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 of U.S. Provisional Patent Application No. 63 / 104,376, filed on October 22, 2020, and U.S. Provisional Patent Application No. 63 / 181,075, filed on April 28, 2021, under 35 U.S.C. § 119(e), the entire contents of which are hereby incorporated by reference for all purposes.

[0002] Sequence Listing This application includes a sequence listing that was electronically submitted in ASCII format, the entire contents of which are hereby incorporated by reference. The name of the ASCII copy created on September 20, 2021 is 1348 - WO - PCT_SL.txt, and it is 569,532 bytes in size.

Background Art

[0003] Interleukin - 2 (IL - 2, NCBI Gene ID: 3558) is an immunoregulatory cytokine that plays an important role in the generation, differentiation, survival, and homeostasis of immune cells. IL - 2 is + CD4 +It has therapeutic potential for the treatment of cancer and chronic viral diseases through its immune-stimulatory effects on T cells and NK cells. However, this is impaired by its ability to preferentially stimulate and expand regulatory T (Treg) cells that suppress the immune system. In addition to this preferential activity against immunosuppressive Treg cells, IL-2 has a very short half-life in humans, requires frequent dosing, and can also induce life-threatening toxicities. The short half-life complicates the ability to administer a dose of IL-2 that has a reduced or minimal immunosuppressive effect but is sufficient to induce the desired immune stimulation to avoid toxicity, presenting a significant challenge for the treatment of patients. See, for example, 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 Documents

[0004]

