-2 mutein containing the substitutions L80F, R81D, L85V, I86V, and I92F (numbered relative to human wild-type IL-2 (SEQ ID NO: 2)) is used in combination with pembrolizumab. In some embodiments, an IL-2 mutein containing the substitutions L80F, R81D, L85V, I86V, and I92F (numbered relative to human wild-type IL-2 (SEQ ID NO: 2)) is used in combination with cemiplimab. In some embodiments, an IL-2 mutein containing the substitutions L80F, R81D, L85V, I86V, and I92F (numbered relative to human wild-type IL-2 (SEQ ID NO: 2)) is used in combination with BMS-936558. In some embodiments, an IL-2 mutein containing the substitutions L80F, R81D, L85V, I86V, and I92F (numbered relative to human wild-type IL-2 (SEQ ID NO: 2)) is used in combination with MDX-1106. In some embodiments, an IL-2 mutein containing the substitutions L80F, R81D, L85V, I86V, and I92F (numbered relative to human wild-type IL-2 (SEQ ID NO: 2)) is used in combination with ONO-4538. In some embodiments, an IL-2 mutein containing the substitutions L80F, R81D, L85V, I86V, and I92F (numbered relative to human wild-type IL-2 (SEQ ID NO: 2)) is used in combination with AMP224. In some embodiments, an IL-2 mutein containing the substitutions L80F, R81D, L85V, I86V, and I92F (numbered relative to human wild-type IL-2 (SEQ ID NO: 2)) is used in combination with CT-011.In some embodiments, an IL-2 mutein comprising the substitutions L80F, R81D, L85V, I86V, and I92F (numbered relative to human wild-type IL-2 (SEQ ID NO:2)) is used in combination with MK-3475. In some embodiments, the IL-2 mutein further comprises the substitution F42A, where the numbering is relative to wild-type human IL-2 of SEQ ID NO:2. In some embodiments, the IL-2 mutein further comprises the substitution Y45A, where the numbering is relative to wild-type human IL-2 of SEQ ID NO:2. In some embodiments, the IL-2 mutein further comprises the substitution E62A, where the numbering is relative to wild-type human IL-2 of SEQ ID NO:2.

[0347] In some embodiments, the IL-2 mutein of the bispecific IL-2 cytokine fusion comprises the substitutions L80F, R81D, L85V, I86V, and I92F (numbered relative to human wild-type IL-2 (SEQ ID NO:2)) and is used in combination with any of the mentioned antibodies. In some embodiments, the IL-2 mutein further comprises the substitution F42A, where the numbering is relative to wild-type human IL-2 of SEQ ID NO:2. In some embodiments, the IL-2 mutein further comprises the substitution Y45A, where the numbering is relative to wild-type human IL-2 of SEQ ID NO:2. In some embodiments, the IL-2 mutein further comprises the substitution E62A, where the numbering is relative to wild-type human IL-2 of SEQ ID NO:2. In some embodiments, the IL-2 mutein further comprises the substitution E62A, where the numbering is relative 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 sequence of the IL-2 mutein is 90% identical to any one of SEQ ID NO:2, SEQ ID NO:6-10, or SEQ ID NO:16. In some embodiments, the IL-2 mutein comprises any one of SEQ ID NO:5 for 5-1, SEQ ID NO:6 for 5-2, SEQ ID NO:7 for 6-6, SEQ ID NO:8 for A2, SEQ ID NO:9 for B1, SEQ ID NO:10 for B11, SEQ ID NO:11 for C5, SEQ ID NO:12 for D10, SEQ ID NO:13 for E10, SEQ ID NO:14 for G8, SEQ ID NO:15 for H4, and SEQ ID NO:16 for H9. In some embodiments, the IL-2 mutein used in combination with an anti-PD-1 antibody is a fusion mutein as described herein. In some embodiments, the IL-2 mutein used in combination with an anti-PD-1 antibody is a fusion mutein as described herein.

