Interleukin-2-Fc fusion proteins and methods of use

Abstract

To provide interleukin-2-Fc fusion proteins and methods of use.SOLUTION: Provided are serum half-life extended-IL-2 variant (IL-2v) heterodimers having reduced binding to the IL2Rα (CD25) subunit. In particular, Fc-IL-2v heterodimers are provided and methods for making and using, e.g. for enhancing an immune response, e.g. in the prevention and treatment of viral infections and cancer. In various embodiments. IL-2v is truncated at the N-terminus by at least 5 amino acids relative to wild-type IL-2 and binds to the interleukin-2 receptor alpha subunit (IL-2RA; CD25) with reduced binding affinity in comparison to wild-type IL-2 (wt IL-2).SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 63 / 104,376, filed October 22, 2020, and U.S. Provisional Patent Application No. 63 / 181,075, filed April 28, 2021, which are incorporated by reference herein in their entireties for all purposes.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy created on September 20, 2021 is titled 1348-WO-PCT_SL.txt and is 569,532 bytes in size. [Background technology]

[0003] Interleukin-2 (IL-2, NCBI gene ID: 3558) is an immunoregulatory cytokine that plays an important role in the development, differentiation, survival, and homeostasis of immune cells. IL-2 is a cytokine that mediates the proliferation and differentiation of CD4 + and CD8 +Through its immunostimulatory effects on T cells and NK cells, IL-2 has therapeutic potential for the treatment of cancer and chronic viral diseases; however, this is compromised by its ability to preferentially stimulate and expand regulatory T (Treg) cells, which suppress the immune system. In addition to this preferential activity on immunosuppressive Treg cells, IL-2 has a very short half-life in humans, requiring frequent dosing and potentially inducing life-threatening toxicities. The short half-life complicates the ability to administer sufficient doses of IL-2 to induce the desired immune stimulation with reduced or minimal immunosuppressive effects while also avoiding toxicity, presenting a significant challenge for patient treatment. See, e.g., Schwartz, et al., "Managing toxicities of high-dose interleukin-2," in Oncology (Williston Park) (2002) Nov;16(11 Suppl 13):11-20. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Schwartz, et al., “Managing toxicities of high-dose interleukin-2,” in Oncology (Williston Park) (2002) Nov;16(11 Suppl 13):11-20 Summary of the Invention [Means for solving the problem]

[0005] In one aspect, provided is an interleukin-2 variant (IL-2v). In various embodiments, IL-2v is truncated by at least five amino acids at the N-terminus compared to wild-type IL-2 and binds to the interleukin-2 receptor alpha subunit (IL-2RA; CD25) with reduced binding affinity compared to wild-type IL-2 (wt IL-2). In some embodiments, IL-2v binds to IL-2RA with an equilibrium dissociation constant (KD) of at least 60 μM (e.g., 60 μM or greater). In some embodiments, IL-2v binds to the interleukin-2 receptor subunit beta (IL-2RB; CD122) and the interleukin-2 receptor subunit alpha (IL-2RB; CD122). In some embodiments, IL-2v binds to a complex with the IL-2 receptor subunit gamma (IL-2RG; CD132) with a KD of less than 150 nM, such as less than 1.5 nM, for example, less than 120 pM, for example, less than 100 pM, for example, less than 80 pM, for example, less than 75 pM, for example, less than 70 pM. In some embodiments, IL-2v binds to a complex with the IL-2 receptor subunit gamma (IL-2RG; CD132) with a KD of less than 150 nM, for example, ... In some embodiments, IL-2v promotes CD8+ T cell proliferation at or above the concentration that induces 50% of maximal signal transducer and activator of transcription (STAT5) activation or signaling in regulatory T (Treg) cells (EC5 0) is at least 1000-fold, such as at least 1500-fold, for example at least 1700-fold, such as at least 2000-fold, for example at least 2500-fold higher than the EC50 for STAT5 activation or signaling of wt IL-2, or IL-2v of any one of SEQ ID NOs: 43 and 44. In some embodiments, the concentration at which IL-2v induces the EC50 for IL-2Rαβγ-mediated STAT5 activation or signaling (e.g., measured as STAT5 activation of CTLL2 cells) is at least 2500-fold, such as at least 5000-fold, for example at least 7500-fold, such as at least 10,000-fold, for example at least 15,000-fold, for example at least 20,000-fold higher than the EC50 for STAT5 activation or signaling of wt IL-2, or IL-2v of any one of SEQ ID NOs: 43 and 44. In some embodiments, IL-2v induces the EC50 for IL-2Rαβγ-mediated STAT5 activation or signaling (e.g., measured as STAT5 activation of CTLL2 cells) is at least 2500-fold, such as at least 5000-fold, for example at least 7500-fold, such as at least 10,000-fold, for example at least 15,000-fold, for example at least 20,000-fold higher than the EC50 for STAT5 activation or signaling of wt IL-2, or IL-2v of any one of SEQ ID NOs: 43 and 44. The concentration inducing 50% of macroproliferation (EC50) is at least 10-fold, such as at least 12-fold, such as at least 15-fold, such as at least 16-fold, such as at least 18-fold, such as at least 20-fold higher, as measured, for example, using KHYG-1 cells, compared to the EC50 for proliferation of wt IL-2 or an IL-2v of any one of SEQ ID NOs: 43 and 44. In some embodiments, the IL-2v comprises a serine at position 125 (C125) and at least two or at least three substitutions at amino acid positions selected from the group consisting of R38, F42, Y45, E61, and E62, where the position numbers are relative to the IL-2v of SEQ ID NO: 44. In some embodiments, the IL-2v comprises a serine at position 125 (C125) and at least two or at least three substitutions at amino acid positions selected from the group consisting of R38, F42, Y45, and E62, where the positions are numbered relative to the IL-2v of SEQ ID NO: 44. In some embodiments, the IL-2v comprises a serine at position 125 (C125) and at least two or at least three substitutions at amino acid positions selected from the group consisting of R38, F42, and E62, where the positions are numbered relative to the IL-2v of SEQ ID NO: 44. In some embodiments, the IL-2v does not comprise an amino acid substitution at one or more positions selected from the group consisting of Y45, E61, E68, and L72. In some embodiments, the IL-2v does not comprise an amino acid substitution at one or more or all of positions selected from the group consisting of D20, Y45, E61, E68, V69, L72, A73, L80, R81, L85, L86, 187, 192, and Q126. In some embodiments, the IL-2v does not comprise an amino acid substitution at one or more or all of positions selected from the group consisting of H16, D20, E61, N88, and V91. In some embodiments, the IL-2v is PEGylated. In one aspect, provided are fusion proteins. In some embodiments, the fusion protein comprises a serum half-life extending polypeptide operably linked to an interleukin-2 variant (IL-2v), wherein IL-2v is truncated by at least five amino acids at the N-terminus compared to wild-type IL-2 and binds to the interleukin-2 receptor alpha subunit (IL-2RA; CD25) with reduced binding affinity compared to wild-type IL-2 (wt IL-2). In some embodiments, the serum half-life extending polypeptide is selected from the group consisting of an immunoglobulin fragment crystallizable region (Fc region), serum albumin, an albumin-binding protein or peptide, an IgG, an XTEN polypeptide, a proline / alanine / serine-rich (PAS) polypeptide, or an elastin-like polypeptide. In some embodiments, the serum half-life extending polypeptide is an immunoglobulin fragment crystallizable region (Fc region). In some embodiments, the fusion protein binds to IL-2RA with an equilibrium dissociation constant (KD) of at least 60 μM (e.g., 60 μM or more). In some embodiments, IL-2v binds to the complex of interleukin-2 receptor subunit beta (IL-2RB; CD122) and interleukin-2 receptor subunit gamma (IL-2RG; CD132) with a KD of less than 150 nM, e.g., less than 1.5 nM, e.g., less than 120 pM, e.g., less than 100 pM, e.g., less than 80 pM, e.g., less than 75 pM, e.g., less than 70 pM. In some embodiments, the fusion protein promotes equal or greater proliferation of CD8+ T cells compared to an IL-2v of any one of SEQ ID NOs: 43 and 44, a fusion protein comprising an Fc operably linked to wt IL-2, or a fusion protein of any one of SEQ ID NOs: 117, 118, 161, and 162.In some embodiments, the concentration (EC50) at which the IL-2v fusion protein induces 50% of maximal signal transducer and activator of transcription 5 (STAT5) activation or signaling of regulatory T (Treg) cells is at least 1000-fold, such as at least 1500-fold, such as at least 1700-fold, such as at least 2000-fold, such as at least 2500-fold higher than the EC50 for STAT5 activation or signaling of wt IL-2, or an IL-2v of any one of SEQ ID NOs: 43 and 44, a fusion protein comprising an Fc operably linked to wt IL-2, or a fusion protein of any one of SEQ ID NOs: 117, 118, 161, or 162. In some embodiments, the concentration at which the IL-2v fusion protein induces an EC50 for IL-2Rαβγ-mediated STAT5 activation or signaling (e.g., measured as STAT5 activation of CTLL2 cells) is at least 2500-fold, such as at least 5000-fold, for example, at least 7500-fold, such as at least 10,000-fold, for example, at least 15,000-fold, for example, at least 20,000-fold higher than the EC50 for STAT5 activation or signaling of wt-IL2, or an IL-2v of any one of SEQ ID NOs: 43 and 44, a fusion protein comprising an Fc operably linked to wt IL 2, or a fusion protein of any one of SEQ ID NOs: 117, 118, 161, or 162. In some embodiments, the concentration at which the IL-2v fusion protein induces 50% of maximal proliferation (EC50) of natural killer (NK) cells is at least 10-fold, such as at least 12-fold, such as at least 15-fold, such as at least 16-fold, such as at least 18-fold, such as at least 20-fold higher than the EC50 for proliferation of wt IL-2, or an IL-2v of any one of SEQ ID NOs: 43 and 44, a fusion protein comprising an Fc operably linked to wt IL-2, or a fusion protein of any one of SEQ ID NOs: 117, 118, 161, or 162, e.g., as measured using KHYG-1 cells.In some embodiments, an IL-2v comprises a serine at position 125 (C125) and at least two or at least three substitutions at an amino acid position selected from the group consisting of R38, F42, Y45, E61, and E62, as position numbered relative to an IL-2v of SEQ ID NO: 44. In some embodiments, an IL-2v comprises a serine at position 125 (C125) and at least two or at least three substitutions at an amino acid position selected from the group consisting of R38, F42, Y45, and E62, as position numbered relative to an IL-2v of SEQ ID NO: 44. In some embodiments, an IL-2v comprises a serine at position 125 (C125) and at least two or at least three substitutions at an amino acid position selected from the group consisting of R38, F42, and E62, as position numbered relative to an IL-2v of SEQ ID NO: 44. In some embodiments, IL-2v does not comprise an amino acid substitution at one or more positions selected from the group consisting of Y45, E61, E68, and L72. In some embodiments, IL-2v comprises the amino acid sequence APTSS (SEQ ID NO: 163). In some embodiments, IL-2v is from human wild-type IL-2. In some embodiments, IL-2v comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-42, or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-42. In some embodiments, the Fc region is from human IgG1, IgG2, IgG3, or IgG4. In some embodiments, the Fc region is from human IgG1 or IgG4.In some embodiments, the Fc region comprises a human IgG1 isotype and is selected from the group consisting of N297A, N297G, N297Q, N297G, D265A, L234A, L235A, C226S, C229S, P238S, E233P, L234V, P238A, A327Q, A327G, P329A, P329G, K322A, L234F, L235E, P331S, T394 In some embodiments, the Fc region comprises one or more amino acid substitutions in the Fc region at residue positions selected from the group consisting of: D, A330L, M252Y, S254T, T256E, M428L, N434S, T366W, T366S, L368A, F405L, Y407V, K409R, H435R, Y436F, and any combination thereof, where residue numbering is according to EU numbering. In some embodiments, the Fc region comprises a human IgG1 isotype and comprises one or more amino acid substitutions in the Fc region at residue positions selected from the group consisting of: L234A, L234V, L234F, L235A, L235E, D265A, P329G, P331S, and any combination thereof, where residue numbering is according to EU numbering. In some embodiments, the Fc region comprises a human IgG4 isotype, and comprises one or more amino acid substitutions in the Fc region at residue positions selected from the group consisting of E233P, F234V, F234A, L235A, G237A, E318A, S228P, L235E, T394D, M252Y, S254T, T256E, N297A, N297G, N297Q, T366W, T366S, L368A, F405L, Y407V, K409R, M428L, N434S, H435R, Y436F, and any combination thereof, wherein residue numbering is according to EU numbering. In some embodiments, the Fc region comprises a human IgG4 isotype and comprises one or more amino acid substitutions in the Fc region at residue positions selected from the group consisting of F234V, F234A, L235A, L235E, S228P, and any combination thereof, wherein residue numbering is according to EU numbering.In some embodiments, the Fc region comprises the following amino acids at the indicated positions (EU index numbering): a tyrosine at position 252, a threonine at position 254, and a glutamic acid (YTE) at position 256; or a leucine at position 428 and a serine (LS) at position 434. In some embodiments, the Fc region comprises the following amino acids at the indicated positions (EU index numbering): an arginine at position 435 and a phenylalanine at position 436. In some embodiments, the terminal Fc amino acid residue (e.g., K447) is removed or eliminated. In some embodiments, the Fc region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 45-72, or an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 45-72. In some embodiments, the Fc region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 45, 47, 49, 52, 54, 56, 57, 59, 61, 63, 65, 67, 69, and 71, or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 45, 47, 49, 52, 54, 56, 57, 59, 61, 63, 65, 67, 69, and 71. In some embodiments, the fusion protein comprises, in N-terminal to C-terminal order, an Fc region and IL-2v. In some embodiments, the fusion protein comprises a flexible linker between the Fc region and IL-2v. In some embodiments, the linker has a length of 4 to 50 amino acids, e.g., 5 to 25 amino acids, e.g., 15 to 25 amino acids, In some embodiments, the linker comprises a poly-glycine serine linker of 1 to 10 units, e.g., 1 to 5 units, e.g., 3 to 5 units, selected from GGGS (SEQ ID NO: 265), GGGGS (SEQ ID NO: 264), and combinations thereof.In some embodiments, the fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 75-116 and 119-160, or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 75-116 and 119-160. In some embodiments, the fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 166-171, or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 166-171. In some embodiments, the fusion protein does not specifically bind to any antigen other than an Fc receptor or the complex of interleukin-2 receptor subunit beta (IL-2RB; CD122) and interleukin-2 receptor subunit gamma (IL-2RG; CD132). In some embodiments, the fusion protein comprises an N-terminal signal peptide or leader sequence. In some embodiments, the IL-2v in the fusion protein is not PEGylated.

[0006] Also provided is a homodimer comprising two Fc-IL-2v fusion proteins described above and herein.

[0007] In another aspect, provided is a heterodimer comprising (i) a first Fc-IL-2v fusion protein as described above and herein, comprising a first Fc domain, and (ii) a second Fc-IL-2v fusion protein as described above and herein, comprising a second Fc domain. In another aspect, provided is a heterodimer comprising (i) one (i.e., one) Fc-IL-2v fusion protein as described above and herein, comprising a first Fc domain, and (ii) a second Fc domain that is, for example, empty or does not comprise a targeting moiety or antigen-binding domain. In some embodiments, the first Fc domain and the second Fc domain contain the following amino acid substitutions (EU numbering), respectively: T366W and T366S / L368A / Y407V; T366S / L368A / Y407V and T366W; T366W / S354C and T366S / L368A / Y407V / Y349C; T366S / L368A / Y407V / Y349C and and T366W / S354C; S364H / F405A and Y349T / T394F; Y349T / T394F and S364H / F405A; T350V / L351Y / F405A / Y407V and T350V / T366L / K392L / T394W; T350V / T366L / K392L / T394W and T350V / L351Y / F405A / Y407V; K360D / D399 M / Y407A and E345R / Q347R / T366V / K409V;E345R / Q347R / T366V / K409V and K360D / D399M / Y407A;K409D / K392D and D399K / E356K;D399K / E356K and K409D / K392D;K360E / K409W and Q347R / D399V / F405T;Q347R / D399V / F In some embodiments, one or both of the first Fc domain and the second Fc domain comprises: K360E / K409W and Q347R / D399V / F405T; K360E / K409W / Y349C and Q347R / D399V / F405T / S354C; Q347R / D399V / F405T / S354C and K360E / K409W / Y349C; K370E / K409W and E357N / D399V / F405T; or E357N / D399V / F405T and K370E / K409W. In some embodiments, one or both of the first Fc domain and the second Fc domain comprises:or leucine at position 428 and serine (LS) at position 434. In some embodiments, one or both of the first Fc domain and the second Fc domain comprise the following amino acids at the indicated positions (EU index numbering): arginine at position 435 and phenylalanine at position 436. In some embodiments, one or both of the first Fc domain and the second Fc domain comprise the human IgG4 isotype and comprise one or more amino acid substitutions in the Fc region at residue positions selected from the group consisting of F234V, F234A, L235A, L235E, S228P, and any combination thereof, wherein the numbering of residues is according to EU numbering. In some embodiments, one or both of the first Fc domain and the second Fc domain comprise a human IgG1 isotype and comprise one or more amino acid substitutions in the Fc region at residue positions selected from the group consisting of L234A, L234V, L234F, L235A, L235E, P331S, and any combination thereof, where residue numbering is according to EU numbering. In some embodiments, a terminal Fc amino acid residue (e.g., K447) is removed or eliminated from one or both of the first Fc domain and the second Fc domain. In some embodiments, the first Fc domain and the second Fc domain, respectively, comprise the amino acid sequence set forth below, or are at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, orOr comprising amino acid sequences which are at least 99% identical: SEQ ID NOs: 45 and 46; SEQ ID NOs: 47 and 48; SEQ ID NOs: 49 and 46; SEQ ID NOs: 45 and 51; SEQ ID NOs: 49 and 51; SEQ ID NOs: 52 and 48; SEQ ID NOs: 47 and 53; SEQ ID NOs: 52 and 53; SEQ ID NOs: 54 and 46; SEQ ID NOs: 45 and 55; SEQ ID NOs: 54 and 55; SEQ ID NOs: 56 and 48; SEQ ID NOs: 47 and 50; SEQ ID NOs: 56 and 50; SEQ ID NOs: 57 and 58; SEQ ID NOs: 59 and 60; SEQ ID NOs: 61 and 58; SEQ ID NOs: 57 and 62; SEQ ID NOs: 63 and 64; SEQ ID NOs: 65 and 60; SEQ ID NOs: 59 and 66; SEQ ID NOs: 67 and 68; SEQ ID NOs: 69 and 58; SEQ ID NOs: 57 and 70; SEQ ID NOs: 69 and 70; SEQ ID NOs: 71 and 60; SEQ ID NOs: 59 and 72; or SEQ ID NOs: 71 and 72. In some embodiments, the heterodimer comprises a human IgG4 Fc-IL-2v fusion protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 75-116, or an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 75-116; and a second Fc region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 46, 51, and 55, or an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 46, 51, and 55. In some embodiments, the heterodimer comprises (i) a first amino acid sequence set forth below, or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the first amino acid sequence set forth below; and (ii) a second amino acid sequence set forth below, or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the second amino acid sequence set forth below.and a second Fc region comprising an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a human IgG4. Fc-IL-2v fusion proteins include: SEQ ID NO:75 and SEQ ID NO:46; SEQ ID NO:76 and SEQ ID NO:46; SEQ ID NO:77 and SEQ ID NO:46; SEQ ID NO:78 and SEQ ID NO:46; SEQ ID NO:79 and SEQ ID NO:46; SEQ ID NO:80 and SEQ ID NO:46; SEQ ID NO:81 and SEQ ID NO:46; SEQ ID NO:82 and SEQ ID NO:46; SEQ ID NO:83 and SEQ ID NO:46; SEQ ID NO:84 and SEQ ID NO:46; SEQ ID NO:85 and SEQ ID NO:46; SEQ ID NO:86 and SEQ ID NO:46; SEQ ID NO:87 and SEQ ID NO:46; SEQ ID NO:88 and SEQ ID NO:46; SEQ ID NO:89 and SEQ ID NO:46; SEQ ID NO:90 and SEQ ID NO:46; SEQ ID NO:91 and SEQ ID NO:46; SEQ ID NO:92 and SEQ ID NO:46; SEQ ID NO:93 and SEQ ID NO:46; SEQ ID NO:94 and SEQ ID NO:46; SEQ ID NO:95 and SEQ ID NO:46; SEQ ID NO:96 and SEQ ID NO:46; SEQ ID NO:97 and SEQ ID NO:46; SEQ ID NO:98 and SEQ ID NO:46; SEQ ID NO:99 and SEQ ID NO:46; SEQ ID NO:100 and SEQ ID NO:46; SEQ ID NO:101 and SEQ ID NO:46; SEQ ID NO:102 and SEQ ID NO:46; SEQ ID NO:103 and SEQ ID NO:46; SEQ ID NO:104 and SEQ ID NO:46; SEQ ID NO:105 and SEQ ID NO:46; SEQ ID NO:106 and SEQ ID NO:46; SEQ ID NO:107 and SEQ ID NO:46; SEQ ID NO:108 and SEQ ID NO:46; SEQ ID NO:109 and SEQ ID NO:46; SEQ ID NO:110 and SEQ ID NO:46; SEQ ID NO:111 and SEQ ID NO:46; SEQ ID NO:112 and SEQ ID NO:46; SEQ ID NO:113 and SEQ ID NO:46; SEQ ID NO:114 and SEQ ID NO:46; SEQ ID NO:115 and SEQ ID NO:46; or SEQ ID NO:116 and SEQ ID NO:46. In some embodiments, the heterodimer comprises (i) a first amino acid sequence set forth below, or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the first amino acid sequence set forth below;(ii) a second Fc region comprising a second amino acid sequence set forth below, or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the second amino acid sequence set forth below: SEQ ID NO:80 and SEQ ID NO:46; SEQ ID NO:107 and SEQ ID NO:46; or SEQ ID NO:114 and SEQ ID NO:46, respectively. In some embodiments, the heterodimer comprises a human IgG4 Fc-IL-2v fusion protein comprising: (i) a first amino acid sequence of SEQ ID NO: 114, or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 114; and (ii) a second Fc region comprising an amino acid sequence of SEQ ID NO: 46, or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 46. In some embodiments, the heterodimer is a human IgG1 Fc-IL-2v fusion protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 119-160, or an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 119-160, and an amino acid sequence selected from the group consisting of SEQ ID NOs: 58, 62, and 70, or an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%,and a second Fc region comprising an amino acid sequence at least 97%, at least 98%, or at least 99% identical. In some embodiments, the heterodimer comprises (i) a first amino acid sequence set forth below, or an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the first amino acid sequence set forth below, and (ii) a second amino acid sequence set forth below, or an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, and a second Fc region comprising an amino acid sequence at least 96%, at least 97%, at least 98%, or at least 99% identical to: SEQ ID NO:119 and SEQ ID NO:58; SEQ ID NO:120 and SEQ ID NO:58; SEQ ID NO:121 and SEQ ID NO:58; SEQ ID NO:122 and SEQ ID NO:58; SEQ ID NO:123 and SEQ ID NO:58; SEQ ID NO:124 and SEQ ID NO:58; SEQ ID NO:125 and SEQ ID NO:58; SEQ ID NO:126 and SEQ ID NO:58; SEQ ID NO:127 and SEQ ID NO:58; SEQ ID NO:128 and SEQ ID NO:58; SEQ ID NO:129 and SEQ ID NO:58; SEQ ID NO:130 and SEQ ID NO:58; SEQ ID NO:131 and SEQ ID NO:58; SEQ ID NO:132 and SEQ ID NO:58; SEQ ID NO:133 and SEQ ID NO:58; SEQ ID NO:134 and SEQ ID NO:58; SEQ ID NO:135 and SEQ ID NO:58; SEQ ID NO:136 and SEQ ID NO:58; SEQ ID NO:137 and SEQ ID NO:58; SEQ ID NO:138 and SEQ ID NO:58; SEQ ID NO:139 and SEQ ID NO:58; SEQ ID NO:140 and SEQ ID NO:58; SEQ ID NO:141 and SEQ ID NO:58; SEQ ID NO:142 and SEQ ID NO:58; SEQ ID NO:143 and SEQ ID NO:58; SEQ ID NO:144 and SEQ ID NO:58; SEQ ID NO:145 and SEQ ID NO:58; SEQ ID NO:146 and SEQ ID NO:58; SEQ ID NO:147 and SEQ ID NO:58; SEQ ID NO:148 and SEQ ID NO:58; SEQ ID NO:149 and SEQ ID NO:58; SEQ ID NO:150 and SEQ ID NO:58; SEQ ID NO:151 and SEQ ID NO:58; SEQ ID NO:152 and SEQ ID NO:58; SEQ ID NO:153 and SEQ ID NO:58; SEQ ID NO:154 and SEQ ID NO:58; SEQ ID NO:155 and SEQ ID NO:58; SEQ ID NO:156 and SEQ ID NO:58; SEQ ID NO:157 and SEQ ID NO:58; SEQ ID NO:158 and SEQ ID NO:58; SEQ ID NO:159 and SEQ ID NO:58; or SEQ ID NO:160 and SEQ ID NO:58.In some embodiments, the heterodimer comprises a human IgG1 Fc-IL-2v fusion protein comprising (i) a first amino acid sequence set forth below, or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the first amino acid sequence set forth below; and (ii) a second amino acid sequence set forth below, or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the second amino acid sequence set forth below, respectively: SEQ ID NO:124 and SEQ ID NO:58; SEQ ID NO:151 and SEQ ID NO:58; or SEQ ID NO:158 and SEQ ID NO:58. In some embodiments of the heterodimer, the polypeptide comprising the first Fc domain comprises a first N-terminal signal peptide or leader sequence, and the polypeptide comprising the second Fc domain comprises a second N-terminal signal peptide or leader sequence. In some embodiments, the first N-terminal signal peptide or leader sequence and the second first N-terminal signal peptide or leader sequence are the same. In some embodiments, the first N-terminal signal peptide or leader sequence and the second first N-terminal signal peptide or leader sequence are different. In some embodiments, the heterodimer does not specifically bind to any antigen other than an Fc receptor or a complex of interleukin-2 receptor subunit beta (IL-2RB; CD122) and interleukin-2 receptor subunit gamma (IL-2RG; CD132). In some embodiments, the second Fc domain is not fused to an antigen-binding domain. In some embodiments, neither the first nor the second Fc domain is fused to an antigen-binding domain.In some embodiments, the heterodimer has a serum half-life in humans of at least 6, 9, 12, 15, 18, 21, or 24 hours, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days or more. In some embodiments, a second Fc domain is fused to the antigen-binding domain. In some embodiments, the antigen-binding molecule binds to CD8. In some embodiments, the antigen-binding domain binds to an immune checkpoint protein. In some embodiments, the immune checkpoint protein is CD27, CD70; CD40, CD40LG; CD47, CD48 (SLAMF2), transmembrane and immunoglobulin domain containing 2 (TMIGD2, CD28H), CD84 (LY9B, SLAMF5), CD96, CD160 (NK1, NK28, BY55), MS4A1 (CD20), CD244 (SLAMF4); CD276 (B7H3); V-set domain containing T cell activation inhibitor 1 (VTC). N1, B7H4); V-set immunoregulatory receptor (V-set immunoregulatory receptor, VSIR, B7H5, VISTA; immunoglobulin superfamily member 11 (IGSF11, VSIG3); natural killer cell cytotoxicity receptor 3 ligand 1 (NCR3LG1, B7H6); HERV-H LTR-associating 2 (HHLA2, B7H7); inducible T cell co-stimulator (ICOS, CD278); inducible T cell costimulator ligand (ICOSLG, B7H2); TNF receptor superfamily member 4 (TNF receptor superfamily member, TNFRSF4, OX40; TNF superfamily member 4 (TNFSF4, OX40L); TNFRSF8 (CD30), TNFSF8 (CD30L); TNFRSF10A (CD261, DR4, TRAILR1), TNFRSF9 (CD137), TNFSF9 (CD137L); TNFRSF10B (CD262, DR5, TRAILR2), TNFRSF10 (TRAIL); TNFRSF14 (HVEM, CD270), TNFSF14 (HVEML); CD272 (B and T lymphocyte-associated (BTLA)); TNFRSF17 (BCMA, CD269), TNFSF13B (BAFF); TNFRSF18 (GITR), TNFSF18 (GITRL); MHC class I polypeptide-related sequence A (MICA); MHC class I polypeptide MHC class I polypeptide-related sequence B (MICB); CD274 (CD274, PDL1, PD-L1); programmed cell death 1 (PDCD1, PD1, PD-1); cytotoxic T lymphocyte-associated protein 4(cytotoxic T-lymphocyte associated, CTLA4, CD152);CD80(B 7-1), CD28; nectin cell adhesion molecule 2 (NECTIN2, CD112); CD226 (DNAM-1); poliovirus receptor (PVR) cell adhesion molecule (PVR, CD155); PVR-related immunoglobulin domain containing (PVRIG, CD112R); T cell immunoreceptor with Ig and ITIM domain (TIGIT); T cell immunoglobulin and mucin domain containing 4 (T cell immunoglobulin and mucin domain containing , TIMD4; TIM4); hepatitis A virus cellular receptor 2 (HAVCR2, TIMD3, TIM3); galectin 9 (LGALS 9); lymphocyte activating 3 (LAG3, CD223); signaling lymphocytic activation molecule family member 1 (SLAMF1, SLAM, CD150); lymphocyte antigen 9 (LY9, CD229, SLAMF3); SLAM family member 6 ( SLAMF6, CD352; SLAM family member 7 (SLAMF7, CD319); sialic acid binding Ig-like lectin 7 (S IGLEC7; sialic acid-binding Ig-like lectin 9 (SIGLEC9); UL16 binding protein 1 (ULBP1); UL16 binding protein 2 (ULBP2); UL16 binding protein 3 (ULBP3); retinoic acid early transcript 1E (RAET1E; ULBP4) retinoic acid early transcript 1G (RAET1G; ULBP5); retinoic acid early transcript 1L (RAET1L; ULBP6); killer cell immunoglobulin-like receptor, three Ig domains, and long cytoplasmic tail 1 (killer cell immunoglobulin-like receptor, KIR, CD158E1); CD160; killer cell lectin-like receptor B1 (killer cell lectin-like receptor, KLRB1, CD161; killer cell lectin-like receptor C1 (KLRC1, NKG2A, CD159A); killer cell lectin-like receptor K1 (KLRK1, NKG2D, CD314); killer cell lectin-like receptor C2 (KLRC2, CD159c, NKG2C); killer cell lectin-like receptor C3 (KLRC3, NKG2E); killer cell lectin-like receptor C4 (KLRC4, NKG2F); killer cell immunoglobulin-like receptor, one Ig domain and long cytoplasmic tail 2 (KIR2DL1); killer cell immunoglobulin-like receptor, killer cell immunoglobulin-like receptor, two Ig domains and long cytoplasmic tail 2 (KIR2DL2); killer cell immunoglobulin-like receptor, three Ig domains and long cytoplasmic tail 2 (KIR2DL3); killer cell immunoglobulin-like receptor, three Ig domains and long cytoplasmic tail 1 (KIR3DL1); killer cell lectin-like receptor D1 (KLRD1); killer cell lectin-like receptor G1 (KLRG1; CLEC15A, MAFA, 2F1); sialic acid-binding Ig-like lectin 7 (SIGLEC7); and sialic acid-binding Ig-like lectin 9 (SIGLEC9). In some embodiments, the immune checkpoint protein is selected from the group consisting of CD274 (CD274, PDL1, PD-L1) and programmed cell death 1 (PDCD1, PD1, PD-1).In some embodiments, the antigen binding domain is a major histocompatibility complex (MHC) molecule-presented (pMHC), asialoglycoprotein receptor 1 (ASGR1), asialoglycoprotein receptor 2 (ASGR2), ATP binding cassette (ATPBC), or a combination thereof. ATP-binding cassette (ABC) family transporters (e.g., ATP-binding cassette subfamily B member 1 (ABCB1; P-GP), ATP-binding cassette subfamily B member 4 (ABCB4; MDR3), ATP-binding cassette subfamily C member 1 (ABCC1; MRP1), ATP-binding cassette subfamily C member 2 (ABCC2; MRP2), ATP-binding cassette subfamily C member 3 (ABCC3; MRP3), ATP-binding cassette subfamily C member 4 (ABCC4; MRP4), ATP-binding cassette subfamily G member 2 (Junior blood group; ABCG2; BCRP), and ATP-binding cassette subfamily B member 11 (ABCB11; also known as bile salt export pump, BSEP); solute carrier (SLC) family transporters (e.g., Solute carrier family 10 member 1 (SLC10A1; also known as sodium taurocholate cotransporter) Sodium-taurocholate Co-transporting Polypeptide (NTCP); Solute carrier family 16 member 1 (SLC16A1; MCT1), solute carrier family 22 member 1 (SLC22A1; OCT1), solute carrier family 22 member 3 (SLC22A3; OCT3), solute carrier family 22 member 7 (SLC22A7; OAT2), solute carrier family 27 member 5 (SLC27A5; FATP5), solute carrier organic anion transporter family member 1B1 (SLCO1B1; OATP1B1), solute carrier organic anion transporter family member 1B3 (SLCO1B3; OATP1B3), and solute carrier organic anion transporter family member 2B1 (SLCO2B1; OATP2B1), transferrin receptor 2 (TFR2, TFRC 2) and HBV epitopes (e.g., HBV core 18-27; env 181-193; env 335-343; pol 575-583). In some embodiments, the antigen-binding domain binds to a target selected from the group consisting of human immunodeficiency virus (HIV) gp120, HIV gp41, human CD4, and human interleukin-7 receptor (IL7R; CD127). In some embodiments, the antigen-binding domain binds to a target selected from the group consisting of herpes simple virus (HSV) glycoprotein B (gB), glycoprotein C (gC), glycoprotein D (gD), and glycoprotein E (gE). In embodiments, the antigen-binding domain is selected from the group consisting of CD19; membrane spanning 4-domains A1 (MS4A1); CD20; CD22 (SIGLEC2); CD2 7 (TNFRSF7); TNFRSF8 (CD30); CD33 (SIGLEC3); CD37; CD38; CD40 (TNFRSF5), CD44; CD47; CD48 (SLAMF2); CD52; CD70 (TNFSF7), CD27L; 5'-nucleotidase ecto (NT5E; CD73), ectonucleoside triphosphate diphosphohydrolase 1 (CD39); CD74; CD79B; CD80; CD86; interleukin 3 receptor subunit alpha (IL3RA), prominin 1 (PROM1; CD133); TNFRSF9 (CD137); syndecan 1 (SDC1; CD138); CD200 molecule (CD200); Alpha fetoprotein (AFP), BAG cochaperone 6 (BAG6); MET proto-oncogene, receptor tyrosine kinase (MET); KIT proto-oncogene, receptor tyrosine kinase (KIT); C-type lectin domain family 12 member A (CLEC12A; CD371) C-type lectin domain containing 9A (CLEC9A; CD370); cadherin 3 (CDH3); carbonic anhydrase 6 (CA6); carbonic anhydrase 9 (CA9); carcinoembryonic antigen-associated cell adhesion carcinoembryonic antigen-related cell adhesion molecule 3 (CEACAM3); carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5); carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6); chorionic somatomammotropin hormone 1 (CSH1); coagulation factor III, tissue factor (F3); collectin subfamily member 10 (COLEC10; CLL1); delta-like canonical Notch ligand 3 (DLL3); ectonucleotide pyrophosphatase / phosphodiesterase 3 (ENPP3); ephrin A1 (EFNA1); epidermal growth factor receptor (EGFR; E RBB; HER1; EGFR variant III (EGFRvIII); EPH receptor A2 (EPHA2); epithelial cell adhesion molecule (EPCAM); erb-b2 receptor tyrosine kinase 2 (ERBB2; HER-2 / neu); fibroblast activation protein alpha (FAP); fibroblast fibroblast growth factor receptor 2 (FGFR2); fibroblast growth factor receptor 3 (FGFR3); folate hydrolase 1 (FOLH1); folate receptor 1 (FOLR1); GD2 ganglioside; glycoprotein NMB (GPNMB; osteoactivin); guanylate cyclase 2C (GUCY2C); human papillomavirus (HPV) E6; HP V E7; major histocompatibility complex (MHC) class I-presented neoantigen, major histocompatibility complex (MHC) class II-presented neoantigen, major histocompatibility complex, class I, E (HLA-E); major histocompatibility complex, class I, F (HLA-F); major histocompatibility complex, class I, G (HLA-G); MHC class I polypeptide-related sequence A (MICA); MHC class I polypeptide-related sequence B (MICB); integrin subunit beta 7 (integrin subunit beta, ITGB7); leukocyte immunoglobulin-like receptor Leukocyte immunoglobulin-like receptor B1 (LILRB1; ILT2); leukocyte immunoglobulin-like receptor B2 (LILRB2; ILT4); LY6 / PLAUR domain-containing 3 (LYPD3); glypican 3 (GPC3); KRAS proto-oncogene, GTPase (KRAS); MAGE family MAGE family member A1 (MAGEA1); MAGE family member A3 (MAGEA3); MAGE family member A4 (MAGEA4); MAGE family member A11 (MAGEA11); MAGE family member C1 (MAGEC1); MAGE family member C2 (MAGEC2); MAGE family member C3 (MAGEC3); MAGE family member D1 (MAGED1); MAGE family member D2 (MAGED2); mesothelin (MSLN); whip mucin 1 (MUC1) and its splice variants (e.g., including MUC1 / A, C, D, X, Y, Z, and REP); mucin 16 (MUC16; CA125); natural killer cytotoxicity receptor 3 ligand 1 (NCR3LG1; B7-H6); necdin, a MAGE family member (NDN); nectin cell adhesion molecule 2 (NECTIN2); nectin cell adhesion molecule 4 (NECTIN4); SLIT and NTRK-like family member 6 (SLITRK6); promyelocytic leukemia (PML); and protein tyrosine kinase (TKI). Protein tyrosine kinase 7 (inactive) (PTK7); poliovirus receptor (PVR) cell adhesion molecule (PVR); SLAM family member 6 (SLAMF6); SLAM family member 7 (SLAMF7); sialic acid-binding Ig-like lectin 7 (SIGLEC7); sialic acid-binding Ig-like lectin 9 (SIGLEC9); sialic acid-binding Ig-like lectin 10 (SIGLEC10); signal-regulatory protein alpha (signal regulatory protein alpha (SIRPA); solute carrier family 34 (sodium phosphate), member 2 (SLC34A2); solute carrier family 39 member 6 (SLC39A6); STEAP family member 1 (STEAP1); suppression of tumorigenicity 2 (ST2); TNF receptor superfamily member a target selected from the group consisting of bar 4 (TNFRSF4; OX40); TNF superfamily member 9 (TNFSF9; 4-1BB-L, CD137L); TNFRSF10A (DR4, TRAILR1); TNFRSF10B (DR5, TRAILR2); TNFRSF13B (BAFF); TNFRSF17 (BCMA); TNFRSF18 (GITR); transferrin (TF); transforming growth factor beta 1 (TGFB1) and its isoforms; triggering receptor expressed on myeloid cells (TREM1); triggering receptor expressed on myeloid cells (TREM2); trophoblast glycoprotein (TPBG); trophinin (TRO); tumor-associated calcium signal transducer 2 (TACSTD2); fucosyl GM1; sialyl Lewis adhesion molecule (sLe); and Lewis Y antigen, or a tumor-associated antigen In some embodiments, the antigen binding domain binds to a target antigen or an epitope of a tumor-associated antigen (TAA) that is presented on a major histocompatibility complex (MHC) molecule. In some embodiments, the TAA is a cancer-testis antigen. In some embodiments, the cancer-testis antigen is acrosin binding protein (ACBP). RBP, alpha fetoprotein (AFP), A-kinase anchoring protein 4 (AKAP4), ATPase family AAA domain containing 2 (ATAD) 2), kinetochore scaffold 1 (KNL1; also known as CASC5), Cardiac body protein 55 (CEP55), cancer / testis antigen 1A (CTAG1A; also known as ESO1; CT6.1; LAGE-2; LAGE2A; NY-ESO-1), cancer / testis antigen 1B (CTAG1B; also known as CT6.1, CTAG, CTAG1, ESO1, LAGE-2, LAGE2B, NY-ESO-1), cancer / testis antigen 2 (CTAG2; also known as CAMEL, CT2, CT6.2, CT6.2a, CT6.2b, ESO2, LAGE-1, LAGE2B), CCCTC-binding factor like (CTCFL), catenin alpha 2 (CT NNA2), cancer / testis antigen 83 (CT83), cyclin A1 (CCNA1), DEAD-box helicase 43 (DDX43), developmental pluripotency associated 2 (DPPA2), fetal and adult testis expressed 1 (FATE1), FMR1 adjacent (FMR1NB) , HORMA domain containing 1 (HORMAD1), Inst Insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3), leucine zipper protein 4 (LUZP4), lymphocyte antigen 6 family member K (LY6K), and Maelstrom spermatogenesis transposon. Maelstrom spermatogenic transposon silencer (MAEL), MAGE family member A1 (MAGEA1); MAGE family member A3 (MAGEA3); MAGE family member A4 (MAGEA4); MAGE family member A11 (MAGEA11); MAGE family member C1 (MAGEC1); MAGE family member C2 (MAGEC2); MAGE family member D1 (MAGED1); MAGE family member D2 (MAGED2), kinesin family member 20B (KIF20B; also known as MPHOSPH1), NUF2 component of the NDC80 kinetochore complex (NUF2). F2), nuclear RNA export factor 2 (NXF2), PAS domain-containing repressor 1 (PASD1), PDZ binding kinase (PBK), piwi-like RNA-mediated gene silencing 2 (PIWIL-2), preferentially expressed antigen in melanoma (PRAME), sperm associated antigen 9 (SPAG9), sperm protein associated with the nucleus (SPANXA1), SPANX family member A2 (SPANXA2), SPANX family member C (SPANXC), SPANX family member D (SPANXD), SSX family member 1 (SSX1), SSX family member 2 (SSX2), synaptonemal complex protein 3 (SPANXA1), and SPANX family member A2 (SPANXA2). protein, SYCP3), testis-expressed intercellular bridge forming factor 14 (TEX14), transcription factor Dp family member 3 (TFDP3), serine protease 50 (P RSS50 (also known as TSP50), TTK protein kinase (TTK), and zinc finger protein 165 (ZNF16 5).