Non-Patent Document 1

Summary of the Invention

Means for Solving the Problems

[0005] In one aspect, provided is an interleukin-2 variant (IL-2v). In various embodiments, the IL-2v has at least 5 amino acids cleaved 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 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, the 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 IL-2v promotes the proliferation of CD8+ T cells equal to or greater than that of wild-type (wt) IL-2 or an IL 2v of either of SEQ ID NOs: 43 and 44. In some embodiments, the concentration (EC5 at which the IL-2v induces 50% of the maximum signal transduction and transcriptional activation of signal transducer and activator of transcription (STAT5) in regulatory T (Treg) cells 0) is at least 1000-fold, for example at least 1500-fold, for example at least 1700-fold, for example at least 2000-fold, for example at least 2500-fold higher compared to the EC50 for STAT5 activation or signal transduction of wt IL-2 or either one of the IL-2v of SEQ ID NOs: 43 and 44. In some embodiments, the concentration at which IL-2v induces an EC50 for IL-2Rαβγ-mediated STAT5 activation or signal transduction (measured as STAT5 activation in CTLL2 cells, for example) is at least 2500-fold, for example at least 5000-fold, for example at least 7500-fold, for example at least 10,000-fold, for example at least 15,000-fold, for example at least 20,000-fold higher compared to the EC50 for STAT5 activation or signal transduction of wt IL-2 or either one of the IL-2v of SEQ ID NOs: 43 and 44. In some embodiments, IL-2v is the most The concentration (EC50) that induces 50% of the large proliferation is, for example, measured using KHYG-1 cells and is at least 10-fold, for example, at least 12-fold, for example, at least 15-fold, for example, at least 16-fold, for example, at least 18-fold, for example, at least 20-fold higher compared to the EC50 for the proliferation of wt IL-2 or any one of the IL-2v of SEQ ID NOs: 43 and 44. In some embodiments, IL-2v comprises 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 with respect to the IL-2v of SEQ ID NO: 44. In some embodiments, IL-2v comprises 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 position numbers are with respect to the IL-2v of SEQ ID NO: 44. In some embodiments, IL-2v comprises 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 position numbers are with respect to the IL-2v of SEQ ID NO: 44. In some embodiments, IL-2v does not contain amino acid substitutions at one or more positions selected from the group consisting of Y45, E61, E68, and L72. Some embodiments are that IL-2v does not contain amino acid substitutions at one or more or all of the positions selected from the group consisting of D20, Y45, E61, E68, V69, L72, A73, L80, R81, L85, L86, I87, I92, and Q126. Some embodiments are that IL-2v does not contain amino acid substitutions at one or more or all of the positions selected from the group consisting of H16, D20, E61, N88, and V91. In some embodiments, IL-2v is PEGylated. In one aspect, provided is a fusion protein. In some embodiments, the fusion protein comprises a serum half-life extending polypeptide operably linked to an interleukin-2 variant (IL-2v), wherein the IL-2v has at least 5 amino acids cleaved 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, albumin-binding protein or peptide, IgG, XTEN polypeptide, proline / alanine / serine rich (PAS) polypeptide, and 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 greater). In some embodiments, the 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 equivalent 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 the maximal signal transduction and transcriptional activation of signal transducer and activator of transcription 5 (STAT5) or signal transduction is at least 1000-fold, for example, at least 1500-fold, for example, at least 1700-fold, for example, at least 2000-fold, for example, at least 2500-fold higher compared to the EC50 for STAT5 activation or signal transduction 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. In some embodiments, the concentration at which the IL-2v fusion protein induces the EC50 of IL-2Rαβγ-mediated STAT5 activation or signal transduction (measured as STAT5 activation in CTLL2 cells, for example) is at least 2500-fold, for example, at least 5000-fold, for example, at least 7500-fold, for example, at least 10,000-fold, for example, at least 15,000-fold, for example, at least 20,000-fold higher compared to the EC50 for STAT5 activation or signal transduction of wt-IL2, or IL-2v of any one of SEQ ID NOs: 43 and 44, a fusion protein comprising Fc operably linked to wt IL2, or a fusion protein of any one of SEQ ID NOs: 117, 118, 161, or 162. In some embodiments, the concentration (EC50) at which the IL-2v fusion protein induces 50% of the maximal proliferation of natural killer (NK) cells, measured using, for example, KHYG-1 cells, is at least 10-fold, for example, at least 12-fold, for example, at least 15-fold, for example, at least 16-fold, for example, at least 18-fold, for example, at least 20-fold higher compared to the EC50 for the 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.In some embodiments, IL-2v comprises 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, wherein the position numbers relate to IL-2v of SEQ ID NO: 44. In some embodiments, IL-2v comprises 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, wherein the position numbers relate to IL-2v of SEQ ID NO: 44. In some embodiments, IL-2v comprises 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, wherein the position numbers relate to IL-2v of SEQ ID NO: 44. In some embodiments, IL-2v does not comprise amino acid substitutions 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 the human IgG1 isotype and includes one or more amino acid substitutions in the Fc region at residue positions 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, T394D, A330L, M252Y, S254T, T256E, M428L, N434S, T366W, T366S, L368A, F405L, Y407V, K409R, H435R, Y436F, and any combination thereof, wherein the residue numbering follows EU numbering. In some embodiments, the Fc region comprises the human IgG1 isotype and includes 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, wherein the residue numbering follows EU numbering. In some embodiments, the Fc region comprises the human IgG4 isotype and includes 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 the residue numbering follows EU numbering. In some embodiments, the Fc region comprises the human IgG4 isotype and includes 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 residue numbering follows EU numbering.In some embodiments, the Fc region contains, at the indicated positions (EU index numbering), the following amino acids: 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). In some embodiments, the Fc region contains, at the indicated positions (EU index numbering), the following amino acids: arginine at position 435 and phenylalanine at position 436. In some embodiments, the terminal Fc amino acid residue (e.g., K447) is removed or excluded. 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 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: 45-72. 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 order from the N-terminus to the C-terminus, 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, such as 5 to 25 amino acids, such as 15 to 25 amino acids. In some embodiments, the linker comprises 1 to 10 units, such as 1 to 5 units, such as 3 to 5 units of a poly-glycine serine linker 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 at least 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 the complex of the Fc receptor or 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] Further provided is a homodimer comprising two Fc-IL-2v fusion proteins as described above and herein.