[0348] In some embodiments, the IL-2 cytokine fusion comprises a sequence selected from the group consisting of: a. SEQ ID NOs: 271, 272, and 273; b. SEQ ID NOs: 274, 275, and 276; c. SEQ ID NOs: 283, 284, and 285, and d. SEQ ID NOs: 365, 366, and 367.

[0349] In some embodiments, the IL-2 cytokine fusion comprises the sequences of SEQ ID NOs: 271, 272, and 273.

[0350] In some embodiments, the IL-2 cytokine fusion comprises the sequences of SEQ ID NOs: 274, 275, and 276.

[0351] In some embodiments, the IL-2 cytokine fusion comprises the sequences of SEQ ID NOs: 283, 284, and 285.

[0352] In some embodiments, the IL-2 cytokine fusion comprises the sequences of SEQ ID NOs: 365, 366, and 367. mPD1 IgG-MDNA109FEAA S125 (KiH)*

[0353] Mutations: F42A, E62A, L80F, R81D, L85V, I86V, I92F, C125S [ka] Gene 2: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 272) Gene 3: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 273)

[0354] huPD1 IgG-MDNA109FEAA S125 (KiH)* Mutations: F42A, E62A, L80F, R81D, L85V, I86V, I92F, C125S [ka] Gene 2: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 275) Gene 3: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 276)

[0355] mPD1 IgG-MDNA413 R39 / Q111 * Mutations: L10V, E12A, L39R, V18I, R65D, D87S, T88S, L101F, K104R, K105T [ka] Gene 3: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 278)

[0356] huPD1 IgG-MDNA413 R39 / Q111 * Mutations: L10V, E12A, L39R, V18I, R...

Claims

1. (A) comprising the amino acid sequences of (i) SEQ ID NOs: 274, 275, and 276; (ii) SEQ ID NOs: 283, 284, and 285; (iii) SEQ ID NOs: 475, 476, and 477; (iv) SEQ ID NOs: 478, 479, and 480; or (v) SEQ ID NOs: 493, 494, and 495; (B) (i) an amino acid sequence based on IL-2 from Table 2 or Table 4, and (ii) any one amino acid sequence from Tables 3, 8, 9, or 10; (C) (i) an amino acid sequence based on IL-4 from Table 9, and (ii) any one amino acid sequence from Tables 2, 3, 4, 8, or 10; (D) (i) an amino acid sequence based on IL-13 from Table 8, and (ii) any one amino acid sequence from Tables 2, 3, 4, 9, or 10; (E) (i) an amino acid sequence based on IL-7, IL-12, IL-15, IL-18, or IL-33 from Table 10, and (ii) any one amino acid sequence from Tables 2, 3, 4, 8, or 9; (F) comprising the amino acid sequence of SEQ ID NOs: 395, 484, 501, 502, 503, 504, 505, 506, 507, or 508 and the IL-2-based amino acid sequence of Table 2; (G) comprising the amino acid sequence of SEQ ID NOs. 395, 484, 501, 502, 503, 504, 505, 506, 507, or 508 and the IL-7, IL-12, IL-15, or IL-18, IL-33 based amino acid sequences of Table 10; (H) comprising the amino acid sequence of SEQ ID NOs: 395, 484, 501, 502, 503, 504, 505, 506, 507, or 508 and any one of the amino acid sequences in Tables 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, or 39; (I) The amino acid sequences of SEQ ID NOs: 395, 484, 501, 502, 503, 504, 505, 506, 507, or 508, and SEQ ID NOs: 6 (H9-F42A), 7 (H9-K43N), 8 (H9-F42A / Y45A, H9-FYAA), 9 (H9-F42A / E62A, H9-FEAA), 10 (H9-F42A / Y45A / E62A, H9-FYEAAAA), 20, SEQ ID NOs: 21, an amino acid sequence selected from the group consisting of SEQ ID NOs: 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 (MDNA109 or H9), 32, 33, 34, 35, and 146 (F42A, E62A, L80F, R81D, L85V, I86V, I92F, and C125S); (J) Containing one or more amino acid sequences from any one of Tables 2, 3, 4, 8, 9, or 10, and containing one or more cytokine binding moieties from Tables 2, 3, 4, 8, 9, or 10; (K) comprising one or more amino acid sequences from any one of Tables 5, 6, 7, 11, 12, 13, 15, or 39, and comprising one or more cytokine-binding moieties from Tables 5, 6, 7, 11, 12, 13, 15, or 39; or (L) Sequence numbers 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, 1 82, 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, 21 3, 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, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, A bifunctional molecule containing one or more amino acid sequences from among 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, or 508.