[0008] In a further aspect, provided is a conjugate comprising an IL-2v, an Fc-IL-2v fusion protein, a homodimer, or a heterodimer, as described above and herein, linked to a therapeutic agent. In some embodiments, the therapeutic agent is covalently attached to, e.g., the IL-2v, Fc-IL-2v fusion protein, homodimer, or heterodimer. In some embodiments, the therapeutic agent is a small organic compound. In some embodiments, the therapeutic agent is selected from GS-4224 and GS-4416. In some embodiments, the therapeutic agent is a pattern recognition receptor (PRR), e.g., Toll-like receptor (TLR), RIG-I-like receptor (RLR), N-terminal receptor (N-terminal receptor), or IL-2v fusion protein (Fc-IL-2v fusion protein). NOD-like receptor (NLR), AIM2-like receptor (ALR), C-type lectin receptor (CLR), DNA receptor In some embodiments, the therapeutic agent is an agonist or activator of a toll-like receptor (TLR), DExD / H-box helicase 58 (DDX58; also known as RIG-I), or stimulator of interferon genes (STING) receptor. In some embodiments, the TLR agonist or activator is selected from the group consisting of a TLR2 agonist, a TLR3 agonist, a TLR4 agonist, a TLR5 agonist, a TLR7 agonist, a TLR8 agonist, and a TLR9 agonist. In some embodiments, the TLR7 agonist is selected from the group consisting of vesatolimod (GS-9620), DS-0509, LHC-165, TMX-101 (imiquimod), RO7020531, and JNJ-4964, and / or the TLR8 agonist is selected from the group consisting of sergantolimod (GS-9688) and NKTR-262 (a dual TLR7 / TLR8 agonist).

[0009] In a further aspect, provided is a polynucleotide encoding an IL-2v described above and herein, an Fc-IL-2v fusion protein described above and herein, or a homodimer described above and herein. In some embodiments, the polynucleotide comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 175-212, or a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 175-212. Also provided is a polynucleotide or multiple polynucleotides encoding the Fc-IL-2v fusion protein and second Fc region of a heterodimer described above and herein. In some embodiments, the polynucleotide encoding the Fc-IL-2v fusion protein comprises a nucleic acid selected from the group consisting of SEQ ID NOs: 175-212, or a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 175-212. In some embodiments, the polynucleotide or polynucleotides encoding the second Fc region comprise a nucleic acid selected from the group consisting of SEQ ID NOs: 214-215, or a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 214-215. In some embodiments, the polynucleotide or polynucleotides are selected from the group consisting of DNA, cDNA, RNA, or mRNA. Also provided is an expression cassette or expression cassettes comprising one or more regulatory sequences operably linked to the polynucleotide or polynucleotides described above and herein.

[0010] In a further aspect, provided is a vector comprising a polynucleotide or polynucleotides, or expression cassette, as described above and herein. In some embodiments, the vector is a plasmid vector or a viral vector. In some embodiments, the viral vector comprises an oncolytic viral vector. In some embodiments, the viral vector comprises a DNA virus or an RNA virus. In some embodiments, the viral vector is selected from the group consisting of Adenoviridae (e.g., adenovirus), Arenaviridae (e.g., lymphocytic choriomeningitis mam arenavirus, Karimum arenavirus (also known as Pichindemum arenavirus), Poxviridae (e.g., vaccinia virus), Herpesviridae (e.g., herpesvirus, e.g., HSV-1), Parvoviridae (e.g., parvovirus H1), Reoviridae (e.g., reovirus), Retroviridae (e.g., lentivirus), Picornaviridae (e.g., Coxsackievirus, Seneca Valley virus, poliovirus), Paramyxoviridae (e.g., measles virus, Newcastle disease virus), and the vector is from a viral family selected from the group consisting of Rhabdoviridae (e.g., Vesicular stomatitis virus (VSV)), Togaviridae (e.g., Alphavirus, Sindbis virus), and Enteroviridae (e.g., Echovirus). Also provided is a lipoplex, e.g., a lipid nanoparticle (LNP), comprising a polynucleotide or polynucleotides, expression cassette, or vector described above and herein.

[0011] In a further aspect, provided is a cell or cell population comprising a polynucleotide or polynucleotides, expression cassette, or vector described above and herein, wherein the cell or cell population expresses IL-2v, an Fc-IL-2v fusion protein, homodimer, or heterodimer described above and herein. In some embodiments, the cell or cell population is a eukaryotic cell. In some embodiments, the cell or cell population comprises a mammalian cell, an insect cell, a plant cell, or a yeast cell. In some embodiments, the mammalian cell is a Chinese Hamster Ovary (CHO) cell. In some embodiments, the mammalian cell is a human cell. In some embodiments, the cell is a human embryonic kidney cell.

[0012] In a further aspect, provided are methods for producing Fc-IL-2 fusion protein heterodimers. In some embodiments, the methods comprise: (a) culturing a cell or cell population, as described above and herein, transformed with at least one polynucleotide or polynucleotides encoding an Fc-IL-2v fusion protein described herein, or an expression cassette or cassettes described herein, in a cell culture under conditions sufficient to express the Fc-IL-2 fusion protein heterodimer molecule; and (b) isolating or purifying the Fc-IL-2 fusion protein heterodimer molecule from the cell culture. In some embodiments, the Fc-IL-2 fusion polypeptide and the Fc polypeptide are expressed and assembled in the same cell. In some embodiments, the isolating or purifying step comprises Protein A chromatography. In some embodiments, the isolating or purifying step further comprises in-stream or immediate pH neutralization of the Protein A chromatography eluate. In some embodiments, the isolating or purifying step further comprises anion exchange chromatography. In some embodiments, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the Fc-IL-2 fusion protein heterodimeric molecules are isolated or purified (e.g., in monodisperse form). In some embodiments, at least 95%, 96%, 97%, 98%, 99% or more of the Fc-IL-2 fusion protein heterodimeric molecules are isolated or purified (e.g., in monodisperse form). In some embodiments, at least 98%, 99% or more of the Fc-IL-2 fusion protein heterodimeric molecules are isolated or purified (e.g., in monodisperse form). In some embodiments, the cells or cell populations are cultured in a culture volume of at least 2 L, e.g., at least 5 L, 10 L, 50 L, 100 L, 150 L, 200 L, 250 L, or more. In some embodiments, the method further comprises formulating the Fc-IL-2 fusion protein heterodimer molecule into a sterile pharmaceutical composition suitable for administration to a human subject.

[0013] In a further aspect, provided is a pharmaceutical composition comprising an IL-2v, Fc-IL-2v fusion protein, homodimer, heterodimer, conjugate, polynucleotide or polynucleotides, expression cassette, vector, or lipoplex (e.g., LNP) described herein and a pharmaceutically acceptable carrier. In some embodiments, the composition comprises an aqueous formulation. In some embodiments, the pharmaceutical composition has a concentration of 0.05 mg / ml to 50 mg / ml, for example, 0.05 mg / ml to 20 mg / ml, for example, 0.1 mg / ml to 40 mg / ml, for example, 1.0 mg / ml to 30 mg / ml, for example, 0.05 mg / ml to 0.06 mg / ml, 0.07 mg / ml, 0.08 mg / ml, 0.09 mg / ml, 0.1 mg / ml, 0.2 mg / ml, 0.3 mg / ml, 0.4 mg / ml, 0.5 mg / ml, 0.6 mg / ml, 0.7 mg / ml, 0.8 mg / ml, 0.9 mg / ml, 1.0 mg / ml, 1.5 mg / ml, 2.0 mg / ml, 2.5 mg / ml, In some embodiments, the composition comprises IL-2v, Fc-IL-2v fusion protein, homodimer, heterodimer, and / or conjugate at a concentration in the range of 1.0 mg / ml, 3.0 mg / ml, 3.5 mg / ml, 4.0 mg / ml, 4.5 mg / ml, 5.0 mg / ml, 6 mg / ml, 7 mg / ml, 8 mg / ml, 9 mg / ml, 10 mg / ml, 11 mg / ml, 12 mg / ml, 13 mg / ml, 14 mg / ml, 15 mg / ml, 16 mg / ml, 17 mg / ml, 18 mg / ml, 19 mg / ml, 20 mg / ml, 25 mg / ml, 30 mg / ml, 35 mg / ml, 40 mg / ml, 45 mg / ml, or 50 mg / ml. In some embodiments, the composition is lyophilized. In some embodiments, the pharmaceutical composition further comprises one or more additional therapeutic agents. In some embodiments, the composition is lyophilized. In some embodiments, the pharmaceutical composition further comprises one or more additional therapeutic agents. In some embodiments, the composition is lyophilized. In some embodiments, the pharmaceutical composition further comprises a second and a third therapeutic agent.

[0014] In a further aspect, there is provided a method for inducing an immune response against human hepatitis B virus (HBV) in a subject in need thereof. Also provided are methods for treating and / or preventing human hepatitis B virus (HBV) in a subject in need thereof. In some embodiments, the anti-HBV method comprises administering to the subject a therapeutically effective amount of an IL-2v, Fc-IL-2v fusion protein, homodimer, heterodimer, conjugate, polynucleotide or polynucleotides, expression cassette, vector, lipoplex (e.g., LNP), or pharmaceutical composition described herein. In some embodiments, the subject is infected with, suspected of being infected with, or at risk of being infected with HBV. In some embodiments, the subject is asymptomatic. In some embodiments, the subject is chronically infected with HBV. In some embodiments, the subject exhibits or is experiencing one or more symptoms selected from liver failure, liver cancer, liver fibrosis, and cirrhosis. In some embodiments, the human is acutely infected with HBV. In some embodiments, the subject exhibits or experiences one or more symptoms selected from jaundice, a visible network of dilated blood vessels in the skin, dark (e.g., orange or brown) urine, pale stools, fever, persistent fatigue, malaise, abdominal pain, ascites, loss of appetite, nausea, and vomiting. In some embodiments, the subject is co-infected with hepatitis D virus (HDV). In some embodiments, the subject is not receiving antiviral therapy, or antiviral therapy is discontinued prior to administration of IL-2v, Fc-IL-2v fusion protein, homodimer, heterodimer, conjugate, polynucleotide or polynucleotides, expression cassette, vector, or lipoplex (e.g., LNP), or pharmaceutical composition. In some embodiments, antiviral therapy is discontinued after one or more administrations of IL-2v, Fc-IL-2v fusion protein, homodimer, heterodimer, conjugate, polynucleotide, vector, lipoplex (e.g., LNP), and / or pharmaceutical composition. In some embodiments, the method may further comprise co-administering one or more antiviral agents to the subject. In some embodiments, the one or more antiviral agents are lamivudine (LAM), adefovir dipivoxil (ADV), or a combination thereof. , entecavir (ETV), telbivudine (LdT), tenofovir disoproxen Xylfumarate (tenofovir disoproxil fumarate, TDF), tenofovir alafen In some embodiments, the method further comprises the use of an HBV antigen inhibitor (e.g., an HBV core antigen (HBcAg) inhibitor, an HBV surface antigen (HBsAg) inhibitor, an HBx inhibitor, or an HBV cytotoxic T cell inhibitor to specifically kill HBV-infected cells. HBV E antigen inhibitors), anti-HBV antigen antibodies, inhibitory nucleic acids targeting HBV (e.g., antisense oligonucleotides, short interfering RNAs, iRNA, DNA-directed RNA interference (ddRNA) i)), gene editors targeting HBV (e.g., CRISPR-Cas (e.g., Cas9, Cas12, Cascade, Cas13), zinc finger nucleases, homing endonucleases, homing meganucleases (e.g., ARCUS), synthetic nucleases, TALENs), covalently closed circular DNA (cccDNA) inhibitors, HBsAg secretion or assembly inhibitors, HBV virus The method further comprises co-administering to the subject a tumor entry inhibitor, and one or more therapeutic agents selected from the group consisting of CAR-T and T-cell bispecific (redirected T-cells).

[0015] In a further aspect, provided are methods of activating latent viral reservoirs in a subject infected with human immunodeficiency virus (HIV). Also provided are methods of treating and / or preventing human immunodeficiency virus (HIV) in a subject in need thereof. In some embodiments, the anti-HIV method comprises administering to the subject a therapeutically effective amount of an IL-2v, Fc-IL-2v fusion protein, homodimer, heterodimer, conjugate, polynucleotide or polynucleotides, expression cassette, vector, lipoplex (e.g., LNP), or pharmaceutical composition described herein. In some embodiments, the method further comprises administering to the subject an additional therapeutic agent. In some embodiments, the method further comprises administering to the subject one or more anti-HIV broadly neutralizing antibodies. In some embodiments, the one or more anti-HIV broadly neutralizing antibodies bind to an epitope or region of gp120 selected from the group consisting of: (i) the third variable loop (V3) and / or the high mannose patch comprising the N332 oligomannose glycan; (ii) the second variable loop (V2) and / or the Env trimer vertex; (iii) the CD4 binding site (CD4bs); (iv) the gp120 / gp41 interface; or (v) the silent face of gp120.In some embodiments, the one or more anti-HIV broadly neutralizing antibodies bind to an epitope or region of gp120 in the third variable loop (V3) and / or the high mannose patch comprising the N332 oligomannose glycan, and are selected from the group consisting of GS-9722, PGT-121, PGT-121.414, PGT-122, PGT-123, PGT-124, PGT-125, PGT-126, PGT-128, PGT-130, PGT-133, PGT-134, PGT-135, PGT-136, PGT-137, PGT-138, PGT-139, PGT-200, PGT-201, PGT-202, PGT-203, PGT-204, PGT-205, PGT-206, PGT-207, PGT-208, PGT-209, PGT-210, PGT-211, PGT-212, PGT-213, PGT-214, PGT-215, PGT-216, PGT-217, PGT-218, PGT-219, PGT-220, PGT-221, PGT-222.414, PGT-222, PGT-223, PGT-224, PGT-225, PGT-226, PGT-228, PGT-229, PGT-230, PGT-231, PGT-232, PGT-233, PGT-234, PGT-235, PGT-236, PGT-237, PGT-238, PGT-239, PGT-240, PGT-241, PGT-242, PGT-243, PGT-244 competes with or comprises the VH and VL regions from an antibody selected from the group consisting of GT-138, PGT-139, 10-1074, 10-1074-J, VRC24, 2G12, BG18, 354BG8, 354BG18, 354BG42, 354BG33, 354BG129, 354BG188, 354BG411, 354BG426, DH270.1, DH270.6, PGDM12, VRC41.01, PGDM21, PCDN-33A, BF520.1, and VRC29.03. In some embodiments, the one or more broadly neutralizing anti-HIV antibodies bind to an epitope or region of gp120 in the second variable loop (V2) and / or Env trimer apex and compete with or comprise the VH and VL regions from an antibody selected from the group consisting of PG9, PG16, PGC14, PGG14, PGT-142, PGT-143, PGT-144, PGT-145, CH01, CH59, PGDM1400, CAP256, CAP256-VRC26.08, CAP256-VRC26.09, CAP256-VRC26.25, PCT64-24E, and VRC38.01.In some embodiments, the one or more broadly neutralizing anti-HIV antibodies bind to an epitope or region of gp120 in the CD4 binding site (CD4bs), and are selected from the group consisting of 3BNC117, GS-9723, 3BNC60, b12, F105, VRC01, VRC07, VRC07-523, VRC03, VRC06, and VRC06b01. competes with or comprises the VH and VL regions from an antibody selected from the group consisting of VRC08, VRC0801, NIH45-46, VRC-PG04, PGV04; CH103, 44-VRC13.01, 1NC9, 12A12, N6, N49-P7, NC-Cow1, IOMA, CH235 and CH235.12, N49P6, N49P7, N49P11, N49P9, and N60P25. In some embodiments, the one or more broadly neutralizing anti-HIV antibodies bind to an epitope or region of gp120 at the gp120 / gp41 interface and compete with or include VH and VL regions from an antibody selected from the group consisting of PGT-151, CAP248-2B, 35O22, 8ANC195, ACS202, VRC34, and VRC34.01. In some embodiments, the one or more broadly neutralizing anti-HIV antibodies bind to an epitope or region of gp120 silent face and compete with or include VH and VL regions from an antibody selected from VRC-PG05 and SF12. In some embodiments, the one or more broadly neutralizing anti-HIV antibodies bind to an epitope or region of gp41 in the membrane proximal region (MPER). In some embodiments, the one or more broadly neutralizing anti-HIV antibodies bind to an epitope or region of gp41 in the membrane proximal region (MPER) and compete with or comprise the VH and VL regions from an antibody selected from the group consisting of 10E8, 10E8v4, 10E8-5R-100cF, 4E10, DH511.11P, 2F5, 7b2, and LN01. In some embodiments, the one or more broadly neutralizing anti-HIV antibodies bind to an epitope or region of a gp41 fusion peptide and compete with or comprise the VH and VL regions from an antibody selected from the group consisting of VRC34 and ACS202.In some embodiments, the subject is antiretroviral therapy (ART) naive or A. ART is discontinued prior to administration of IL-2v, an Fc-IL-2v fusion protein, a homodimer, a heterodimer, a conjugate, a polynucleotide or polynucleotides, an expression cassette, a vector, a lipoplex (e.g., an LNP), or a pharmaceutical composition. In some embodiments, ART is discontinued after one or more administrations of an IL-2v, an Fc-IL-2v fusion protein, a homodimer, a heterodimer, a conjugate, a polynucleotide or polynucleotides, an expression cassette, a vector, a lipoplex (e.g., an LNP), or a pharmaceutical composition. In some embodiments, the method further comprises administering one or more antiretroviral therapy (ART) agents to the subject. In some embodiments, the subject is chronically infected with HIV.

[0016] In a further aspect, provided are methods of enhancing, improving, and / or increasing the response to vaccine therapy in a subject in need thereof. In some embodiments, the vaccine enhancement method comprises co-administering to a subject (1) an effective amount of an IL-2v, Fc-IL-2v fusion protein, homodimer, heterodimer, conjugate, polynucleotide or polynucleotides, expression cassette, vector, lipoplex (e.g., LNP), or pharmaceutical composition described herein, and (2) an effective amount of a vaccine. In some embodiments, the vaccine is selected from the group consisting of an antiviral vaccine, an antibacterial vaccine, and an anticancer vaccine. In some embodiments, the vaccine is selected from the group consisting of hepatitis A virus (HAV), hepatitis B virus (HBV), human immunodeficiency virus (HIV), cytomegalovirus (CMV), herpes simplex virus (HSV), Epstein-Barr virus (EBV), human orthovirus (HPV), or human IL-2v. In some embodiments, the vaccine is an antiviral vaccine against a virus selected from the group consisting of pneumovirus or human respiratory syncytial virus (RSV), human papillomavirus (HPV), varicella-zoster virus, measles virus, mumps virus, poliovirus vaccine, influenza virus, paramyxovirus, rotavirus, Zika virus, dengue virus, Ebola virus, and coronavirus (e.g., betacoronavirus, e.g., severe acute respiratory syndrome-associated coronavirus, e.g., SARS-CoV2). In some embodiments, the vaccine is an antiviral vaccine against a virus selected from the group consisting of mycobacterium tuberculosis, pertussis, tetanus, diphtheria, meningococcus, pneumococcus, influenza virus, or a strain of influenza virus. The present invention includes an antibacterial vaccine against a bacterium selected from the group consisting of Haemophilus influenza, cholera, typhoid, and anthrax. In some embodiments, the method includes a prime-boost regimen comprising administering a priming composition at a first time point and administering one or more boosting compositions at one or more subsequent time points. In some embodiments, the priming composition comprises an IL-2v, an Fc-IL-2v fusion protein, a homodimer, a heterodimer, a conjugate, a polynucleotide or polynucleotides, an expression cassette, a vector, a lipoplex (e.g., LNP), or a pharmaceutical composition described herein. In some embodiments, the one or more boosting compositions comprise an IL-2v, an Fc-IL-2v fusion protein, a homodimer, a heterodimer, a conjugate, a polynucleotide or polynucleotides, an expression cassette, a vector, a lipoplex (e.g., LNP), or a pharmaceutical composition described herein. In some embodiments, the priming composition and the boosting composition are the same. In some embodiments, the base composition and the reinforcement composition are different.

[0017] In a further aspect, provided are methods of preventing, reducing, and / or inhibiting the recurrence, growth, proliferation, migration, and / or metastasis of a cancer cell or cancer cell population in a subject in need thereof. In some embodiments, the anti-cancer method comprises administering to the subject a therapeutically effective amount of IL-2v, Fc-IL-2v fusion protein, homodimer, heterodimer, conjugate, polynucleotide or polynucleotides, expression cassette, vector, lipoplex (e.g., LNP), or pharmaceutical composition described herein. In some embodiments, the IL-2v, Fc-IL-2v fusion protein, homodimer, heterodimer, conjugate, polynucleotide, vector, lipoplex (e.g., LNP), and / or pharmaceutical composition is co-administered with one or more anti-neoplastic or chemotherapeutic agents. In some embodiments, the one or more antineoplastic or chemotherapeutic agents are nucleoside analogs (e.g., 5-fluorouracil, gemcitabine, cytarabine, cladribine, pentostatin, fludarabine), taxanes (e.g., paclitaxel, nab-paclitaxel, docetaxel, cabazitaxel), platinum coordination complexes (cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin, picoplatin, satraplatin), or the like. dicycloplatin, eptaplatin, lobaplatin dihydrofolate reductase (DHFR) inhibitors (e.g., methotrexate, trimetrexate, pemetrexed), topoisomerase inhibitors (e.g., doxorubicin, daunorubicin, dactinomycin, eniposide, epirubicin, etoposide, idarubicin, irinotecan, mitochondrial infusion), xantrone, pixantrone, sobuzoxane, topotecan, irinotecan, MM-398 (liposomal irinotecan), vosaroxin and GPX-150, aldoxorubicin, AR-67, mavereltinib, AST-2818, avitinib (ACEA-0010), irofulven (MGI-114)), alkylating agents (e.g., nitrogen mustards (e.g., cyclophosphamide, chlormethine, uramustine or uracil mustard, melphalan, chlorambucil, ifosfamide, bendamustine, temozolomide, carmustine), nitrosoureas (e.g., carmustine, lomustine, streptozocin), alkyl sulfonates (e.g., busulfan)), and mixtures thereof. In some embodiments, the IL-2v, Fc-IL-2v fusion protein, homodimer, heterodimer, conjugate, polynucleotide, vector, lipoplex (e.g., LNP), and / or pharmaceutical composition is co-administered with a FOLFOX regimen, a FOLFIRI regimen, a FOLFOXIRI regimen, or a FOLFIRINOX regimen. ... SF7), CD27L); 5'-nucleotidase ecto (NT5E; CD73), ectonucleoside triphosphate diphosphohydrolase 1 (CD39); CD74; CD79B; CD80; CD86; interleukin-3 receptor subunit alpha (IL3RA), prominin 1 (PROM1; CD133); TNFRSF9 (CD137); syndecan 1 (SDC1; CD138); CD200 molecule (CD200); alphafeto (AFP), BAG cochaperone 6 (BAG6); MET proto-oncogene, receptor tyrosine kinase (MET);KIT proto-oncogene, receptor tyrosine kinase (KIT); C-type lectin domain family 12 member A (CLEC12A; CD371); C-type lectin domain-containing 9A (CLEC9A; CD370); cadherin 3 (CDH3); carbonic anhydrase 6 (CA6); carbonic anhydrase 9 (CA9); carcinoembryonic antigen-related cell adhesion molecule 3 (CEACAM3); carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5); carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6); chorionic somatomammotropic hormone 1 (CSH1); coagulation factor III, tissue factor (F3); collectin subfamily member 10 (COLEC10; CLL1); delta-like canonical notch ligand 3 (DLL3); ectonucleotide pyrophosphatase / phospholipase Phosphodiesterase 3 (ENPP3); ephrin A1 (EFNA1); epidermal growth factor receptor (EGFR; ERBB; HER1); EGFR variant III (EGFRvIII); EPH receptor A2 (EPHA2); epithelial cell adhesion molecule (EPCAM); erb-b2 receptor tyrosine kinase 2 (ERBB2; HER-2 / neu); fibroblast activation protein alpha (FAP); fibroblast growth factor receptor 2 (FGFR2); fibroblast growth factor receptor 3 (FGFR3); folate hydrolase 1 (FOLH1); folate receptor 1 (FOLR1); GD2 ganglioside; glycoprotein NMB (GPNMB; osteoactivin); guanylate cyclase 2C (GUCY2C); human papillomavirus (HPV) E6; HPV E7; major histocompatibility complex (MHC) class I-presented neoantigen, major histocompatibility complex (MHC) class II-presented neoantigen, major histocompatibility complex, class I, E (HLA-E); major histocompatibility complex, class I, F (HLA-F); major histocompatibility complex, class I, G (HLA-G); MHC class I polypeptide-related sequence A (MICA); MHC class I polypeptide-related sequence B (MICB); integrin subunit β7 (ITGB7); leukocyte immunoglobulin-like receptor B1 (LILRB1; ILT2); leukocyte immunoglobulin-like receptor B2 (LILRB2; ILT4); LY6 / PLAUR domain-containing 3 (LYPD3); glypican 3 (GPC3); KRAS proto-oncogene, GTPase (KRAS);MAGE family member A1 (MAGEA1); MAGE family member A3 (MAGEA3); MAGE family member A4 (MAGEA4); MAGE family member A11 (MAGEA11); MAGE family member C1 (MAGEC1); MAGE family member C2 (MAGEC2); MAGE family member C3 (MAGEC3); MAGE family member D1 (MAGED1); MAGE family member D2 (MAGED2); mesothelin (MSLN); mucin 1 (MUC1); and their supraspinal proteins. isovariants (e.g., including MUC1 / A, C, D, X, Y, Z, and REP); mucin 16 (MUC16; CA125); natural killer cytotoxicity receptor 3 ligand 1 (NCR3LG1; B7-H6); necdin, a MAGE family member (NDN); nectin cell adhesion molecule 2 (NECTIN2); nectin cell adhesion molecule 4 (NECTIN4); SLIT and NTRK-like family member 6 (SLITRK6); promyelocytic leukemia (PML); protein tyrosine kinase 7 (inactive) (PTK7); poliovirus receptor (PVR) cell Cellular adhesion molecule (PVR); SLAM family member 6 (SLAMF6); SLAM family member 7 (SLAMF7); sialic acid-binding Ig-like lectin 7 (SIGLEC7); sialic acid-binding Ig-like lectin 9 (SIGLEC9); sialic acid-binding Ig-like lectin 10 (SIGLEC10); signal-regulatory protein alpha (SIRPA); solute carrier family 34 (sodium phosphate), member 2 (SLC34A2); solute carrier family 39 member 6 (SLC39A6); STEAP family member 1 (STEAP1); tumorigenic Suppression of 2 (ST2); TNF receptor superfamily member 4 (TNFRSF4; OX40); TNF superfamily member 9 (TNFSF9; 4-1BB-L, CD137L); TNFRSF10A (DR4, TRAILR1); TNFRSF10B (DR5, TRAILR2); TNFRSF13B (BAFF); TNFRSF17 (BCMA); TNFRSF18 (GITR); transferrin (TF); transforming growth factor beta 1 (TGFB1) and its isoforms; triggering receptor expressed on myeloid cells 1 (TREM1);The immunotherapy is co-administered with one or more antibodies or antigen-binding antibody fragments thereof or antibody-drug conjugates thereof, CD3-targeting multispecific molecules, NK cell-activating receptor-targeting multispecific molecules, or non-immunoglobulin antigen-binding domains, or antibody mimetic proteins, directed against one or more targets or tumor-associated antigens (TAA) selected from the group consisting of triggering receptor expressed on myeloid cells 2 (TREM2); trophoblast glycoprotein (TPBG); trophinin (TRO); tumor-associated calcium signaling factor 2 (TACSTD2); fucosyl-GM1; sialyl Lewis adhesion molecule (sLe); and Lewis Y antigen. In some embodiments, the one or more antibodies or antigen-binding antibody fragments thereof or antibody-drug conjugates thereof, CD3-targeting multispecific molecules, NK cell-activating receptor-targeting multispecific molecules, or non-immunoglobulin antigen-binding domains, or antibody mimetic proteins bind to an epitope of the target or tumor-associated antigen (TAA) presented on a major histocompatibility complex (MHC) molecule. In some embodiments, the NK cell activating receptor is selected from the group consisting of CD16, NKp30, NKp44, NKp46, NKp80, and NKG2D. In some embodiments, the IL-2v, Fc-IL-2v fusion protein, homodimer, heterodimer, conjugate, polynucleotide, vector, lipoplex (e.g., LNP), and / or pharmaceutical composition is activated by natural killer (NK) cells, NK-T cells, T cells, cytokine-induced killer (CIK) cells, macrophages (MAC) cells, tumor infiltrating lymphocytes (TIL), and; and one or more cell therapies selected from the group consisting of dendritic cells (DCs). In some embodiments, the one or more cell therapies comprise a T cell therapy selected from the group consisting of α / β TCR T cells, γ / δ TCR T cells, regulatory T (Treg) cells, and TRuC™ T cells. In some embodiments, the one or more cell therapies comprise an NK cell therapy, e.g., comprising NK-92 cells. In some embodiments, the one or more cell therapies comprise cells that are autologous, syngeneic, or allogeneic to the subject. In some embodiments, the one or more cell therapies comprise cells comprising a chimeric antigen receptor (CAR). In some embodiments, the cells in the cell therapy CD19; transmembrane 4 domain A1 (MS4A1; CD20); CD22 (SIGLEC2); CD27 (TNFRSF7); TNFRSF8 (CD30); CD33 (SIGLEC3); CD37; CD38; CD40 (TNFRSF5); CD44; CD47; CD48 (SLAMF2); CD52; CD70 (TNFSF7), CD27L; 5'-nucleotidase ecto- (NT5E; CD73), ectonucleoside triphosphate diphosphohydrolase 1 (CD39); CD74; CD79B; CD80; CD86; interleukin-3 receptor subunit alpha (IL3RA), prominin 1 (PROM1; CD133) TNFRSF9 (CD137); syndecan 1 (SDC1; CD138); CD200 molecule (CD200); alphafetoprotein (AFP), BAG cochaperone 6 (BAG6); MET proto-oncogene, receptor tyrosine kinase (MET); KIT proto-oncogene, receptor tyrosine kinase (KIT); C-type lectin domain family 12 member A (CLEC12A; CD371); C-type lectin domain-containing 9A (CLEC9A; CD370); cadherin 3 (CDH3); carbonic anhydrase 6 (CA6); carbonic anhydrase 9 (CA9); carcinoembryonic antigen-related cell adhesion molecule 3 (CEACAM3); carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5); carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6); chorionic somatomammotropic hormone 1 (CSH1); coagulation factor III, tissue factor (F3); collectin subfamily member 10 (COLE) C10; CLL1); delta-like canonical Notch ligand 3 (DLL3); ectonucleotide pyrophosphatase / phosphodiesterase 3 (ENPP3); ephrin A1 (EFNA1); epidermal growth factor receptor (EGFR; ERBB; HER1); EGFR variant III (EGFRvIII); EPH receptor A2 (EPHA2); epithelial cell adhesion molecule (EPCAM); erb-b2 receptor tyrosine kinase 2 (ERBB2; HER-2 / neu); fibroblast activation protein alpha (FAP); fibroblast growth factor receptor 2 (FGFR2); fibroblast growth factor receptor 3 (FGFR3); folate hydrolase 1 (FOLH1); folate receptor 1 (FOLR1); GD2 ganglioside; glycoprotein NMB (GPNMB; osteoactivin); guanylate cyclase 2C (GUCY2C); human papillomavirus (HPV) E6; HPV E7; major histocompatibility complex (MHC) class I-presented neoantigen, major histocompatibility complex (MHC) class II-presented neoantigen, major histocompatibility complex, class I, E (HLA-E); major histocompatibility complex, class I, F (HLA-F); major histocompatibility complex, class I, G (HLA-G); MHC class I polypeptide-related sequence A (MICA); MHC class I polypeptide-related sequence B (MICB); integrin subunit β7 (ITGB7); leukocyte immunoglobulin-like receptor B1 (LILRB1; ILT2);Leukocyte immunoglobulin-like receptor B2 (LILRB2; ILT4); LY6 / PLAUR domain-containing 3 (LYPD3); glypican 3 (GPC3); KRAS proto-oncogene, GTPase (KRAS); MAGE family member A1 (MAGEA1); MAGE family member A3 (MAGEA3); MAGE family member A4 (MAGEA4); MAGE family member A11 (MAGEA11); MAGE family member C1 (MAGEC1); MAGE family member C2 (MAGEC2); MAGE family member C3 (MAGEC3); MAGE family member D1 (MAGED1); MAGE family member D2 (MAGED2); mesothelin (MSLN); mucin 1 (MUC1) and its splice variants (e.g., including MUC1 / A, C, D, X, Y, Z, and REP); mucin 16 (MUC16; CA125); natural killer cytotoxicity receptor 3 ligand 1 (NCR3LG1; B7-H6); necdin, a MAGE family member (NDN); nectin cell adhesion molecule 2 (NECTIN2); nectin cell adhesion molecule 4 (NECTIN4) ;SLIT and NTRK-like family member 6 (SLITRK6); promyelocytic leukemia (PML); protein tyrosine kinase 7 (inactive) (PTK7); poliovirus receptor (PVR) cell adhesion molecule (PVR); SLAM family member 6 (SLAMF6); SLAM family member 7 (SLAMF7); sialic acid-binding Ig-like lectin 7 (SIGLEC7); sialic acid-binding Ig-like lectin 9 (SIGLEC9); sialic acid-binding Ig-like lectin 10 (SIGLEC10); signal-regulatory protein alpha (SIRPA) solute carrier protein Milli-34 (sodium phosphate), member 2 (SLC34A2); solute carrier family 39 member 6 (SLC39A6); STEAP family member 1 (STEAP1); suppressor of tumorigenicity 2 (ST2); TNF receptor superfamily member 4 (TNFRSF4; OX40); TNF superfamily member 9 (TNFSF9; 4-1BB-L, CD137L); TNFRSF10A (DR4, TRAILR1); TNFRSF10B (DR5, TRAILR2); TNFRSF13B (BAFF); TNFRSF17 (BCMA);The cells of the cell therapy bind to a target or tumor-associated antigen (TAA) selected from the group consisting of TNFRSF18 (GITR); transferrin (TF); transforming growth factor beta 1 (TGFB1) and its isoforms; triggering receptor expressed on myeloid cells 1 (TREM1); triggering receptor expressed on myeloid cells 2 (TREM2); trophoblast glycoprotein (TPBG); trophinin (TRO); tumor-associated calcium signaling factor 2 (TACSTD2); fucosyl-GM1; sialyl Lewis adhesion molecule (sLe); and Lewis Y antigen. In some embodiments, the cells of the cell therapy bind to an epitope of the target or tumor-associated antigen (TAA) presented on a major histocompatibility complex (MHC) molecule. In some embodiments, the TAA is a cancer-testis antigen. In some embodiments, the cancer testis antigen is acrosin binding protein (ACRBP), alpha fetoprotein (AFP), A kinase anchoring protein 4 (AKAP4), ATPase family AAA domain containing 2 (ATAD2), kinetochore scaffold 1 (KNL1; also known as CASC5), centrosomal protein 55 (CEP55), cancer / testis antigen 1A (CTAG1A; also known as ESO1; CT6.1; LAGE-2; LAGE2A; NY-ESO-1), cancer / testis antigen 1B (CTAG1B; also known as CT6.1, CTAG, CTAG1, ESO1, LAGE-2), or NY-ESO-1. , LAGE2B, NY-ESO-1), cancer / testis antigen 2 (CTAG2; also known as CAMEL, CT2, CT6.2, CT6.2a, CT6.2b, ESO2, LAGE-1, LAGE2B), CCCTC-binding factor-like (CTCFL), catenin alpha 2 (CTNNA2), cancer / testis antigen 83 (CT83), cyclin A1 (CCNA1), DEAD-box helicase 43 (DDX43), developmental pluripotency associated 2 (DPPA2), fetal and adult testis expressed 1 (FATE1), FMR1 adjacent (FMR1NB), HORMA domain containing 1 (HORMAD1), insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3), leucine zipper protein 4 (LUZP4), lymphocyte antigen 6 family member K (LY6K), maelstrom spermatogenesis transposon silencer (MAEL), MAGE family member A1 (MAGEA1);MAGE family member A3 (MAGEA3); MAGE family member A4 (MAGEA4); MAGE family member A11 (MAGEA11); MAGE family member C1 (MAGEC1); MAGE family member C2 (MAGEC2); MAGE family member D1 (MAGED1); MAGE family member D2 (MAGED2), kinesin family member 20B (KIF20B; also known as MPHOSPH1), NUF2 component of the NDC80 kinetochore complex (NUF2), nuclear export factor 2 (NXF2), PAS domain-containing repressor 1 (PASD1), PDZ-binding kinase (PBK), piwi-like RNA-mediated gene silencing 2 (PIWIL-2), melanoma preferred expressed antigen (PRAME), sperm-associated antigen 9 (SPAG9), nuclear X-linked family member A1-related sperm protein (SPANXA1), SPANX family member A2 (SPANXA2), SPANX family member C (SPANXC), SPANX family member D (SPANXD), SSX family member 1 (SSX1), SSX family member 2 (SSX2), synaptonemal structural protein 3 (SYCP3), testis-expressed intercellular bridge forming factor 14 (TEX14), transcription factor Dp family member 3 (TFDP3), serine protease 50 (PRSS50, also known as TSP50), TTK protein kinase (TTK), and zinc finger protein 165 (ZNF165). In some embodiments, the IL-2v, Fc-IL-2v fusion protein, homodimer, heterodimer, conjugate, polynucleotide, vector, lipoplex (e.g., LNP), and / or pharmaceutical composition is co-administered with a targeted E3 ligase ligand conjugate. In some embodiments, the IL-2v, Fc-IL-2v fusion protein, homodimer, heterodimer, conjugate, polynucleotide, vector, lipoplex (e.g., LNP), and / or pharmaceutical composition is co-administered with a protein tyrosine phosphatase, non-receptor type 11 (PTPN11 or SHP2), myeloid cell leukemia sequence 1 (MCL1) apoptosis regulator; 5'-nucleotidase ecto (NT5E or CD73), ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1 or CD39), transforming growth factor beta 1 (TGFB1 or TGFβ), heme oxygenase 1 (heme oxygenase, HMOX1, HO -1 or HO1), heme oxygenase 2 (HMOX2, HO-2 or HO2), vascular endothelial growth factor A (VEGFA or VEGF), erb-b2 receptor tyrosine kinase 2 (ERBB2, HER2, HER2 / neu, or CD340), epidermal growth factor receptor (EGFR, ERBB, ERBB1, or HER1), ALK receptor tyrosine kinase (ALK, CD246), poly(ADP-ribose) polymerase 1 (PARP1), poly(ADP-ribose) polymerase 2 (poly(ADP-ribose)polymerase, PARP2), TCDD-inducible poly(ADP-ribose)polymerase (TCDD-inducible poly(ADP-ribose)polymerase, TIPARP, PARP7), Icrin-dependent kinase 4 (CDK4), cyclin-dependent CDK6, TNF receptor superfamily member 14 (TNFRSF14, HVEM, CD270), CC motif chemokine receptor 2 (CCR2, CD192), and CC motif chemokine receptor 5 (CCR2, CD192). CXCR5, CD195), CC motif chemokine receptor 8 (CCR8, CDw198), CC motif chemokine receptor 2 (CXCR2, CD182), CC motif chemokine receptor 3 (CXCR3, CD182, CD183), CC motif chemokine receptor 4 (CXCR4, CD184), arginase (ARG1, ARG2), carbonic anhydrase (CA1, CA2, CA3, CA4, CA5A, CA5B, CA6, CA7, CA8, CA9, CA10, CA11, CA12, CA13, CA14), prostaglandin-endoperoxide synthase 1 (prostaglandin-endoperoxide synthase 1), and CXCR5. Prostaglandin-endoperoxide synthase 2 (PTGS2, COX-2), secretory phospholipase A2, prostaglandin E synthase (PTGES, PGES), arachidonate 5-lipoxygenase (ALOX5, 5-LOX), soluble epoxide hydrolase 2 (EPHX2), indoleamine 2,3-dioxygenase 1 (IDO1), indoleamine 2,3-dioxygenase 2 (IDO2), hypoxia inducible factor 1 subunit alpha (HIF1A), and Ndjiopo Etin-1 (angiopoietin, ANGPT1), endothelial TEK tyrosine kinase (TIE-2, TEK), Janus kinase 1 (JAK1), catenin beta 1 (catenin beta, CTNNB1), histone deacetylase 9 (HDAC9), 5'-3' exoribonuclease 1 (XRN1), and / or WRN RecQ-like helicase (WRN). In some embodiments, the inhibitor comprises an antibody or antigen-binding fragment thereof or an antibody-drug conjugate thereof, a CD3-targeted multispecific molecule, an NK cell-activating receptor-targeted multispecific molecule, a non-immunoglobulin antigen-binding molecule, or an antibody mimetic protein. In some embodiments, the inhibitor comprises an inhibitory nucleic acid. In some embodiments, the inhibitor comprises a small organic molecule. In some embodiments, the inhibitor of 5'-nucleotidase ecto (NT5E or CD73) is selected from the group consisting of MEDI9447 (oleclumab), CPI-006, BMS-986179, IPH5301, TJ4309 (TJD5), NZV-930, AB-680, PSB-12379, PSB-12441, PSB-12425, CB-708, and PBF-16622. In some embodiments, the inhibitor of CCR2 and / or CCR5 is selected from the group consisting of BMS-813160, PF-04136309, and CCX-872. In some embodiments, the inhibitor of MCL1 is selected from the group consisting of GS-9716, tapotoclax (AMG-176), AMG-397, S-64315, AZD-5991, 483-LM, A 1210477, UMI-77, JKY-5-037, PRT-1419, and APG-3526. In some embodiments, the inhibitor of PTPN11 or SHP2 is selected from the group consisting of TNO155 (SHP-099), RMC-4550, JAB-3068, and RMC-4630. In some embodiments, the inhibitor of Janus kinase 1 (JAK1) is selected from the group consisting of filgotinib, tofacitinib, baricitinib, and ABT-494. In some embodiments, the IL-2v, Fc-IL-2v fusion protein, homodimer, heterodimer, conjugate, polynucleotide, vector, lipoplex (e.g., LNP), and / or pharmaceutical composition is co-administered with an oncolytic viral vector.In some embodiments, the oncolytic viral vector comprises a DNA virus or an RNA virus. In some embodiments, the viral vector is from a viral family selected from the group consisting of Adenoviridae (e.g., adenovirus), Arenaviridae (e.g., Lymphocytic choriomeningitis Mam arenavirus, Karimum arenavirus (also known as Pichindemum arenavirus), Poxviridae (e.g., vaccinia virus), Herpesviridae (e.g., herpesvirus, e.g., HSV-1), Parvoviridae (e.g., parvovirus H1), Reoviridae (e.g., reovirus), Picornaviridae (e.g., Coxsackievirus, Seneca Valley virus, poliovirus), Paramyxoviridae (e.g., measles virus, Newcastle disease virus (NDV)), Rhabdoviridae (e.g., vesicular stomatitis virus (VSV)), Togaviridae (e.g., alphavirus, Sindbis virus), Enteroviridae (e.g., echovirus). In some embodiments, the subject has cancer. In some embodiments, the subject is in remission of cancer.In some embodiments, the subject is diagnosed with a hematological cancer, e.g., a leukemia (e.g., acute myelogenous leukemia (AML), acute lymphoblastic leukemia (ALL), B-cell ALL, myelodysplastic syndrome (MDS), myeloproliferative disease (MPD), chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), anaplastic leukemia), lymphoma (e.g., small lymphocytic lymphoma (SLL), mantle cell lymphoma (MCL), follicular lymphoma (FL), T-cell lymphoma, B-cell lymphoma, diffuse large B-cell lymphoma), or a leukemia (e.g., idiopathic leukemia ... lymphoma (DLBCL), marginal zone lymphoma (MZL), Waldenstrom's macroglobulinemia (WM), and / or myeloma (e.g., multiple myeloma (M. In some embodiments, the subject has a cancer selected from the group consisting of an epithelial tumor (e.g., carcinoma, squamous cell carcinoma, basal cell carcinoma, squamous intraepithelial neoplasia), a ductal tumor (e.g., adenocarcinoma, adenoma, adenomyoma), a mesenchymal or soft tissue tumor (e.g., sarcoma, rhabdomyosarcoma, leiomyosarcoma, liposarcoma, fibrosarcoma, dermatofibrosarcoma, neurofibrosarcoma, fibrous histiocytoma, angiosarcoma, angiomyxoma, leiomyoma, chondroma, chondrosarcoma, alveolar soft part sarcoma, epithelioid hemangioendothelioma, Spitz tumor, synovial sarcoma), and lymphoma. In some embodiments, the subject is diagnosed with bone (e.g., ameloblastoma, aneurysmal bone cyst, angiosarcoma, chondroblastoma, chondroma, chondromyxoid fibroma, chondrosarcoma, chordoma, dedifferentiated chondrosarcoma, enchondroma, epithelioid hemangioendothelioma, fibrous dysplasia, giant cell tumor of bone, hemangioma and related lesions, osteoblastoma, osteochondroma, osteosarcoma, osteoid, osteoma, periosteal chondroma, desmoid, Ewing's sarcoma); lips and oral cavity (e.g., odontogenic ameloblastoma, oral leukoplakia, oral squamous cell carcinoma, primary oral mucosal melanoma); salivary glands (e.g., pleomorphic salivary adenoma, salivary gland adenoid cystic carcinoma, salivary gland mucoepidermoid carcinoma, salivary gland Warthin's tumor); esophagus (e.g., Barrett's esophagus, dysplasia, and adenocarcinoma); gastrointestinal tract (e.g., stomach (e.g., gastric adenoma, primary gastric lymphoma, gastrointestinal stromal tumor, tumor (GIST), metastatic deposits, gastric carcinoid, gastric sarcoma, neuroendocrine carcinoma, primary gastric squamous cell carcinoma, gastric adenoacanthomas), small intestine and smooth muscle (e.g., intravenous leiomyomatosis), colon (e.g., colorectal adenocarcinoma), rectum, anus); pancreas (e.g., serous neoplasms (including microcystic or macrocystic serous cystadenoma, solid serous cystadenoma, Von Hippel-Landau (VHL)-associated serous cystic neoplasm, serous cystadenocarcinoma); mucinous cystic neoplasms (MCN), intraductal papillary mucinous neoplasms mucinous neoplasm (IPMN), intraductal oncocytic papillary neoplasm (IOPN), intraductal tubular neoplasm, cystic acinar neoplasm ( pancreatic adenocarcinoma (including acinar cell cystadenoma, acinar cell cystadenocarcinoma, and pancreatic adenocarcinoma), invasive pancreatic ductal adenocarcinoma (including tubular adenocarcinoma and adenosquamous carcinoma), mucinous carcinoma, medullary carcinoma, hepatoid carcinoma, signet ring cell carcinoma, undifferentiated carcinoma, undifferentiated carcinoma with osteoclast-like giant cells, acinar cell carcinoma, neuroendocrine tumor, neuroendocrine microadenoma, neuroendocrine tumor (NET), neuroendocrine carcinoma (NEC) (including small cell or large cell NEC), islet cell adenoma, gastrinoma, glucagonoma, serotoninoma, somatostatinoma, VIPoma, solid-pseudopapillary neoplasm (SPN), and pancreatoblastoma; gallbladder (e.g., cancer of the gallbladder and extrahepatic bile duct, intrahepatic cholangiocarcinoma); neuroendocrine (e.g., adrenocortical carcinoma, carcinoid tumor, pheochromocytoma, pituitary adenoma); thyroid (e.g., poorly differentiated (anaplastic) carcinoma, medullary carcinoma, oncocytic tumor, papillary carcinoma, adenocarcinoma); liver (e.g., adenoma, mixed hepatocellular and cholangiocarcinoma, fibrolamellar carcinoma, hepatoblastoma, hepatocellular carcinoma, mesenchymal tumor, nested stromal epithelial tumor, undifferentiated carcinoma; hepatocellular carcinoma, intrahepatic cholangiocarcinoma, cholangiocyst adenocarcinoma, epithelioid hemangioendothelioma, angiosarcoma, embryonal sarcoma, rhabdomyosarcoma, solitary fibrous tumor, teratoma, yolk sac tumor, carcinosarcoma, rhabdoid tumor); kidney (e.g., ALK-rearranged renal cell carcinoma, chromophobe renal cell carcinoma) carcinoma, clear cell renal carcinoma, clear cell sarcoma, metanephric adenoma, metanephric adenofibroma, mucinous tubular and spindle cell renal carcinoma, renal tumor, nephroblastoma (Wilms' tumor), papillary adenoma, papillary renal cell carcinoma, renal oncocytoma, renal cell carcinoma, succinate dehydrogenase-deficient renal cell carcinoma carcinoma), collecting duct carcinoma); breast (e.g., invasive ductal carcinoma (including, but not limited to, acinic cell carcinoma, adenoid cystic carcinoma, apocrine adenocarcinoma, cribriform carcinoma, glycogen-rich / clear cell, inflammatory carcinoma, lipid-rich carcinoma, medullary carcinoma, metaplastic carcinoma, micropapillary carcinoma, mucinous carcinoma, neuroendocrine carcinoma, malignant oncocytoma, papillary carcinoma, sebaceous gland carcinoma, secretory carcinoma, tubular carcinoma); lobular carcinoma (pleomorphic carcinoma, signet ring cell carcinoma); peritoneal (e.g., mesothelioma; primary peritoneal carcinoma);Female reproductive tissues (ovaries (e.g., choriocarcinoma, epithelial tumors, germ cell tumors, sex cord-stromal tumors), fallopian tubes (e.g., serous adenocarcinoma, mucinous carcinoma, endometrioid adenocarcinoma, clear cell adenocarcinoma, transitional cell carcinoma, squamous cell carcinoma, undifferentiated carcinoma, Mullerian tumor, adenosarcoma, leiomyosarcoma, teratoma, germ cell tumor, choriocarcinoma, trophoblastic tumor), uterus (e.g., cervical cancer, endometrial polyps, endometrial hyperplasia, intraepithelial carcinoma (EIC)), endometrial cancer (e.g., endometrioid carcinoma, serous carcinoma, clear cell carcinoma, mucinous carcinoma, squamous cell carcinoma, transitional carcinoma, small cell carcinoma, undifferentiated carcinoma, mesenchymal neoplasm), leiomyomas (e.g., endometrial stromal nodules, leiomyosarcoma, endometrial stromal sarcoma) sarcoma:ESS), mesenchymoma), mixed epithelial and mesenchymoma (e.g., glandular line; Fibroma, carcinofibroma, adenosarcoma, carcinosarcoma (malignant mixed mesodermal sarcoma (MMMT)), endometrial stromal tumor, endometrial malignancy Endometrial malignant mullerian mixed tumor, gestational trophoblastic tumor (including partial hydatiform mole, complete hydatiform mole, invasive hydatiform mole, placenta accreta tumor), vulva, and vagina; male reproductive Organ tissues (including prostate, testis (e.g., germ cell tumor, spermatocytic seminoma), penis); bladder (e.g., squamous cell carcinoma, urothelial carcinoma, bladder urothelial carcinoma); brain (e.g., glioma (e.g., astrocytoma (including non-invasive, low-grade, poorly differentiated, glioblastoma; oligodendroglioma, ependymoma), meningioma, ganglioglioma, schwannoma, craniopharyngioma, chordoma, non-Hodgkin's lymphoma (NHL), indolent non-Hodgkin's lymphoma (NHL)), lymphoma (iNHL), refractory iNHL, pituitary tumor; eye (e.g., retinocytoma, retinoblastoma, intraocular melanoma, posterior uveal melanoma, iris hamartoma); head and neck (e.g., nasopharyngeal carcinoma, endolymphatic sac tumor (ELST), epidermoid carcinoma, laryngeal cancer (including squamous cell carcinoma (SCC) (e.g., glottic carcinoma, supraglottic carcinoma, subglottic carcinoma, and combined laryngeal carcinoma), carcinoma in situ, verrucous, spindle cell, and basaloid SCC, undifferentiated carcinoma, laryngeal adenocarcinoma, adenoid cystic carcinoma, neuroendocrine carcinoma, and laryngeal sarcoma)), head and neck paraganglioma (e.g., carotid body, paraganglioma, vagus nerve); thymus (e.g., thymoma); heart (e.g., cardiac myxoma); lung (e.g., small cell carcinoma (SCLC), non-small cell lung cancer) carcinoma:NSCLC) (squamous cell carcinoma: SCC), adenocarcinoma and large cell carcinoma, carcinoid (typical or atypical), carcinosarcoma, pulmonary blastoma, giant cell carcinoma, spindle cell carcinoma, pleuropulmonary blastoma; lymphoma (e.g., lymphomas (including Hodgkin's lymphoma, non-Hodgkin's lymphoma (NHL), indolent non-Hodgkin's lymphoma (iNHL), refractory iNHL), Epstein-Barr virus (EBV)-associated lymphoproliferative disorders (including B-cell lymphoma and T-cell lymphoma (e.g., Burkitt's lymphoma); large B-cell lymphoma lymphoma, diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma, indolent B-cell lymphoma, low-grade B-cell lymphoma, fibrin-associated diffuse large cell lymphoma; primary effusion lymphoma; plasmablastic lymphoma; extranodal NK / T-cell lymphoma, nasal type; peripheral T-cell lymphoma, cutaneous T-cell lymphoma, angioimmunoblastic T-cell lymphoma; follicular T-cell lymphoma; systemic T-cell lymphoma); lymphangioleiomyomatosis; central nervous system system:CNS) (e.g., glioma (astrocytoma) (e.g., pilocytic astrocytoma, pilocytic myxoid astrocytoma, subependymal giant cell astrocytoma, pleomorphic xanthoastrocytoma, diffuse astrocytoma, fibrous astrocytoma, large round cell astrocytoma) tumors, protoplasmic astrocytomas, anaplastic astrocytomas, glioblastomas (e.g., giant cell glioblastoma, gliosarcoma, glioblastoma multiforme), and gliomatosis cerebri), oligodendrogliomas (e.g., oligodendrogliomas, anaplastic oligodendrogliomas), oligoastrocytic tumors (e.g., oligoastrocytoma, anaplastic oligoastrocytoma), ependymal tumors (e.g., subependymoma, myxopapillary ependymoma, ependymomas (e.g., cellular, papillary, clear cell, elongated ependymal cell), poorly differentiated ependymoma), optic gliomas, and non-gliomas (including, e.g., choroid plexus tumors, neuronal and mixed neuronal-glial tumors, pineal tumors, embryonal tumors, medulloblastomas, meningeal tumors, primary CNS lymphomas, germ cell tumors, pituitary adenomas, cranial and paraspinal nerve tumors, and stellar region tumors); neurofibromas, meningiomas, peripheral nerve sheath tumors, and neuroblastomas Neuroendocrine tissue (including, but not limited to, neuroblastoma, ganglioneuroblastoma, ganglioneuroneuroma, trisomy 19 ependymoma); neuroendocrine tissue (including, for example, preganglionic system (adrenal medulla (pheochromocytoma) and extra-adrenal paraganglioma (extra-adrenal paraganglioma)); skin (including, for example, clear cell hidradenoma, cutaneous benign fibrous histiocytoma, cylindroma, hidradenoma, melanoma (cutaneous melanoma, mucosal melanoma), pilomatricoma, Spitz tumor); and soft tissue (including, for example, invasive angiomyxoma, alveolar rhabdomyosarcoma, alveolar soft part sarcoma, angiofibroma, angiomatoid fibrous histiocytoma, synovial sarcoma, biphasic synovial sarcoma, clear cell sarcoma, dermatofibrosarcoma protuberans, desmoid fibromatosis, small round cell tumor, desmoplastic small round cell tumor, elastofibroma, embryonal rhabdomyosarcoma, Ewing's sarcoma / primitive neurectodermal tumors). tumor:PNET), extraskeletal myxoid chondrosarcoma, extraskeletal osteosarcoma, paraspinal sarcoma, inflammatory myofibroblastic tumor, lipoblastoma, lipoma, chondroid lipoma, liposarcoma / malignant lipoma-like tumor, liposarcoma, myxoid liposarcoma, fibromyxoid sarcoma, lymphangioleiomyoma, malignant myoepithelioma, soft tissue melanoma, myoepithelial carcinoma, myoepithelioma, myxoinflammatory fibroblastic sarcoma, undifferentiated sarcoma, hemangiopericytoma, rhabdomyosarcoma, non-rhabdomyosarcoma soft tissue sarcoma soft tissue sarcoma (NRSTS), soft tissue leiomyosarcoma, undifferentiated sarcoma, well-differentiated type In some embodiments, the subject has a solid tumor in a tissue or organ, or a solid tumor arising therefrom, selected from the group consisting of: liposarcoma. In some embodiments, the subject has a blood cancer selected from the group consisting of melanoma, leukemia, acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL); lymphoma; B-cell non-Hodgkin's lymphoma; and multiple myeloma (MM); or a blood cancer selected from the group consisting of melanoma; head and neck; ovarian mesothelioma; endometrial; prostate; sarcoma; neuroblastoma; liver; lung; breast; esophageal, gastric, and pancreatic. In some embodiments, the subject has a cancer selected from the group consisting of lung cancer, colon cancer, breast cancer, prostate cancer, cervical cancer, and head and neck cancer. In some embodiments, the subject is chemotherapy-naive or has not received chemotherapy. In some embodiments, the subject has received a lymphodepleting chemotherapy regimen. In some embodiments, the subject has a blood cancer selected from the group consisting of: melanoma; head and neck; ovarian mesothelioma; endometrial; prostate; sarcoma; neuroblastoma; liver; lung; breast; esophageal, stomach, and pancreatic cancer ... In the present invention, the subject may or may not have bone marrow cells depleted.

[0018] With respect to antiviral, anticancer, and vaccine-enhanced combination therapy methods, in some embodiments, the IL-2v, Fc-IL-2v fusion protein, homodimer, heterodimer, conjugate, polynucleotide, vector, lipoplex (e.g., LNP), and / or pharmaceutical composition is co-administered with one or more additional therapeutic agents. In some embodiments, the one or more additional therapeutic agents comprise one or more agonists or activators of one or more toll-like receptors (TLRs). In some embodiments, the TLR agonists or activators are selected from the group consisting of a TLR2 agonist, a TLR3 agonist, a TLR4 agonist, a TLR5 agonist, a TLR7 agonist, a TLR8 agonist, and a TLR9 agonist. In some embodiments, the TLR7 agonist is selected from the group consisting of GS9620 (vesatolimod), R848 (resiquimod), DS-0509, LHC-165, TMX-101 (imiquimod), RO7020531, and JNJ-4964, and / or the TLR8 agonist is selected from the group consisting of sergantolimod (GS-9688), R848 (resiquimod), and NKTR-262 (a dual TLR7 / TLR8 agonist). In some embodiments, the cytokine or chemokine therapy induces the proliferation or activation of α / β T cells, γ / δ T cells, NK-T cells, NK cells, and / or dendritic cells. In some embodiments, the one or more immunostimulatory cytokines or chemokines are co-administered with one or more immunostimulatory cytokines or chemokines that promote or increase the production of IL-10, IL-12, IL-18, gamma chain-dependent cytokines (e.g., IL-4, IL-7, IL-9, IL-15, and IL-21), fms-related tyrosine kinase 3 (fms-related tyrosine kinase 3), or IL-16. kinase, FLT3 ligand (FLT3L; FLT3LG; NCBI Gene ID: 2323), interferon (IFN)-α, IFN-β, PEGylated interferon (e.g., PEG-IFN-α2a and / or PEG-IFN-α2b), IFN-γ, CXCL9 / Mig (monokine induced by interferon-γ), CXCL10 / IP10 (interferon-γ-inducible 10 kDa protein), and CXCL11 / I-TAC (interferon-inducible T cell α-chemoattractant), CXCL4 / PF4 (platelet factor 4), monocyte chemoattractant protein 2 (MCP-2), macrophage inflammatory protein 1 alpha (MIP-1α), macrophage inflammation Mitotic inflammation protein 1 beta (MIP-1β), and regulated on activation normal T expressed and secreted protein ( In some embodiments, the one or more additional therapeutic agents comprise one or more interleukin receptor agonists of an interleukin receptor selected from IL-10, IL-12, IL-18, and a gamma chain-dependent cytokine (e.g., IL-4, IL-7, IL-9, IL-15, and IL-21). In some embodiments, the one or more additional therapeutic agents comprise one or more cytokines selected from the group consisting of IL-10, IL-12, IL-18, a gamma chain-dependent cytokine (e.g., IL-4, IL-7, IL-9, IL-15, and IL-21), IFN-α, IFN-β, pegylated interferon (e.g., PEG-IFN-α2a and / or PEG-IFN-α2b), IFN-γ, and variants thereof. In some embodiments, the one or more additional therapeutic agents comprise one or more innate immune activators. In some embodiments, the one or more innate immune activators are selected from the group consisting of fms-related tyrosine kinase 3 (FLT3, also known as CD135, FLK-2, FLK2, STK1), stimulator of interferon genes (STING) receptor, DExD / H-box helicase 58 (DDX58;In some embodiments, the one or more innate immune activators comprise one or both of GS-3583 and GS-9992. In some embodiments, the one or more additional therapeutic agents comprise immunotherapy, immune stimulatory therapy, cytokine therapy, chemokine therapy, cell therapy, gene therapy, and combinations thereof. In some embodiments, the immunotherapy comprises co-administration of one or more antagonists or inhibitors of inhibitory immune checkpoint proteins or receptors and / or one or more activators or agonists of stimulatory immune checkpoint proteins or receptors. In some embodiments, the one or more immune checkpoint proteins or receptors are CD27, CD70; CD40, CD40LG; CD47, CD48 (SLAMF2), transmembrane and immunoglobulin domain containing 2 (TMIGD2, CD28H), CD84 (LY9B, SLAMF5), CD96, CD160 (NK1, NK28, BY55), MS4A1 (CD20), CD244 (SLAMF4); CD276 (B7H3); V-set domain-containing inhibitor of T-cell activation 1 (VTCN1, B7H4); V-set immunoregulatory receptor (VSIR, B7H5, VISTA); immunoglobulin superfamily member 11 (IGSF11, VSIG3); natural killer cytotoxicity receptor 3 ligand 1 (NCR3LG1, B7H6); HERV-H LTR-associated 2 (HHLA2, B7H7); inducible T-cell costimulator (ICOS, CD278); inducible T-cell costimulator ligand (ICOSLG, B7H2); TNF receptor superfamily member 4 (TNFRSF4, OX40); TNF superfamily member 4 (TNFSF4, OX40L); TNFRSF8 (CD30), TNFSF8 (CD30L);TNFRSF10A (CD261, DR4, TRAILR1), TNFRSF9 (CD137), TNFSF9 (CD137L); TNFRSF10B (CD262, DR5, TRAILR2), TNFRSF10 (TRAIL); TNFRSF14 (HVEM, CD270), TNFSF14 (HVEML); CD272 (B and T lymphocyte-associated (BTLA)); TNFRSF17 (BCMA, CD269), TNFSF13B (BAFF); TNFRSF18 (GITR), TNFSF18 (GITRL); MHC class I polypeptide-related sequence A (M ICA; MHC class I polypeptide-related sequence B (MICB); CD274 (CD274, PDL1, PD-L1); programmed cell death 1 (PDCD1, PD1, PD-1); cytotoxic T lymphocyte-associated protein 4 (CTLA4, CD152); CD80 (B7-1), CD28; nectin cell adhesion molecule 2 (NECTIN2, CD112); CD226 (DNAM-1); poliovirus receptor (PVR) cell adhesion molecule (PVR, CD155); PVR-related immunoglobulin domain-containing (PVRIG, CD112R); containing Ig and ITIM domains T cell immunoreceptor (TIGIT); T cell immunoglobulin and mucin domain containing 4 (TIMD4; TIM4); hepatitis A virus cellular receptor 2 (HAVCR2, TIMD3, TIM3); galectin 9 (LGALS9); lymphocyte activation 3 (LAG3, CD223); signaling lymphocyte activation molecule family member 1 (SLAMF1, SLAM, CD150); lymphocyte antigen 9 (LY9, CD229, SLAMF3); SLAM family member 6 (SLAMF6, CD352); SLAM family member 7 (SLAMF7, CD319) sialic acid-binding Ig-like lectin 7 (SIGLEC7); sialic acid-binding Ig-like lectin 9 (SIGLEC9); UL16-binding protein 1 (ULBP1); UL16-binding protein 2 (ULBP2); UL16-binding protein 3 (ULBP3); retinoic acid early transcript 1E (RAET1E; ULBP4); retinoic acid early transcript 1G (RAET1G; ULBP5); retinoic acid early transcript 1L (RAET1L; ULBP6); killer cell immunoglobulin-like receptor, three Ig domains, and long cytoplasmic tail 1 (KIR, CD158E1); CD160;Killer cell lectin-like receptor B1 (KLRB1, CD161); killer cell lectin-like receptor C1 (KLRC1, NKG2A, CD159A); killer cell lectin-like receptor K1 (KLRK1, NKG2D, CD314); killer cell lectin-like receptor C2 (KLRC2, CD159c, NKG2C); killer cell lectin-like receptor C3 (KLRC3, NKG2E); killer cell lectin-like receptor C4 (KLRC4, NKG2F); killer cell immunoglobulin-like receptor, one Ig domain, and long cytoplasmic tail 2 (KIR2DL1); killer cell immunoglobulin-like Killer cell immunoglobulin-like receptor, two Ig domains, and long cytoplasmic tail 2 (KIR2DL2); killer cell immunoglobulin-like receptor, three Ig domains, and long cytoplasmic tail 2 (KIR2DL3); killer cell immunoglobulin-like receptor, three Ig domains, and long cytoplasmic tail 1 (KIR3DL1); killer cell lectin-like receptor D1 (KLRD1); killer cell lectin-like receptor G1 (KLRG1; CLEC15A, MAFA, 2F1); sialic acid-binding Ig-like lectin 7 (SIGLEC7); and sialic acid-binding Ig-like lectin 9 (SIGLEC9). In some embodiments, the immunotherapy comprises co-administration of one or more blockers or inhibitors of one or more T cell inhibitory immune checkpoint proteins or receptors. In some embodiments, the T cell inhibitory immune checkpoint protein or receptor is CD274 (CD274, PDL1, PD-L1); programmed cell death 1 ligand 2 (PDCD1LG2, PD-L2, CD273); programmed cell death 1 (PDCD1, PD1, PD-1); cytotoxic T lymphocyte-associated protein 4 (CTLA4, CD152); CD276 (B7H3); V-set domain-containing inhibitor of T-cell activation 1 (VTCN1, B7H4); V-set immunoregulatory receptor (VSIR, B7H5, VISTA); immunoglobulin superfamily member 11 (IGSF11, VSIG3); TNFRSF14 (HVEM, CD270), TNFSF14 (HVEML); CD272 (B and T lymphocyte-associated (BTLA)); PVR-related immunoglobulin domain-containing (PVRIG, CD112R); T cell immunoreceptor with Ig and ITIM domains (TIGIT);Lymphocyte activation 3 (LAG3, CD223); Hepatitis A virus cellular receptor 2 (HAVCR2, TIMD3, TIM3); Galectin 9 (LGALS9); Killer cell immunoglobulin-like receptor, three Ig domains and long cytoplasmic tail 1 (KIR, CD158E1); Killer cell immunoglobulin-like receptor, one Ig domain and long cytoplasmic tail 2 (KIR2DL1); Killer cell immunoglobulin-like receptor, two Ig domains and long cytoplasmic tail 2 (KIR2DL2); Killer cell immunoglobulin-like receptor, three Ig domains and long cytoplasmic tail 2 (KIR2DL3); and Killer cell immunoglobulin-like receptor, three Ig domains and long cytoplasmic tail 1 (KIR3DL1). In some embodiments, the immunotherapy comprises co-administration of one or more agonists or activators of one or more T cell stimulatory immune checkpoint proteins or receptors. In some embodiments, the T cell stimulatory immune checkpoint protein or receptor is selected from the group consisting of CD27, CD70; CD40, CD40LG; inducible T cell costimulatory factor (ICOS, CD278); inducible T cell costimulatory factor ligand (ICOSLG, B7H2); TNF receptor superfamily member 4 (TNFRSF4, OX40); TNF superfamily member 4 (TNFSF4, OX40L); TNFRSF9 (CD137), TNFSF9 (CD137L); TNFRSF18 (GITR), TNFSF18 (GITRL); CD80 (B7-1), CD28; nectin cell adhesion molecule 2 (NECTIN2, CD112); CD226 (DNAM-1); poliovirus receptor (PVR) cell adhesion molecule (PVR, CD155). In some embodiments, the immunotherapy comprises co-administration of one or more blockers or inhibitors of one or more NK cell inhibitory immune checkpoint proteins or receptors. In some embodiments, the NK cell inhibitory immune checkpoint protein or receptor is killer cell immunoglobulin-like receptor, three Ig domains and long cytoplasmic tail 1 (KIR, CD158E1); killer cell immunoglobulin-like receptor, one Ig domain and long cytoplasmic tail 2 (KIR2DL1);Killer cell immunoglobulin-like receptor, two Ig domains and long cytoplasmic tail 2 (KIR2DL2); killer cell immunoglobulin-like receptor, three Ig domains and long cytoplasmic tail 2 (KIR2DL3); killer cell immunoglobulin-like receptor, three Ig domains and long cytoplasmic tail 1 (KIR3DL1); CD160; killer cell lectin-like receptor B1 (KLRB1, CD161); killer cell lectin-like receptor C1 (KLRC1, NKG2A, CD159A); killer cell lectin-like receptor D1 (KLRD1, CD94), killer cell lectin-like receptor G1 (KLRG1; CLEC15A, MAFA, 2F1); sialic acid-binding Ig-like lectin 7 (SIGLEC7); and sialic acid-binding Ig-like lectin 9 (SIGLEC9). In some embodiments, the immunotherapy comprises co-administering one or more agonists or activators of one or more NK cell-stimulatory immune checkpoint proteins or receptors. In some embodiments, the NK cell-stimulatory immune checkpoint proteins or receptors are CD16, CD226 (DNAM-1), killer cell lectin-like receptor K1 (KLRK1, NKG2D, CD314); and SLAM family member 7 (SLAMF7). In some embodiments, the one or more immune checkpoint inhibitors comprise a proteinaceous (e.g., antibody) inhibitor of PD-L1 (CD274), PD-1 (PDCD1), or CTLA4. In some embodiments, the one or more immune checkpoint proteins or receptors are selected from the group consisting of CD274 (CD274, PDL1, PD-L1) and programmed cell death 1 (PDCD1, PD1, PD-1). In some embodiments, C; Proteinaceous (e.g., antibody) inhibitors of CTLA4 include ipilimumab, tremelimumab, BMS-986218, AGEN1181, AGEN1884 (zalifrelimab), BMS-986249, MK-1308, REGN-4659, ADU-1604, CS-1002, BCD-145, APL-509, JS-007, BA-3071, ONC-392, AGEN-2041, JHL-1155, KN-044, CG-0161, ATOR-1144, PBI-5D3H5, and FPT-155 (CTLA4 / PD-L1 / CD28). , PF-06936308 (PD-1 / CTLA4), MGD-019 (PD-1 / CTLA4), KN-046 (PD-1 / CTLA4), MEDI-5752 (CTLA4 / PD-1), XmAb-20717 (PD-1 / CTLA4), and AK-104 (CTLA4 / PD-1).In some embodiments, proteinaceous (e.g., antibody) inhibitors of programmed cell death 1 (PDCD1; NCBI Gene ID: 5133; CD279, PD-1, PD1) include but are not limited to zimberelimab (AB122, GLS-010, WBP-3055), pembrolizumab (KEYTRUDA®, MKN-3475, SCH900475), nivolumab (OPDIVO®, BMS-936558, MDX-1106), cemiplimab (LIBTAYO®; cemiplimab-rwlc, REGN-2810), pidilizumab (CT-011), AMG-404, MEDI0680 (AMP-514), spartalizumab (PDR001 ), tislelizumab (BGB-A317), toripalimab (JS-001), genolimzumab (CBT-501, APL-501, GB226), SHR-1201, camrelizumab (SHR-1210), sintilimab (TYVYT®, IBI-308), dostallimab (TSR-042, WBP-285), lambrolizumab (MK-3475); sasanlimab (PF-06801591), cetrelimab (JNJ-63723283), selplulimab (HLX-10), retifanlimab (MGA-012), balstilimab (AGEN2034), prorugolimab (BCD 100), budigalimab (ABBV-181), vopratelimab (JTX-4014), AK-103 (HX-008), AK-105, CS 1003, BI-754091, LZM-009, Sym-021, BAT-1306, PD1-PIK, tevotelimab (MGD013; PD-1 / LAG-3), RO-7247669(PD-1 / LAG-3), FS-118(LAG-3 / PD-L1), RO-7121661(PD1 / TIM-3), RG7769(PD -1 / TIM-3), PF-06936308 (PD1 / CTLA4), MGD-019 (PD-1 / CTLA4), KN-046 (PD1 / CTLA4), XmAb-20717 (PD1 / CTLA4), AK-104 (CTLA4 / PD-1), and MEDI-5752 (CTLA4 / PD-1).In some embodiments, the proteinaceous (e.g., antibody) inhibitor of the CD274 molecule (NCBI Gene ID: Gene ID:29126; B7-H, B7H1, PD-L1) is selected from the group consisting of atezolizumab (TECENTRIQ®), avelumab (BAVENCIO®; MSB0010718C), embafolimab (ASC22), durvalumab (IMFINZI®; MEDI-4736), BMS-936559 (MDX1105), cosibelimab (CK-301), lodapolimab (LY3300054), and the like. Ga Garivulimab (BGB A333), embafolimab (KN035), opucolimab (HLX20), manelimab (BCD135), CX-072, CBT-502 (TQB2450), MSB-2311, SHR-1316, sugemalimab (CS-1001; WBP3155), A167 (KL-A167, HBM9167), STI-A1015 (IMC-001), FAZ-053, BMS-936559 (MDX1105 ), INCB086550, GEN-1046 (PD-L1 / 4-1BB), FPT-155 (CTLA4 / PD-L1 / CD28), M7824 (PD-L1 / TGFβ-EC domain), CA-170 (PD-L1 / VISTA), CDX-527 (CD27 / PD-L1), LY-3415244 (TIM-3 / PDL1), INBRX-105 (4-1BB / PDL1), and GNS-1480 (PD-L1 / EGFR). In some embodiments, the one or more immune checkpoint inhibitors comprise a small molecule inhibitor of CD274 (PDL1, PD-L1), programmed cell death 1 (PDCD1, PD1, PD-1), or CTLA4. In some embodiments, the small molecule inhibitor of CD274 or PDCD1 is selected from the group consisting of GS-4224, GS-4416, INCB086550, and MAX10181. In some embodiments, the small molecule inhibitor of CTLA4 comprises BPI-002. In some embodiments, the immunomodulator The therapy includes co-administering one or more agents that selectively deplete regulatory T (Treg) cells. In some embodiments, the one or more agents that selectively deplete effector regulatory T (Treg) cells include C-C motif chemokine receptor 4 (CCR4), C-C motif chemokine receptor 7 (CCR7), C-C motif chemokine receptor 8 (CCR8), C-X-C motif chemokine receptor 4 (CXCR4; CD184), TNFRSF4 (OX40), TNFRSF18 (GITR, CD357), TNFRSF9 (4-1BB, CD137), cytotoxic T lymphocyte-associated protein 4 (CTLA4, CD152), programmed cell death 1 (PDCD1, PD-1), sialyl Lewis x (CD15s), CD 27, ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1; CD39), protein tyrosine phosphatase receptor type C (PTPRC; CD45), neural cell adhesion molecule 1 (NCAM1; CD56), selectin L (SELL; CD62L), integrin subunit αE (ITGAE; CD103), interleukin 7 receptor (IL7R; CD127), CD40 ligand (CD40LG; CD154), folate receptor α (FOLR1), folate receptor β (FOLR2), leucine-rich repeat containing 32 (LRRC32; GARP), IKAROS family zinc finger 2 (IKAROS family zinc finger, IKZF2; HELIOS), inducible T-cell costimulatory factor (ICOS; CD278), lymphocyte activation factor 3 (LAG3; CD223), transforming growth factor beta 1 (TGFB1), hepatitis A virus cellular receptor 2 (HAVCR2; CD366; TIM3), T cell immunoreceptor with Ig and ITIM domains (TIGIT), TNF receptor superfamily member 1B (CD120b; TNFR2), IL-2RA (CD25), and combinations thereof. In some embodiments, the immunotherapy comprises co-administration of one or more agents that selectively deplete suppressor myeloid cells.In some embodiments, the suppressive myeloid cells are selected from tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs). In some embodiments, the one or more agents that selectively deplete suppressive myeloid cells comprise an antibody or antigen-binding fragment thereof that selectively binds to a cell surface receptor selected from the group consisting of colony stimulating factor 1 receptor (CSF1R), CC-motif chemokine receptor 2 (CCR2), CC-motif chemokine ligand 2 (CCL2), target receptor expressed on myeloid cells 2 (TREM2), complement C5a receptor 1 (C5AR1), and mixtures thereof. In some embodiments, the one or more agents that selectively deplete suppressor myeloid cells include an agent that inhibits nuclear receptor subfamily 1 group H member 3 (NR1H3; LXRA) or nuclear receptor subfamily 1 group A member 2 (NR1H2; LXRB).In some embodiments, the one or more additional therapeutic agents are selected from the group consisting of mitogen-activated protein kinase kinase kinase kinase 1 (MAP4K1) (also known as Hematopoietic Progenitor Kinase, HPK1), phosphatidylinositol-4,5-bisphosphate 3-kinase comprising catalytic subunit alpha (PIK3CA), catalytic subunit beta (PIK3CB), catalytic subunit gamma (PIK3CG), and catalytic subunit delta (PIK3CD), diacylglycerol kinase alpha (DGKA, DAGK, DAGK1, or DGK-alpha), T cell immunoreceptor with Ig and ITIM domains (TIGIT), X-linked inhibitor of apoptosis (XIAP, BIRC4, IAP-3), baculoviral IAP repeat-containing 2 (IAP repeat 2), and baculoviral IAP repeat 3 (IAP repeat 3). In some embodiments, the one or more additional therapeutic agents include an inhibitor or antagonist of a baculoviral IAP repeat-containing (BIRC2, cIAP1), a baculoviral IAP repeat-containing 3 (BIRC3, cIAP2), a baculoviral IAP repeat-containing 5 (BIRC5, survivin), or a cytokine inducible SH2-containing protein (CISH). In some embodiments, the one or more additional therapeutic agents include an activator or agonist of a toll-like receptor (TLR); a stimulator of interferon genes (STING) receptor; an inducible T-cell costimulator (ICOS, CD278); and / or a member of the TNF receptor superfamily (TNFRSF).In some embodiments, the TNF receptor superfamily (TNFRSF) members are TNFRSF1A, TNFRSF1B, TNFRSF4 (OX40), TNFRSF5 (CD40), TNFRSF6 (FAS), TNFRSF7 (CD27), TNFRSF8 (CD30), TNFRSF9 (4-1BB, CD137), TNFRSF10A (CD261, DR4, TRAILR1), TNFRSF10B (CD262, DR5, TRAILR2), TNFRSF10C (CD263, TRAILR3), ), TNFRSF10D (CD264, TRAILR4), TNFRSF11A (CD265, RANK), TNFRSF11B, TNFRSF12A (CD266), TNFRSF13B (CD267), TNFRSF13C (CD268), TNFRSF16 (NGFR, CD271), TNFRSF17 (BCMA, CD269), TNFRSF18 (GITR, CD357), TNFRSF19, TNFRSF21 (CD358, DR6), and TNFRSF25 (DR3). In some embodiments, the TNFRSF4 (OX40 or CD134) activator or agonist comprises INCAGN1949, tavolimab (MEDI0562), pogalizumab (MOXR0916 / RG7888), MEDI6469, BMS986178, PF-04518600, GSK3174998, IBI101, ATOR-1015, ABBV-368, or SL-279252; the TNFRSF9 (4-1BB or CD137) activator or agonist comprises urelumab, BMS-663513, utomilumab (PF-05082566), CTX-471, MP-0310, ADG-106, ATOR-1017, AGEN2373, or QL1806, and / or TNFRSF18 (GITR or CD357) activators or agonists include GWN323, MEDI1873, MK-1248, MK-4166, TRX518, INCAGN1876, BMS-986156, BMS-986256, AMG-228, ASP1951 (PTZ522), FPA-154, or OMP-336B11.In some embodiments, the method comprises co-administering molecules that simultaneously bind to TNF receptor superfamily member 4 (TNFRSF4, OX40, or CD134) and TNF receptor superfamily member 18 (TNFRSF18, GITR, or CD357). In some embodiments, the method comprises co-administering a molecule selected from the group consisting of AGEN1884 (zalifrelimab), AGEN1181, AGEN2034 (balstilimab), AGEN1307, AGEN1327, AGEN1777, AGEN2373, AGEN1223, and GS-1423.