2. (i) Compared to wild-type IL-13, comprising the following substitutions: L10H, E15R, R86T, D87G, T88R, and R108K, or Compared to wild-type IL-13, the following substitutions are included: L10V, E12A, V18I, R65D, D87S, T88S, L101F, K104R, and K105T; (ii) Compared to wild-type IL-13, it contains the following substitutions: L10H, E15R, R86T, D87G, T88R, and R108K, or Compared to wild-type IL-13, it includes the following substitutions: L10V, E12A, V18I, R65D, D87S, T88S, L101F, K104R, and K105T. Compared to wild-type IL-13, it further includes the R39 polymorphism and / or the Q111 polymorphism; (iii) Compared to wild-type IL-2, the following substitutions are included: L80F, R81D, L85V, I86V, I92F; (iv) Compared to wild-type IL-2, comprising the following substitutions L80F, R81D, L85V, I86V, and I92F, and further comprising the following substitutions compared to wild-type IL-2: F42A and E62A, C125S, or F42A, E62A, and C125S; (v) Compared to wild-type IL-4, the following substitutions are included: R121K, Y124F, S125R; or (vi) The bifunctional molecule according to claim 1, comprising the following substitutions compared to wild-type IL-4: K117R, T118V, R121Q, D122S, Y124W, S125F, S128G, S129A.

3. The bifunctional molecule according to claim 1 or 2, further comprising an Fc domain, albumin, an anti-PD1 antibody, or an anti-CD3 antibody.

4. (i) huPD1 IgG-MDNA109FEAAC125 (KiH), huAntiPD1-MDNA109FEAA-T3A-C125S (1:1 KIH), Anti-huPD1-MDNA109 (KIH), huPD1-MDNA109FEAA (KiH), huPD1 IgG-MDNA132 L39 / Q111 (KiH), huPD1 IgG-MDNA413 R39 / Q111, MDNA413R39 / Q111-Fc (1:1 KIH), huPD1 IgG-MDNA109FEAAS125 (KiH), MDNA109FEAA-Fc-MDNA132.15 (2:1:1 KIH), MDNA132.15-Fc-MDNA413 (1:1:2 KIH), or huPD1-MDNA109FEAA(KiH)*; (ii) containing Sequence ID 395 (MDNA132.15); (iii) Includes sequence number 484 (MDNA132R.15); (iv) containing Sequence ID 501 (MDNA132-Q111); (v) containing Sequence ID No. 502 (MDNA132-R111); (vi) containing Sequence ID 503 (cpMDNA132.15-Q111); (vii) containing Sequence ID 504 (cpMDNA132.15-R111); (viiii) containing Sequence ID 505 (cpMDNA132.15-Q111-PE); (ix) containing Sequence ID 506 (cpMDNA132.15-R111-PE); (x) containing Sequence ID 507 (MDNA132.15-Q111-PE); or (xi) A bifunctional molecule according to any one of claims 1 to 3, comprising Sequence ID No. 508 (MDNA132.15-R111-PE).

5. A bifunctional molecule according to any one of claims 1 to 4, (i) an IL-2-based sequence comprising an IL-2-based sequence having increased binding affinity to CD122 (IL-2Rβ) compared to wild-type human IL-2, or an IL-2-based sequence comprising an IL-2-based sequence having increased binding ability to IL-2Rβ compared to wild-type human IL-2; (ii) containing an IL-2 based sequence in which IL2Rα binding is suppressed or completely absent; (iii) comprising an IL-2-based sequence in which IL2Rα binding is suppressed or in which IL2Rα binding is completely absent, The IL-2-based sequence further comprises the following amino acid substitutions: F42A, E62A, or F42A and E62A (numbering is based on wild-type human IL-2 of Sequence ID No. 2): (iv) comprising an IL-2-based sequence in which IL-2Rα binding is suppressed and / or in which IL-2Rα binding is absent, and the IL-2-based sequence further comprises amino acid substitutions F42A, E62A, or F42A and E62A (numbering is based on wild-type human IL-2 of SEQ ID NO: 2), and the bifunctional molecule exhibits reduced binding affinity to CD25 (IL-2Rα), induces proliferation of immune cells (including CD8 T cells and NK cells), induces activation of effector immune cells (including CD8 T cells and NK cells), exhibits reduced binding affinity to CD25 (IL-2Rα) and induces proliferation of immune cells (including CD8 T cells and NK cells), or exhibits reduced binding affinity to CD25 (IL-2Rα) and induces proliferation of immune cells (including CD8 T cells and NK cells), and induces activation of effector immune cells (CD8 It induces the activation of T cells and NK cells; (v) comprising an IL-2-based sequence in which IL2Rα binding is suppressed and / or in which IL2Rα binding is absent, wherein the IL-2-based sequence further comprises amino acid substitutions F42A, E62A, or F42A and E62A (numbering is based on wild-type human IL-2 of SEQ ID NO: 2), and The bifunctional molecule includes an IL-2-based sequence that exhibits reduced binding affinity to CD25 (IL-2Rα), induces proliferation of immune cells (including CD8 T cells and NK cells), induces activation of effector immune cells (including CD8 T cells and NK cells), exhibits reduced binding affinity to CD25 (IL-2Rα) and induces proliferation of immune cells (including CD8 T cells and NK cells), or exhibits reduced binding affinity to CD25 (IL-2Rα), induces proliferation of immune cells (including CD8 T cells and NK cells), induces activation of effector immune cells (including CD8 T cells and NK cells), and has reduced binding affinity to CD25 compared to wild-type human IL-2; (vi) comprising an IL-2-based sequence in which IL-2Rα binding is suppressed and / or in which IL-2Rα binding is absent, and the IL-2-based sequence further comprises amino acid substitutions F42A, E62A, or F42A and E62A (numbering is based on wild-type human IL-2 of SEQ ID NO: 2), and the bifunctional molecule exhibits reduced binding affinity to CD25 (IL-2Rα), induces proliferation of immune cells (including CD8 T cells and NK cells), induces activation of effector immune cells (including CD8 T cells and NK cells), exhibits reduced binding affinity to CD25 (IL-2Rα) and induces proliferation of immune cells (including CD8 T cells and NK cells), or exhibits reduced binding affinity to CD25 (IL-2Rα) and induces proliferation of immune cells (including CD8 T cells and NK cells), and induces activation of effector immune cells (CD8 A IL-2-based sequence that induces activation of T cells and NK cells, and has reduced binding affinity to CD25 compared to wild-type human IL-2, and induces limited activity or no activity at all with respect to the proliferation, activation, or proliferation and activation of immunosuppressive regulatory T cells (Treg); (vii) Binds to IL-2R and PD1 on target cells; (viiii) Containing IL-2R and PD1 on target cells, and comprising a cytokine binding moiety and an anti-PD1 antibody, (a) Induce activation of tumor-infiltrating CD8+ T cells, and (b) Prevent the exhaustion of tumor-infiltrating CD8+ T cells, as in (a); (ix) Binds to IL-2R and PD1 on target cells, and comprises a cytokine binding moiety and an anti-PD1 antibody, (a) Induce activation of tumor-infiltrating CD8+ T cells, and (b) The same tumor-infiltrating CD8+ T cells as in (a) are prevented from becoming exhausted, and the cytokine binding portion and the anti-PD1 antibody are covalently bound; (x) Conjugates IL-2R and PD1 on target cells, and comprises a cytokine binding moiety and an anti-PD1 antibody, (a) Induce activation of tumor-infiltrating CD8+ T cells, and (b) The same tumor-infiltrating CD8+ T cells are prevented from becoming exhausted as in (a), the cytokine binding portion and the anti-PD1 antibody are covalently bound, and the tumor-infiltrating CD8+ T cells are analyzed for the expression of one or more of the following markers: inhibitory PD1 receptor, TIM3, and cytotoxic granzyme B; (xi) Conjugates IL-2R and PD1 on target cells, and comprises a cytokine binding moiety and an anti-PD1 antibody, (a) Induce activation of tumor-infiltrating CD8+ T cells, and (b) The same as in (a), the exhaustion of tumor-infiltrating CD8+ T cells is prevented, the cytokine binding moiety and the anti-PD1 antibody are covalently bound, and the tumor-infiltrating CD8+ T cells are analyzed for the expression of one or more of the following markers: inhibitory PD1 receptor, TIM3, and cytotoxic granzyme B, and either induce a decrease in the expression of the inhibitory PD1 receptor, or induce a decrease in the expression of TIM3 in CD8+ T cells compared to untreated cells or cells treated with the non-covalently bound cytokine binding moiety and the anti-PD1 antibody, or induce a decrease in the expression of the inhibitory PD1 receptor and induce a decrease in the expression of TIM3 in CD8+ T cells compared to untreated cells or cells treated with the non-covalently bound cytokine binding moiety and the anti-PD1 antibody; (xi) Containing IL-2R and PD1 on target cells, and comprising a cytokine binding moiety and an anti-PD1 antibody, (a) Induce activation of tumor-infiltrating CD8+ T cells, and (b) The same tumor-infiltrating CD8+ T cell exhaustion as in (a), wherein the cytokine binding portion and the anti-PD1 antibody are covalently bound, and the tumor-infiltrating CD8+ T cells are analyzed for the expression of one or more of the following markers: inhibitory PD1 receptor, TIM3, and cytotoxic granzyme B, and induce a decrease in the expression of the inhibitory PD1 receptor, or induce a decrease in the expression of TIM3 in CD8+ T cells compared to untreated cells or cells treated with the cytokine binding portion and anti-PD1 antibody that are not covalently bound, or the inhibitory P A bifunctional molecule comprising: (xiiii) a target cell, which binds to IL-2R and CD3 and comprises a cytokine binding moiety and an anti-CD3 antibody; or (xiiii) a target cell, which binds to IL-2R and CD3 and comprises a cytokine binding moiety and an anti-CD3 antibody.