[0019] With respect to antiviral, anticancer, and vaccine enhancement methods, in some embodiments, the IL-2v, Fc-IL-2v fusion protein, homodimer, heterodimer, conjugate, polynucleotide, vector, lipoplex (e.g., LNP), and / or pharmaceutical composition is administered systemically or locally, e.g., via a route selected from intravenous, subcutaneous, intramuscular, intradermal, intratumoral, and mucosal (e.g., oral, intranasal, rectal, intravaginal). In some embodiments, the IL-2v, Fc-IL-2v fusion protein, homodimer, heterodimer, conjugate, polynucleotide, vector, lipoplex (e.g., LNP), and / or pharmaceutical composition and the one or more additional therapeutic agents are administered by the same or different routes of administration. In various embodiments, the IL-2v, Fc-IL-2v fusion protein, homodimer, heterodimer, conjugate, polynucleotide, vector, lipoplex (e.g., LNP), and / or pharmaceutical composition and the one or more additional therapeutic agents are co-administered according to the same schedule (e.g., co-administration at the same time intervals) or according to different schedules (e.g., co-administration at different time intervals).In some embodiments, the IL-2v, Fc-IL-2v fusion protein, homodimer, heterodimer, conjugate, polynucleotide, vector, lipoplex (e.g., LNP), and / or pharmaceutical composition is administered at a dose in the range of 0.5 μg / kg to 1000 μg / kg, for example, in the range of 1 μg / kg to 500 μg / kg, for example, in the range of 10 μg / kg to 300 μg / kg, for example, in the range of 30 μg / kg to 600 μg / kg, for example, at least 0.5 μg / kg per dose to a maximum of 0.2 μg / kg per dose, 0.3 μg / kg, 0.4 μg / kg, 0.5 μg / kg, 0.6 μg / kg, 0.7 μg / kg, 0.8 μg / kg, 0.9 μg / kg, 1 μg / kg, 1.5 μg / kg, 2 μg / kg, 2.5μg / kg, 3μg / kg, 3.5μg / kg, 4μg / kg, 4.5μg / kg, 5μg / kg, 6μg / kg, 7μg / kg, 8μg / kg, 9μg / kg, 10μ g / kg, 15μg / kg, 20μg / kg, 25μg / kg, 30μg / kg, 40μg / kg, 50μg / kg, 60μg / kg, 70μg / kg, 80μg / kg, 90μ g / kg, 100μg / kg, 110μg / kg, 120μg / kg, 130μg / kg, 140μg / kg, 150μg / kg, 200μg / kg, 250μg / kg, 300μg / kg, 400μg / kg, 500μg / kg, 600μg / kg, 700μg / kg, 800μg / kg, 900μg / kg, and 1000μg / kg. In some embodiments, the IL-2v, Fc-IL-2v fusion protein, homodimer, heterodimer, conjugate, polynucleotide, vector, lipoplex (e.g., LNP), and / or pharmaceutical composition is administered at a dose ranging from 0.02 mg to 100 mg, e.g., 0.04 mg to 80 mg, e.g., at least 0.02 mg per dose to up to 0.03 mg, 0.04 mg, 0.05 mg, 0.1 mg, 0.5 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 10 mg, 12 mg, 15 mg, 20 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, or 100 mg per dose.In various embodiments, the methods comprise administering multiple doses at predetermined intervals of IL-2v, Fc-IL-2v fusion proteins, homodimers, heterodimers, conjugates, polynucleotides, vectors, lipoplexes (e.g., LNPs), and / or pharmaceutical compositions, optionally with one or more additional therapeutic agents. In various embodiments, the methods comprise multiple administrations of IL-2v, Fc-IL-2v fusion protein, homodimer, heterodimer, conjugate, polynucleotide, vector, lipoplex (e.g., LNP), and / or pharmaceutical composition, optionally with one or more additional therapeutic agents, over a period of at least 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months or more. In some embodiments, the methods comprise one or more administrations of IL-2v, Fc-IL-2v fusion protein, homodimer, heterodimer, conjugate, polynucleotide, vector, lipoplex (e.g., LNP), and / or pharmaceutical composition, optionally with one or more additional therapeutic agents, at predetermined intervals of at least one week and up to at least two weeks, three weeks, one month, two months, three months, four months, five months, or six months apart. In some embodiments, the IL-2v, Fc-IL-2v fusion protein, homodimer, heterodimer, conjugate, polynucleotide, vector, lipoplex (e.g., LNP), and / or pharmaceutical composition is administered weekly (i.e., QW), once every two weeks (i.e., once every other week or once every two weeks, i.e., Q2W), once every three weeks (i.e., once every three weeks, i.e., Q3W), once monthly (i.e., QM), or once every two months (i.e., once every other month or once every two months, i.e., Q2M), once every three months (Q3M), once every four months (Q4M), once every five months (Q5M), once every six months (Q6M), or less frequently.In some embodiments, the IL-2v, Fc-IL-2v fusion protein, homodimer, heterodimer, conjugate, polynucleotide, vector, lipoplex (e.g., LNP), and / or pharmaceutical composition is administered subcutaneously two or more times at one or more intervals between once every other week (i.e., once every other week or once every two weeks, i.e., Q2W) and once every three weeks (i.e., once every three weeks, i.e., Q3W). In some embodiments, the IL-2v, Fc-IL-2v fusion protein, homodimer, heterodimer, or conjugate has a serum half-life in humans of at least 6, 9, 12, 15, 18, 21, or 24 hours, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days or more. In some embodiments, the subject or mammal is a human.

[0020] In another aspect, provided are kits. In various embodiments, the kits comprise one or more unit doses of IL-2v, Fc-IL-2v fusion proteins, homodimers, heterodimers, conjugates, polynucleotides, vectors, lipoplexes (e.g., LNPs), and / or pharmaceutical compositions described above and herein. In some embodiments, the one or more unit doses are in a single container or in two or more separate containers. In some embodiments, the kits comprise one or more containers selected from the group consisting of vials, ampoules, and pre-filled syringes. In some embodiments, the kits comprise one or more containers containing the fusion protein and / or homodimer in an aqueous solution. In some embodiments, the aqueous solution has a concentration of 0.05 mg / ml to 50 mg / ml, for example, 0.05 mg / ml to 20 mg / ml, for example, 0.1 mg / ml to 40 mg / ml, for example, 1.0 mg / ml to 30 mg / ml, for example, 0.05 mg / ml to 0.06 mg / ml, 0.07 mg / ml, 0.08 mg / ml, 0.09 mg / ml, 0.1 mg / ml, 0.2 mg / ml, 0.3 mg / ml, 0.4 mg / ml, 0.5 mg / ml, 0.6 mg / ml, 0.7 mg / ml, 0.8 mg / ml, 0.9 mg / ml, 1.0 mg / ml, 1.5 mg / ml, 2.0 mg / ml, 2.5 mg / ml, 3.0 mg / ml, 3.5 mg / ml, In various embodiments, the one or more unit doses comprise IL-2v, Fc-IL-2v fusion protein, homodimer, heterodimer, conjugate, polynucleotide, vector, lipoplex (e.g., LNP), and / or pharmaceutical composition at a concentration in the range of 4.0 mg / ml, 4.5 mg / ml, 5.0 mg / ml, 6 mg / ml, 7 mg / ml, 8 mg / ml, 9 mg / ml, 10 mg / ml, 11 mg / ml, 12 mg / ml, 13 mg / ml, 14 mg / ml, 15 mg / ml, 16 mg / ml, 17 mg / ml, 18 mg / ml, 19 mg / ml, 20 mg / ml, 25 mg / ml, 30 mg / ml, 35 mg / ml, 40 mg / ml, 45 mg / ml, or 50 mg / ml. In various embodiments, the one or more unit doses are the same or different. In some embodiments, each unit dose ranges from 0.5 μg / kg to 1000 μg / kg, for example, from 1 μg / kg to 500 μg / kg, for exampleIn the range of 10 μg / kg to 300 μg / kg, for example, in the range of 30 μg / kg to 600 μg / kg, for example, at least 0.5 μg / kg per dose to at most 0.2 μg / kg per dose, 0.3 μg / kg, 0.4 μg / kg, 0.5 μg / kg, 0.6 μg / kg, 0.7 μg / kg, 0.8 μg / kg, 0.9 μg / kg, 1 μg / kg, 1.5 μg / kg, 2 μg / kg, 2.5 μg / kg, 3 μg / kg, 3.5 μg / kg, 4 μg / kg, 4.5 μg / kg, 5 μg / kg, 6 μg / kg, 7 μg / kg, 8 μg / kg, 9 μg / kg g / kg, 10μg / kg, 15μg / kg, 20μg / kg, 25μg / kg, 30μg / kg, 40μg / kg, 50μg / kg, 60μg / kg, 70μg / kg, 80μg / kg, 90μg / kg, 100μg / kg, 110μg / kg, 120μg / kg, 130μg / kg, 140μg / kg, 150μg / kg, 200μg / kg, 250μg / kg, 300μg / kg, 400μg / kg, 500μg / kg, 600μg / kg, 700μg / kg, 800μg / kg, 900μg / kg, 1000μg / kg. In some embodiments, each unit dose is in the range of 0.02 mg to 100 mg, e.g., 0.04 mg to 80 mg, e.g., at least 0.02 mg per dose to at most 0.03 mg, 0.04 mg, 0.05 mg, 0.1 mg, 0.5 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 10 mg, 12 mg, 15 mg, 20 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, or 100 mg per dose. In some embodiments, the kit further comprises one or more unit doses of one or more additional therapeutic agents. In some embodiments, the kit comprises one or more unit doses of one or more antiviral agents, e.g., against HBV, HIV, HSV, or coronavirus. In some embodiments, the kit comprises one or more agonists or activators of one or more toll-like receptors (TLRs). In some embodiments, the TLR agonist or activator is selected from the group consisting of a TLR2 agonist, a TLR3 agonist, a TLR4 agonist, a TLR5 agonist, a TLR7 agonist, a TLR8 agonist, and a TLR9 agonist. In some embodiments, the TLR7 agonist is selected from the group consisting of GS9620 (vesatolimod),and / or the TLR8 agonist is selected from the group consisting of sergantolimod (GS-9688), R848 (resiquimod), and NKTR-262 (a dual TLR7 / TLR8 agonist). In some embodiments, the kit comprises one or more immunostimulatory cytokines or chemokines that promote or increase the proliferation or activation of α / β T cells, γ / δ T cells, NK-T cells, NK cells, and / or dendritic cells. In some embodiments, the one or more immunostimulatory cytokines or chemokines are selected from the group consisting of IL-10, IL-12, IL-18, gamma chain-dependent cytokines (e.g., IL-4, IL-7, IL-9, IL-15, and IL-21), fms-related tyrosine kinase 3 (FLT3) ligand (FLT3L; FLT3LG; NCBI Gene ID: 2323), interferon (IFN)-α, IFN-β, PEGylated interferon (e.g., PEG-IFN-α2a and / or PEG-IFN-α2b), IFN-γ, CXCL9 / Mi, and / or IL-10. g (monokine induced by interferon-γ), CXCL10 / IP10 (interferon-γ-induced 10 kDa protein), and CXCL11 / I-TAC (interferon-induced T cell alpha-chemoattractant), CXCL4 / PF4 (platelet factor 4), monocyte chemoattractant protein 2 (MCP-2), macrophage inflammatory protein 1 alpha (MIP-1α), macrophage inflammatory protein 1 beta (MIP-1β), and regulatory protein for normal T-expressed and secreted activation and secretion (RANTES). In some embodiments, the kit comprises one or more interleukin receptor agonists of an interleukin receptor selected from IL-10, IL-12, IL-18, and gamma chain-dependent cytokines (e.g., IL-4, IL-7, IL-9, IL-15, and IL-21). In some embodiments, the kits include IL-10, IL-12, IL-18, gamma chain-dependent cytokines (e.g., IL-4, IL-7, IL-9, IL-15, and IL-21), IFN-α, IFN-β, PEGylated interferons (e.g.,In some embodiments, the kit comprises one or more cytokines selected from the group consisting of PEG-IFN-α2a and / or PEG-IFN-α2b), IFN-γ, and variants thereof. In some embodiments, the kit comprises one or more innate immune activators. In some embodiments, the kit comprises an agonist of a receptor selected from the group consisting of fms-related tyrosine kinase 3 (FLT3, also known as CD135, FLK-2, FLK2, STK1), stimulator of interferon genes (STING) receptor, DExD / H-box helicase 58 (DDX58; also known as RIG-I), NLR family pyrin domain-containing 3 (NLRP3), and nucleotide-binding oligomerization domain-containing 2 (NOD2). In some embodiments, the kit comprises one or both of GS-3583 or GS-9992. In some embodiments, the kit comprises one or more antagonists or inhibitors of inhibitory immune checkpoint proteins or receptors and / or one or more activators or agonists of stimulatory immune checkpoint proteins or receptors. In some embodiments, the one or more immune checkpoint proteins or receptors are CD27, CD70; CD40, CD40LG; CD47, CD48 (SLAMF2), transmembrane and immunoglobulin domain containing 2 (TMIGD2, CD28H), CD84 (LY9B, SLAMF5), CD96, CD160 (NK1, NK28, BY55), MS4A1 (CD20), CD244 (SLAMF4); CD276 (B7H3); V-set domain-containing inhibitor of T-cell activation 1 (VTCN1, B7H4); V-set immunoregulatory receptor (VSIR, B7H5, VISTA); immunoglobulin superfamily member 11 (IGSF11, VSIG3); natural killer cytotoxicity receptor 3 ligand 1 (NCR3LG1, B7H6); HERV-H LTR-associated 2 (HHLA2, B7H7); inducible T cell costimulatory factor (ICOS, CD278); inducible T cell costimulatory factor ligand (ICOSLG, B7H2); TNF receptor superfamily member 4 (TNFRSF4, OX40); TNF superfamily member 4 (TNFSF4, OX40L); TNFRSF8 (CD30), TNFSF8 (CD30L); TNFRSF10A (CD261, DR4,TRAILR1), TNFRSF9 (CD137), TNFSF9 (CD137L); TNFRSF10B (CD262, DR5, TRAILR2), TNFRSF10 (TRAIL); TNFRSF14 (HVEM, CD270), TNFSF14 (HVEML); CD272 (B and T lymphocyte-associated (BTLA)); TNFRSF17 (BCMA, CD269), TNFSF13B (BAFF); TNFRSF18 (GITR), TNFSF18 (GITRL); MHC class I polypeptide-related sequence A (MICA); MHC class I polypeptide-related sequence B (MICB); CD274 (CD274, PDL1, PD-L1); programmed cell death 1 (PDCD1, PD1, PD-1); cytotoxic T lymphocyte-associated protein 4 (CTLA4, CD152); CD80 (B7-1), CD28; nectin cell adhesion molecule 2 (NECTIN2, CD112); CD226 (DNAM-1); poliovirus receptor (PVR) cell adhesion molecule (PVR, CD155); PVR-related immunoglobulin domain-containing (PVRIG, CD112R); T cell adhesion molecule with Ig and ITIM domains T-cell immunoreceptor (TIGIT); T-cell immunoglobulin and mucin domain containing 4 (TIMD4; TIM4); hepatitis A virus cellular receptor 2 (HAVCR2, TIMD3, TIM3); galectin 9 (LGALS9); lymphocyte activation 3 (LAG3, CD223); signaling lymphocyte activation molecule family member 1 (SLAMF1, SLAM, CD150); lymphocyte antigen 9 (LY9, CD229, SLAMF3); SLAM family member 6 (SLAMF6, CD352); SLAM family member 7 (SLAMF7, CD319; sialic acid-binding Ig-like lectin 7 (SIGLEC7); sialic acid-binding Ig-like lectin 9 (SIGLEC9); UL16-binding protein 1 (ULBP1); UL16-binding protein 2 (ULBP2); UL16-binding protein 3 (ULBP3); retinoic acid early transcript 1E (RAET1E; ULBP4); retinoic acid early transcript 1G (RAET1G; ULBP5); retinoic acid early transcript 1L (RAET1L; ULBP6); killer cell immunoglobulin-like receptor, three Ig domains, and long cytoplasmic tail 1 (KIR,CD158E1; CD160; killer cell lectin-like receptor B1 (KLRB1, CD161); killer cell lectin-like receptor C1 (KLRC1, NKG2A, CD159A); killer cell lectin-like receptor K1 (KLRK1, NKG2D, CD314); killer cell lectin-like receptor C2 (KLRC2, CD159c, NKG2C); killer cell lectin-like receptor C3 (KLRC3, NKG2E); killer cell lectin-like receptor C, killer cell immunoglobulin-like receptor, 1 Ig domain and long cytoplasmic tail 2 (KIR2DL1); killer cell immunoglobulin-like receptor, 2 Ig domains and long cytoplasmic tail 2 (KIR2DL2); killer cell immunoglobulin-like receptor, 3 Ig domains and long cytoplasmic tail 2 (KIR2DL3); killer cell immunoglobulin-like receptor, 3 Ig domains and long cytoplasmic tail 1 (KIR3DL1); killer cell lectin-like receptor D1 (KLRD1); killer cell lectin-like receptor G1 (KLRG1; CLEC15A, MAFA, 2F1); sialic acid-binding Ig-like lectin 7 (SIGLEC7); and sialic acid-binding Ig-like lectin 9 (SIGLEC9). In some embodiments, the kit comprises one or more blockers or inhibitors of one or more T cell inhibitory immune checkpoint proteins or receptors.In some embodiments, the T cell inhibitory immune checkpoint protein or receptor is CD274 (CD274, PDL1, PD-L1); programmed cell death 1 ligand 2 (PDCD1LG2, PD-L2, CD273); programmed cell death 1 (PDCD1, PD1, PD-1); cytotoxic T lymphocyte-associated protein 4 (CTLA4, CD152); CD276 (B7H3); V-set domain-containing inhibitor of T-cell activation 1 (VTCN1, B7H4); V-set immunoregulatory receptor (VSIR, B7H5, VISTA); immunoglobulin superfamily member 11 (IGSF11, VSIG3); TNFRSF14 (HVEM, CD270), TNFSF14 (HVEML); CD272 (B and T lymphocyte-associated (BTLA)); PVR-associated immunoglobulin domain-containing (PVRI) G, CD112R); T cell immunoreceptor with Ig and ITIM domains (TIGIT); lymphocyte activation 3 (LAG3, CD223); hepatitis A virus cell receptor 2 (HAVCR2, TIMD3, TIM3); galectin 9 (LGALS9); killer cell immunoglobulin-like receptor, three Ig domains, and long cytoplasmic tail 1 (KIR, CD158E1); killer cell immunoglobulin-like receptor, one Ig domain, and long cytoplasmic tail 2 (KIR2DL1); killer cell immunoglobulin-like receptor, two Ig domains, and long cytoplasmic tail 2 (KIR2DL2); killer cell immunoglobulin-like receptor, three Ig domains, and long cytoplasmic tail 2 (KIR2DL3); and killer cell immunoglobulin-like receptor, three Ig domains, and long cytoplasmic tail 1 (KIR3DL1). In some embodiments, the kit comprises one or more agonists or activators of one or more T cell stimulatory immune checkpoint proteins or receptors.In some embodiments, the T cell stimulatory immune checkpoint protein or receptor is selected from the group consisting of CD27, CD70; CD40, CD40LG; inducible T cell costimulatory factor (ICOS, CD278); inducible T cell costimulatory factor ligand (ICOSLG, B7H2); TNF receptor superfamily member 4 (TNFRSF4, OX40); TNF superfamily member 4 (TNFSF4, OX40L); TNFRSF9 (CD137), TNFSF9 (CD137L); TNFRSF18 (GITR), TNFSF18 (GITRL); CD80 (B7-1), CD28; nectin cell adhesion molecule 2 (NECTIN2, CD112); CD226 (DNAM-1); poliovirus receptor (PVR) cell adhesion molecule (PVR, CD155). In some embodiments, the kit further comprises one or more blockers or inhibitors of one or more NK cell inhibitory immune checkpoint proteins or receptors. In some embodiments, the NK cell inhibitory immune checkpoint protein or receptor is selected from the group consisting of killer cell immunoglobulin-like receptor, three Ig domains, and a long cytoplasmic tail 1 (KIR, CD158E1); killer cell immunoglobulin-like receptor, one Ig domain, and a long cytoplasmic tail 2 (KIR2DL1); killer cell immunoglobulin-like receptor, two Ig domains, and a long cytoplasmic tail 2 (KIR2DL2); killer cell immunoglobulin-like receptor, three Ig domains, and a long cytoplasmic tail 2 (KIR2DL3); killer cell These include immunoglobulin-like receptor, three Ig domains, and long cytoplasmic tail 1 (KIR3DL1); CD160; killer cell lectin-like receptor B1 (KLRB1, CD161); killer cell lectin-like receptor C1 (KLRC1, NKG2A, CD159A); killer cell lectin-like receptor D1 (KLRD1, CD94); killer cell lectin-like receptor G1 (KLRG1; CLEC15A, MAFA, 2F1); sialic acid-binding Ig-like lectin 7 (SIGLEC7); and sialic acid-binding Ig-like lectin 9 (SIGLEC9). In some embodiments, the kit comprises one or more agonists or activators of one or more NK cell-stimulatory immune checkpoint proteins or receptors.In some embodiments, the NK cell-stimulatory immune checkpoint proteins or receptors are CD16, CD226 (DNAM-1), and killer cell lectin-like receptor K1 (KLRK1, NKG2D, CD314); and SLAM family member 7 (SLAMF7). In some embodiments, the kit comprises a proteinaceous (e.g., antibody) inhibitor of PD-L1 (CD274), PD-1 (PDCD1), or CTLA4. In some embodiments, the kit comprises a proteinaceous (e.g., antibody) inhibitor of PD-L1 (CD274) or PD-1 (PDCD1). In some embodiments, proteinaceous (e.g., antibody) inhibitors of CTLA4 include ipilimumab, tremelimumab, BMS-986218, AGEN1181, AGEN1884 (zalifrelimab), BMS-986249, MK-1308, REGN-4659, ADU-1604, CS-1002, BCD-145, APL-509, JS-007, BA-3071, ONC-392, AGEN2041, JHL-1155, KN-0 44, CG-0161, ATOR-1144, PBI-5D3H5, FPT-155 (CTLA4 / PD-L1 / CD28), PF-06936308 (PD-1 / CTLA4), MGD-019 (PD-1 / CTLA4), KN-046 (PD-1 / CTLA4), MEDI-5752 (CTLA4 / PD-1), XmAb-20717 (PD-1 / CTLA4), and AK-104 (CTLA4 / PD-1).In some embodiments, proteinaceous (e.g., antibody) inhibitors of programmed cell death 1 (PDCD1; NCBI Gene ID: 5133; CD279, PD-1, PD1) include, but are not limited to, zimberelimab (AB122, GLS-010, WBP-3055), pembrolizumab (KEYTRUDA®, MKN-3475, SCH900475), nivolumab (OPDIVO®, BMS-936558, MDX-1106), cemiplimab (LIBTAYO®; cemiplimab-rwlc, REGN-2810), pidilizumab (CT-011), AMG-404, MEDI0680 (AMP-514), spam Rutalizumab (PDR001), tislelizumab (BGB-A317), toripalimab (JS-001), genolimuzumab (CBT-501, APL-501, GB226), SHR-1201, camrelizumab (SHR-1210), sintilimab (TYVYT®, IBI-308), dostallimab (TSR-042, WBP-285), lambrolizumab (MK-3475); sasanlimumab (PF-06801591), cetrelimab (JNJ-63723283), selplulimab (HLX-10), retifanlimab (MGA-012), balstilimab (AGEN2034), prorugolimab (BCD 100), budigalimab (ABBV-181), vopratelimab (JTX-4014), AK-103 (HX-008), AK-105, CS 1003, BI-754091, LZM-009, Sym-021, BAT-1306, PD1-PIK, tevotelimab (MGD013; PD-1 / LAG-3), RO-7247669(PD-1 / LAG-3), FS-118(LAG-3 / PD-L1), RO-7121661(PD1 / TIM-3), RG7769(PD -1 / TIM-3), PF-06936308 (PD1 / CTLA4), MGD-019 (PD-1 / CTLA4), KN-046 (PD1 / CTLA4), XmAb-20717 (PD1 / CTLA4), AK-104 (CTLA4 / PD-1), and MEDI-5752 (CTLA4 / PD-1).In some embodiments, the proteinaceous (e.g., antibody) inhibitor of the CD274 molecule (NCBI Gene ID: Gene ID:29126; B7-H, B7H1, PD-L1) is selected from the group consisting of atezolizumab (TECENTRIQ®), avelumab (BAVENCIO®; MSB0010718C), embafolimab (ASC22), durvalumab (IMFINZI®; MEDI-4736), BMS-936559 (MDX1105), cosibelimab (CK-301), lodapolimab (LY3300054), and the like. GaRivlimab (BGB A333), embafolimab (KN035), opcolimab (HLX20), manelimab (BCD135), CX-072, CBT-502 (TQB2450), MSB-2311, SHR-1316, sugemalimab (CS-1001; WBP3155), A167 (KL-A167, HBM9167), STI-A1015 (IMC-001), FAZ-053, BMS-936559 (MDX1105), INCB08655 In some embodiments, the kit comprises a small molecule inhibitor of CD274 (PDL1, PD-L1), programmed cell death 1 (PDCD1, PD1, PD-1), or CTLA4. In some embodiments, the small molecule inhibitor of CD274 or PDCD1 is selected from the group consisting of GS-4224, GS-4416, INCB086550, and MAX10181. In some embodiments, the small molecule inhibitor of CTLA4 comprises BPI-002. In some embodiments, the kit comprises one or more agents that selectively deplete regulatory T (Treg) cells.In some embodiments, the one or more agents that selectively deplete effector regulatory T (Treg) cells are selected from the group consisting of C-C motif chemokine receptor 4 (CCR4), C-C motif chemokine receptor 7 (CCR7), C-C motif chemokine receptor 8 (CCR8), C-X-C motif chemokine receptor 4 (CXCR4; CD184), TNFRSF4 (OX40), TNFRSF18 (GITR, CD357), TNFRSF9 (4-1BB, CD137), cytotoxic T lymphocyte-associated protein 4 (CTLA4, CD152), programmed cell death 1 (PDCD1, P D-1), sialyl Lewis x (CD15s), CD27, ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1; CD39), protein tyrosine phosphatase receptor type C (PTPRC; CD45), neural cell adhesion molecule 1 (NCAM1; CD56), selectin L (SELL; CD62L), integrin subunit α E (ITGAE; CD103), interleukin 7 receptor (IL7R; CD127), CD40 ligand (CD40LG; CD154), folate receptor α (FOLR1), folate receptor β (FOLR2), leucine lysate. The present invention also includes an antibody or antigen-binding fragment thereof that selectively binds to a cell surface receptor selected from the group consisting of: T cell repeat-containing 32 (LRRC32; GARP), IKAROS family zinc finger 2 (IKZF2; HELIOS), inducible T cell costimulatory factor (ICOS; CD278), lymphocyte activation 3 (LAG3; CD223), transforming growth factor beta 1 (TGFB1), hepatitis A virus cellular receptor 2 (HAVCR2; CD366; TIM3), T cell immunoreceptor with Ig and ITIM domains (TIGIT), TNF receptor superfamily member 1B (CD120b; TNFR2), IL-2RA (CD25), and combinations thereof. In some embodiments, the kit includes a kit of mitogen-activated protein kinase kinase kinase kinase 1 (MAP4K1) (also known as hematopoietic progenitor kinase 1 (HPK1)), phosphatidylinositol-4,5-bisphosphate 3-kinase including catalytic subunit alpha (PIK3CA), catalytic subunit beta (PIK3CB), catalytic subunit gamma (PIK3CG), and catalytic subunit delta (PIK3CD), diacylglycerol kinase alpha (DGKA, DAGK, DAGK1), and phosphokinase alpha (PIK3CA, DAGK2, DAGK3). or DGK-alpha), T cell immunoreceptor with Ig and ITIM domains (TIGIT), X-linked inhibitor of apoptosis (XIAP, BIRC4, IAP-3), baculoviral IAP repeat-containing 2 (BIRC2, cIAP1), baculoviral IAP repeat-containing 3 (BIRC3, cIAP2), baculoviral IAP repeat-containing 5 (BIRC5, survivin), or cytokine-inducible SH2-containing protein (CISH). In some embodiments, the kit includes one or more additional therapeutic agents, including an inhibitor or antagonist of a toll-like receptor (TLR); an interferon gene stimulator (STING) receptor; an inducible T cell costimulator (ICOS, CD278); and / or a TNF receptor superfamily (TNFRSF) member.In some embodiments, the TNF receptor superfamily (TNFRSF) members are TNFRSF1A, TNFRSF1B, TNFRSF4 (OX40), TNFRSF5 (CD40), TNFRSF6 (FAS), TNFRSF7 (CD27), TNFRSF8 (CD30), TNFRSF9 (4-1BB, CD137), TNFRSF10A (CD261, DR4, TRAILR1), TNFRSF10B (CD262, DR5, TRAILR2), TNFRSF10C (CD263, TRAILR3), ), TNFRSF10D (CD264, TRAILR4), TNFRSF11A (CD265, RANK), TNFRSF11B, TNFRSF12A (CD266), TNFRSF13B (CD267), TNFRSF13C (CD268), TNFRSF16 (NGFR, CD271), TNFRSF17 (BCMA, CD269), TNFRSF18 (GITR, CD357), TNFRSF19, TNFRSF21 (CD358, DR6), and TNFRSF25 (DR3). In some embodiments, the kit includes a TNFRSF4 (OX40 or CD134) activator or agonist selected from INCAGN1949, tavolimab (MEDI0562), pogalizumab (MOXR0916 / RG7888), MEDI6469, BMS-986178, PF-04518600, GSK3174998, IBI101, ATOR-1015, ABBV-368, or SL-279252, urelumab, BMS-663513, utomilumab (PF-05082566), CTX-471, MP-0310, A and / or a TNFRSF9 (4-1BB or CD137) activator or agonist selected from DG-106, ATOR-1017, AGEN2373, or QL1806, and / or a TNFRSF18 (GITR or CD357) activator or agonist selected from GWN323, MEDI1873, MK-1248, MK-4166, TRX518, INCAGN1876, BMS-986156, BMS-986256, AMG-228, ASP1951 (PTZ522), FPA-154, or OMP-336B11. In some embodiments, the kit comprises a molecule that simultaneously binds to TNF receptor superfamily member 4 (TNFRSF4, OX40, or CD134) and TNF receptor superfamily member 18 (TNFRSF18, GITR, or CD357). In some embodiments, the kit comprises a molecule selected from the group consisting of AGEN1884 (zalifrelimab), AGEN1181, AGEN2034 (balstilimab), AGEN1307, AGEN2373, AGEN1223, and GS-1423.

[0021] With respect to kits comprising therapeutic agents for anti-HBV combination therapy, in some embodiments, the one or more antiviral agents are selected from the group consisting of lamivudine (LAM), adefovir dipivoxil (ADV), entecavir (ETV), telbivudine (LdT), tenofovir disoproxil fumarate (TDF), tenofovir alafenamide (TAF or VEMLIDY®), ledipasvir plus sofosbuvir (HARVONI®), and PEGylated interferon (e.g., PEG-IFN-α2a and / or PEG-IFN-α2b). In some embodiments, the kit further comprises one or more unit doses of one or more therapeutic agents selected from the group consisting of an HBV antigen inhibitor (e.g., an HBV core antigen (HBcAg) inhibitor, an HBV surface antigen (HBsAg) inhibitor, an HBx inhibitor, an HBV E antigen inhibitor), an anti-HBV antigen antibody, an inhibitory nucleic acid targeting HBV (e.g., an antisense oligonucleotide, a small interfering RNA (siRNA), a DNA-directed RNA interference (ddRNAi)), a gene editor targeting HBV (e.g., a CRISPR-Cas (e.g., Cas9, Cas12, Cascade, Cas13), a zinc finger nuclease, a homing endonuclease, a homing meganuclease (e.g., ARCUS), a synthetic nuclease, a TALEN), a covalently closed circular DNA (cccDNA) inhibitor, an HBsAg secretion or assembly inhibitor, an HBV viral entry inhibitor, and a CAR-T and a T-cell bispecific (redirected T cell) for specifically killing HBV-infected cells.

[0022] Regarding kits containing therapeutic agents for anti-HBV combination therapy, in some embodiments, the kit includes one or more unit doses of one or more antiretroviral agents. In some embodiments, the kit includes one or more broadly neutralizing anti-HIV antibodies. In some embodiments, the one or more broadly neutralizing anti-HIV antibodies bind to an epitope or region of gp120 selected from the group consisting of: (i) the third variable loop (V3) and / or the high mannose patch comprising the N332 oligomannose glycan, (ii) the second variable loop (V2) and / or the Env trimer vertex, (iii) the CD4 binding site (CD4bs), (iv) the gp120 / gp41 interface, or (v) the silent face of gp120. In some embodiments, the one or more anti-HIV broadly neutralizing antibodies bind to an epitope or region of gp120 in the third variable loop (V3) and / or the high mannose patch comprising the N332 oligomannose glycan, and are selected from the group consisting of GS-9722, PGT-121, PGT-121.414, PGT-122, PGT-123, PGT-124, PGT-125, PGT-126, PGT-128, PGT-130, PGT-133, PGT-134, PGT-135, PGT-136, PGT-137, PGT-138, PGT-139, PGT-200, PGT-201, PGT-202, PGT-203, PGT-204, PGT-205, PGT-206, PGT-207, PGT-208, PGT-209, PGT-210, PGT-211, PGT-212, PGT-213, PGT-214, PGT-215, PGT-216, PGT-217, PGT-218, PGT-219, PGT-220, PGT-221, PGT-222.414, PGT-222, PGT-223, PGT-224, PGT-225, PGT-226, PGT-228, PGT-229, PGT-230, PGT-231, PGT-232, PGT-233, PGT-234, PGT-235, PGT-236, PGT-237, PGT-238, PGT-239, PGT-240, PGT-241, PGT-242, PGT-243, PGT-244 competes with or comprises the VH and VL regions from an antibody selected from the group consisting of GT-138, PGT-139, 10-1074, 10-1074-J, VRC24, 2G12, BG18, 354BG8, 354BG18, 354BG42, 354BG33, 354BG129, 354BG188, 354BG411, 354BG426, DH270.1, DH270.6, PGDM12, VRC41.01, PGDM21, PCDN-33A, BF520.1, and VRC29.03.In some embodiments, the one or more broadly neutralizing anti-HIV antibodies bind to an epitope or region of gp120 in the second variable loop (V2) and / or Env trimer apex and compete with or comprise the VH and VL regions from an antibody selected from the group consisting of PG9, PG16, PGC14, PGG14, PGT-142, PGT-143, PGT-144, PGT-145, CH01, CH59, PGDM1400, CAP256, CAP256-VRC26.08, CAP256-VRC26.09, CAP256-VRC26.25, PCT64-24E, and VRC38.01. In some embodiments, the one or more anti-HIV broadly neutralizing antibodies bind to an epitope or region of gp120 in the CD4 binding site (CD4bs), including 3BNC117, GS-9723, 3BNC60, b12, F105, VRC01, VRC07, VRC07-523, VRC03, VRC06, VRC06b01. competes with or comprises the VH and VL regions from an antibody selected from the group consisting of VRC08, VRC0801, NIH45-46, VRC-PG04, PGV04; CH103, 44-VRC13.01, 1NC9, 12A12, N6, N49-P7, NC-Cow1, IOMA, CH235 and CH235.12, N49P6, N49P7, N49P11, N49P9, and N60P25. In some embodiments, the one or more broadly neutralizing anti-HIV antibodies bind to an epitope or region of gp120 at the gp120 / gp41 interface and compete with or include VH and VL regions from an antibody selected from the group consisting of PGT-151, CAP248-2B, 35O22, 8ANC195, ACS202, VRC34, and VRC34.01. In some embodiments, the one or more broadly neutralizing anti-HIV antibodies bind to an epitope or region of gp120 silent face and compete with or include VH and VL regions from an antibody selected from VRC-PG05 and SF12. In some embodiments, the one or more broadly neutralizing anti-HIV antibodies bind to an epitope or region of gp41 in the membrane proximal region (MPER). In some embodiments, the one or more broadly neutralizing anti-HIV antibodies bind to an epitope or region of gp41 in the membrane proximal region (MPER) and compete with or comprise the VH and VL regions from an antibody selected from the group consisting of 10E8, 10E8v4, 10E8-5R-100cF, 4E10, DH511.11P, 2F5, 7b2, and LN01. In some embodiments, the one or more broadly neutralizing anti-HIV antibodies bind to an epitope or region of a gp41 fusion peptide and compete with or comprise the VH and VL regions from an antibody selected from the group consisting of VRC34 and ACS202.

[0023] Regarding kits containing therapeutic agents for vaccine-enhanced combination therapy, in some embodiments, the kit comprises one or more unit doses of a vaccine. In some embodiments, the vaccine is selected from the group consisting of an antiviral vaccine, an antibacterial vaccine, and an anticancer vaccine. In some embodiments, the vaccine comprises an antiviral vaccine against a virus selected from the group consisting of hepatitis A virus (HAV), hepatitis B virus (HBV), human immunodeficiency virus (HIV), cytomegalovirus (CMV), herpes simplex virus (HSV), Epstein-Barr virus (EBV), human orthopneumovirus or human respiratory syncytial virus (RSV), human papillomavirus (HPV), varicella-zoster virus, measles virus, mumps virus, poliovirus vaccine, influenza virus, paramyxovirus, rotavirus, Zika virus, dengue virus, Ebola virus, and a coronavirus (e.g., a betacoronavirus, e.g., a severe acute respiratory syndrome-associated coronavirus, e.g., SARS-CoV2). In some embodiments, the vaccine is directed against Mycobacterium tuberculosis, Pertussis, Included are antibacterial vaccines against bacteria selected from the group consisting of tetanus, diphtheria, meningococcus, pneumococcus, Haemophilus influenza, cholera, typhoid, and anthrax.

[0024] With respect to kits containing therapeutic agents for anti-cancer combination therapy, in some embodiments, the kits include one or more unit doses of one or more antineoplastic or chemotherapeutic agents. In some embodiments, the kits include nucleoside analogs (e.g., 5-fluorouracil, gemcitabine, cytarabine, cladribine, pentostatin, fludarabine), taxanes (e.g., paclitaxel, nab-paclitaxel, docetaxel, cabazitaxel), platinum coordination complexes (cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthroline), and the like. dihydrofolate reductase (DHFR) inhibitors (e.g., methotrexate, trimetrexate, pemetrexed), topoisomerase inhibitors (e.g., doxorubicin, daunorubicin, dactinomycin, eniposide, epirubicin, etoposide, idarubicin, irinotecan, mitochondrial infusion), and one or more unit doses of one or more antineoplastic or chemotherapeutic agents selected from the group consisting of xantrone, pixantrone, sobuzoxane, topotecan, irinotecan, MM-398 (liposomal irinotecan), vosaroxin and GPX-150, aldoxorubicin, AR-67, mavereltinib, AST-2818, avitinib (ACEA-0010), irofulven (MGI-114)), alkylating agents (e.g., nitrogen mustards (e.g., cyclophosphamide, chlormethine, uramustine or uracil mustard, melphalan, chlorambucil, ifosfamide, bendamustine, temozolomide, carmustine), nitrosoureas (e.g., carmustine, lomustine, streptozocin), alkyl sulfonates (e.g., busulfan)), and mixtures thereof. In some embodiments, the kit comprises: CD19; transmembrane 4 domain A1 (MS4A1; CD20); CD22 (SIGLEC2); CD27 (TNFRSF7); TNFRSF8 (CD30); CD33 (SIGLEC3); CD37; CD38; CD40 (TNFRSF5), CD44; CD47; CD48 (SLAMF2); CD52; CD70 (TNFSF7), CD27L; 5'-nucleotidase ecto (NT5E;CD73), ectonucleoside triphosphate diphosphohydrolase 1 (CD39), CD74; CD79B; CD80; CD86; interleukin-3 receptor subunit alpha (IL3RA); prominin 1 (PROM1; CD133); TNFRSF9 (CD137); syndecan 1 (SDC1; CD138); CD200 molecule (CD200); alphafetoprotein (AFP), BAG cochaperone 6 (BAG6); MET proto-oncogene, receptor tyrosine kinase (MET); KIT Oncogenic gene, receptor tyrosine kinase (KIT); C-type lectin domain family 12 member A (CLEC12A; CD371); C-type lectin domain-containing 9A (CLEC9A; CD370); cadherin 3 (CDH3); carbonic anhydrase 6 (CA6); carbonic anhydrase 9 (CA9); carcinoembryonic antigen-related cell adhesion molecule 3 (CEACAM3); carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5); carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6); chorionic somatomammotropin pin hormone 1 (CSH1); coagulation factor III, tissue factor (F3); collectin subfamily member 10 (COLEC10; CLL1); delta-like canonical notch ligand 3 (DLL3); ectonucleotide pyrophosphatase / phosphodiesterase 3 (ENPP3); ephrin A1 (EFNA1); epidermal growth factor receptor (EGFR; ERBB; HER1); EGFR variant III (EGFRvIII); EPH receptor A2 (EPHA2); epithelial cell adhesion molecule (EP CAM); erb-b2 receptor tyrosine kinase 2 (ERBB2; HER-2 / neu); fibroblast activation protein alpha (FAP); fibroblast growth factor receptor 2 (FGFR2); fibroblast growth factor receptor 3 (FGFR3); folate hydrolase 1 (FOLH1); folate receptor 1 (FOLR1); GD2 ganglioside; glycoprotein NMB (GPNMB; osteoactivin); guanylate cyclase 2C (GUCY2C); human papillomavirus (HPV) E6; HPV E7; major histocompatibility complex (MHC) class I-presented neoantigen, major histocompatibility complex (MHC) class II-presented neoantigen, major histocompatibility complex, class I, E (HLA-E); major histocompatibility complex, class I, F (HLA-F);Major histocompatibility complex, class I, G (HLA-G); MHC class I polypeptide-related sequence A (MICA); MHC class I polypeptide-related sequence B (MICB); integrin subunit β7 (ITGB7); leukocyte immunoglobulin-like receptor B1 (LILRB1; ILT2); leukocyte immunoglobulin-like receptor B2 (LILRB2; ILT4); LY6 / PLAUR domain-containing 3 (LYPD3); glypican 3 (GPC3); KRAS proto-oncogene, GTPase (KRAS); MAGE family member A1 (MAGEA1); MAGE family member A3 (MAGEA3); MAGE family member A4 (MAGEA4); MAGE family member A11 (MAGEA11); MAGE family member C1 (MAGEC1); MAGE family member C2 (MAGEC2); MAGE family member C3 (MAGEC3); MAGE family member D1 (MAGED1); MAGE family member D2 (MAGED2); mesothelin (MSLN); mucin 1 (MUC1), and its splice variants (e.g., MUC1 / A, C, , D, X, Y, Z, and REP); mucin 16 (MUC16; CA125); natural killer cytotoxicity receptor 3 ligand 1 (NCR3LG1; B7-H6); necdin, a MAGE family member (NDN); nectin cell adhesion molecule 2 (NECTIN2); nectin cell adhesion molecule 4 (NECTIN4); SLIT and NTRK-like family member 6 (SLITRK6); promyelocytic leukemia (PML); protein tyrosine kinase 7 (inactive) (PTK7); poliovirus receptor (PVR) cell adhesion molecule (PVR); SLAM family member 6 (SLAMF6); SLAM family member 7 (SLAMF7); sialic acid-binding Ig-like lectin 7 (SIGLEC7); sialic acid-binding Ig-like lectin 9 (SIGLEC9); sialic acid-binding Ig-like lectin 10 (SIGLEC10); signal-regulatory protein alpha (SIRPA) solute carrier family 34 (sodium phosphate), member 2 (SLC34A2); solute carrier family 39 member 6 (SLC39A6); STEAP family member 1 (STEAP1); suppressor of tumorigenicity 2 (ST2);TNF receptor superfamily member 4 (TNFRSF4; OX40); TNF superfamily member 9 (TNFSF9; 4-1BB-L, CD137L); TNFRSF10A (DR4, TRAILR1); TNFRSF10B (DR5, TRAILR2); TNFRSF13B (BAFF); TNFRSF17 (BCMA); TNFRSF18 (GITR); transferrin (TF); transforming growth factor beta 1 (TGFB1) and its isoforms; triggering receptor expressed on myeloid cells 1 (TREM1); triggering receptor expressed on myeloid cells 2 (TREM2); trophoblast glycoprotein (TPBG); trophinin (TRO); tumor-associated calcium signaling factor 2 (TACSTD2); fucosyl-GM1; sialyl Lewis adhesion molecule (sLe);and Lewis Y antigen. In some embodiments, the kit comprises one or more antibodies or antigen-binding antibody fragments thereof or antibody-drug conjugates thereof, CD3-targeting multispecific molecules, NK cell-activating receptor-targeting multispecific molecules, or non-immunoglobulin antigen-binding domains, or antibody mimetic proteins, directed against one or more targets or tumor-associated antigens (TAA) selected from the group consisting of: IL-16, IL-17, IL-21, IL-18, IL-19, IL-20, IL-31, IL-41, IL-42, IL-43, IL-44, IL-45, IL-56, IL-57, IL-58, IL-59, IL-60, IL-61, IL-62, IL-63, IL-64, IL-65, IL-66, IL-67, IL-68, IL-69, IL-70, IL-71, IL-72, IL-73, IL-74, IL-75, IL-76, IL-77, IL-78, IL-79, IL-80, IL-81, IL-82, IL-83, IL-84, IL-85, IL-85, IL-86, IL-87, IL-88, IL-89 ... In some embodiments, the kit comprises one or more immune cell populations selected from the group consisting of natural killer (NK) cells, NK-T cells, T cells, cytokine-induced killer (CIK) cells, macrophage (MAC) cells, tumor-infiltrating lymphocytes (TIL), and dendritic cells (DC). In some embodiments, the kit comprises a T cell population selected from the group consisting of alpha / beta TCR T cells, gamma / delta TCR T cells, regulatory T (Treg) cells, and TRuC™ T cells. In some embodiments, the one or more cell therapies comprise NK cell therapy comprising NK-92 cells. In some embodiments, the cells are allogeneic to the intended recipient. In some embodiments, the one or more immune cell populations comprise one or more chimeric antigen receptors (CARs). In some embodiments, one or more CARs are selected from the group consisting of CD19; transmembrane 4 domain A1 (MS4A1; CD20); CD22 (SIGLEC2); CD27 (TNFRSF7); TNFRSF8 (CD30); CD33 (SIGLEC3); CD37; CD38; CD40 (TNFRSF5), CD44; CD47; CD48 (SLAMF2); CD52; CD70 (TNFSF7), CD27L; 5'-nucleotidase ecto (NT5E;CD73), ectonucleoside triphosphate diphosphohydrolase 1 (CD39), CD74; CD79B; CD80; CD86; interleukin-3 receptor subunit alpha (IL3RA), prominin 1 (PROM1; CD133); TNFRSF9 (CD137); syndecan 1 (SDC1; CD138); CD200 molecule (CD200); alphafetoprotein (AFP), BAG cochaperone 6 (BAG6); MET proto-oncogene, receptor tyrosine kinase (MET); K IT proto-oncogene, receptor tyrosine kinase (KIT); C-type lectin domain family 12 member A (CLEC12A; CD371); C-type lectin domain-containing 9A (CLEC9A; CD370); cadherin 3 (CDH3); carbonic anhydrase 6 (CA6); carbonic anhydrase 9 (CA9); carcinoembryonic antigen-related cell adhesion molecule 3 (CEACAM3); carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5); carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6); chorionic sarcoma coagulation factor III, tissue factor (F3); collectin subfamily member 10 (COLEC10; CLL1); delta-like canonical notch ligand 3 (DLL3); ectonucleotide pyrophosphatase / phosphodiesterase 3 (ENPP3); ephrin A1 (EFNA1); epidermal growth factor receptor (EGFR; ERBB; HER1); EGFR variant III (EGFRvIII); EPH receptor A2 (EPHA2); epithelial Intercellular adhesion molecule (EPCAM); erb-b2 receptor tyrosine kinase 2 (ERBB2; HER-2 / neu); fibroblast activation protein alpha (FAP); fibroblast growth factor receptor 2 (FGFR2); fibroblast growth factor receptor 3 (FGFR3); folate hydrolase 1 (FOLH1); folate receptor 1 (FOLR1); GD2 ganglioside; glycoprotein NMB (GPNMB; osteoactivin); guanylate cyclase 2C (GUCY2C); human papillomavirus; HPV E6; HPV E7; major histocompatibility complex (MHC) class I-presented neoantigens, major histocompatibility complex (MHC) class II-presented neoantigens, major histocompatibility complex, class I, E (HLA-E); major histocompatibility complex, class I, F (HLA-F); major histocompatibility complex, class I, G (HLA-G); MHC class I polypeptide-related sequence A (MICA); MHC class I polypeptide-related sequence B (MICB); integrin subunit β7 (ITGB7); leukocyte immunoglobulin-like receptor B1 (LILRB1; ILT2); leukocyte immunoglobulin-like receptor B2 (LILRB2; ILT4); LY6 / PLAUR domain-containing 3 (LYPD3); glypican 3 (GPC3); KRAS proto-oncogene, GTPase (KRAS); MAGE family MAGE family member A1 (MAGEA1); MAGE family member A3 (MAGEA3); MAGE family member A4 (MAGEA4); MAGE family member A11 (MAGEA11); MAGE family member C1 (MAGEC1); MAGE family member C2 (MAGEC2); MAGE family member C3 (MAGEC3); MAGE family member D1 (MAGED1); MAGE family member D2 (MAGED2); mesothelin (MSLN); mucin 1 (MUC1) and its splice variants (e.g., including MUC1 / A, C, D, X, Y, Z, and REP); mucin 16 (MUC16; CA125); natural killer cytotoxicity receptor 3 ligand 1 (NCR3LG1; B7-H6); necdin,MAGE family member (NDN); nectin cell adhesion molecule 2 (NECTIN2); nectin cell adhesion molecule 4 (NECTIN4); SLIT and NTRK-like family member 6 (SLITRK6); promyelocytic leukemia (PML); protein tyrosine kinase 7 (inactive) (PTK7); poliovirus receptor (PVR) cell adhesion molecule (PVR); SLAM family member 6 (SLAMF6); SLAM family member 7 (SLAMF7); sialic acid-binding Ig-like lectin 7 (SIGLEC7); sialic acid-binding Ig-like lectin 9 (SIGLEC9); sialic acid-binding Ig-like lectin 10 (SIGLEC10); signal-regulatory protein alpha (SIRPA) solute carrier family 34 (sodium phosphate), member 2 (SLC34A2); solute carrier family 39 member 6 (SLC39A6); STEAP family member 1 (STEAP1); suppressor of tumorigenicity 2 (ST2) ; TNF receptor superfamily member 4 (TNFRSF4; OX40); TNF superfamily member 9 (TNFSF9; 4-1BB-L, CD137L); TNFRSF10A (DR4, TRAILR1); TNFRSF10B (DR5, TRAILR2); TNFRSF13B (BAFF); TNFRSF17 (BCMA); TNFRSF18 (GITR); transferrin (TF); transforming growth factor beta 1 (TGFB1) and its isoforms; triggering receptor expressed on myeloid cells 1 (TREM1); triggering receptor expressed on myeloid cells 2 (TREM2); trophoblast glycoprotein (TPBG); trophinin (TRO); tumor-associated calcium signaling factor 2 (TACSTD2); fucosyl-GM1; sialyl Lewis adhesion molecule (sLe); and Lewis Y antigen. In some embodiments, one or more CARs bind to an epitope of a target or tumor-associated antigen (TAA) presented on a major histocompatibility complex (MHC) molecule. In some embodiments, the TAA is a cancer-testis antigen. In some embodiments, the cancer-testis antigen is acrosin-binding protein (ACRBP), alpha-fetoprotein (AFP), A-kinase anchoring protein 4 (AKAP4),ATPase family AAA domain containing 2 (ATAD2), kinetochore scaffold 1 (KNL1; also known as CASC5), centrosomal protein 55 (CEP55), cancer / testis antigen 1A (CTAG1A; also known as ESO1; CT6.1; LAGE-2; LAGE2A; NY-ESO-1), cancer / testis antigen 1B (CTAG1B; also known as CT6.1, CTAG, CTAG1, ESO1, LAGE-2, LAGE2B, NY-ESO-1), cancer / testis antigen 2 (CTAG2; also known as CAMEL) , CT2, CT6.2, CT6.2a, CT6.2b, ESO2, LAGE-1, LAGE2B), CCCTC-binding factor-like (CTCFL), catenin alpha 2 (CTNNA2), cancer / testis antigen 83 (CT83), cyclin A1 (CCNA1), DEAD-box helicase 43 (DDX43), developmental pluripotency associated 2 (DPPA2), fetal and adult testis expressed 1 (FATE1), FMR1 adjacent (FMR1NB), HORMA domain containing 1 (HORMAD1), insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3), leucine zipper protein 4 (LUZP4), lymphocyte antigen 6 family member K (LY6K), maelstrom spermatogenesis transposon silencer (MAEL), MAGE family member A1 (MAGEA1); MAGE family member A3 (MAGEA3); MAGE family member A4 (MAGEA4); MAGE family member A11 (MAGEA11); MAGE family member C1 (MAGEC1); MAGE family member C2 (MAGEC2); MAGE family member D1 (MAGED1); MAGE family member Bar D2 (MAGED2), kinesin family member 20B (KIF20B; also known as MPHOSPH1), NUF2 component of the NDC80 kinetochore complex (NUF2), nuclear export factor 2 (NXF2), PAS domain-containing repressor 1 (PASD1), PDZ-binding kinase (PBK), piwi-like RNA-mediated gene silencing 2 (PIWIL-2), melanoma preferentially expressed antigen (PRAME), sperm-associated antigen 9 (SPAG9), nuclear X-linked family member A1-related sperm protein (SPANXA1), SPANX family member A2 (SPANXA2), SPANX family member C (SPANXC),The E3 ligase ligand conjugate is selected from the group consisting of SPANX family member D (SPANXD), SSX family member 1 (SSX1), SSX family member 2 (SSX2), synaptonemal structural protein 3 (SYCP3), testis-expressed 14 intercellular bridge forming factor (TEX14), transcription factor Dp family member 3 (TFDP3), serine protease 50 (PRSS50, also known as TSP50), TTK protein kinase (TTK), and zinc finger protein 165 (ZNF165). In some embodiments, the kit comprises one or more unit doses of a targeted E3 ligase-ligand conjugate. In some embodiments, the kit includes one or more unit doses of a protein tyrosine phosphatase, non-receptor type 11 (PTPN11 or SHP2), myeloid cell leukemia sequence 1 (MCL1) apoptosis regulator, 5'-nucleotidase ecto (NT5E or CD73), ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1 or CD39), transforming growth factor beta 1 (TGFB1 or TGFβ), heme oxygenase 1 (HMOX1, HO-1 or HO1), heme oxygenase 2 (HMOX2, HO-2 or HO2), vascular endothelial growth factor A (VEGFA or VEGF), erb-b2 receptor tyrosine kinase 2 (ERBB2, HER2, HER2 / neu, or CD340), epidermal growth factor receptor (EGFR, ERBB, ERBB1, or HER1), ALK receptor tyrosine kinase Poly(ADP-ribose) polymerase (ALK, CD246), poly(ADP-ribose) polymerase 1 (PARP1), poly(ADP-ribose) polymerase 2 (PARP2), TCDD-inducible poly(ADP-ribose) polymerase (TIPARP, PARP7), cyclin-dependent kinase 4 (CDK4), cyclin-dependent kinase 6 (CDK6), TNF receptor superfamily member 14 (TNFRSF14, HVEM, CD270), CC-motif chemokine receptor 2 (CCR2, CD192), CC-motif chemokine receptor 5 (CCR5, CD195), CC-motif chemokine receptor 8 (CCR8, CD198), CC-motif chemokine receptor 2 (CXCR2, CD182), CC-motif chemokine receptor 3 (CXCR3, CD182, CD183), CC-motif chemokine receptor 4 (CXCR4,CD184), arginase (ARG1, ARG2), carbonic anhydrase (CA1, CA2, CA3, CA4, CA5A, CA5B, CA6, CA7, CA8, CA9, CA10, CA11, CA12, CA13, CA14), prostaglandin-endoperoxide synthase 1 (PTGS1, COX-1), prostaglandin-endoperoxide synthase 2 (PTGS2, COX-2), secretory phospholipase A2, prostaglandin E synthase (PTGES, PGES), arachidonate-5-lipoxygenase (ALOX5) , 5-LOX), soluble epoxide hydrolase 2 (EPHX2), indoleamine 2,3-dioxygenase 1 (IDO1), indoleamine 2,3-dioxygenase 2 (IDO2), hypoxia-inducible factor 1 subunit alpha (HIF1A), angiopoietin 1 (ANGPT1), endothelial TEK tyrosine kinase (TIE-2, TEK), Janus kinase 1 (JAK1), catenin beta 1 (CTNNB1), histone deacetylase 9 (HDAC9), 5'-3' exoribonuclease 1 (XRN1), and / or WRN RecQ-like helicase (WRN). In some embodiments, the inhibitor of 5'-nucleotidase ecto (NT5E or CD73) is selected from the group consisting of MEDI9447 (oleclumab), CPI-006, BMS-986179, IPH5301, TJ4309 (TJD5), NZV-930, AB-680, PSB-12379, PSB-12441, PSB-12425, CB-708, and PBF-1662. In some embodiments, the inhibitor of CCR2 and / or CCR5 is selected from the group consisting of BMS-813160, PF-04136309, and CCX-872. In some embodiments, the inhibitor of MCL1 is selected from the group consisting of GS-9716, tapotoclax (AMG-176), AMG-397, S-64315, AZD-5991, 483-LM, A 1210477, UMI-77, JKY-5-037, and PRT-1419. In some embodiments, the inhibitor of PTPN11 or SHP2 is selected from the group consisting of TNO155 (SHP-099), RMC-4550, JAB-3068,and RMC-4630. In some embodiments, the inhibitor of Janus kinase 1 (JAK1) is selected from the group consisting of filgotinib, tofacitinib, baricitinib, and ABT-494.

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, exemplary methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present application, including definitions, will control. The materials, methods, and examples are illustrative only and are not intended to be limiting.

[0026] Other features and advantages of the invention will become apparent from the following detailed description and claims. [Brief explanation of the drawings]

[0027] [Figure 1-1] Shown are (A) the IL-2 receptor quaternary complex, (B) the IL-2 interface with key residues selected for mutation, and (C-L) graphs depicting the interactions of these key residues with surrounding residues from IL-2Rα and IL-2. [Figure 1-2] Shown are (A) the IL-2 receptor quaternary complex, (B) the IL-2 interface with key residues selected for mutation, and (C-L) graphs depicting the interactions of these key residues with surrounding residues from IL-2Rα and IL-2. [Figure 1-3] Shown are (A) the IL-2 receptor quaternary complex, (B) the IL-2 interface with key residues selected for mutation, and (C-L) graphs depicting the interactions of these key residues with surrounding residues from IL-2Rα and IL-2. [Figure 1-4]Shown are (A) the IL-2 receptor quaternary complex, (B) the IL-2 interface with key residues selected for mutation, and (C-L) graphs depicting the interactions of these key residues with surrounding residues from IL-2Rα and IL-2. [Figure 1-5] Shown are (A) the IL-2 receptor quaternary complex, (B) the IL-2 interface with key residues selected for mutation, and (C-L) graphs depicting the interactions of these key residues with surrounding residues from IL-2Rα and IL-2. [Figure 1-6] Shown are (A) the IL-2 receptor quaternary complex, (B) the IL-2 interface with key residues selected for mutation, and (C-L) graphs depicting the interactions of these key residues with surrounding residues from IL-2Rα and IL-2. [Figure 1-7] Shown are (A) the IL-2 receptor quaternary complex, (B) the IL-2 interface with key residues selected for mutation, and (C-L) graphs depicting the interactions of these key residues with surrounding residues from IL-2Rα and IL-2. [Figure 1-8] Shown are (A) the IL-2 receptor quaternary complex, (B) the IL-2 interface with key residues selected for mutation, and (C-L) graphs depicting the interactions of these key residues with surrounding residues from IL-2Rα and IL-2. [Figure 1-9] Shown are (A) the IL-2 receptor quaternary complex, (B) the IL-2 interface with key residues selected for mutation, and (C-L) graphs depicting the interactions of these key residues with surrounding residues from IL-2Rα and IL-2. [Figure 1-10] Shown are (A) the IL-2 receptor quaternary complex, (B) the IL-2 interface with key residues selected for mutation, and (C-L) graphs depicting the interactions of these key residues with surrounding residues from IL-2Rα and IL-2. [Figure 1-11] Shown are (A) the IL-2 receptor quaternary complex, (B) the IL-2 interface with key residues selected for mutation, and (C-L) graphs depicting the interactions of these key residues with surrounding residues from IL-2Rα and IL-2. [Figure 1-12] Shown are (A) the IL-2 receptor quaternary complex, (B) the IL-2 interface with key residues selected for mutation, and (C-L) graphs depicting the interactions of these key residues with surrounding residues from IL-2Rα and IL-2.

[0028] [Figure 2]

[0023] Figure 1 shows a ribbon diagram of the engineered heterodimeric Fc-IL-2v fusion proteins described herein. Chain 1 is a variant IgG4 Fc subunit fused to IL-2v via a flexible polypeptide linker. Chain 2 is a complementary variant IgG4 Fc subunit that preferentially heterodimerizes with chain 1.

[0029] [Figure 3] 1 shows the in vitro potency of Fc-IL-2 variant fusion proteins with single substitutions in IL-2 at the IL-2Rα binding interface of CTLL-2 cells.

[0030] [Figure 4-1] Figure 1 shows the Biacore binding responses obtained after injecting different concentrations of Fc-IL-2 variant fusion proteins over the IL-2Rα surface. Binding was tested at concentrations up to 20.0 μM in a two-fold concentration series for all samples except for 104.46 (F), 111.46 (M), and 113.46 (O), which were tested up to 12.3 μM, 14.8 μM, and 16.1 μM, respectively. [Figure 4-2] Figure 1 shows the Biacore binding responses obtained after injecting different concentrations of Fc-IL-2 variant fusion proteins over the IL-2Rα surface. Binding was tested at concentrations up to 20.0 μM in a two-fold concentration series for all samples except for 104.46 (F), 111.46 (M), and 113.46 (O), which were tested up to 12.3 μM, 14.8 μM, and 16.1 μM, respectively. [Figure 4-3]Figure 1 shows the Biacore binding responses obtained after injecting different concentrations of Fc-IL-2 variant fusion proteins over the IL-2Rα surface. Binding was tested at concentrations up to 20.0 μM in a two-fold concentration series for all samples except for 104.46 (F), 111.46 (M), and 113.46 (O), which were tested up to 12.3 μM, 14.8 μM, and 16.1 μM, respectively. [Figure 4-4] Figure 1 shows the Biacore binding responses obtained after injecting different concentrations of Fc-IL-2 variant fusion proteins over the IL-2Rα surface. Binding was tested at concentrations up to 20.0 μM in a two-fold concentration series for all samples except for 104.46 (F), 111.46 (M), and 113.46 (O), which were tested up to 12.3 μM, 14.8 μM, and 16.1 μM, respectively. [Figure 4-5] Figure 1 shows the Biacore binding responses obtained after injecting different concentrations of Fc-IL-2 variant fusion proteins over the IL-2Rα surface. Binding was tested at concentrations up to 20.0 μM in a two-fold concentration series for all samples except for 104.46 (F), 111.46 (M), and 113.46 (O), which were tested up to 12.3 μM, 14.8 μM, and 16.1 μM, respectively. [Figure 4-6] Figure 1 shows the Biacore binding responses obtained after injecting different concentrations of Fc-IL-2 variant fusion proteins over the IL-2Rα surface. Binding was tested at concentrations up to 20.0 μM in a two-fold concentration series for all samples except for 104.46 (F), 111.46 (M), and 113.46 (O), which were tested up to 12.3 μM, 14.8 μM, and 16.1 μM, respectively. [Figure 4-7] Figure 1 shows the Biacore binding responses obtained after injecting different concentrations of Fc-IL-2 variant fusion proteins over the IL-2Rα surface. Binding was tested at concentrations up to 20.0 μM in a two-fold concentration series for all samples except for 104.46 (F), 111.46 (M), and 113.46 (O), which were tested up to 12.3 μM, 14.8 μM, and 16.1 μM, respectively. [Figure 4-8]Figure 1 shows the Biacore binding responses obtained after injecting different concentrations of Fc-IL-2 variant fusion proteins over the IL-2Rα surface. Binding was tested at concentrations up to 20.0 μM in a two-fold concentration series for all samples except for 104.46 (F), 111.46 (M), and 113.46 (O), which were tested up to 12.3 μM, 14.8 μM, and 16.1 μM, respectively. [Figure 4-9] Figure 1 shows the Biacore binding responses obtained after injecting different concentrations of Fc-IL-2 variant fusion proteins over the IL-2Rα surface. Binding was tested at concentrations up to 20.0 μM in a two-fold concentration series for all samples except for 104.46 (F), 111.46 (M), and 113.46 (O), which were tested up to 12.3 μM, 14.8 μM, and 16.1 μM, respectively. [Figure 4-10] Figure 1 shows the Biacore binding responses obtained after injecting different concentrations of Fc-IL-2 variant fusion proteins over the IL-2Rα surface. Binding was tested at concentrations up to 20.0 μM in a two-fold concentration series for all samples except for 104.46 (F), 111.46 (M), and 113.46 (O), which were tested up to 12.3 μM, 14.8 μM, and 16.1 μM, respectively. [Figure 4-11] Figure 1 shows the Biacore binding responses obtained after injecting different concentrations of Fc-IL-2 variant fusion proteins over the IL-2Rα surface. Binding was tested at concentrations up to 20.0 μM in a two-fold concentration series for all samples except for 104.46 (F), 111.46 (M), and 113.46 (O), which were tested up to 12.3 μM, 14.8 μM, and 16.1 μM, respectively. [Figure 4-12] Figure 1 shows the Biacore binding responses obtained after injecting different concentrations of Fc-IL-2 variant fusion proteins over the IL-2Rα surface. Binding was tested at concentrations up to 20.0 μM in a two-fold concentration series for all samples except for 104.46 (F), 111.46 (M), and 113.46 (O), which were tested up to 12.3 μM, 14.8 μM, and 16.1 μM, respectively. [Figure 4-13]Figure 1 shows the Biacore binding responses obtained after injecting different concentrations of Fc-IL-2 variant fusion proteins over the IL-2Rα surface. Binding was tested at concentrations up to 20.0 μM in a two-fold concentration series for all samples except for 104.46 (F), 111.46 (M), and 113.46 (O), which were tested up to 12.3 μM, 14.8 μM, and 16.1 μM, respectively. [Figure 4-14] Figure 1 shows the Biacore binding responses obtained after injecting different concentrations of Fc-IL-2 variant fusion proteins over the IL-2Rα surface. Binding was tested at concentrations up to 20.0 μM in a two-fold concentration series for all samples except for 104.46 (F), 111.46 (M), and 113.46 (O), which were tested up to 12.3 μM, 14.8 μM, and 16.1 μM, respectively. [Figure 4-15] Figure 1 shows the Biacore binding responses obtained after injecting different concentrations of Fc-IL-2 variant fusion proteins over the IL-2Rα surface. Binding was tested at concentrations up to 20.0 μM in a two-fold concentration series for all samples except for 104.46 (F), 111.46 (M), and 113.46 (O), which were tested up to 12.3 μM, 14.8 μM, and 16.1 μM, respectively. [Figure 4-16] Figure 1 shows the Biacore binding responses obtained after injecting different concentrations of Fc-IL-2 variant fusion proteins over the IL-2Rα surface. Binding was tested at concentrations up to 20.0 μM in a two-fold concentration series for all samples except for 104.46 (F), 111.46 (M), and 113.46 (O), which were tested up to 12.3 μM, 14.8 μM, and 16.1 μM, respectively. [Figure 4-17] Figure 1 shows the Biacore binding responses obtained after injecting different concentrations of Fc-IL-2 variant fusion proteins over the IL-2Rα surface. Binding was tested at concentrations up to 20.0 μM in a two-fold concentration series for all samples except for 104.46 (F), 111.46 (M), and 113.46 (O), which were tested up to 12.3 μM, 14.8 μM, and 16.1 μM, respectively. [Figure 4-18]Figure 1 shows the Biacore binding responses obtained after injecting different concentrations of Fc-IL-2 variant fusion proteins over the IL-2Rα surface. Binding was tested at concentrations up to 20.0 μM in a two-fold concentration series for all samples except for 104.46 (F), 111.46 (M), and 113.46 (O), which were tested up to 12.3 μM, 14.8 μM, and 16.1 μM, respectively.

[0031] [Figure 5] 1 shows the in vitro potency of Fc-IL-2 variant fusion proteins with two or three substitutions in IL-2 at the IL-2Rα binding interface of CTLL-2 cells.

[0032] [Figure 6-1] Figure 1 shows the in vitro potency of Fc-IL-2 variant fusion proteins with two or three amino acid substitutions in IL-2 at the IL-2Rα binding interface on STAT5 activation of CD8+ T cells (A) and Treg (B) cells. [Figure 6-2] Figure 1 shows the in vitro potency of Fc-IL-2 variant fusion proteins with two or three amino acid substitutions in IL-2 at the IL-2Rα binding interface on STAT5 activation of CD8+ T cells (A) and Treg (B) cells.

[0033] [Figure 7] 1 shows the in vitro potency of Fc-IL-2 variant fusion proteins with two or three substitutions in IL-2 at the IL-2Rα binding interface on KHYG-1 cells.

[0034] [Figure 8-1] 1 shows the in vitro potency of Fc-IL-2 variant fusion proteins with two or three amino acid substitutions in IL-2 at the IL-2Rα binding interface on CD8+ T cell (A) and NK cell (B) proliferation. [Figure 8-2]1 shows the in vitro potency of Fc-IL-2 variant fusion proteins with two or three amino acid substitutions in IL-2 at the IL-2Rα binding interface on CD8+ T cell (A) and NK cell (B) proliferation.

[0035] [Figure 9-1] Figure 1 shows the in vitro potency of Fc-IL-2 variant fusion proteins on STAT5 activation in cynomolgus monkey CD8+ T cells (A) and Treg cells (B). [Figure 9-2] Figure 1 shows the in vitro potency of Fc-IL-2 variant fusion proteins on STAT5 activation in cynomolgus monkey CD8+ T cells (A) and Treg cells (B).

[0036] [Figure 10] 1 shows the in vitro potency of murine surrogate Fc-IL-2 variant fusion proteins on CTLL-2 cells.

[0037] [Figure 11] 1 shows the in vitro efficacy of mouse surrogate Fc-IL-2 variant fusion proteins on Ba / F3 cells.

[0038] [Figure 12] 1 shows single-dose pharmacokinetic (PK) curves for Fc-IL-2v heterodimers 107.46 (circles), 108.46 (x), 113.46 (triangles), and 114.46 (squares) in cynomolgus monkeys.

[0039] [Figure 13-1] 1 shows the multiple dose PK values ​​of Fc-IL-2v heterodimers 107.46 (A) and 114.46 (B) after multiple subcutaneous administration to cynomolgus monkeys. [Figure 13-2] 1 shows the multiple dose PK values ​​of Fc-IL-2v heterodimers 107.46 (A) and 114.46 (B) after multiple subcutaneous administration to cynomolgus monkeys.

[0040] [Figure 14-1] 1 shows the effect of repeated subcutaneous doses of Fc-IL-2v heterodimers 107.46 and 114.46 on the number of cynomolgus monkey CD8+ T cells (A) and Treg cells (B). [Figure 14-2] 1 shows the effect of repeated subcutaneous doses of Fc-IL-2v heterodimers 107.46 and 114.46 on the number of cynomolgus monkey CD8+ T cells (A) and Treg cells (B).

[0041] [Figure 15-1] The in vitro effects of Fc-IL-2v heterodimer 114.46 on HBV-specific IFN-γ+ CD8+ T cells (A) and Ki67+ CD8+ T cells (B) are shown. [Figure 15-2] The in vitro effects of Fc-IL-2v heterodimer 114.46 on HBV-specific IFN-γ+ CD8+ T cells (A) and Ki67+ CD8+ T cells (B) are shown.

[0042] [Figure 16] Figure 1 shows survival of B16-F10 tumor-bearing mice treated with the murine surrogate Fc-IL-2v heterodimer 168.250.

[0043] [Figure 17] Individual B16-F10 tumor volumes in mice treated with the murine surrogate Fc-IL-2v heterodimer 168.250 are shown.

[0044] [Figure 18] 1 shows the mean CT26 tumor volume in mice treated with murine surrogate Fc-IL-2v heterodimer 171.250 alone and in combination with an anti-PD-1 antibody.

[0045] [Figure 19] 1 shows survival of CT26 tumor-bearing mice treated with murine surrogate Fc-IL-2v heterodimer 171.250 alone and in combination with an anti-PD-1 antibody.

[0046] [Figure 20-1] Figure 1 shows the in vivo antiviral effect of murine surrogate Fc-IL-2v heterodimer 171.250 alone and in combination with αPD-L1 in LCMV-infected mice. Liver LCMV titers (A). Liver memory CD8+ T cells in LCMV mice (B). Serum LCMV titers (C). [Figure 20-2] Figure 1 shows the in vivo antiviral effect of murine surrogate Fc-IL-2v heterodimer 171.250 alone and in combination with αPD-L1 in LCMV-infected mice. Liver LCMV titers (A). Liver memory CD8+ T cells in LCMV mice (B). Serum LCMV titers (C). [Figure 20-3] Figure 1 shows the in vivo antiviral effect of murine surrogate Fc-IL-2v heterodimer 171.250 alone and in combination with αPD-L1 in LCMV-infected mice. Liver LCMV titers (A). Liver memory CD8+ T cells in LCMV mice (B). Serum LCMV titers (C).

[0047] [Figure 21-1] HBsAg (A), HBeAg (B), and HBV DNA (C) levels after treatment of AAV-HBV-infected mice with murine Fc-IL-2v heterodimer 167.250 and anti-PD-L1 antibody. [Figure 21-2] HBsAg (A), HBeAg (B), and HBV DNA (C) levels after treatment of AAV-HBV-infected mice with murine Fc-IL-2v heterodimer 167.250 and anti-PD-L1 antibody. [Figure 21-3] HBsAg (A), HBeAg (B), and HBV DNA (C) levels after treatment of AAV-HBV-infected mice with murine Fc-IL-2v heterodimer 167.250 and anti-PD-L1 antibody.

[0048] [Figure 22]Figure 1 shows the numbers of HBcAg-specific and HBsAg-specific IFN-γ+HBV-specific T cells in AAV-HBV-infected mice after treatment with murine Fc-IL-2v heterodimer 167.250 and anti-PD-L1 antibody. DETAILED DESCRIPTION OF THE INVENTION

[0049] 1. Introduction Provided are fusion proteins comprising a serum half-life extending polypeptide operably linked to a variant, mutant, or non-naturally occurring IL-2 (IL-2v). In various embodiments, the IL-2v of the fusion protein is truncated by at least five amino acids at its N-terminus compared to wild-type IL-2 (i.e., does not contain amino acid residues corresponding to amino acid residues 1-5 of mature wild-type IL-2 (wt IL-2)) and binds to the interleukin-2 receptor alpha subunit (IL-2RA; CD25) with reduced binding affinity compared to wt IL-2. The IL-2 variant fusion proteins described herein possess several structural features that significantly reduce dosing frequency while enhancing their safety and therapeutic efficacy. Additionally, these features contribute to improved manufacturability through high-level production of soluble product using expression and purification platforms typically used for monoclonal antibody production. For example, the Fc-IL-2v fusion protein described herein was engineered to have negligible affinity for IL-2Rα, which is highly expressed on Treg cells, by introducing point mutations into IL-2 at the IL-2 / IL-2Rα interface. Additionally, the use of a heterodimeric Fc allowed for the fusion of a single copy of IL-2v to the dimeric Fc. This fusion design mimics the monovalent nature of native IL-2 and avoids the potential for avidity-driven binding to the IL-2 receptor. One feature that enhances manufacturability is the introduction of a mutation into one subunit of the Fc heterodimer to disrupt Protein A binding and avoid co-purification of the desired heterodimeric product with the corresponding homodimeric contaminant.The use of an IgG4-derived Fc, which naturally lacks the ability to activate complement and has reduced Fc gamma receptor (FcγR) binding compared to IgG1, in combination with additional mutations to further minimize FcγR binding, eliminated the potential for antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), or complement-dependent cytotoxicity (CDC). These Fc modifications The modification may be combined with additional changes in the IL-2v protein to further enhance manufacturability, i.e., substituting an unpaired cysteine ​​residue with serine to prevent undesirable aggregation or modification that may occur at this unpaired cysteine ​​residue. The unpaired cysteine ​​within the Fc domain may be substituted with serine, for example (e.g., at a position corresponding to position 136 in any one of SEQ ID NOS: 45-56 or 141-143). Furthermore, deletion of the first five residues of the mature native IL-2 sequence eliminated a potential O-glycosylation site on threonine (T3) to improve manufacturing control. Deletion of the first five residues of mature native IL-2 eliminates the need for substitution of T3 with a residue that cannot be O-glycosylated (e.g., T3A) to reduce the potential for sequence-dependent immunogenicity. The serum half-life-extended IL-2v molecules described herein provide safer, more effective, and less frequently administered IL-2-based therapeutics that can be applied to the treatment of a wider variety of diseases than is currently the case.

[0050] Among other things, the present disclosure identifies the source of the problem of determining the minimum combination of amino acid substitutions in wild-type (wt) IL-2 that can abolish binding to IL-2Rα sufficiently to minimize stimulation of effector CD4+ and CD8+ T cells and immunosuppressive Treg cells preferentially over NK cells. Various literature reports describing IL-2 substitution studies have not provided comprehensive analyses of the contributions of individual amino acid side chains to IL-2α binding, due to the limited set of IL-2 residues evaluated in any single study or, for example, when replacing an amino acid with an amino acid bearing an opposite charge, due to the drastic nature of the substitutions reported in such studies. The present disclosure provides, among other things, particularly useful and effective constructs that include variant IL-2 moieties. The present disclosure provides IL-2v constructs that combine the minimum number of substitutions capable of eliciting the desired biology (i.e., limiting substitutions to either alanine or glycine while minimizing stimulation of immunosuppressive Treg cells preferentially over effector immune cells). For example, the present disclosure provides heterodimeric Fc-IL-2v fusion proteins comprising the amino acid substitutions R38G, F42A, and E62A in IL-2. In some embodiments, the provided heterodimeric Fc-IL-2v fusion proteins may further comprise (i) an amino acid substitution of C125S and / or (ii) a deletion of the first five residues of the mature, native IL-2 sequence. While not wishing to be bound by any particular theory, the present disclosure notes that such a C125S substitution may be particularly useful and / or effective in reducing aggregation. Similarly, while not wishing to be bound by any particular theory, the present disclosure notes that the deletion of the first five residues removes a potential O-glycosylation site at threonine (T3), which, in some embodiments, may be useful and / or effective in improving manufacturing controls and reducing the risk of sequence-dependent immunogenicity.

[0051] In some embodiments, the heterodimeric Fc-IL-2v fusion proteins described herein comprising R38G, F42A, and E62A are characterized by one, two, three, four, five or more (e.g., all) of the following: (i) very weak or essentially undetectable binding to IL-2Rα compared to Fc-IL-2 fusion proteins with a native IL-2Rα binding interface, while retaining binding to IL-2Rβγ (see Example 8); (ii) a binding affinity to IL-2Rα compared to Fc-IL-2 fusion proteins with a native IL-2Rα binding interface; (iii) lower potency in STAT5 activation of CTLL-2 cells expressing IL-2Rαβγ compared to Fc-IL-2 proteins or Fc-IL-2v heterodimers with R38G and E62A or F42A and E62A substitutions (see Example 9); (iv) lower potency of STAT5 activation in human Treg cells compared to activation by Fc-IL-2 fusion proteins with the native IL-2Rα binding interface (see Example 10); (v) lower potency of STAT5 activation in human Treg cells compared to activation by Fc-IL-2 fusion proteins with the native IL-2Rα binding interface (see Example 11); (v) similar proliferation of human CD8+ T cells and NK cells with an Fc-IL-2 fusion protein having the native IL-2Rα binding interface (see Example 12); (vi) less potent activation of STAT5 in non-human primate Treg cells with an Fc-IL-2 fusion protein having the native IL-2Rα binding interface (see Example 13); (vii) a greater proliferation of human CD8+ T cells and NK cells with an Fc-IL-2 fusion protein having the native IL-2Rα binding interface (see Example 14); (viii) increased in vivo drug exposure in non-human primates compared to an equivalent dose of an Fc-IL-2v heterodimer with F42A and E62A substitutions (see Examples 17 and 18); or (ix) lower in vivo proliferation of Treg cells compared to an Fc-IL-2v heterodimer with F42A and E62A substitutions (Example 19).

[0052] IL-2 variants have been the subject of significant research for over 20 years, with much effort focused on developing variants with reduced affinity for IL-2Rα (e.g., compared to wild-type IL-2 as set forth in SEQ ID NO: 216) as part of the trimeric IL-2Rαβγ. Positions of particular focus include R38, F42, K43, Y45, E61, and E62, which were determined in studies of analogs utilizing binding assays for IL-2Rα (see, e.g., Ju, et al., 1990, The Biology and Clinical Applications of Interleukin-2, ed. Rees, RC (Oxford Univ. Press, Oxford), pp. 7-14). Various reports have described substitutions at one or more of these positions and evaluated their effect on IL-2Rα binding; however, these studies often utilized drastic substitutions to the native side chain, e.g., substituting a native amino acid with a charged side chain for one with a side chain carrying the opposite charge, or, e.g., substituting a native amino acid with an uncharged side chain for one with a charged side chain (see Sauve et al., Proc. Natl. Acad. Sci. USA, 88:4636, 1991; Heaton, et al., Cancer Res. 53(11):2597-602, 1993; Wang, et al. Eur J Immunol. 25(5):1212-6, 1995; Vazquez-Lombardi, et al., Nat Commun. 12;8:15373, 2017). Such drastic changes may seem favorable in terms of disrupting binding, but may increase the risk of introducing sequence-dependent immunogenicity.IL-2 variant molecules can be immunogenic in the clinic (see Satyanarayana, C&EN Magazine, "IL-2 treatment can be dangerous. Here's how drug firms are trying to fix it," Apr. 4, 2021, 99(12) (cen.acs.org / pharmaceuticals / biologics / safer-IL2-cancer-immunotherapy-autoimmunity / 99 / i12)). Therefore, it would be advantageous to reduce the risk of immunogenicity by incorporating fewer amino acid substitutions into the IL-2 molecule. In addition, no studies have systematically compared the effects of a large panel of single-point substitutions and / or their combinations, making it difficult to design minimally substituted IL-2 variants that achieve the dual goal of limiting the stimulation of immunosuppressive Treg cells preferentially over effector CD4+ and CD8+ T cells and NK cells, while simultaneously limiting the total number and nature of substitutions to reduce the likelihood that the variant will be immunogenic in humans.