6. A bifunctional molecule according to any one of claims 1 to 5, (i) comprising an IL-13-based sequence exhibiting increased binding affinity to IL-13Rα1 and decreased binding affinity to IL-13Rα2; (ii) comprising an IL-13-based sequence exhibiting increased binding affinity to IL-13Rα1 and decreased binding affinity to IL-13Rα2, wherein the increase in binding affinity to 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, or 120-fold or greater, and the decrease in binding affinity to IL-13Rα2 is at least 1 / 30, 1 / 40, 1 / 50, or 1 / 60 or less; (iii) comprising an IL-13-based sequence exhibiting increased binding affinity to IL-13Rα1 and decreased binding affinity to IL-13Rα2, and inhibiting pSTAT6 signaling by at least 20%, at least 30%, at least 40%, or at least 50%; (iv) comprising an IL-13-based sequence exhibiting increased binding affinity to IL-13Rα1 and decreased binding affinity to IL-13Rα2, and inhibiting IL-13-induced TF-1 proliferation by at least 20%, at least 30%, at least 40%, or at least 50%; (v) comprising an IL-13-based sequence exhibiting increased binding affinity to IL-13Rα1 and decreased binding affinity to IL-13Rα2, and inhibiting IL-4 or IL-13-induced M2 polarization of macrophages by at least 20%, at least 30%, at least 40%, or at least 50%; or (vi) A bifunctional molecule containing an IL-4-based sequence in which specific binding to type I or type II IL-4R is increased by at least 5, 8, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, or 120 times compared to natural IL-4.