[0053] In particular, the present disclosure surprisingly demonstrates that multiple substitutions in IL-2, such as a G at position 38, an A at position F42, and an A at position E62, are particularly useful and / or effective. Indeed, results from previous studies evaluating multiple substitutions in IL-2 variants have shown that at least four substitutions are required at positions and / or substitutions different from a G at position 38, an A at position F42, or an A at position E62. Indeed, studies of multiple substitutions in IL-2 variants have shown that at least four substitutions are required to maintain CD8+ T cell and NK cell proliferation similar to wt IL-2, while significantly reducing Treg proliferation and IL-2Rα binding compared to wt IL-2.

[0054] For example, IL-2 variants were evaluated by combining substitutions containing A at positions 38, 42, 45, and 62 (Carmenate et al., J Immunol. 190(12)6230-6238, 2013) to determine their ability to stimulate CD8+ T cell and NK cell proliferation, reduce Treg proliferation, and reduce IL-2Rα binding. The results showed that IL-2 variants containing R38A, F42A, Y45A, and E62A significantly reduced Treg proliferation and IL-2Rα binding compared to wild-type IL-2 while stimulating CD8+ T cell and NK cell proliferation similarly to wild-type IL-2. Therefore, at least four substitutions, including an A at position 45, were clearly required to significantly reduce Treg proliferation and IL-2Rα binding while stimulating CD8+ T cell and NK cell proliferation. In contrast, the present disclosure provides an IL-2 variant that contains a G at position 38, an A at position 42, and an A at position E62, but does not contain an A substitution at position 45.

[0055] In another example, the IL-2 variant described above containing a combination of As at positions 38, 42, 45, and 62 (Carmenate, et al., 2013, supra) was further evaluated to assess the contribution of each substitution to impaired IL-2Rα binding (Rojas, et al., J Mol Recognit. 28(4):261-8, 2015). Results of the binding assay showed that single IL-2 substitutions R38A and Y45A still bound to IL-2Rα (34% and 4% of wtIL-2, respectively), while single IL-2 substitutions F42A and E62A resulted in negligible IL-2Rα binding (each less than 1% of wtIL-2). The results of the proliferation assay showed that the single IL-2 substitutions, R38A and Y45A, still caused proliferation in IL-2Rα-expressing CTLL-2 cells (48.2% and 7.1% of wild-type, respectively), whereas the single IL-2 substitutions, F42A and E62A, caused negligible proliferation in CTLL-2 cells (2.0% and 1.3%, respectively), and the combination of all four IL-2 substitutions (R38A, F42A, Y45A, and E62A) caused undetectable CTLL-2 cell proliferation. These results indicate that (i) at least four mutations are required to sufficiently reduce IL-2Rα binding for undetectable proliferation of IL-2Rα-expressing CTLL-2 cells, and (ii) among these four mutations, R38A is the least conducive to IL-2Rα binding and the highest proliferation in CTLL-2 cells. Unlike the work of Carmenate et al., the present disclosure demonstrated that R38G was a more effective single mutation at reducing IL-2Rα binding than R38A, and thus an IL-2 variant with only three substitutions, including R38G in combination with F42A and E62A, was sufficient to induce the desired biology on primary human immune cells by attenuating STAT5 activation in CTLL-2 cells by over 30,000-fold compared to a comparable molecule with the native IL-2Rα binding interface, and by minimizing stimulation of immunosuppressive Treg cells preferentially over effector T cells and NK cells.

[0056] Additionally, a study by Roche (see, e.g., WO 2012 / 107417) describes the development of IL-2 variants associated with heterodimeric IgG fusion molecules with antigen-binding domains specific for antigens, including, for example, tumor antigens. This study shows, inter alia, that (i) a substitution of G at position L72 in IL-2 is required for the desired loss of IL-2Rα binding and reduced human Treg activation, associated with substitutions F42A and Y45A, (ii) desirable IgG IL-2 fusions have a T3A substitution in IL-2 to eliminate an O-glycosylation site, and (iii) desirable IgG IL-2 fusions have a C125A substitution to avoid intermolecular disulfide bridges. The present disclosure demonstrates that IL-2 variants containing substitutions at positions R38G, F42A, and E62A reduced STAT5 activation of CTLL-2 cells by more than 30,000-fold, while IL-2 variants with substitutions at F42A, Y45A, and L72G reduced STAT5 activation of CTLL-2 cells by only 3,800-fold compared to comparable molecules with the native IL-2Rα binding interface. In addition, as shown in Example 10, IL-2 variants containing either the native IL-2Rα binding interface or the R38G / F42A / E62A or F42A / Y45A / L72G triple substitutions had comparable activity in activating CD8+ T cells. However, while both of these triple-substituted variants significantly reduced activity against Treg cells, the R38G / F42A / E62A-containing variant was superior, exhibiting a more modest difference between activity against Treg versus effector CD8 cells (EC50 values ​​of 3.0 nM and 10.7 nM, respectively) compared to the F42A / Y45A / L72G-containing variant (EC50 values ​​of 1.2 nM and 9.8 nM, respectively). Furthermore, the present disclosure demonstrates that deletion of the first five N-terminal amino acids of IL-2 can be used as a preferred strategy to eliminate O-glycosylation sites, as it avoids any mutagenesis of the native IL-2 sequence for this purpose, which may increase the risk of potential immunogenicity in humans.

[0057] Furthermore, a recent study reported by Cugene (see, e.g., WO 2020 / 252418) described the development of IL-2 variants related to bivalent IL-2 homodimeric Fc fusions, and found, inter alia, that (i) none of the A, F, or G substitutions at position R38 dramatically improved specificity for IL-2Rβγ compared to the equivalently fused IL-2Rαβγ, and (ii) among the substitutions tested, R38A (EC50 of 3.23) was more useful in improving such specificity than any of the others (R38G and R38F). (iii) substitutions of F, H, L, and A at position E62 all improved specificity for IL-2Rβγ compared to the equivalently fused IL-2Rαβγ; (iv) among the substitutions tested, E62F (EC50 of 151) was more useful than any of the others in improving such specificity (EC50s of 2.57, 2.38, and 60.5 for E26H, E62L, and E62A, respectively); and (iii) desirable IL-2-Fc fusions do not have an S residue at position 125. Indeed, the reference IL-2 used in the Cugene study apparently naturally contains S125, and while Cugene recommends substituting away from S (particularly using the S125I substitution), the present disclosure substitutes for S (particularly using the C125S substitution). In contrast to this recent work by Cugene, the present disclosure provides an IL-2 Fc fusion protein variant containing G at position 38, A at position 42, A at position 62, and S at position 125.

[0058] It will be apparent to those skilled in the art that the available teachings prior to this disclosure, whether individually or together, have led to the development of IL-2 variants that differ from those described herein, particularly in relation to Fc fusions. 2. Variant interleukin-2 (IL-2v) protein a. Variant IL-2 (IL-2v) with reduced binding affinity to the IL-2 receptor alpha subunit (IL-2RA)

[0059] In terms of functional attributes, for example, the variant IL-2 (IL-2v) domain of the fusion proteins described herein generally binds to the alpha subunit of the IL-2 receptor (IL-2RA) with reduced affinity, e.g., with a KD of at least 60 μM. The alpha subunit of the IL-2 receptor can be human, non-human primate, or murine. Human IL-2RA (also known as CD25; IDDM10, IL-2R, IMD41, TCGFR, p55) has been assigned NCBI gene ID: 3559. Mouse il2ra (also known as CD25; Il2r; Ly-43) has been assigned NCBI gene ID: 16184. Rhesus IL-2RA has been assigned NCBI gene ID: 574300. Macaca fascicularis (cynomolgus or crab-eating macaque) IL-2RA is assigned NCBI gene ID: 102123605. In some embodiments, IL-2v has an equilibrium dissociation constant (K) of at least 60 μM (e.g., 60 μM or greater). D ) binds to IL-2RA. Further, in some embodiments, for example, a variant IL-2 (IL-2v) domain of a fusion protein described herein, in a complex of interleukin-2 receptor subunit beta (IL-2RB; CD122) and interleukin-2 receptor subunit gamma (IL-2RG; CD132), has a Kd of less than 150 nM, such as less than 1.5 nM, such as less than 120 pM, such as less than 100 pM, such as less than 80 pM, such as less than 75 pM, such as less than 70 pM, in some embodiments, for example, a variant IL-2 (IL-2v) domain of a fusion protein described herein, in a complex of interleukin-2 receptor subunit beta (IL-2RB; CD122) and interleukin-2 receptor subunit gamma (IL-2RG; CD132), under conditions equivalent to that of wild-type IL-2, as determined in a cell line harboring an artificial Fc-fused IL2Rβ / IL2Rγ heterodimer described herein. D Kd within 10-fold, for example, 9-fold, 8-fold, 7-fold, 6-fold, 5-fold, 4-fold, 3-fold, 2-fold, or less DThe complex of interleukin-2 receptor subunit beta (IL-2RB; CD122) and interleukin-2 receptor subunit gamma (IL-2RG; CD132) can be human, non-human primate, or murine. Human IL-2RB (also known as CD122, IL15RB, IMD63, and P70-75) has been assigned NCBI gene ID: 3560, and human IL-2RG (also known as P64; CIDX; IMD4; CD132; SCIDX; IL-2RG; SCIDX1) has been assigned NCBI gene ID: 3561. Mouse il2rb (also known as p70; CD122; IL15R beta; Il-2R beta; IL-15R beta; Il-2 / 15R beta) is assigned NCBI gene ID: 16185, and mouse il2rg (also known as gc; p64; [g]c; CD132; gamma(c)) is assigned NCBI gene ID: 16186. Rhesus IL-2RB is assigned NCBI gene ID: 696331, and rhesus IL-2RG is assigned NCBI gene ID: 641338. Cynomolgus IL-2RB is assigned NCBI gene ID: 102138714, and rhesus IL-2RG is assigned NCBI gene ID: 102144912. Binding affinity can be determined according to any method in the art. One method for determining binding affinity is surface plasmon resonance (SPR).

[0060] In various embodiments, for example, the variant IL-2 (IL-2v) domain of the fusion proteins described herein promotes or induces equal or greater proliferation of CD8+ T cells compared to wt IL-2, the IL-2v of any one of SEQ ID NOs: 43 and 44. Additionally, in some embodiments, for example, the IL-2v of the fusion proteins described herein promotes or induces equal or greater proliferation of CD8+ T cells at the concentration that induces 50% of maximal signal transducer and activator of transcription 5 (STAT5) activation or signaling in regulatory T (Treg) cells (EC 50 ) is the EC value for STAT5 activation or signaling of wt IL-2 or IL-2v of any one of SEQ ID NOs: 43 and 44 50The expression level of T cells is at least 1000-fold, e.g., at least 1500-fold, e.g., at least 1700-fold, e.g., at least 2000-fold, e.g., at least 2500-fold higher than that of T cells in the control group. See, e.g., Gilmour, et al., Proc Natl Acad Sci USA (1995) 92(23):10772-6; Gaffen, Cytokine (2001) 14(2):63-77; Varker, et al., Clin Cancer Res (2006) 12(19):5850-8. "Regulatory T cells" (Treg; Treg cells), also known as "suppressive T cells," are a subpopulation of T cells that are immunosuppressive and generally suppress or downregulate the induction and proliferation of effector T cells. Tregs express the surface biomarkers CD4 and CD25 (IL-2 receptor alpha chain) and the intracellular DNA-binding biomarker FOXP3. Human Foxp3+CD4+ T cells have been divided into three subfractions based on the expression levels of Foxp3 and the cell surface molecules CD25 and CD45RA. The Foxp3hiCD45RA-CD25hi and Foxp3loCD45RA+CD25lo phenotypes correspond to suppressive Treg cells, whereas the Foxp3loCD45RA-CD25lo fraction marks activated T effector (Teff) cells without suppressive activity. In addition, in healthy subjects, Compared with Treg cells from cancer patients, Treg cells from cancer patients are typically characterized by distinct expression profiles of chemokine receptors, such as CCR4, CXCR4, and CCR5, which promote migration to tumors in response to corresponding chemokine ligands derived from the tumor microenvironment (see, e.g., Liu, et al., FEBS J. (2016) 283(14):2731-48 and Miyara, et al., Immunity (2009) 30, 899-911).

[0061] In some embodiments, for example, IL-2v of the fusion proteins described herein inhibits 50% (EC 50) is the EC 200 for STAT5 activation or signaling of wt IL-2 or any one of IL-2v of SEQ ID NOs: 43 and 44. 50 The antibody titer is at least 2500 times, for example, at least 5000 times, for example, at least 7500 times, for example, at least 10,000 times, for example, at least 15,000 times, for example, at least 20,000 times higher than that of the antibody titer. See, for example, Gilmour, et al., Proc Natl Acad Sci USA (1995) 92(23):10772-6; Gaffen, Cytokine (2001) 14(2):63-77; Ortega, et al., J Immunol. (1984) 133(4):1970-5; Gillis, et al., J Immunol. (1978) 120(6):2027-32.

[0062] In some embodiments, for example, IL-2v of the fusion proteins described herein is administered at a concentration that induces 50% of the maximal proliferation of natural killer (NK) cells (EC 50 ) is the EC 20 value for proliferation of wt IL-2 or IL-2v of any one of SEQ ID NOs: 43 and 44, measured using, for example, KHYG-1 cells. 50 The activity is at least 10 times, for example, at least 12 times, for example, at least 15 times, for example, at least 16 times, for example, at least 18 times, for example, at least 20 times higher than that of the control. See, for example, Suck, et al., Exp Hematol (2005) Oct; 33(10): 1160-71; Yagita, et al., Leukemia (2000) 14(5): 922-30; cell line KHYG-1 has DSMZ number: ACC725; ExPASy cynomolgus monkey KHYG-1 (CVCL_2976); CellBank Australia CODE: JCRB0156.

[0063] "Polypeptide variants," as that term is used herein, are polypeptides that typically differ from the polypeptides specifically disclosed herein by one or more substitutions, deletions, additions, and / or insertions. Such variants may be naturally occurring or may be synthetically produced, for example, by modifying one or more of the above polypeptide sequences described herein, by assessing one or more biological activities of the polypeptides described herein, and / or using any of several techniques well known in the art.

[0064] The term "variant" may also refer to any naturally occurring or genetically engineered molecule containing one or more nucleotide or amino acid mutations. In one embodiment, the multispecific antigen-binding molecule is a bispecific antigen-binding molecule. In one embodiment, the multispecific antigen-binding molecule is a bispecific antibody. For example, somatic variants may include all related naturally occurring antibodies that are part of or derived from the same B-cell lineage. Genetically engineered variants may include all single or combinatorial mutations made to the antibody.

[0065] With respect to structural attributes, for example, the IL-2v domain of the fusion proteins described herein generally does not include the first five amino acid residues corresponding to amino acid positions 1-5 of wt IL-2 (e.g., does not include the amino acid APTSS sequence (SEQ ID NO: 163)). As used herein, the numbering of a given amino acid polymer or nucleic acid polymer refers to a sequence in which the position of any given polymer building block (e.g., amino acid, nucleotide, also commonly referred to as "residue") is designated by reference to the same or equivalent position (e.g., based on optimal alignment or consensus sequence) in a selected amino acid or nucleic acid polymer, rather than the actual numerical position of the building block in the given polymer. " In other words, for example, in the IL-2v domain of the IL-2v and IL-2v fusion proteins described herein, the first five amino acids corresponding to wild-type or native mature IL-2 have been truncated. For fusion proteins based on human IL-2, the numbering of the IL-2 positions is done with reference to mature human IL-2 (NCBI gene ID: 3558) shown below, or with reference to SEQ ID NO: 44 (IL-2v with serine at position 125 (C125S)): APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 216).

[0066] In various embodiments, the IL-2v domain is from (e.g., based on or derived from) human wild-type IL-2 or wild-type IL-2 of a non-human primate. For fusion proteins based on rhesus IL-2, the numbering of the IL-2 positions is done with reference to mature rhesus IL-2 (NCBI Gene ID: 708017) shown below: APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRDTKDLISNINVIVLELKGSETTLMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 217).

[0067] For cynomolgus IL-2 based fusion proteins, the numbering of the IL-2 positions is done with reference to mature cynomolgus IL-2 (NCBI Gene ID: 102129830) shown below: APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELRHLQCLEEELKPLEEVLNLAQSKSFHLRDTKDLISNINVIVLELKGSETTLMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 262).

[0068] In some embodiments, IL-2v comprises a serine at position 125 (C125) and at least two or at least three substitutions (e.g., with glycine or alanine) at an amino acid position selected from the group consisting of R38, F42, Y45, E61, E62, and L72. In some embodiments, IL-2v comprises a serine at position 125 (C125) and at least two or at least three, no more than three, no more than two substitutions (e.g., with glycine or alanine) at an amino acid position selected from the group consisting of R38, F42, Y45, E61, and E62. In some embodiments, IL-2v comprises a serine at position 125 (C125) and at least two or at least three, no more than three, no more than two substitutions (e.g., with glycine or alanine) at an amino acid position selected from the group consisting of R38, F42, Y45, and E62. In some embodiments, IL-2v comprises a serine at position 125 (C125) and at least two or at least three, no more than three, no more than two substitutions (e.g., to glycine or alanine) at an amino acid position selected from the group consisting of R38, F42, and E62. In some embodiments, IL-2v comprises a serine at position 125 (C125) and at least two or at least three, no more than three, no more than two substitutions at an amino acid position selected from the group consisting of R38G, F42A, Y45G, E61A, E62A, and L72G. In some embodiments, IL-2v comprises a serine at position 125 (C125) and at least two or at least three, no more than three, no more than two substitutions at an amino acid position selected from the group consisting of R38G, F42A, Y45G, E61A, and E62A. In some embodiments, IL-2v comprises a serine at position 125 (C125) and at least two or at least three, no more than three, no more than two substitutions at an amino acid position selected from the group consisting of R38G, F42A, Y45G, and E62A. In some embodiments, IL-2v comprises a serine at position 125 (C125) and at least two or at least three, no more than three, no more than two substitutions at an amino acid position selected from the group consisting of R38G, F42A, and E62A. The foregoing position numbers refer to the IL-2v of SEQ ID NO:44.In some embodiments, the IL-2v does not comprise an amino acid substitution at one or more or all of positions selected from the group consisting of D20, Y45, E61, E68, V69, L72, A73, L80, R81, L85, L86, 187, 192, and Q126. In some embodiments, the IL-2v does not comprise an amino acid substitution at one or more or all of positions selected from the group consisting of H16, D20, E61, N88, and V91.

[0069] In some embodiments, IL-2v comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-42, or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-42. Exemplary IL-2v sequences based on wild-type human IL-2, including SEQ ID NOs: 1-44, are provided in Table A. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4]

[0070] Modifications can be made in the structures of the IL-2v and IL-2v fusion polynucleotides and polypeptides described herein to also obtain functional molecules that encode variant or derivative polypeptides with desirable characteristics. When it is desired to alter the amino acid sequence of a polypeptide to produce an equivalent or even improved variant or portion of a polypeptide described herein, one of skill in the art will typically change one or more of the codons in the encoding DNA sequence.

[0071] For example, certain amino acids can be substituted for other amino acids in a protein's structure without appreciable loss of its ability to bind to other polypeptides (e.g., antigens) or cells. Because it is the binding ability and properties of a protein that define its biological functional activity, certain amino acid sequence substitutions can be made in the protein sequence, and of course, the underlying DNA coding sequence, and still obtain a protein with similar properties. Thus, it is contemplated that various changes can be made in the polypeptide sequences of the disclosed antibodies and antigen-binding fragments thereof, or the corresponding DNA sequences encoding such polypeptides, without appreciable loss of their biological utility or activity.

[0072] Often, a polypeptide variant contains one or more conservative substitutions, which are substitutions of an amino acid with another amino acid of similar properties that one skilled in the art of peptide chemistry would expect to leave the secondary structure and hydropathic properties of the polypeptide unchanged.

[0073] As used herein, "identity" refers to the percentage of identical nucleotides or amino acid residues at corresponding positions in two or more sequences when the sequences are aligned, i.e., taking into account gaps and insertions to maximize sequence matching. Sequences are generally aligned for maximum correspondence over a designated region, e.g., a region of at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or more amino acids or nucleotides in length, and may extend to the entire length of the reference polypeptide or polynucleotide sequence. For sequence comparison, typically, one sequence serves as a reference sequence, and test sequences are compared to that sequence. When using a sequence comparison algorithm, test and reference sequences are input into a computer program, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Otherwise, standard parameters can be used. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence based on the designated program parameters.

[0074] When comparing polynucleotide and polypeptide sequences, two sequences are said to be "identical" if, when aligned for maximum correspondence as described below, the sequence of nucleotides or amino acids in the two sequences is identical. Comparison between two sequences is typically performed by comparing over a comparison window to identify and compare local regions of sequence similarity. As used herein, a "comparison window" refers to at least 20 contiguous positions, usually 30-75 contiguous positions, 40-50 contiguous positions, or a segment spanning the entire length of a sequence; a sequence within the comparison window can be compared to a reference sequence over the same number of contiguous positions after the two sequences are optimally aligned.

[0075] Optimal alignment of sequences for comparison may be performed using the Megalign program in the Lasergene suite of bioinformatics software (DNASTAR, Inc., Madison, WI) using default parameters. This program implements several alignment schemes described in the following references: Dayhoff, "MO" (1978) A model of evolutionary change in protein-Matrices for detecting distant relationships.In Dayhoff, MO (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington DC Vol. 5, Suppl. 3, pp. 345-358; Hein J. (1990) Unified Approach to Alignment and Phylogenes, pp. 626-645 Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, CA; Higgins, DGand Sharp, PM (1989) CABIOS 5:151-153; Myers, EWand Muller W. (1988) CABIOS 4:11-17; Robinson, ED (1971) Comb. Theor 77:105; Santou, N. Nes, M. (1987) Mol. Biol. Evol. 4:406-425; Sneath, PHA and Sokal, RR (1973) Numerical Taxonomy-the Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco, CA; Wilbur, WJand Lipman, DJ (1983) Proc. Natl. Acad., Sci. USA80:726-730.

[0076] Alternatively, optimal alignment of sequences for comparison may be performed by the local identity algorithm of Smith and Waterman (1981), Add. APL. Math. 2:482, by the identity alignment algorithm of Needleman and Wunsch (1970), J. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman (1988), Proc. Natl. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, BLAST, FASTA, and TFASTA, Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, WI), or by inspection.

[0077] Examples of suitable algorithms for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nucl. Acids Res. 25:3389-3402 and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively. BLAST and BLAST 2.0 can be used, for example, with the parameters described herein to determine percent sequence identity for the polynucleotides and polypeptides described herein. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (blast.ncbi.nlm.nih.gov / Blast.cgi).

[0078] In one illustrative example, the cumulative score is calculated based on a parameter M (reward score for a pair of matching residues) for nucleotide sequences. The word hits can be calculated using a score (X; always > 0) and N (penalty score for mismatched residues; always < 0). Extension of the word hits in each direction is stopped when: the cumulative alignment score falls by an amount X from its maximum achieved value; the cumulative score falls below zero due to the accumulation of alignments of one or more negative-scoring residues; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11 and an expectation (E) of 10, while BLOSUM62 uses as defaults a scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915) alignment, (B) of 50, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

[0079] For amino acid sequences, a scoring matrix can be used to calculate the cumulative score. Extension of the word hits in each direction is halted when: the cumulative alignment score falls off by an amount X from its maximum achieved value; the cumulative score falls below zero due to the accumulation of alignments of one or more negative-scoring residues; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.

[0080] In one approach, "percent sequence identity" is determined by comparing two optimally aligned sequences over a comparison window of at least 20 positions. The portion of the polynucleotide or polypeptide sequence in the comparison window may contain additions or deletions (i.e., gaps) of up to 20 percent, typically 5-15 percent, or 10-12 percent, compared to the reference sequence (which does not contain additions or deletions) for optimal alignment of the two sequences. This percentage is calculated by determining the number of positions where the same nucleic acid base or amino acid residue occurs in both sequences to obtain the number of matched positions, dividing this number by the total number of positions in the reference sequence (i.e., the window size), and multiplying the result by 100 to obtain the percentage sequence identity.

[0081] In some embodiments, IL-2v, serum half-life extended IL-2v, e.g., Fc-IL-2v fusion proteins, and homodimers and heterodimers thereof, do not comprise a signal peptide. In some embodiments, IL-2v, serum half-life extended IL-2v, e.g., Fc-IL-2v fusion proteins, and homodimers and heterodimers thereof comprise an N-terminal signal peptide. The signal peptide can be an endogenous signal peptide (e.g., from a native or wild-type IL-2 protein) or from a heterologous polypeptide. In various embodiments, the signal peptide or leader sequence is from a source protein selected from a serum protein, an immunoglobulin, a cytokine, a chemokine, a chaperone protein, an invariant protein, and a protein that targets proteins to the lysosomal compartment. In various embodiments, the signal peptide or leader sequence is from a protein selected from colony stimulating factor 2 (CSF2, GM-CSF), tissue-type plasma membrane endothelial growth factor (TGF-EGFR), or a fusion protein. activators of phospholipase A (PLAT, t-PA), CC motif chemokine ligand 7 (CCL7, MCP-3), CXC motif chemokine ligand 10 (CXC motif chemokine ligand, CXCL10, IP-10), CD74 (p33; DHLAG; HLA In various embodiments, the signal peptide or leader sequence is from a source protein selected from colony-stimulating factor 2 (CSF2, GM-CSF), immunoglobulin kappa; Ia-GAMMA, invariant chain, and serum albumin (ALB). In some embodiments, the signal peptide is from a serum albumin signal peptide (e.g., comprising the amino acid sequence KWVTFISLLFLFSSAYS (SEQ ID NO:218)). In various embodiments, the signal peptide or leader sequence is selected from the amino acid sequence of any one of SEQ ID NOs:218-231, or a sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs:218-231. Exemplary signal sequences that can be used in the present IL-2v, serum half-life extended IL-2v, e.g., Fc-IL-2v fusion proteins, and homodimers and heterodimers thereof are provided in Table B. [Table 2]

[0082] The signal peptide can be designed to be cleaved after secretion from cells to form a mature fusion protein, for example.A modified human serum albumin signal peptide for secreting proteins in cells that can be used in expressing the present fusion protein is described, for example, in Attallah, et al., Protein Expr Purif. (2017) 132: 27-33.Additional signal peptide sequences for use in expressing the fusion proteins described herein are described, for example, in Kober, et al., Biotechnol Bioeng. (2013) 110(4): 1164-73.

[0083] In certain embodiments, the IL-2v domain comprises or is derived from a mouse or murine IL-2 sequence. Mus musculus IL-2 is identified as NCBI gene ID 16183. For fusion proteins based on mouse IL-2, the numbering of the IL-2 positions refers to mature mouse IL-2 as shown below: APTSSSTSSSTAEAQQQQQQQQQQQHLEQLLMDLQELLSRMENYRNLKLPRMLTFKFYLPKQATELKDLQCLEDELGPLRHVLDLTQSKSFQLEDAENFISNIRVTVVKLKGSDNTFECQFDDESATVVDFLRRWIAFCQSIISTSPQ (SEQ ID NO: 234).

[0084] Exemplary IL-2v domains based on or derived from wild-type mouse IL-2 that have reduced binding to mouse IL-2RA (also known as il2ra, CD25; Il2r; Ly-43; NCBI Gene ID: 16184) are provided in Table D. Generally, the IL-2v domain of the fusion proteins described herein does not include the first 23 amino acid residues of the mature wild-type mouse IL-2 sequence (e.g., does not include amino acids APTSSTSSSSTAEAQQQQQQQQ (SEQ ID NO: 235)). In some embodiments, the mouse IL-2v includes an alanine at position 140 (C140) and at least one, two, or at least three substitutions at amino acid positions selected from the group consisting of R52, F56, Y59, E76, and L86, where the position numbering is with respect to the sequence of mature mouse IL-2 represented by SEQ ID NO: 234. In some embodiments, murine IL-2v comprises an alanine at position 140 (C140) and at least one, two, or at least three substitutions with alanine or glycine at an amino acid position selected from the group consisting of R52, F56, Y59, E76, and L86, where the position numbering is with respect to the sequence of mature murine IL-2 represented by SEQ ID NO: 234. In some embodiments, murine IL-2v comprises an amino acid sequence corresponding to residues 250-375 of an amino acid sequence selected from the group consisting of SEQ ID NOs: 166-171, or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to an amino acid sequence corresponding to residues 250-375 of an amino acid sequence selected from the group consisting of SEQ ID NOs: 166-171.

[0085] As needed or desired, the IL-2v polypeptides described herein may or may not be PEGylated. B. Serum half-life extending polypeptide

[0086] The fusion proteins described herein comprise a variant IL-2 that binds to IL-2RA with reduced binding affinity and a serum half-life extending polypeptide. In some embodiments, the fusion protein comprises, from N- to C-terminus, a serum half-life extending polypeptide (e.g., an Fc region) and IL-2v. In some embodiments, the fusion protein comprises, from N- to C-terminus, IL-2v and a serum half-life extending polypeptide (e.g., an Fc region). For example, polypeptides that can be used to extend the serum half-life of another polypeptide via conjugation or fusion are known in the art and can be used in the present fusion proteins. Exemplary serum half-life extending polypeptides that can be conjugated or fused to IL-2v described herein include, but are not limited to, an immunoglobulin fragment crystallizable region (Fc region), one or more serum albumin moieties, an albumin-binding protein or peptide, an IgG, an XTEN polypeptide, a proline / alanine / serine-rich (PAS) polypeptide, or an elastin-like polypeptide. IL-2v conjugated or fused to one or more serum albumin moieties, albumin-binding proteins or peptides, IgG, XTEN polypeptides, proline / alanine / serine-rich (PAS) polypeptides, elastin-like polypeptides need not dimerize (e.g., form homodimers or heterodimers). One or more serum albumin moieties, albumin-binding proteins or peptides, IgG, XTEN polypeptides, proline / alanine / serine-rich (PAS) polypeptides, elastin-like polypeptides can be conjugated or fused to either or both the N-terminus or C-terminus of IL-2v.Exemplary XTEN protein polymers that can be used in the present IL-2v fusion proteins are described, for example, in Schellenberger, et al., Nat Biotechnol. 2009 Dec;27(12):1186-90; Podust, et al., Journal of Controlled Release 240(2016)52-66; WO 2010 / 091122; WO 2011 / 123813; WO 2013 / 130683; WO 2016 / 077505; and WO 2017 / 197048. Exemplary proline / alanine / serine-rich (PAS) polypeptides that can be used in the present IL-2v fusion proteins are described, for example, in Schlapschy, et al., Protein Eng Des. Sel. (2013) 26(8):489-501 and Breibeck, et al., Biopolymers. (2018) Jan;109(1).doi:10.1002 / bip.23069, WO 2016122806 and WO 2016130451. The foregoing references are incorporated herein by reference in their entirety for all purposes.

[0087] In some embodiments, the serum half-life extending polypeptide is an immunoglobulin fragment crystallizable region (Fc region). Generally, the Fc domain is composed of or derived from the same species as the IL-2 domain (e.g., human, dog, cat, mouse, or monkey). In some embodiments, the Fc region is from human IgG1, IgG2, IgG3, or IgG4. In some embodiments, the Fc region is from human IgG1 or IgG4.

[0088] In some embodiments, the Fc modifications can promote one or more of an increase in the serum half-life of the molecule or a decrease in antibody effector function. In other embodiments, certain of these modifications reduce antibody effector function and increase antibody half-life. In some embodiments, the Fc-IL-2v fusion proteins described herein comprise two or more, three or more, four or more, five or more, six or more, six or fewer, five or fewer, four or fewer, three or fewer, two or fewer, or one modified Fc amino acid residue. Exemplary amino acid substitutions are described below.

[0089] In some embodiments, the Fc domain of the fusion protein does not include the hinge region, which is truncated or deleted in whole or in part. The structural hinge region of human IgG1, IgG2, and IgG4 antibodies is a 19-23 amino acid peptide linker containing two to four cysteine ​​residues, genetically encoded on the hinge exon along with the 5' end of the CH2 exon, and allows disulfide bridges between the first and second Fc domains (Roux, et al., J. Immunol. (1998) 161:4083). The structural hinge region is composed of amino acid residue positions 216-238 (EU numbering) or 226-251 (Kabat numbering) (identified at imgt.org). In some embodiments, the Fc region comprises or is derived from a human IgG4 isotype and does not include the amino acid sequence ESKYGPPCPPCP (SEQ ID NO: 236). In some embodiments, the Fc region comprises or is derived from a human IgG1 isotype and does not comprise the amino acid sequence EPKSCDKTHTCPPCP (SEQ ID NO: 237) or EPKSCDKTHTCPPCPAPELL (SEQ ID NO: 238). Fc mutations that increase serum half-life

[0090] In some embodiments, the Fc region comprises an amino acid modification that promotes an increase in the serum half-life of the fusion protein. Mutations that increase the half-life of antibodies have been described. In one embodiment, the constant region of the Fc-IL-2v fusion protein described herein comprises a methionine to tyrosine substitution at position 252 (EU numbering), a serine to threonine substitution at position 254 (EU numbering), and a threonine to glutamic acid substitution at position 256 (EU numbering). See, e.g., U.S. Patent No. 7,658,921. This type of variant, termed the "YTE variant," exhibits a four-fold increased half-life compared to the wild-type version of the same antibody (Dall'Acqua, et al., J Biol Chem, 281:23514-24 (2006); Robbie, et al., J Biol Chem, 281:23514-24 (2006)). al., Antimicrob Agents Chemotherap., 57(12):6147-6153 (2013)). In certain embodiments, the Fc-IL-2v fusion proteins described herein comprise an IgG constant domain containing one, two, three, or more amino acid substitutions among amino acid residues at positions 251-257, 285-290, 308-314, 385-389, and 428-436 (EU numbering). Alternatively, M428L and N434S ("LS") substitutions can increase the pharmacokinetic half-life of the fusion protein. In other embodiments, the Fc-IL-2v fusion proteins described herein contain M428L and N434S substitutions (EU numbering). In other embodiments, the Fc-IL-2v fusion proteins described herein contain T250Q and M428L (EU numbering) mutations. In other embodiments, the Fc-IL-2v fusion proteins described herein comprise H433K and N434F (EU numbering) mutations. Fc mutations that reduce or eliminate effector activity

[0091] In some embodiments, the Fc-IL-2v fusion proteins described herein can have an Fc domain with amino acid substitutions that reduce or eliminate Fc effector functions (including, for example, antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC)).

[0092] In some embodiments, the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to reduce or eliminate the antibody's effector function. For example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320, and 322 (EU numbering) can be replaced with a different amino acid residue to reduce the affinity of the fusion protein for an effector ligand. The effector ligand for which affinity is altered can be, for example, an Fc receptor (e.g., residue positions 234, 235, 236, 237, 297 (EU numbering)) or a C1 complement component (e.g., residue positions 297, 318, 320, 322 (EU numbering)). U.S. Patent Nos. 5,624,821 and 5,648,260, both by Winter et al.

[0093] Fc modifications that reduce or eliminate effector function include substitutions, insertions, and deletions at one or more positions, including, for example, 234, 235, 236, 237, 267, 269, 325, and 328, e.g., 234G, 235G, 236R, 237K, 267R, 269R, 325L, and 328R (EU numbering). Additionally, Fc variants can include 236R / 328R. Other modifications to reduce FcγR and complement interaction include substitutions at positions 297A, 234A, 235A, 318A, 228P, 236E, 268Q, 309L, 329G, 330S, 331S, 220S, 226S, 229S, 238S, 233P, and 234V (EU numbering). These and other modifications are reviewed in Strohl (2009) Current Opinion in Biotechnology 20:685-691; see also Schlothauer, et al., Protein Eng Des Sel. (2016) 29(10):457-466. Effector function (both ADCC and complement activation) can be reduced while maintaining neonatal FcR binding (while maintaining half-life) by mutating IgG residues at one or more positions 233-236 and 327-331 (e.g., E233P, L234V, L235A, optionally G236A, A327G, A330S, and P331S in IgG1; E233P, F234V, L235A, optionally G236A in IgG4; A330S and P331S (EU numbering) in IgG2). See Armour et al. (1999) Eur. J. Immunol. 29:2613; WO 99 / 58572. Other mutations that reduce effector function include L234A and L235A in IgG1 (Alegre et al. (1994) Transplantation 57:1537); V234A and G237A in IgG2 (Cole (1997) J. Immunol. 159:3613; see also U.S. Patent No. 5,834,597), and S228P and L235E in IgG4 (Reddy et al. (2000) J. Immunol. 164:1925). Another combination of mutations to reduce effector function in human IgG1 includes L234F, L235E, and P331S. Oganesyan et al. (2008) Acta Crystallogr. D. Biol. Crystallogr. 64:700. See generally Labrijn et al. (2008) Curr. Op. Immunol. 20:479. Additional mutations found to reduce effector function in the context of Fc(IgG1) fusion proteins (abatacept) include C226S, C229S, and P238S (EU numbering). See Davis et al. (2007) J. Immunol. 34:2204.

[0094] ADCC activity can be reduced by modifying the Fc region. In certain embodiments, sites that affect binding to Fc receptors, such as sites other than the salvage receptor binding site, may be removed. In other embodiments, the Fc region may be modified to remove the ADCC site. Exemplary ADCC sites have been described for IgG1 (Sarmay, et al. (1992) Molec. Immunol. 29(5):633-9). In one embodiment, the G236R and L328R variants of human IgG1 effectively eliminate FcγR binding (Horton, et al. (2011) J. Immunol. 186:4223 and Chu, et al. (2008) Mol. Immunol. 45:3926). In other embodiments, Fc regions with reduced FcγR binding include amino acid substitutions L234A, L235E, and G237A. Gross, et al. (2001) Immunity 15:289. Modifications in the IgG Fc region identified in WO 88 / 007089 to reduce binding to FcγRI such that ADCC is reduced (e.g., 234A; 235E; 236A; G237A) can be used in the fusion proteins of the invention. See also Duncan & Winter (1988) Nature 332:563; Chappel et al. (1991) Proc. Nat'l Acad. Sci. (USA) 88:9036, and Sondermann et al. (2000) Nature 406:267 (discussion of the effect of these mutations on FcγRII binding).

[0095] CDC activity can also be reduced by modifying the Fc region. Mutations at IgG1 positions D270, K322, P329, and P331, specifically the alanine mutations D270A, K322A, P329A, and P331A, significantly reduce the ability of the corresponding antibodies to bind C1q and activate complement (Idusogie et al. (2000) J. Immunol. 164:4178, WO 99 / 51642). Modifications at position 331 of IgG1 (e.g., P331S) have been shown to reduce complement binding (Tao et al. et al. (1993) J. Exp. Med. 178:661; Xu Y, et al. J. Biol. Chem. 1994. 269:3469-74; and Canfield & Morrison (1991) J. Exp. Med. 173:1483. In another example, one or more amino acid residues within amino acid positions 231-239 are altered to thereby reduce the ability of the antibody to fix complement (WO 94 / 29351). Modifications of the IgG Fc region identified in WO 88 / 007089 that reduce or eliminate binding to complement component C1q and thus reduce or eliminate CDC (e.g., E318A or V / K320A and K322A / Q) can be used in the fusion proteins of the invention.

[0096] In some embodiments, the Fc with reduced complement fixation has the amino acid substitutions A330S and P331S. Gross et al. (2001) Immunity 15:289.

[0097] Other Fc variants with reduced ADCC and / or CDC have been described by Glaesner et al. al. (2010) Diabetes Metab. Res. Rev. 26:287 (F234A and L235A to reduce ADCC and ADCP in IgG4); Hutchins et al. (1995) Proc. Nat'l Acad. Sci. (USA) 92:11980 (F234A, G237A, and E318A in IgG4); An et al. (2009) MAbs 1:572, and US Patent Application Publication No. 2007 / 0148167 (H268Q, V309L, A330S, and P331S in IgG2); McEarchern et al. (2007) Blood 109:1185 (C226S, C229S, E233P, L234V, L235A in IgG1); Vafa et al. al. (2014) Methods 65:114 (V234A, G237A, P238S, H268A, V309L, A330S, P331S in IgG2) (EU numbering).

[0098] In certain embodiments, the fusion protein has an Fc that has essentially no effector function, e.g., the Fc has reduced or eliminated binding to FcγR, reduced or eliminated complement fixation, e.g., is effectorless. An exemplary effector-less IgG1 Fc contains the following five mutations: L234A, L235E, G237A, A330S, and P331S (EU numbering) (Gross et al. (2001) Immunity 15:289). These five substitutions may be combined with N297A to also eliminate glycosylation. Mutations that promote heterodimerization

[0099] In some embodiments, the first and second Fc domains have mutations that promote heterodimerization. Mutations in Fc domain pairs that facilitate or promote heterodimerization are reviewed in Ha, et al., Front. Immunol. (2016) 7:394. In some embodiments, the first and second Fc domains each contain the following amino acid substitutions (EU numbering) (or vice versa): T366W and T366S / L368A / Y407V; T366W / S354C and T366S / L368A / Y407V / Y349C; S364H / F405A and Y349T / T394F; T350V / L351Y / F405A / Y407V and T350V / T366L / K392. L / T394W; K360D / D399M / Y407A and E345R / Q347R / T366V / K409V; K409D / K392D and D399K / E356K; K360E / K409W and Q347R / D399V / F405T; K360E / K409W / Y349C and Q347R / D399V / F405T / S354C; F405L and K409R; or K370E / K409W and E357N / D399V / F405T.