7. A bifunctional molecule according to any one of claims 1 to 6, a. Sequence IDs 271, 272, and 273, b. Sequence IDs 274, 275, and 276, c. Sequence IDs 283, 284, and 285, and d. Sequence IDs 365, 366, and 367 The bifunctional molecule comprising an IL-2 mutein or IL-2-based sequence containing a sequence selected from the group.

8. A bifunctional molecule according to any one of claims 1 to 7, (i) Covalently conjugate with an antibody selected from the group consisting of dupilumab, nivolumab (OPDIVO®), BMS-936558, MDX-1106, ONO-4538, AMP224, CT-011, and MK-3475 (pembrolizumab or KEYTRUDA®), semiplimab (REGN2810), SHR-1210 (CTR20160175 and CTR20170090), SHR-1210 (CTR20170299 and CTR20170322), JS-001 (CTR20160274), IBI308 (CTR20160735), and BGB-A317 (CTR20160872); or (ii) A bifunctional molecule that covalently binds to an antibody selected from the group consisting of anti-CTLA4mAb, anti-PD-L1 antagonist antibody, anti-LAG-3, agonist antibody targeting immunostimulatory proteins (including anti-CD40mAb), anti-CD137mAb (anti-4-1-BB antibody), lirirumab (anti-KIRmAb, IPH2102 / BMS-986015, which blocks NK cell inhibitory receptors) and PF-05082566 (utomirumab), anti-OX40mAb, anti-GITRmAb, anti-CD27mAb, anti-ICOSmAb, Herceptin, anti-EGFR, anti-VEGF, anti-TIGIT, anti-LAG3, anti-CD8, anti-CD47, anti-SIRS alpha, and anti-CD112R.

9. A composition comprising one or more amino acid sequences from sequence numbers 395, 484, 501, 502, 503, 504, 505, 506, 507, or 508.

10. A nucleic acid encoding a bifunctional molecule or composition according to any one of claims 1 to 9.

11. A vector comprising the nucleic acid described in claim 10.

12. A method for treating a target cancer requiring cancer treatment, comprising administering a bifunctional molecule or composition according to any one of claims 1 to 9.

13. A method for treating a target cancer requiring cancer treatment, comprising administering a nucleic acid encoding a bifunctional molecule or composition according to any one of claims 1 to 9.

14. A method for treating a viral disease requiring treatment of a viral disease, comprising administering a vector comprising a nucleic acid encoding a bifunctional molecule or composition according to any one of claims 1 to 9.

15. A method of treating cancer, including the use of combination therapy, i) Therapeutic antibodies, ii) A bifunctional molecule or composition according to any one of claims 1 to 9, wherein the bifunctional molecule comprises an IL-2, IL-4, or IL-13 based sequence according to any one of claims 1 to 9, The method, including the method described above.

16. A pharmaceutical composition comprising a bifunctional molecule according to any one of claims 1 to 9 and a pharmaceutically acceptable carrier.

17. A pharmaceutical composition comprising an anti-PD-1 antibody or inhibitor, a bifunctional molecule or composition according to any one of claims 1 to 9, and a pharmaceutically acceptable carrier.

18. A pharmaceutical composition comprising an anti-PD-L1 antibody or inhibitor, a bifunctional molecule or composition according to any one of claims 1 to 9, and a pharmaceutically acceptable carrier.

19. A pharmaceutical composition comprising an anti-CD3 antibody or inhibitor, a bifunctional molecule or composition according to any one of claims 1 to 9, and a pharmaceutically acceptable carrier.

20. A pharmaceutical composition comprising a therapeutic antibody or inhibitor according to claim 8, a bifunctional molecule or composition according to any one of claims 1 to 9, and a pharmaceutically acceptable carrier.

21. The pharmaceutical composition according to any one of claims 16 to 20, wherein the difunctional molecule is covalently bonded to an anti-PD-1 antibody or inhibitor, an anti-PD-L1 antibody or inhibitor, an anti-CD3 antibody or inhibitor, or the therapeutic antibody or inhibitor according to claim 8.

Images