[0100] In some embodiments, heterodimerization of the Fc regions of two different heavy chain-containing species can be promoted by so-called "knobs-into-holes" mutations (Atwell et al. 1997. JMB 270:26-35). The "hole" mutations (T366S, L368A, and Y407V) are incorporated into one Fc-containing chain, and the T366W "knob" mutation is incorporated into the other chain. In addition, a C220S mutation can be incorporated into the IgG1 hinge region of the scFv-containing arm to eliminate a free cysteine ​​that would otherwise form a disulfide bond with the corresponding cysteine ​​in the light chain of wild-type IgG1. Cotransfection of such constructs results in the preferential formation of heterodimeric Fc with low levels of homodimeric contamination. Additionally, incorporation of the S354C mutation can be incorporated into an Fc containing a "knob" mutation, and incorporation of the Y349C mutation into an Fc containing a "hole" mutation can optionally be used to create a covalent bond between the two halves of a heterodimeric Fc if additional thermodynamic stability is desired (Merchant et al. 1998. Nat. Biotechnol. 16:677-81).

[0101] To facilitate purification of heterodimeric molecules from contaminating homodimeric products, the H435R or H435R+Y436F mutation to reduce or eliminate Protein A binding can be introduced into one, but not both, Fc-containing chains (Jendeberg, L. et al. 1997 J. Immunol. Methods 201:25-34). This reduces or eliminates Protein A binding of homodimeric contaminants containing those mutations, greatly simplifying purification of the desired heterodimer from the remaining homodimeric contaminants via an additional chromatography step (e.g., ion exchange, e.g., anion exchange). In embodiments incorporating the H435R (or H435R+Y436F) mutation in the first or second Fc region of a heavy chain, if the VH region of the same heavy chain is derived from a VH3 family variable region, this VH region can also contain the amino acid substitutions described herein to reduce or eliminate Protein A binding of the entire heavy chain. IgG1 isotype Fc

[0102] In one embodiment, the Fc region comprises or is derived from human IgG1. In some embodiments, the antibody has a chimeric heavy chain constant region (e.g., having the CH1, hinge, and CH2 regions of IgG4 and the CH3 region of IgG1).

[0103] IgG1 antibodies exist in a variety of allotypes and isoallotypes. In certain embodiments, the Fc-IL-2v fusion proteins described herein comprise an IgG1 heavy chain having the following allotypes: G1m1; nG1m2; G1m3; G1m17,1; G1m17,1,2; G1m3,1; or G1m17. Each of these allotypes or isoallotypes is characterized by the following amino acid residues at the indicated positions within the IgG1 heavy chain constant region (Fc) (EU numbering): G1m1:D356, L358; nG1m1:E356, M358; G1m3:R214, E356, M358, A431; G1m17,1:K214, D356, L358, A431; G1m17,1.2:K214, D356, L358, G431; G1m3,1:R214, D356, L358, A431; and G1m17: K214, E356, M358, A431.

[0104] In a specific embodiment, the IL-2v domain or a truncated fragment thereof is linked directly or via an intervening amino acid sequence (e.g., a GS linker) to the wild-type IgG1m3 sequence or a fragment thereof provided below. EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 74). For example, in various embodiments, an IgG1m3 fragment has the first five residues (EPKSC; SEQ ID NO: 232) removed and has the following sequence: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 233).

[0105] In certain embodiments, the Fc-IL-2v fusion protein has an IgG1 isotype. In some embodiments, the Fc-IL-2v fusion protein contains a human IgG1 constant region. In some embodiments, the human IgG1 Fc region contains one or more modifications. For example, in some embodiments, the Fc region contains one or more amino acid substitutions (e.g., relative to a wild-type Fc region of the same isotype). In some embodiments, the one or more amino acid substitutions are N297A, N297Q (Bolt et al. (1993) Eur J Immunol 23:403-411), D265A, L234A, L235A (McEarchem et al., (2007) Blood, 109:1185-1192), C226S, C229S ... al.,(2007)Blood.109:1185-1192), P238S(Davis et al.,(2007)J Rheumatol,34:2204-2210), E233P,L234V(McEarchern et al. al., (2007) Blood, 109:1185-1192), P238A, A327Q, A327G, P329A (Shields R L. et al., (2001) J Biol Chem. 276(9):6591-604), P329G (Schlothauer, et al., Protein Eng Des Sel. (2016)29(10):457-466), K322A, L234F, L235E (Hezareh, et al. al., (2001) J Virol 75, 12161-12168; Oganesyan et al., (2008). Acta Crystallographica 64, 700-704), P331S (Oganesyan et al., (2008) Acta Crystallographica 64, 700-704), T394D (Wilkinson et al. (2013) MAbs 5(3):406-417), A330L, M252Y, S254T, and / or T256E, where the amino acid positions are according to the EU numbering convention. In certain embodiments, the Fc region further comprises an amino acid deletion at a position corresponding to glycine 236 according to the EU numbering convention.

[0106] In some embodiments, the Fc-IL-2v fusion protein has an IgG1 isotype with a heavy chain constant region containing a C220S amino acid substitution according to the EU numbering convention.

[0107] In some embodiments, the Fc region comprises a human IgG1 isotype and includes the following amino acids: N297A, N297G, N297Q, N297G, D265A, L234A, L235A, C226S, C229S, P238S, E233P, L234V, P238A, A327Q, A327G, P329A, P329G, K322A, L234F, L235E, P331 In some embodiments, the Fc region comprises one or more amino acid substitutions in the Fc region at residue positions selected from the group consisting of: S, T394D, A330L, M252Y, S254T, T256E, M428L, N434S, T366W, T366S, L368A, Y407V, H435R, Y436F, and any combination thereof, where residue numbering is according to EU numbering. In some embodiments, the Fc region comprises a human IgG1 isotype and comprises one or more amino acid substitutions in the Fc region at residue positions selected from the group consisting of: L234A, L234V, L234F, L235A, L235E, P329G, A330L, P331S, and any combination thereof, where residue numbering is according to EU numbering. IgG4 isotype Fc

[0108] For uses where effector functions are to be avoided entirely, e.g., when antigen binding alone is sufficient to produce the desired therapeutic benefit and effector functions only result in (or increase the risk of) unwanted side effects, IgG4 antibodies may be used, or antibodies or fragments may be devised that lack the Fc region or a substantial portion thereof, or the Fc may be mutated to eliminate glycosylation entirely (e.g., N297A). Alternatively, hybrid constructs of human IgG2 (CH1 domain and hinge region) and human IgG4 (CH2 and CH3 domains) have been generated that lack effector functions, lack the ability to bind to FcγR (like IgG2), and are unable to activate complement (like IgG4). (See Rother et al. (2007) Nat. Biotechnol. 25:1256, Mueller et al. (1997) Mol. Immunol. 34:441, and Labrijn et al. (2008) Curr. Op. Immunol. 20:479, which generally discuss Fc modifications to reduce effector function).

[0109] In certain embodiments, the Fc-IL-2v fusion protein has an IgG4 isotype. In some embodiments, the Fc-IL-2v fusion protein contains a human IgG4 constant region. In some embodiments, the human IgG4 constant region comprises an Fc region. In some embodiments, the Fc region contains one or more modifications. For example, in some embodiments, the Fc region contains one or more amino acid substitutions (e.g., relative to a wild-type Fc region of the same isotype). In some embodiments, the one or more amino acid substitutions are selected from E233P, F234V, F234A, L235A, G237A, E318A, S228P, L235E, T394D, M252Y, S254T, T256E, N297A, N297G, N297Q, T366W, T366S, L368A, Y407V, M428L, N434S, H435R, Y436F, and any combination thereof, wherein the amino acid positions are according to the EU numbering convention. See, e.g., Hutchins et al. (1995) Proc Natl Acad Sci USA, 92:11980-11984; Reddy et al., (2000) J Immunol, 164:1925-1933; Angal et al., (1993) Mol Immunol. 30(1):105-8; U.S. Patent No. 8,614,299(B2); Vafa O. et al., (2014) Methods 65:114-126; and Jacobsen et al., J. Biol. Chem. (2017) 292(5):1865-1875. In some embodiments, the Fc region comprises a human IgG4 isotype and comprises one or more amino acid substitutions in the Fc region at residue positions selected from the group consisting of F234V, F234A, L235A, L235E, S228P, and any combination thereof, wherein residue numbering is according to EU numbering.

[0110] In some embodiments, the IgG4 variants of the present disclosure may be combined with one or more mutations according to the EU numbering convention, such as the S228P mutation (Angal et al., (1993) Mol Immunol, 30:105-108) and / or Peters et al., (2012) J Biol Chem. 13;287(29):24525-33). IgG2 isotype Fc

[0111] In certain embodiments, the Fc-IL-2v fusion protein has an IgG2 isotype. In some embodiments, the Fc-IL-2v fusion protein contains a human IgG2 constant region. In some embodiments, the human IgG2 constant region comprises an Fc region. In some embodiments, the Fc region contains one or more modifications. For example, in some embodiments, the Fc region contains one or more amino acid substitutions (e.g., relative to a wild-type Fc region of the same isotype). In some embodiments, the one or more amino acid substitutions are selected from P238S, V234A, G237A, H268A, H268Q, H268E, V309L, N297A, N297G, N297Q, V309L, A330S, P331S, C232S, C233S, M252Y, S254T, and / or T256E, where the amino acid positions are according to the EU numbering convention (Vafa, et al., (2014) Methods 65:114-126).

[0112] In certain embodiments, the Fc-IL-2v fusion proteins described herein comprise L234F, L235E, and D265A mutations, collectively referred to as "FEA." The FEA mutations reduce or eliminate effector function. In certain embodiments, the Fc-IL-2v fusion proteins described herein comprise L234F, L235E, D265A, and F405L mutations, collectively referred to as "FEAL." In certain embodiments, the Fc-IL-2v fusion proteins described herein comprise L234F, L235E, D265A, and a mutation selected from the group consisting of F405L, F405A, F405D, F405E, F405H, F405I, F405K, F405M, F405N, F405Q, F405S, F405T, F405V, F405W, and F405Y. In certain embodiments, the Fc-IL-2v fusion proteins described herein comprise L234F, L235E, D265A, and K409R mutations, collectively referred to as "FEAR." In certain embodiments, FEAR and FEAR comprise the fusion protein described herein. In certain embodiments, the Fc-IL-2v fusion proteins described herein additionally comprise M428L and N434S mutations, collectively referred to as LS. In certain embodiments, the Fc-IL-2v fusion proteins described herein comprise L234F, L235E, D265A, F405L, M428L, and N434S mutations, collectively referred to as "FEALLS." In certain embodiments, the Fc-IL-2v fusion proteins described herein comprise the L234F, L235E, D265A, M428L, and N434S mutations, together with one additional mutation selected from the group consisting of F405L, F405A, F405D, F405E, F405H, F405I, F405K, F405M, F405N, F405Q, F405S, F405T, F405V, F405W, and F405Y. In certain embodiments, the Fc-IL-2v fusion proteins described herein comprise the L234F, L235E, D265A, K409R, M428L, and N434S mutations, collectively referred to as "FEARLS."In certain embodiments, FEALLS and FEARLS comprise a fusion protein described herein. By reducing or abrogating effector functions on the Fc domain of the Fc-IL-2v fusion protein, cells bound by the molecule are not killed by innate effector cells, e.g., NK cells, macrophages.

[0113] In certain embodiments, the one or more modifications are selected from the following Fc amino acid substitutions (EU numbering) or a combination thereof: L234F; L235E; G236A; S239D; F243L; D265E; D265A; S267E; H268F; R292P; N297Q; N297G, N297A; S298A; S324T; I332E; S239D; A330L; L234F; L23 5E;P331S;F243L;Y300L;V305I;P396L;S298A;E333A;K334A;E345R;L235V;F243L;R292P;Y300L;P396L;M 428L;E430G;N434S;G236A, S267E, H268F, S324T, and I332E;G236A, S239D, and I332E;S239D, A330L, I332E;L 234F, L235E, and P331S; F243L, R292P, Y300L, V305I, and P396L; G236A, H268F, S324T, and I332E; S239D, H268F, S324T, and I332E; S298A, E333A, and K334A; L235V, F243L, R292P, Y300L, and P396L; S239D, I332E; S239D, S29 8A, and I332E; G236A, S239D, I332E, M428L, and N434S; G236A, S239D, A330L, I332E, M428L, and N434S; S239D, I332E, G236A, and A330L; M428L and N4343S; M428L, N434S; G236A, S239D, A330L, and I332E; and G236A and I332E. In certain embodiments, the one or more modifications are selected from the group consisting of D265A, L234F, L235E, N297A, N297G, N297Q, and P331S. In certain embodiments, the one or more modifications are selected from N297A and D265A. In certain embodiments, the one or more modifications are selected from L234F and L235E. In certain embodiments, the one or more modifications are selected from L234F, L234E, and D265A. In certain embodiments, the one or more modifications are selected from L234F, L234E, and N297Q.In certain embodiments, the one or more modifications are selected from L234F, L235E, and P331S. In certain embodiments, the one or more modifications are selected from D265A and N297Q. In certain embodiments, the one or more modifications are selected from L234F, L235E, D265A, N297A, N297G, N297Q, and P331S.

[0114] Mutations that reduce Fc receptor binding and find use in the fusion proteins described herein include, for example: N297A; N297G; N297Q; D265A; L234F / L235E; L234F / L235E / N297Q, L234F / L235E / P331S, D265A / N297Q; and L234F / L235E / D265A / N297Q / P331S (all EU numbering). In certain embodiments, the Fc-IL-2v fusion proteins described herein comprise the L234F and L235E mutations. In certain embodiments, the Fc-IL-2v fusion proteins described herein comprise the L234F, L235E, and D265A mutations. In certain embodiments, the Fc-IL-2v fusion proteins described herein comprise the L234F, L235E, and N297Q mutations. In certain embodiments, the Fc-IL-2v fusion proteins described herein comprise the N297A or N297Q mutations. In certain embodiments, the Fc-IL-2v fusion proteins described herein comprise the N297A, N297G, or N297Q mutations, as well as the L234F, L235E, and D265A mutations. In certain embodiments, one, two, three, four, or more amino acid substitutions are introduced in the Fc region to alter the effector function of the antigen-binding molecule. For example, these substitutions are located at positions selected from the group consisting of amino acid residues 234, 235, 236, 237, 265, 297, 318, 320, and 322 (according to EU numbering). These positions can be replaced with different amino acid residues such that the antigen-binding molecule has altered (e.g., reduced) affinity for an effector ligand (e.g., an Fc receptor or C1 component of complement) but retains the antigen-binding ability of the parent antibody. In certain embodiments, the Fc-IL-2v fusion proteins described herein comprise E233P, L234V, L235A, and / or G236A mutations (EU numbering). In some embodiments, the Fc-IL-2v fusion proteins described herein comprise A327G, A330S, and / or P331S mutations (EU numbering).In some embodiments, the Fc-IL-2v fusion proteins described herein comprise a K322A mutation (EU numbering). In some embodiments, the Fc-IL-2v fusion proteins described herein comprise E318A, K320A, and K322A (EU numbering) mutations. In certain embodiments, the Fc-IL-2v fusion proteins described herein comprise a L235E (EU numbering) mutation.

[0115] In some embodiments, the Fc portion of the fusion protein comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the following amino acid sequence: GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 239), [Table 3]

[0116] In some embodiments, a terminal Fc amino acid residue (e.g., K447) is deleted or eliminated. In some embodiments, the Fc region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 45-72, or an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 45-72. In some embodiments, the Fc region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 45, 47, 49, 52, 54, 56, 57, 59, 61, 63, 65, 67, 69, and 71, or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 45, 47, 49, 52, 54, 56, 57, 59, 61, 63, 65, 67, 69, and 71. In some embodiments, the Fc region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 46, 48, 50, 51, 53, 55, 58, 60, and 62, or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 46, 48, 50, 51, 53, 55, 58, 60, and 62. c. linker

[0117] In various embodiments, the IL-2v domain or a truncated fragment thereof is directly linked to, contiguously linked to, or adjacent to the serum half-life extending polypeptide (e.g., the Fc domain). In some embodiments, the IL-2v domain or a truncated fragment thereof is operably linked to the serum half-life extending polypeptide (e.g., the Fc domain) via a linker, e.g., the linker is positioned between the serum half-life extending polypeptide (e.g., the Fc domain) and the IL-2v. Optionally, the linker can be a flexible linker. For example, the linker can be an amino acid sequence comprising 1 to 10 repeats or units, e.g., 1 to 5 repeats or units, e.g., 3 to 5 repeats or units, e.g., 3, 4, or 5 repeats, of the GGGS motif (SEQ ID NO: 265), e.g., 3, 4, or 5 repeats of the GGGGS motif (SEQ ID NO: 264), or a mixture thereof (a "GS linker") (Desplancq et al. 1994, Protein Engineering 7:1027-1033). In some embodiments, the linker has a length of 4 to 50 amino acids, e.g., 5 amino acids to 25 amino acids, e.g., 12 amino acids to 15, 16, 20, or 25 amino acids. In some embodiments, the linker comprises four repeats of the GGGGS motif (SEQ ID NO: 246).

[0118] In certain embodiments, the IL-2v domain or truncated fragment thereof is linked directly or via an intervening amino acid sequence (e.g., a GS linker) to human IgG1 (e.g., a variant IgG1m3 sequence), IgG2, IgG3, or IgG4 with 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions. d. Exemplary Fc-IL-2v Fusion Proteins

[0119] Also provided is an Fc-IL-2v fusion protein comprising an IL-2v domain as described above and herein and an Fc domain as described above and herein.

[0120] Functionally, in various embodiments, the Fc-IL-2v fusion protein has an equilibrium dissociation constant (K) of at least 60 μM (e.g., 60 μM or more), e.g., at least 70 μM, 80 μM, 90 μM, 100 μM or more. D ) binds to IL-2RA (weaker binding affinity or K D In some embodiments, the Fc-IL-2v fusion protein binds to the complex of interleukin-2 receptor subunit beta (IL-2RB; CD122) and interleukin-2 receptor subunit gamma (IL-2RG; CD132) with a K of less than 150 nM, for example, less than 1.5 nM, for example, less than 120 pM, for example, less than 100 pM, for example, less than 80 pM, for example, less than 75 pM, for example, less than 70 pM. D In some embodiments, the Fc-IL-2v fusion protein promotes equal or greater proliferation of CD8+ T cells compared to an IL-2v of any one of SEQ ID NOS: 43 and 44, a fusion protein comprising Fc operably linked to wt IL-2, or a fusion protein of any one of SEQ ID NOS: 117, 118, 161, and 162. In some embodiments, the IL-2v fusion protein binds at a concentration (EC) that induces 50% of maximal signal transducer and activator of transcription 5 (STAT5) activation or signaling in regulatory T (Treg) cells. 50 ) is a fusion protein comprising an Fc operably linked to wt IL-2, or an IL-2v of any one of SEQ ID NOs: 43 and 44, or a fusion protein of any one of SEQ ID NOs: 117, 118, 161, or 162, for STAT5 activation or signaling. 50 In some embodiments, the EC 2 of IL-2Rαβγ-mediated STAT5 activation or signaling (e.g., measured as STAT5 activation of CTLL2 cells) is at least 1000-fold, such as at least 1500-fold, such as at least 1700-fold, such as at least 2000-fold, such as at least 2500-fold higher compared to IL-2Rαβγ-mediated STAT5 activation or signaling (e.g., measured as STAT5 activation of CTLL2 cells). 50The concentration that induces STAT5 activation or signaling is the EC 200 for wt IL-2, or IL-2v of any one of SEQ ID NOS: 43 and 44, a fusion protein comprising Fc operably linked to wt IL-2, or a fusion protein of any one of SEQ ID NOS: 117, 118, 161, or 162. 50 In some embodiments, the IL-2v fusion protein is at least 2500-fold, such as at least 5000-fold, such as at least 7500-fold, such as at least 10,000-fold, such as at least 15,000-fold, such as at least 20,000-fold higher than the concentration at which IL-2v fusion protein induces 50% of maximal proliferation of natural killer (NK) cells (EC 50 ) is measured, for example, using KHYG-1 cells, and the EC for proliferation of wt IL-2, or IL-2v of any one of SEQ ID NOS: 43 and 44, a fusion protein comprising Fc operably linked to wt IL-2, or a fusion protein of any one of SEQ ID NOS: 117, 118, 161, or 162. 50 is at least 10 times, such as at least 12 times, for example at least 15 times, such as at least 16 times, for example at least 18 times, such as at least 20 times higher compared to

[0121] Structurally, in various embodiments, the Fc-IL-2v fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 75-116 and 119-160, or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 75-116 and 119-160. Exemplary Fc-IL-2v fusion proteins based on or derived from human wild-type Fc and human wild-type IL-2 are provided in Table C.

[0122] In various embodiments, the Fc-IL-2v fusion protein is based on or derived from murine wild-type Fc and murine wild-type IL-2 and comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 166-171, or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 166-171. Exemplary Fc-IL-2v fusion proteins based on or derived from murine wild-type Fc and murine wild-type IL-2 are provided in Table D. In various embodiments, the murine Fc-IL-2v fusion protein is in the form of a heterodimer with an Fc domain comprising, for example, the amino acid sequence of SEQ ID NO:250 or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO:250. GPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLGAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLSCAVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMVSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK (SEQ ID NO: 250)

[0123] Generally, the Fc-IL-2v fusion proteins described herein are not fused to a second cytokine, for example, the Fc-IL-2v fusion proteins described herein are not fused to a second interleukin, including a second IL-2, or an interferon.

[0124] In some embodiments, the Fc-IL-2v fusion protein is aglycosylated. In some embodiments, the IL-2v in the Fc-IL-2v fusion protein is aglycosylated. In some embodiments, the Fc region or Fc domain of the Fc-IL-2v fusion protein is glycosylated, e.g., having a single N-linked glycan at position N297 (EU numbering) in one or both of the Fc regions or Fc domains of the Fc-IL-2v heterodimers described herein. [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5] [Table 4-6] [Table 4-7] [Table 4-8] [Table 4-9] [Table 4-10] [Table 4-11] [Table 4-12] [Table 4-13] [Table 4-14] [Table 4-15] [Table 4-16] [Table 4-17] [Table 4-18] [Table 4-19] [Table 4-20] [Table 5-1] [Table 5-2]

[0125] e. conjugate Any of the Fc-IL-2v fusion proteins disclosed herein, or their homodimers or heterodimers, can be conjugated. IL-2v, IL-2v fusion proteins, or their homodimers or heterodimers can be conjugated to polymers (e.g., polyethylene glycol (PEG), polyethyleneimine (PEI) modified with PEG (PEI-PEG), polyglutamic acid (PGA), N-(2-hydroxypropyl) methacrylamide (HPMA)). copolymers), hyaluronic acid, radioactive materials (e.g. 90 Y, 131 I, 125 I, 35 S, 3 H, 121 In, 99Tc), fluorescent substances (e.g., fluorescein and rhodamine), fluorescent proteins, luminescent substances (e.g., luminol), Qdots, haptens, enzymes (e.g., glucose oxidase), metal chelates, biotin, avidin, and polymeric substances such as drugs (including the antiviral and anticancer drugs described herein).

[0126] The above-described conjugated IL-2v, IL-2v fusion protein, or homodimer or heterodimer thereof can be prepared, for example, according to known methods for chemically modifying IL-2v, IL-2v fusion protein, or homodimer or heterodimer thereof described herein. In certain embodiments, a labeling moiety or therapeutic moiety is conjugated to the Fc portion of the fusion protein. Methods for modifying antibody Fc regions are well known in the art (e.g., U.S. Pat. Nos. 5,057,313 and 5,156,840).

[0127] In some embodiments, IL-2v, an IL-2v fusion protein, or a homodimer or heterodimer thereof is conjugated to a drug or therapeutic agent. In various embodiments, the drug is a small organic compound or an inhibitory nucleic acid, e.g., a short inhibitory RNA (siRNA), a microRNA (miRNA). In some embodiments, the drug or therapeutic agent is an anti-neoplastic or chemotherapeutic agent known in the art and described herein. In some embodiments, the drug or therapeutic agent is a bacterial toxin, e.g., diphtheria toxin.

[0128] In some embodiments, the therapeutic agent is a small molecule immune checkpoint inhibitor, e.g., GS-4224 or GS-4416. In some embodiments, the therapeutic agent is an agonist or activator of a pattern recognition receptor (PRR), e.g., a Toll-like receptor (TLR), a RIG-I-like receptor (RLR), a NOD-like receptor (NLR), an AIM2-like receptor (ALR), a C-type lectin receptor (CLR), a DNA receptor, or an RNA receptor. In some embodiments, the therapeutic agent is an agonist or activator of a Toll-like receptor (TLR), DExD / H-box helicase 58 (DDX58; also known as RIG-I), or a stimulator of interferon genes (STING) receptor. In some embodiments, the TLR agonist or activator is selected from the group consisting of a TLR2 agonist, a TLR3 agonist, a TLR4 agonist, a TLR5 agonist, a TLR7 agonist, a TLR8 agonist, and a TLR9 agonist. In some embodiments, the TLR7 agonist is selected from the group consisting of vesatolimod (GS-9620), DS-0509, LHC-165, TMX-101 (imiquimod), RO7020531, and JNJ-4964, and / or the TLR8 agonist is selected from the group consisting of sergantolimod (GS-9688), and / or a dual TLR7 / TLR8 agonist such as NKTR-262, telluritolimod, BDB-001, and CV8102.

[0129] In some embodiments, the drug or therapeutic agent is monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), calicheamicin, ansamitocin, maytansine or an analog thereof (e.g., mertansine / emtansine (DM1), ravtansine / soravtansine (DM4)), an anthracycline (e.g., doxorubicin, daunorubicin, epirubicin, idarubicin), a pyrrolobenzodiazepine (e.g., benzodiazepine), or a benzodiazepine (e.g., benzodiazepine). , PBD) DNA cross-linking agent SC-DR002 (D6.5), duocarmycin, microtubule inhibitor (MTI) (e.g., taxane, vinca alkaloid, epothilone), pyrrolobenzodiazepine (PBD) or its dimer and duocarmycins (A, B1, B2, C1, C2, D, SA, CC-1065). f homodimer

[0130] Also provided are homodimers, hi some embodiments, the homodimers comprise two Fc-IL-2v fusion proteins described above and herein. G heterodimer

[0131] Also provided are heterodimers. In various formats and configurations, the heterodimers can comprise (i) one IL-2v domain, (ii) two IL-2v domains, (iii) one IL-2v domain and an antigen-binding domain, (iv) one IL-2v domain and two antigen-binding domains (binding to the same or different target antigens), or (v) two IL-2v domains and two antigen-binding domains (binding to the same or different target antigens). The heterodimers generally comprise a half-life extending moiety (e.g., an Fc domain, one or more serum albumin moieties, an albumin-binding protein or peptide, an IgG, an XTEN polypeptide, a proline / alanine / serine-rich (PAS) polypeptide, an elastin-like polypeptide). In some embodiments, the heterodimer is monovalent for IL-2v, e.g., having one IL-2v domain. or non-targeted (IL-2βγ receptor complex-targeted) heterodimers

[0132] In some embodiments, the heterodimer comprises two IL-2v domains. Such heterodimers include (i) a first Fc-IL-2v fusion protein described herein comprising a first Fc domain, and (ii) a second Fc-IL-2v fusion protein described herein comprising a second Fc domain. In such non-targeting embodiments, the first Fc domain and the second Fc domain do not comprise or are not fused to an antigen-binding domain. In such non-targeting embodiments, the first Fc domain and the second Fc domain are heterodimerized.

[0133] In some embodiments, the heterodimer comprises one IL-2v domain. Such a heterodimer comprises (i) one (i.e., one) Fc-IL-2v fusion protein comprising a first Fc domain, and (ii) a second Fc domain. In such non-targeting embodiments, the second Fc domain does not comprise or is not fused to an antigen-binding domain. In such non-targeting embodiments, the second Fc domain is "empty" but is heterodimerized to the Fc-IL-2v fusion protein (e.g., using Fc substitution), for example, to promote molecular stability and reduce or prevent homodimerization or assembly of molecules having two IL-2v domains. In some embodiments, neither the first nor the second Fc domain is fused to an antigen-binding domain. Such embodiments of the heterodimer bind with greater affinity and / or specificity to the complex of interleukin-2 receptor subunit beta (IL-2RB; CD122) and interleukin-2 receptor subunit gamma (IL-2RG; CD132) compared to any other antigen or target molecule. In some embodiments of heterodimers having one IL-2v domain and no antigen binding domain, the heterodimer does not specifically bind to any antigen or target molecule other than interleukin-2 receptor subunit beta (IL-2RB; CD122) or the complex of IL-2RB and interleukin-2 receptor subunit gamma (IL-2RG; CD132). The specific binding of the heterodimer is at least 10 7 , 108 , 10 9 , or 10 10 M -1 Specific binding refers to an affinity that is detectably high and distinguishable from nonspecific binding that occurs to at least one unrelated target. Specific binding can be the result of bonds between specific functional groups or the formation of a specific spatial fit (e.g., lock and key type), while nonspecific binding is usually the result of van der Waals forces. Heterodimeric Platform - First and Second Fc Domains

[0134] Generally, a heterodimer has a first Fc domain and a second Fc domain (or a first Fc region and a second Fc region), where the first Fc domain and the second Fc domain are different. To facilitate heterodimerization, in some embodiments, the first Fc domain and the second Fc domain contain the following amino acid substitutions (EU numbering), respectively: T366W and T366S / L368A / Y407V; T366S / L368A / Y407V and T366W; T366W / S354C and T366S / L368A / Y407V / Y349C; T366S / L368A / Y407V and T366W; T366W / S354C and T366S / L368A / Y407V / Y349C; T366S / L368A / Y407V and T366W; 8A / Y407V / Y349C and T366W / S354C;S364H / F405A and Y349T / T394F;Y349T / T394F and S364H / F405A;T350V / L351Y / F405A / Y407V and T350V / T366L / K392L / T394W;T350V / T366L / K392L / T394W and T350V / L351Y / F405A / Y407V; K360D / D399M / Y407A and E345R / Q347R / T366V / K409V;E345R / Q347R / T366V / K409V and K360D / D399M / Y407A;K409D / K392D and D399K / E356K;D399K / E356K and K409D / K392D;K360E / K409W and Q347R / D399V / F405T;Q347R / D3 Includes 99V / F405T and K360E / K409W; K360E / K409W / Y349C and Q347R / D399V / F405T / S354C; Q347R / D399V / F405T / S354C and K360E / K409W / Y349C; K370E / K409W and E357N / D399V / F405T; or E357N / D399V / F405T and K370E / K409W.

[0135] To promote an extended serum half-life of the heterodimer, in some embodiments, one or both of the first Fc domain and the second Fc domain comprise the following amino acids at the indicated positions (EU index numbering): tyrosine at position 252, threonine at position 254, and glutamic acid at position 256 (YTE); or leucine at position 428 and serine at position 434 (LS).

[0136] To facilitate Protein A purification of the heterodimer, in some embodiments, either the first Fc domain or the second Fc domain comprises the following amino acids at the indicated positions (EU index numbering): arginine at position 435 and phenylalanine at position 436. In some embodiments, either the first Fc domain or the second Fc domain comprises an arginine at position 435 and a phenylalanine at position 436, and T366S / L368A / Y407V amino acid substitutions.

[0137] In some embodiments, the effector function of one or both Fc domains of the heterodimer is reduced or eliminated. In some embodiments, one or both of the first Fc domain and the second Fc domain comprise a human IgG4 isotype and comprise one or more amino acid substitutions in the Fc domain or Fc region at residue positions selected from the group consisting of F234V, F234A, L235A, L235E, S228P, and any combination thereof, where residue numbering is according to EU numbering. In some embodiments, one or both of the first Fc domain and the second Fc domain comprise a human IgG1 isotype and comprise one or more amino acid substitutions in the Fc region at residue positions selected from the group consisting of L234A, L234V, L234F, L235A, L235E, P331S, and any combination thereof, where residue numbering is according to EU numbering.

[0138] In some embodiments, a terminal Fc amino acid residue (e.g., K447) is removed or eliminated from one or both of the first and second Fc domains. In some embodiments, the first and second Fc domains each comprise the amino acid sequence set forth below, or comprise an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth below, respectively: SEQ ID NOs: 45 and 46, SEQ ID NOs: 47 and 48, SEQ ID NOs: 49 and 46, SEQ ID NOs: 45 and 51, SEQ ID NOs: 49 and 51, SEQ ID NOs: 52 and 48, SEQ ID NOs: 47 and 53, SEQ ID NOs: 52 and 53, SEQ ID NOs: 54 and 46, SEQ ID NOs: 45 and 55, SEQ ID NOs: 54 and 55, SEQ ID NOs: 56 and 48, SEQ ID NOs: 47 and 50, SEQ ID NOs: 56 and 50, SEQ ID NOs: 57 and 58, SEQ ID NOs: 59 and 60, SEQ ID NOs: 61 and 58, SEQ ID NOs: 57 and 62, SEQ ID NOs: 63 and 64, SEQ ID NOs: 65 and 60, SEQ ID NOs: 59 and 66, SEQ ID NOs: 67 and 68, SEQ ID NOs: 69 and 58, SEQ ID NOs: 57 and 70, SEQ ID NOs: 69 and 70, SEQ ID NOs: 71 and 60, SEQ ID NOs: 59 and 72, or SEQ ID NOs: 71 and 72.

[0139] In some embodiments, the heterodimer comprises a human IgG4 Fc-IL-2v fusion protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 75-116, or an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 75-116; and a second Fc region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 46, 51, and 55, or an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 46, 51, and 55. In some embodiments, the second Fc region comprises the amino acid sequence of SEQ ID NO: 46, or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 46. In some embodiments, the human IgG4 Fc-IL-2v fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 80, 107, and 114, or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 80, 107, and 114.In some embodiments, the human IgG4 Fc-IL-2v fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 80, 107, and 114, or an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 80, 107, and 114, and the second Fc region comprises an amino acid sequence of SEQ ID NO: 46, or an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 46.

[0140] In some embodiments, the heterodimer comprises a human IgG4 Fc region comprising: (i) a first amino acid sequence set forth below, or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the first amino acid sequence set forth below; and (ii) a second amino acid sequence set forth below, or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the second amino acid sequence set forth below. Fc-IL-2v fusion proteins include: SEQ ID NO:75 and SEQ ID NO:46, SEQ ID NO:76 and SEQ ID NO:46, SEQ ID NO:77 and SEQ ID NO:46, SEQ ID NO:78 and SEQ ID NO:46, SEQ ID NO:79 and SEQ ID NO:46, SEQ ID NO:80 and SEQ ID NO:46, SEQ ID NO:81 and SEQ ID NO:46, SEQ ID NO:82 and SEQ ID NO:46, SEQ ID NO:83 and SEQ ID NO:46, SEQ ID NO:84 and SEQ ID NO:46, SEQ ID NO:85 and SEQ ID NO:46, SEQ ID NO:86 and SEQ ID NO:46, SEQ ID NO:87 and SEQ ID NO:46, SEQ ID NO:88 and SEQ ID NO:46, SEQ ID NO:89 and SEQ ID NO:46, SEQ ID NO:90 and SEQ ID NO:46, SEQ ID NO:91 and SEQ ID NO:46, SEQ ID NO:92 and SEQ ID NO:46, SEQ ID NO:93 and SEQ ID NO:46, SEQ ID NO:94 and SEQ ID NO:95. 4 and SEQ ID NO:46, SEQ ID NO:95 and SEQ ID NO:46, SEQ ID NO:96 and SEQ ID NO:46, SEQ ID NO:97 and SEQ ID NO:46, SEQ ID NO:98 and SEQ ID NO:46, SEQ ID NO:99 and SEQ ID NO:46, SEQ ID NO:100 and SEQ ID NO:46, SEQ ID NO:101 and SEQ ID NO:46, SEQ ID NO:102 and SEQ ID NO:46, SEQ ID NO:103 and SEQ ID NO:46, SEQ ID NO:104 and SEQ ID NO:46, SEQ ID NO:105 and SEQ ID NO:46, SEQ ID NO:106 and SEQ ID NO:46, SEQ ID NO:107 and SEQ ID NO:46, SEQ ID NO:108 and SEQ ID NO:46, SEQ ID NO:109 and SEQ ID NO:46, SEQ ID NO:110 and SEQ ID NO:46, SEQ ID NO:111 and SEQ ID NO:46, SEQ ID NO:112 and SEQ ID NO:46, SEQ ID NO:113 and SEQ ID NO:46,SEQ ID NO:114 and SEQ ID NO:46, SEQ ID NO:115 and SEQ ID NO:46, or SEQ ID NO:116 and SEQ ID NO:46. In some embodiments, the heterodimer comprises a human IgG4 Fc-IL-2v fusion protein comprising: (i) a first amino acid sequence set forth below, or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the first amino acid sequence set forth below; and (ii) a second Fc region comprising a second amino acid sequence set forth below, or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the second amino acid sequence set forth below: SEQ ID NO:80 and SEQ ID NO:46, SEQ ID NO:107 and SEQ ID NO:46, or SEQ ID NO:114 and SEQ ID NO:46, respectively.

[0141] In some embodiments, the heterodimer comprises or consists of a human IgG4 Fc-IL-2v fusion protein comprising: (i) a first amino acid sequence of SEQ ID NO: 114, or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 114; and (ii) a second Fc region comprising an amino acid sequence of SEQ ID NO: 46, or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 46. In some embodiments, the heterodimer (i) comprises amino acid substitutions R38G, F42A, and E62A in a first amino acid sequence of SEQ ID NO: 114, or in an IL-2 domain (i.e., the amino acid at position 281 of SEQ ID NO: 114 is G, the amino acid at position 285 of SEQ ID NO: 114 is A, and the amino acid at position 305 of SEQ ID NO: 114 is A), and does not contain residues corresponding to amino acids 1-5 of wild-type human IL-2 (e.g., does not contain APTSS (SEQ ID NO: 163)), and has an identical sequence identity to SEQ ID NO: 114 at least 80%, at least 85%, at least 90%, at least 91%, at least and (ii) a second Fc region comprising the amino acid sequence of SEQ ID NO:46, or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:46.In some embodiments, the heterodimer comprises, or consists of, (i) a first amino acid sequence of SEQ ID NO: 114, or an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 114, and that comprises R38G, F42A, and E62A amino acid substitutions in the IL-2 domain (i.e., the amino acid at position 281 of SEQ ID NO: 114 is G, the amino acid at position 285 is A, and the amino acid at position 305 of SEQ ID NO: 114 is A), and that does not contain residues corresponding to amino acids 1-5 of wild-type human IL-2 (e.g., does not contain APTSS (SEQ ID NO: 163)); and (ii) a second Fc region comprising the amino acid sequence of SEQ ID NO: 46, or an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 46. In some embodiments, the heterodimer comprises or consists of a human IgG4 Fc-IL-2v fusion protein comprising (i) a first amino acid sequence of SEQ ID NO: 114 and (ii) a second Fc region comprising the amino acid sequence of SEQ ID NO: 46. In some embodiments, the heterodimer does not specifically bind to any antigen other than an Fc receptor or the complex of interleukin-2 receptor subunit beta (IL-2RB; CD122) and interleukin-2 receptor subunit gamma (IL-2RG; CD132). In some embodiments, the heterodimer does not comprise an immunoglobulin antigen-binding domain (i.e., does not comprise an immunoglobulin heavy or light chain variable region, i.e., does not comprise a VH or VL). In some embodiments, the IL-2 domain does not comprise amino acid substitutions other than or in addition to R38G, F42A, E62A, and C125S compared to wild-type human IL-2.

[0142] In some embodiments, the heterodimer comprises a human IgG1 Fc-IL-2v fusion protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 119-160, or an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 119-160; and a second Fc region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 58, 62, and 70, or an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 58, 62, and 70. In some embodiments, the second Fc region comprises the amino acid sequence of SEQ ID NO: 58, or an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 58. In some embodiments, the human IgG1 Fc-IL-2v fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 124, 151, and 158, or an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 124, 151, and 158.In some embodiments, the human IgG1 Fc-IL-2v fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 124, 151, and 158, or an amino acid sequence which is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 124, 151, and 158, and the second Fc region comprises an amino acid sequence of SEQ ID NO: 58, or an amino acid sequence which is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 58.

[0143] In some embodiments, the heterodimer comprises a human IgG4 Fc region comprising: (i) a first amino acid sequence set forth below, or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the first amino acid sequence set forth below; and (ii) a second amino acid sequence set forth below, or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the second amino acid sequence set forth below. Fc-IL-2v fusion proteins include: SEQ ID NO:119 and SEQ ID NO:58, SEQ ID NO:120 and SEQ ID NO:58, SEQ ID NO:121 and SEQ ID NO:58, SEQ ID NO:122 and SEQ ID NO:58, SEQ ID NO:123 and SEQ ID NO:58, SEQ ID NO:124 and SEQ ID NO:58, SEQ ID NO:125 and SEQ ID NO:58, SEQ ID NO:126 and SEQ ID NO:58, SEQ ID NO:127 and SEQ ID NO:58, SEQ ID NO:128 and SEQ ID NO:58, SEQ ID NO:129 and SEQ ID NO:58, SEQ ID NO:130 and SEQ ID NO:58, SEQ ID NO:131 and SEQ ID NO:58, SEQ ID NO:132 and SEQ ID NO:58, SEQ ID NO:133 and SEQ ID NO:58, SEQ ID NO:134 and SEQ ID NO:58, SEQ ID NO:135 and SEQ ID NO:58, SEQ ID NO:136 and SEQ ID NO:58, SEQ ID NO:137 7 and SEQ ID NO:58, SEQ ID NO:138 and SEQ ID NO:58, SEQ ID NO:139 and SEQ ID NO:58, SEQ ID NO:140 and SEQ ID NO:58, SEQ ID NO:141 and SEQ ID NO:58, SEQ ID NO:142 and SEQ ID NO:58, SEQ ID NO:143 and SEQ ID NO:58, SEQ ID NO:144 and SEQ ID NO:58, SEQ ID NO:145 and SEQ ID NO:58, SEQ ID NO:146 and SEQ ID NO:58, SEQ ID NO:147 and SEQ ID NO:58, SEQ ID NO:148 and SEQ ID NO:58, SEQ ID NO:149 and SEQ ID NO:58, SEQ ID NO:150 and SEQ ID NO:58, SEQ ID NO:151 and SEQ ID NO:58, SEQ ID NO:152 and SEQ ID NO:58, SEQ ID NO:153 and SEQ ID NO:58, SEQ ID NO:154 and SEQ ID NO:58, SEQ ID NO:155 and SEQ ID NO:58, SEQ ID NO:156 and SEQ ID NO:58,SEQ ID NO:157 and SEQ ID NO:58, SEQ ID NO:158 and SEQ ID NO:58, SEQ ID NO:159 and SEQ ID NO:58, or SEQ ID NO:160 and SEQ ID NO:58. In some embodiments, the heterodimer comprises a human IgG4 Fc-IL-2v fusion protein comprising: (i) a first amino acid sequence set forth below, or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the first amino acid sequence set forth below; and (ii) a second Fc region comprising a second amino acid sequence set forth below, or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the second amino acid sequence set forth below: SEQ ID NO:124 and SEQ ID NO:58; SEQ ID NO:151 and SEQ ID NO:58; or SEQ ID NO:158 and SEQ ID NO:58, respectively.

[0144] Polypeptide sequences of exemplary first and second Fc domain pairs are provided in Table E. [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4] [Table 6-5] [Table 6-6] [Table 6-7] [Table 6-8]

[0145] Optionally, one or both of the first Fc domain-containing polypeptide and / or the second Fc domain-containing polypeptide may comprise an N-terminal signal peptide or leader sequence. In embodiments in which both the first Fc domain and / or the second Fc domain-containing polypeptide may comprise an N-terminal signal peptide or leader sequence, the first and second N-terminal signal peptides or leader sequences may be the same or different. Targeted Heterodimers

[0146] Also provided are targeted heterodimers. In some embodiments, the targeted heterodimer comprises a single Fc-IL-2v fusion protein dimerized with a fusion protein comprising a second Fc domain fused to the antigen-binding domain. In some embodiments, the targeted heterodimer comprises a first fusion protein comprising an antigen-binding domain fused to an Fc-IL-2v fusion protein comprising a first Fc domain dimerized with a second fusion protein comprising a second antigen-binding domain fused to a second Fc domain. In some embodiments, the targeted heterodimer comprises a first fusion protein comprising a first antigen-binding domain fused to a first Fc-IL-2v fusion protein dimerized with a second fusion protein comprising a second antigen-binding domain fused to a second Fc-IL-2v fusion protein. Embodiments of targeted heterodimers comprising first and second antigen-binding domains can be bispecific, binding to first and second target molecules, where the target molecules are the same or different.

[0147] Optionally, the first and / or second Fc domain can be fused directly to the antigen-binding domain or via a linker and / or hinge region. In some embodiments, a fusion protein comprising a second Fc domain fused to an antigen-binding domain comprises, in N- to C-terminal order, an antigen-binding domain and an Fc region. In some embodiments, a fusion protein comprising a second Fc domain fused to an antigen-binding domain comprises, in N- to C-terminal order, an Fc region and an antigen-binding domain. Such targeting heterodimers typically comprise a single antigen-binding domain. In heterodimers comprising a second antigen-binding domain, the second antigen-binding domain can be fused to the N-terminus of the Fc-IL-2v fusion protein. Heterodimers with one or two (first and second) antigen-binding domains enable IL-2v to be directed near an antigen or target of interest. Targeted IL-2v heterodimers have been shown to be effective against anti-cancer and anti-viral (e.g., hepatitis B virus (HBV; NCBI: txid10407); human immunodeficiency virus (HIV; e.g., HIV-1; NCBI: txid11676); herpesviridae (NCBI: txid10292) (including herpes simplex virus (HSV), HSV-1 (NCBI: txid102980) and HSV-2 (NCBI: txid10310)); cytomegalovirus (CMV; NCBI: txid10358); varicella-zoster virus (Varicella zoster virus, These antibodies are useful in several therapeutic areas, including VZV (NCBI:txid10335); Epstein-Barr virus (EBV; NCBI:txid10376); and severe acute respiratory syndrome (SARS)-associated coronavirus (NCBI:txid694009), including SARS-CoV2 (NCBI:txid2697049).

[0148] In some embodiments, the targeting moiety or domain comprises an antibody fragment. An "antibody fragment" comprises a portion of an intact antibody, e.g., the antigen-binding or variable region of the intact antibody. Examples of antibody fragments include single-chain antibody molecules (e.g., scFv; sc(Fv)2, Fab, F(ab)2, Fab', F(ab')2, Facb, and Fv fragments; diabodies; linear antibodies (e.g., Zapata et al., Protein Eng. 8(10):1057-1062 (1995)); and multispecific antibodies formed from antibody fragments. A "single-chain Fv" or "scFv" or "sFv" antibody fragment comprises the heavy chain variable region (VH) domain and the light chain variable region (LCH) domain of an antibody. The VH and VL comprise a single chain variable fragment (VH) domain, and these domains are present in a single polypeptide chain. The VH and VL are generally connected by a peptide linker. In other examples, the linker can be a single amino acid. In some examples, the linker can be a chemical bond. The VH and VL can be arranged in any order. Examples of arrangements include [VH]linker[VL]; or [VL]linker[VH]; see, e.g., Hustone et al., Proc. Natl. Acad. Sci. USA, 85:5879-5883 (1988), and Pluckthun, "The Pharmacology of Monoclonal Antibodies," Vol. 113, Ed Resenburg and Moore, Springer Verlag, New York, pp. 269-315, (1994). In some embodiments, the antibody fragment comprises a Fab or a single-chain variable fragment (scFv).

[0149] In some embodiments, the targeting moiety or domain comprises a non-immunoglobulin or antibody mimetic protein. Examples of non-immunoglobulin or antibody mimetic protein targeting moieties or domains include, but are not limited to, adnectins, affibody molecules, affilins, affimers, affitins, alphabodies, anticalins, peptide aptamers, armadillo repeat proteins (ARMs), atrimers, avimers, designed ankyrin repeat proteins (DARPins®), fynomers, knottins, Kunitz domain peptides, monobodies, and nanoCLAMPs. Non-immunoglobulin or antibody mimetic protein targeting moieties or domains for use in the Fc-IL-2v fusion protein heterodimers described herein can be found, for example, in Zhang, et al., Methods Mol Biol. 2017;1575:3-13; Ta, et al., Future Med Chem. 2017 8;9(12):1301-1304; Yu, et al., Annu Rev Anal Chem (Palo Alto Calif). 2017 Jun. 12;10(1):293-320, Baloch, et al., Crit Rev Biotechnol.2016;36(2):268-75, and Bruce, et al., Chembiochem.2016 Oct 17;17(20):1892-1899. In some embodiments, the targeting moiety or antigen-binding domain is a peptide, e.g., a cyclic or cyclized peptide. In some embodiments, the targeting moiety or antigen-binding domain is from the extracellular domain of a cell surface receptor.

[0150] In some embodiments, the targeting moiety or antigen-binding domain has T-cell receptor (TCR)-like binding properties and binds to an epitope of a target (e.g., a tumor-associated antigen (TAA) or an intracellularly expressed viral protein) presented on a major histocompatibility complex (MHC) molecule. Therapeutic areas that benefit from immune stimulation

[0151] In some embodiments, the first and / or second antigen-binding domain binds to a CD8 alpha molecule (CD8A; NCBI gene ID: 925; CD8, Leu2, p32) and competes with or comprises a VH and VL region from an antibody selected from the group IAB22M2C, OKT8. Immune Checkpoint Proteins

[0152] Useful antigen targets in the context of several therapeutic areas, including the prevention and treatment of cancer and viral infections, include immune checkpoint proteins, and thus, in some embodiments, the first and / or second antigen-binding domains bind to immune checkpoint proteins. Examples of immune checkpoint proteins or receptors include CD27 (NCBI Gene ID: 939), CD70 (NCBI Gene ID: 970), CD40 (NCBI Gene ID: 958), CD40LG (NCBI Gene ID: 959), CD47 (NCBI Gene ID: 961), CD48 (SLAMF2; NCBI Gene ID: 962), transmembrane and immunoglobulin domain-containing 2 (TMIGD2, CD28H; NCBI Gene ID: 126259), CD84 (LY9B, SLAMF5; NCBI Gene ID: 8832), CD96 (NCBI Gene ID: 10225), CD160 (also known as NK1, NK28, BY55; NCBI Gene ID: 111 26), MS4A1 (CD20), NCBI Gene ID: 931), CD244 (SLAMF4), NCBI Gene ID: 51744), CD276 (B7H3), NCBI Gene ID: 80381), V-set domain-containing T-cell activation inhibitor 1 (VTCN1, B7H4; NCBI Gene ID: 79679), V-set immunoregulatory receptor (VSIR, B7H5, VISTA; NCBI Gene ID: 64115), immunoglobulin superfamily member 11 (IGSF11, VSIG3; NCBI Gene ID: 152404), natural killer cytotoxicity receptor 3 ligand 1 (NCR3LG1, B7H6; NCBI Gene ID: 374383), HERV-H LTR-associated 2 (HHLA2, B7H7; NCBI gene ID: 11148), inducible T cell costimulator (ICOS, CD278; NCBI gene ID: 29851), inducible T cell costimulator ligand (ICOSLG, B7H2; NCBI gene ID: 23308), TNF receptor superfamily member 4 (TNFRSF4, OX40; NCBI gene ID: 7293), TNF superfamily member 4 (TNFSF4, OX40L;NCBI Gene ID: 7292), TNFRSF8 (CD30), NCBI Gene ID: 943), TNFSF8 (CD30L), NCBI Gene ID: 944), TNFRSF10A (CD261, DR4, TRAILR1; NCBI Gene ID: 8797), TNFRSF9 (CD137), NCBI Gene ID: 3604), TNFSF9 (CD137L), NCBI Gene ID: 8744), TNFRSF10B (CD262, DR5, TRAILR2; NCBI Gene ID: 8795), TNFRSF10 (TRAIL; NCBI Gene ID: 8 743), TNFRSF14 (HVEM, CD270; NCBI Gene ID: 8764), TNFSF14 (HVEML; NCBI Gene ID: 8740), CD272 (B and T lymphocyte-associated (BTLA), NCBI Gene ID: 151888), TNFRSF17 (BCMA, CD269; NCBI Gene ID: 608), TNFSF13B (BAFF; NCBI Gene ID: 10673), TNFRSF18 (GITR; NCBI Gene ID: 8784), TNFSF18 (GITRL; NCBI Gene ID: 8995), MHC class I polypeptide MHC class I polypeptide-related sequence A (MICA; NCBI gene ID: 100507436), MHC class I polypeptide-related sequence B (MICB; NCBI gene ID: 4277), CD274 (CD274, PDL1, PD-L1; NCBI gene ID: 29126), programmed cell death 1 (PDCD1, PD1, PD-1; NCBI gene ID: 5133), cytotoxic T lymphocyte-associated protein 4 (CTLA4, CD152; NCBI gene ID: 1493), CD80 (B7-1; NCBI gene ID: 941), CD28 (NCBI gene ID: 940), and Nectin cell adhesion molecule 2 (NECTIN2, CD112; NCBI Gene ID: 5819), CD226 (DNAM-1, NCBI Gene ID: 10666), poliovirus receptor (PVR) cell adhesion molecule (PVR, CD155; NCBI Gene ID: 5817), PVR-related immunoglobulin domain-containing (PVRIG, CD112R; NCBI Gene ID: 79037), T cell immunoreceptor with Ig and ITIM domains (TIGIT; NCBI Gene ID: 201633), T cell immunoglobulin and mucin domain-containing 4 (TIMD4, TIM4;NCBI Gene ID: 91937), hepatitis A virus cellular receptor 2 (HAVCR2, TIMD3, TIM3...

Claims

1. (1) a human IgG4 Fc-IL-2 variant (human IgG4 Fc-IL-2v) fusion protein comprising a first Fc region, the human IgG4 Fc-IL-2v fusion protein comprising the amino acid sequence set forth in SEQ ID NO:114; and (2) a polypeptide comprising a second Fc region comprising the amino acid sequence set forth in SEQ ID NO:

46. (1) The human IgG4 Fc-IL-2v fusion protein comprising the first Fc region comprises a first N-terminal signal peptide or leader sequence selected from the amino acid sequences of any one of SEQ ID NOs:218 to 231, and (2) The heterodimer according to claim 1, wherein the polypeptide comprising the second Fc region comprises a second N-terminal signal peptide or leader sequence selected from the amino acid sequences of any one of SEQ ID NOs: 218 to 231.

3. The heterodimer of claim 2 , wherein the first N-terminal signal peptide or leader sequence and the second N-terminal signal peptide or leader sequence are the same.

4. The heterodimer of claim 2 , wherein the first N-terminal signal peptide or leader sequence and the second N-terminal signal peptide or leader sequence are different.

5. The heterodimer according to any one of claims 1 to 4, wherein the heterodimer specifically binds to an antigen consisting of an Fc receptor and a complex of interleukin 2 receptor subunit beta (IL-2RB; CD122) and interleukin 2 receptor subunit gamma (IL-2RG; CD132).

6. The heterodimer of any one of claims 1 to 5, wherein the second Fc region is not fused to an antigen binding domain.

7. The heterodimer of any one of claims 1 to 6, wherein neither the first nor the second Fc region is fused to an antigen binding domain.

8. 8. The heterodimer of any one of claims 1 to 7, wherein the heterodimer comprises a serum half-life in humans of at least 6, 9, 12, 15, 18, 21, 24 hours, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days or more.

9. A polynucleotide or a plurality of polynucleotides encoding a polypeptide comprising the human IgG4Fc-IL-2v fusion protein and the second Fc region of the heterodimer of any one of claims 1 to 8.

10. 10. The polynucleotide or polynucleotides of claim 9, wherein the polynucleotide or polynucleotides are selected from the group consisting of DNA, cDNA, RNA, or mRNA.

11. 11. An expression cassette or expression cassettes comprising one or more regulatory sequences operably linked to the polynucleotide or polynucleotides of claim 9 or claim 10.

12. A cell or cell population comprising the polynucleotide or polynucleotides of claim 9 or claim 10, the expression cassette or expression cassettes of claim 11, said cell or cell population expressing the heterodimer of any one of claims 1 to 8.

13. The cell or cell population of claim 12, wherein the cell or cell population is a eukaryotic cell.

14. 14. The cell or cell population of claim 12 or claim 13, wherein the cell or cell population comprises a mammalian cell, an insect cell, a plant cell, or a yeast cell.

15. 15. The cell or cell population of claim 14, wherein the mammalian cell is a Chinese Hamster Ovary (CHO) cell.

16. The cell or cell population of claim 14, wherein the mammalian cell is a human cell.

17. 17. The cell or cell population of claim 16, wherein the cell is a human embryonic kidney cell.

18. A method for producing a heterodimer, wherein the heterodimer comprises: (1) a human IgG4 Fc-IL-2 variant (human IgG4 Fc-IL-2v) fusion protein comprising a first Fc region; and (2) a polypeptide comprising a second Fc region; The method further comprising: a) culturing a cell or a cell population according to any one of claims 12 to 17, transformed with a polynucleotide or polynucleotides according to claim 9 or claim 10, or an expression cassette or expression cassettes according to claim 11, in a cell culture under conditions sufficient to express said heterodimeric molecule; b) isolating or purifying said heterodimeric molecule from said cell culture.

19. 19. The method of claim 18, wherein (1) the human IgG4 Fc-IL-2v fusion protein and (2) the polypeptide comprising the second Fc region are expressed and assembled in the same cell.

20. 20. The method of claim 18 or claim 19, wherein the isolation or purification step comprises Protein A chromatography.

21. 21. The method of claim 20, wherein the isolation or purification step further comprises in-stream or instantaneous pH neutralization of the Protein A chromatography eluate.

22. 22. The method of claim 20 or claim 21, wherein the isolation or purification step further comprises anion exchange chromatography.

23. 23. The method of any one of claims 18-22, wherein at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the heterodimeric molecules are isolated or purified.

24. 24. The method of any one of claims 18 to 23, wherein the cell or cell population is cultured in a culture volume of at least 2L, at least 5L, 10L, 50L, 100L, 150L, ​​200L, 250L, or more.

25. The method of any one of claims 18 to 24, further comprising formulating said heterodimeric molecule into a sterile pharmaceutical composition suitable for administration to a human subject.

26. A pharmaceutical composition comprising the heterodimer of any one of claims 1 to 8, the polynucleotide or polynucleotides of claim 9 or claim 10, the expression cassette or expression cassettes of claim 11, and a pharma- ceutically acceptable carrier.

27. 27. The pharmaceutical composition of claim 26, wherein the composition comprises an aqueous formulation.

28. 0.05mg / ml to 50mg / ml, 0.05mg / ml to 20mg / ml, 0.1mg / ml to 40mg / ml, 1.0mg / ml ~30mg / ml, 0.05mg / ml ~0.06mg / ml, 0.07mg / ml, 0.08mg / ml, 0.09mg / ml, 0. 1mg / ml, 0.2mg / ml, 0.3mg / ml, 0.4mg / ml, 0.5mg / ml, 0.6mg / ml, 0.7mg / ml, 0.8mg / ml, 0.9mg / ml, 1.0mg / ml, 1.5mg / ml, 2.0mg / ml, 2.5mg / ml, 3.0mg / ml 28. The pharmaceutical composition of claim 26 or claim 27, comprising the heterodimer at a concentration in the range of: 1., 3.5 mg / ml, 4.0 mg / ml, 4.5 mg / ml, 5.0 mg / ml, 6 mg / ml, 7 mg / ml, 8 mg / ml, 9 mg / ml, 10 mg / ml, 11 mg / ml, 12 mg / ml, 13 mg / ml, 14 mg / ml, 15 mg / ml, 16 mg / ml, 17 mg / ml, 18 mg / ml, 19 mg / ml, 20 mg / ml, 25 mg / ml, 30 mg / ml, 35 mg / ml, 40 mg / ml, 45 mg / ml, or 50 mg / ml.

29. 30. The pharmaceutical composition of claim 28, wherein the composition is lyophilized.

30. 30. The pharmaceutical composition of any one of claims 26 to 29, further comprising one or more additional therapeutic agents.

31. The pharmaceutical composition of any one of claims 26 to 30, further comprising a second therapeutic agent.

32. The pharmaceutical composition of any one of claims 26 to 31, further comprising a second and a third therapeutic agent.

33. 33. A composition comprising the heterodimer of any one of claims 1 to 8, the polynucleotide or polynucleotides of claim 9 or claim 10, the expression cassette or expression cassettes of claim 11, or the pharmaceutical composition of any one of claims 26 to 32, for use in a subject in need of preventing, reducing and / or inhibiting recurrence, growth, proliferation, migration and / or metastasis of a cancer cell or cancer cell population, wherein said use comprises administering said composition to said subject.

34. 34. The composition of claim 33, wherein said use comprises administering said composition simultaneously with one or more anti-neoplastic or chemotherapeutic agents.

35. 35. The composition of claim 34, wherein the one or more antineoplastic or chemotherapeutic agents are selected from the group consisting of nucleoside analogs, taxanes, platinum coordination complexes, dihydrofolate reductase (DHFR) inhibitors, topoisomerase inhibitors, vosaroxin and GPX-150, aldoxorubicin, AR-67, mavereltinib, AST-2818, avitinib (ACEA-0010), irofulven (MGI-114), alkylating agents, and mixtures thereof.

36. 36. The composition of any one of claims 33 to 35, wherein said use comprises administering said composition simultaneously with a FOLFOX, FOLFIRI, FOLFOXIRI, or FOLFIRINOX regimen.

37. The composition of any one of claims 33 to 36, wherein the subject has cancer.

38. The composition of any one of claims 33 to 37, wherein the subject is in remission of cancer.

39. The composition of any one of claims 33 to 38, wherein the subject has a solid tumor.

40. 40. The composition of claim 39, wherein the tumor is a malignant tumor.

41. 41. The composition of claim 39 or claim 40, wherein the tumor is a metastatic tumor.

42. 42. The composition of any one of claims 39 to 41, wherein the subject has a cancer selected from the group consisting of epithelial tumors, ductal tumors, mesenchymal or soft tissue tumors, and lymphomas.

43. 43. The composition of any one of claims 39 to 42, wherein the subject has a solid tumor in, or arising from, a tissue or organ selected from the group consisting of bone; lips and oral cavity; esophagus; gastrointestinal tract, stomach, rectum, anus; pancreas; gallbladder; neuroendocrine; thyroid; liver; kidney; breast; ovaries, vulva, vagina; prostate, testes, penis; bladder; brain; eye; head and neck; thymus; heart; lung; lymphatic; central nervous system (CNS); neuroendocrine tissue; skin; and soft tissue.

44. 44. The composition of any one of claims 39-43, wherein the subject has a solid tumor cancer selected from the group consisting of melanoma; head and neck; ovarian; mesothelioma; endometrial; prostate; sarcoma; neuroblastoma; liver; lung; breast; esophageal, gastric, and pancreatic.

45. 45. The composition of any one of claims 39 to 44, wherein the subject has a cancer selected from the group consisting of lung cancer, colorectal cancer, breast cancer, prostate cancer, cervical cancer, and head and neck cancer.

46. The composition of any one of claims 33 to 45, wherein the subject is chemotherapy naive or has not received chemotherapy.

47. The composition of any one of claims 33 to 46, wherein the subject has undergone a lymphocyte-depleting chemotherapy regimen.

48. The composition of any one of claims 33 to 46, wherein the subject has bone marrow cells or is not bone marrow cell depleted.

49. 49. The composition of any one of claims 33 to 48, wherein said use comprises administering said heterodimer, said polynucleotide or polynucleotides, and / or said pharmaceutical composition simultaneously with one or more additional therapeutic agents.

50. 50. The composition of claim 49, wherein the one or more additional therapeutic agents comprise an inhibitor in the form of an antibody against PD-L1 (CD274), PD-1 (PDCD1), or CTLA4.

51. Said inhibitor in the form of an antibody against CTLA4 is selected from the group consisting of ipilimumab, tremelimumab, BMS-986218, AGEN1181, AGEN1884 (zalifrelimab), BMS-986249, MK-1308, REGN-4659, ADU-1604, CS-1002, BCD-145, APL-509, JS-007, BA-3071, ONC-392, AGEN-2041, JHL-1155, KN-044, CG-0161, ATO 51. The composition of claim 50, wherein the antibody is selected from the group consisting of R-1144, PBI-5D3H5, FPT-155 (CTLA4 / PD-L1 / CD28), PF-06936308 (PD-1 / CTLA4), MGD-019 (PD-1 / CTLA4), KN-046 (PD-1 / CTLA4), MEDI-5752 (CTLA4 / PD-1), XmAb-20717 (PD-1 / CTLA4), and AK-104 (CTLA4 / PD-1).

52. Said inhibitors in the form of antibodies against programmed cell death 1 (PDCD1; NCBI gene ID: 5133; CD279, PD-1, PD1) are selected from the group consisting of zimberelimab (AB122, GLS-010, WBP-3055), pembrolizumab (KEYTRUDA®, MK-3475, SCH900475), nivolumab (OPDIVO®, BMS-936558, MDX-1106), cemiplimab (LIBTAYO®; cemiplimab-rwlc, REGN-2810), pidilizumab (CT-011), AMG-404, MEDI0680 (AMP-514), spartalizumab (P DR001), tislelizumab (BGB-A317), toripalimab (JS-001), genolimuzumab (CBT-501, APL-501, GB226), SHR-1201, camrelizumab (SHR-1210), sintilimab (TYVYT®, IBI-308), dostallimab (TSR-042, WBP-285), lambrolizumab (MK-3475); sasanlimumab (PF-06801591), cetrelimab (JNJ-63723283), serplulimab (HLX-10), retifanlimab (MGA-012), balstilimab (AGEN2034), prorugolimab (BCD 100), budigalimab (ABBV-181), bopratelimab (JTX-4014), AK-103 (HX-008), AK-105, CS-1003, BI-754091, LZM-009, Sym-021, BAT-1306, PD1-PIK, tebotelimab (MGD013; PD-1 / LAG-3), RO-7247669 (PD-1 / LAG-3), FS-118 (LAG-3 / PD-L1), RO-71 51. The composition of claim 50, wherein the antibody is selected from the group consisting of 21661 (PD1 / TIM-3), RG7769 (PD-1 / TIM-3), PF-06936308 (PD1 / CTLA4), MGD-019 (PD-1 / CTLA4), KN-046 (PD1 / CTLA4), XmAb-20717 (PD1 / CTLA4), AK-104 (CTLA4 / PD-1), and MEDI-5752 (CTLA4 / PD-1).

53. Said inhibitor in the form of an antibody against the CD274 molecule (NCBI gene ID: gene ID: 29126; B7-H, B7H1, PD-L1) is one of the following: atezolizumab (TECENTRIQ®), avelumab (BAVENCIO®; MSB0010718C), embafolimab (ASC22), durvalumab (IMFINZI®; MEDI-4736), BMS-936559 (MDX1105), cosibelimab (CK-301), lodapolimab (LY3300054), garivulimab (BGB A333), embafolimab (KN035), opucolimab (HLX20), manelimab (BCD135), CX-072, CBT-502 (TQB2450), MSB-2311, SHR-1316, sugemalimab (CS-1001; WBP3155), A167 (KL-A167, HBM9167), STI-A1015 (IMC-001), FAZ-053, BMS-936559 (MDX1105), INCB086550, GEN-1 51. The composition of claim 50, wherein the antibody is selected from the group consisting of 046 (PD-L1 / 4-1BB), FPT-155 (CTLA4 / PD-L1 / CD28), M7824 (PD-L1 / TGFβ-EC domain), CA-170 (PD-L1 / VISTA), CDX-527 (CD27 / PD-L1), LY-3415244 (TIM-3 / PDL1), INBRX-105 (4-1BB / PDL1), and GNS-1480 (PD-L1 / EGFR).

54. 54. The composition of any one of claims 33 to 53, wherein the heterodimer, the polynucleotide or polynucleotides, and / or the pharmaceutical composition are administered systemically or locally.

55. 55. The composition of any one of claims 33 to 54, wherein the heterodimer, the polynucleotide or polynucleotides, and / or the pharmaceutical composition are administered via a route selected from intravenous, subcutaneous, intramuscular, intradermal, intratumoral, and mucosal.

56. 56. The composition of any one of claims 33 to 55, wherein the heterodimer, the polynucleotide or polynucleotides, and / or the pharmaceutical composition, and one or more additional therapeutic agents are administered by the same route of administration.

57. 57. The composition of any one of claims 33 to 56, wherein the heterodimer, the polynucleotide or polynucleotides, and / or the pharmaceutical composition, and one or more additional therapeutic agents are administered by different routes of administration.

58. 58. The composition of any one of claims 33 to 57, wherein the heterodimer, the polynucleotide or polynucleotides, and / or the pharmaceutical composition, and one or more additional therapeutic agents are co-administered according to the same schedule.

59. 59. The composition of any one of claims 33 to 58, wherein the heterodimer, the polynucleotide or polynucleotides, and / or the pharmaceutical composition, and one or more additional therapeutic agents are co-administered according to different schedules.

60. wherein the heterodimer, the polynucleotide or polynucleotides, and / or the pharmaceutical composition is in the range of 0.5 μg / kg to 1000 μg / kg, in the range of 1 μg / kg to 500 μg / kg, in the range of 10 μg / kg to 300 μg / kg, in the range of 30 μg / kg to 600 μg / kg, at least 0.5 μg / kg per dose to at most 0.2 μg / kg per dose, 0.3 μg / kg, 0.4 μg / kg, 0.5 μg / kg, 0.6 μg / kg, 0.7 μg / kg, 0.8 μg / kg, 0.9 μg / kg, 1 μg / kg, 1.5 μg / kg, 2 μg / kg, 2.5 μg / kg, 3 μg / kg, 3.5 μg / kg, 4 μg / kg, 4.5 μg / kg, 5 μg / kg, 6μg / kg, 7μg / kg, 8μg / kg, 9μg / kg, 10μg / kg, 15μg / kg, 20μg / kg, 25μg / kg, 30μg / kg, 40μg / kg , 50μg / kg, 60μg / kg, 70μg / kg, 80μg / kg, 90μg / kg, 100μg / kg, 110μg / kg, 120μg / kg, 130μg / k 60. The composition of any one of claims 33 to 59, administered at a dose of: 140 μg / kg, 150 μg / kg, 200 μg / kg, 250 μg / kg, 300 μg / kg, 400 μg / kg, 500 μg / kg, 600 μg / kg, 700 μg / kg, 800 μg / kg, 900 μg / kg, 1000 μg / kg.

61. 61. The composition of any one of claims 33-60, wherein the heterodimer, the polynucleotide or polynucleotides, and / or the pharmaceutical composition is administered at a dose ranging from 0.02 mg to 100 mg, 0.04 mg to 80 mg, at least 0.02 mg per dose and up to 0.03 mg per dose, 0.04 mg, 0.05 mg, 0.1 mg, 0.5 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 10 mg, 12 mg, 15 mg, 20 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, or 100 mg.

62. 62. The composition of any one of claims 33 to 61, wherein said use comprises administering said heterodimer, said polynucleotide or polynucleotides, and / or said pharmaceutical composition multiple times at predetermined intervals, optionally with one or more additional therapeutic agents.

63. 63. The composition of any one of claims 33 to 62, wherein said use comprises administration over a period of at least 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months or more.

64. 64. The composition of any one of claims 33 to 63, wherein said use comprises one or more administrations at predetermined intervals spaced at least 1 week up to at least 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months apart.

65. 65. The composition of any one of claims 33 to 64, wherein the heterodimer, the polynucleotide or polynucleotides, and / or the pharmaceutical composition are administered weekly (i.e., QW), biweekly (i.e., once every other week or once every two weeks, i.e., Q2W), once every three weeks (i.e., once every three weeks, i.e., Q3W), monthly (i.e., QM), or bimonthly (i.e., once every other month or once every two months, i.e., Q2M), once every three months (Q3M), once every four months (Q4M), once every five months (Q5M), once every six months (Q6M), or less frequently.

66. 66. The composition of any one of claims 33-65, wherein the heterodimer, the polynucleotide or polynucleotides, and / or the pharmaceutical composition are administered subcutaneously two or more times at one or more intervals between once every other week (i.e., once every other week or once every two weeks, i.e., Q2W) to once every three weeks (i.e., once every three weeks, i.e., Q3W).

67. 67. The composition of any one of claims 33-66, wherein the heterodimer has a serum half-life in humans of at least 6, 9, 12, 15, 18, 21, 24 hours, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days or more.

68. The composition of any one of claims 33 to 67, wherein the subject is a human.

69. 32. A kit comprising one or more unit doses of the heterodimer of any one of claims 1 to 8, the polynucleotide or polynucleotides of claim 9 or claim 10, the expression cassette or expression cassettes of claim 11, or the pharmaceutical composition of any one of claims 26 to 32.

70. 70. The kit of claim 69, wherein the one or more unit doses are in a single container.

71. 70. The kit of claim 69, wherein the one or more unit doses are in two or more separate containers.

72. 72. The kit of any one of claims 69 to 71, comprising one or more containers selected from the group consisting of vials, ampoules, and pre-filled syringes.

73. 73. The kit of any one of claims 69 to 72, comprising one or more containers comprising said human IgG4 Fc-IL-2v fusion protein and / or said heterodimer in an aqueous solution.

74. The aqueous solution contains 0.05 mg / ml to 50 mg / ml, 0.05 mg / ml to 20 mg / ml, 0.1 mg / ml to 40 mg / ml, 1.0 mg / ml ~ 30mg / ml, 0.05mg / ml ~ 0.06mg / ml, 0.07mg / ml, 0.08mg / ml, 0.09mg / ml, 0.1m g / ml, 0.2mg / ml, 0.3mg / ml, 0.4mg / ml, 0.5mg / ml, 0.6mg / ml, 0.7mg / ml, 0.8mg / ml l, 0.9mg / ml, 1.0mg / ml, 1.5mg / ml, 2.0mg / ml, 2.5mg / ml, 3.0mg / ml, 3.5mg / ml, 4 74. The kit of claim 73, comprising the heterodimer, the polynucleotide or polynucleotides, and / or the pharmaceutical composition at a concentration in the range of: 0 mg / ml, 4.5 mg / ml, 5.0 mg / ml, 6 mg / ml, 7 mg / ml, 8 mg / ml, 9 mg / ml, 10 mg / ml, 11 mg / ml, 12 mg / ml, 13 mg / ml, 14 mg / ml, 15 mg / ml, 16 mg / ml, 17 mg / ml, 18 mg / ml, 19 mg / ml, 20 mg / ml, 25 mg / ml, 30 mg / ml, 35 mg / ml, 40 mg / ml, 45 mg / ml, or 50 mg / ml.

75. The kit of any one of claims 69 to 74, wherein the one or more unit doses are the same.

76. The kit of any one of claims 69 to 75, wherein the one or more unit doses are different.

77. Each unit dose may be in the range of 0.5 μg / kg to 1000 μg / kg, 1 μg / kg to 500 μg / kg, 10 μg / kg to 300 μg / kg, 30 μg / kg to 600 μg / kg, at least 0.5 μg / kg per dose to a maximum of 0.2 μg / kg per dose, 0.3 μg / kg, 0.4 μg / kg, 0.5 μg / kg, 0.6 μg / kg, 0.7 μg / kg, 0.8 μg / kg, 0.9 μg / kg, 1 μg / kg, 1.5 μg / kg, 2 μg / kg, 2.5 μg / kg, 3 μg / kg, 3.5 μg / kg, 4 μg / kg, 4.5 μg / kg, 5 μg / kg, 6 μg / kg, 7 μg / kg, 8 μg / kg, 9 μg / kg, 10 μg / kg, 11 μg / kg, 12 μg / kg, 13 μg / kg, 14 μg / kg, 15 μg / kg, 16 μg / kg, 17 μg / kg, 18 μg / kg, 19 μg / kg, 20 μg / kg, 21 μg / kg, 22 μg / kg, 23 μg / kg, 24 μg / kg, 25 μg / kg, 26 μg / kg, 27 μg / kg, 28 μg / kg, 29 μg / kg, 30 μg / kg, 31 μg / kg, 32 μg / kg, 33 μg / kg, 34 μg / kg, 35 μg / kg, 36 μg / kg, 37 μg / kg, 38 μg / kg, 39 μg / kg, 40 μ μg / kg, 9 μg / kg, 10 μg / kg, 15 μg / kg, 20 μg / kg, 25 μg / kg, 30 μg / kg, 40 μg / kg, 50 μg / kg , 60μg / kg, 70μg / kg, 80μg / kg, 90μg / kg, 100μg / kg, 110μg / kg, 120μg / kg, 130μg / kg , 140μg / kg, 150μg / kg, 200μg / kg, 250μg / kg, 300μg / kg, 400μg / kg, 500μg / kg, 600μ 77. The kit according to any one of claims 69 to 76, which is 700 μg / kg, 700 μg / kg, 800 μg / kg, 900 μg / kg, 1000 μg / kg.

78. 78. The kit of any one of claims 69-77, wherein each unit dose is in the range of 0.02 mg to 100 mg, 0.04 mg to 80 mg, at least 0.02 mg per dose to a maximum of 0.03 mg per dose, 0.04 mg, 0.05 mg, 0.1 mg, 0.5 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 10 mg, 12 mg, 15 mg, 20 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, or 100 mg.

79. 79. The kit of any one of claims 69-78, further comprising one or more unit doses of one or more additional therapeutic agents.

80. 80. The kit of claim 79, comprising one or more unit doses of one or more anti-neoplastic or chemotherapeutic agents.

81. 81. The kit of claim 80, wherein the one or more unit doses of the one or more antineoplastic or chemotherapeutic agents are selected from the group consisting of nucleoside analogs, taxanes, platinum coordination complexes, dihydrofolate reductase (DHFR) inhibitors, topoisomerase inhibitors, vosaroxin and GPX-150, aldoxorubicin, AR-67, mavereltinib, AST-2818, avitinib (ACEA-0010), irofulven (MGI-114), alkylating agents, nitrosoureas, alkyl sulfonates, and mixtures thereof.

82. 80. The kit of claim 79, comprising an inhibitor in the form of an antibody against PD-L1 (CD274), PD-1 (PDCD1), or CTLA4.

83. Said inhibitor in the form of an antibody against CTLA4 is selected from the group consisting of ipilimumab, tremelimumab, BMS-986218, AGEN1181, AGEN1884 (zalifrelimab), BMS-986249, MK-1308, REGN-4659, ADU-1604, CS-1002, BCD-145, APL-509, JS-007, BA-3071, ONC-392, AGEN2041, JHL-1155, KN-044, CG-0161, ATO 83. The kit of claim 82, wherein the antibody is selected from the group consisting of R-1144, PBI-5D3H5, FPT-155 (CTLA4 / PD-L1 / CD28), PF-06936308 (PD-1 / CTLA4), MGD-019 (PD-1 / CTLA4), KN-046 (PD-1 / CTLA4), MEDI-5752 (CTLA4 / PD-1), XmAb-20717 (PD-1 / CTLA4), and AK-104 (CTLA4 / PD-1).

84. Said inhibitors in the form of antibodies against programmed cell death 1 (PDCD1; NCBI gene ID: 5133; CD279, PD-1, PD1) are selected from the group consisting of zimberelimab (AB122, GLS-010, WBP-3055), pembrolizumab (KEYTRUDA®, MK-3475, SCH900475), nivolumab (OPDIVO®, BMS-936558, MDX-1106), cemiplimab (LIBTAYO®; cemiplimab-rwlc, REGN-2810), pidilizumab (CT-011), AMG-404, MEDI0680 (AMP-514), spartalizumab (P DR001), tislelizumab (BGB-A317), toripalimab (JS-001), genolimuzumab (CBT-501, APL-501, GB226), SHR-1201, camrelizumab (SHR-1210), sintilimab (TYVYT®, IBI-308), dostallimab (TSR-042, WBP-285), lambrolizumab (MK-3475); sasanlimumab (PF-06801591), cetrelimab (JNJ-63723283), serplulimab (HLX-10), retifanlimab (MGA-012), balstilimab (AGEN2034), prorugolimab (BCD 100), budigalimab (ABBV-181), bopratelimab (JTX-4014), AK-103 (HX-008), AK-105, CS-1003, BI-754091, LZM-009, Sym-021, BAT-1306, PD1-PIK, tebotelimab (MGD013; PD-1 / LAG-3), RO-7247669 (PD-1 / LAG-3), FS-118 (LAG-3 / PD-L1), RO-7121 661 (PD-1 / TIM-3), RG7769 (PD-1 / TIM-3), PF-06936308 (PD-1 / CTLA4), MGD-019 (PD-1 / CTLA4), KN-046 (PD-1 / CTLA4), XmAb-20717 (PD-1 / CTLA4), AK-104 (CTLA4 / PD-1), and MEDI-5752 (CTLA4 / PD-1).

85. Said inhibitor in the form of an antibody against the CD274 molecule (NCBI gene ID: gene ID: 29126; B7-H, B7H1, PD-L1) is one of the following: atezolizumab (TECENTRIQ®), avelumab (BAVENCIO®; MSB0010718C), embafolimab (ASC22), durvalumab (IMFINZI®; MEDI-4736), BMS-936559 (MDX1105), cosibelimab (CK-301), lodapolimab (LY3300054), garivulimab (BGB A333), embafolimab (KN035), opucolimab (HLX20), manelimab (BCD135), CX-072, CBT-502 (TQB2450), MSB-2311, SHR-1316, sugemalimab (CS-1001; WBP3155), A167 (KL-A167, HBM9167), STI-A1015 (IMC-001), FAZ-053, BMS-936559 (MDX1105), INCB086550, GEN-1 046 (PD-L1 / 4-1BB), FPT-155 (CTLA4 / PD-L1 / CD28), M7824 (PD-L1 / TGFβ-EC domain), CA-170 (PD-L1 / VISTA), CDX-527 (CD27 / PD-L1), LY-3415244 (TIM-3 / PDL1), INBRX-105 (4-1BB / PDL1), and GNS-1480 (PD-L1 / EGFR).

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