[0007] In another aspect, provided is a heterodimer comprising: (i) the first Fc-IL-2v fusion protein described above and herein that includes a first Fc domain; and (ii) the second Fc-IL-2v fusion protein described above and herein that includes a second Fc domain. In another aspect, provided is a heterodimer comprising: (i) one (i.e., a single) Fc-IL-2v fusion protein described above and herein that includes a first Fc domain; and (ii) a second Fc domain that is, for example, empty or does not include a targeting moiety or antigen-binding domain. In some embodiments, the first Fc domain and the second Fc domain each have the following amino acid substitutions (EU numbering): T366W and T366S / L368A / Y407V; T366S / L368A / Y407V and T366W; T366W / S354C and T366S / L368A / Y407V / Y349C; T366S / L368A / 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 / D399V / 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. In some embodiments, one or both of the first Fc domain and the second Fc domain areAt the indicated positions (EU index numbering), it contains the following amino acids: tyrosine at position 252, threonine at position 254, and glutamate at position 256 (YTE); or leucine at position 428 and serine at position 434 (LS). In some embodiments, one or both of the first Fc domain and the second Fc domain contain, at the indicated positions (EU index numbering), the following amino acids: 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 contain the human IgG4 isotype and include 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, with residue numbering according to EU numbering. In some embodiments, one or both of the first Fc domain and the second Fc domain contain the human IgG1 isotype and include 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, with residue numbering according to EU numbering. In some embodiments, the terminal Fc amino acid residue (e.g., K447) is removed or excluded 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 each contain the amino acid sequence described below, or each has 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%comprising an amino acid sequence that is at least 99% identical: SEQ ID NO: 45 and 46; SEQ ID NO: 47 and 48; SEQ ID NO: 49 and 46; SEQ ID NO: 45 and 51; SEQ ID NO: 49 and 51; SEQ ID NO: 52 and 48; SEQ ID NO: 47 and 53; SEQ ID NO: 52 and 53; SEQ ID NO: 54 and 46; SEQ ID NO: 45 and 55; SEQ ID NO: 54 and 55; SEQ ID NO: 56 and 48; SEQ ID NO: 47 and 50; SEQ ID NO: 56 and 50; SEQ ID NO: 57 and 58; SEQ ID NO: 59 and 60; SEQ ID NO: 61 and 58; SEQ ID NO: 57 and 62; SEQ ID NO: 63 and 64; SEQ ID NO: 65 and 60; SEQ ID NO: 59 and 66; SEQ ID NO: 67 and 68; SEQ ID NO: 69 and 58; SEQ ID NO: 57 and 70; SEQ ID NO: 69 and 70; SEQ ID NO: 71 and 60; SEQ ID NO: 59 and 72; or SEQ ID NO: 71 and 72. In some embodiments, the heterodimer is an amino acid sequence selected from the group consisting of SEQ ID NOs: 75-116, 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: 75-116, and an amino acid sequence selected from the group consisting of SEQ ID NOs: 46, 51, and 55, 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, 51, and 55, and a second Fc region. In some embodiments, the heterodimer is (i) the first amino acid sequence described 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 described below, and (ii) the second amino acid sequence described below, or an amino acid sequence that is at least 80%,A human IgG4 Fc-IL-2v fusion protein comprising: 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, respectively, to: 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 is (i) the first amino acid sequence described 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 described below, and(ii) a second Fc region comprising the second amino acid sequence described 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 described below: a human IgG4 Fc-IL-2v fusion protein, each comprising: 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. In some embodiments, the heterodimer is (i) the 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) 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, and a second Fc region comprising: a human IgG4 Fc-IL-2v fusion protein. In some embodiments, the heterodimer is an amino acid sequence selected from the group consisting of SEQ ID NOs: 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 at least 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 119-160, and a human IgG1 Fc-IL-2v fusion protein, and an amino acid sequence selected from the group consisting of SEQ ID NOs: 58, 62, and 70, 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%,comprising a second Fc region having an amino acid sequence that is at least 97%, at least 98%, or at least 99% identical. In some embodiments, the heterodimer comprises (i) the first amino acid sequence described 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 described below, and (ii) the second amino acid sequence described 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%, A human IgG1 Fc-IL-2v fusion protein, each comprising a second Fc region comprising an amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical: 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 (i) the first amino acid sequence described 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 described below, and (ii) the second amino acid sequence described 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 described below, and a second Fc region comprising the same; 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 the complex of the Fc receptor or 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 Fc domain 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, 24 hours, for example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days or more. In some embodiments, the 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 ( VSIR, B7H5, VISTA); Immunoglobulin superfamily member 11 (immunoglobulin superfamily, IGSF11, VSIG3); Natural killer cell cytotoxicity receptor 3 ligand 1 (natural killer cell cytotoxicity receptor 3 ligand, NCR3LG1, B7H6); HERV-H LTR-associated 2 (HERV-H LTR-associating, HHLA2, B7H7); Inducible T cell co-stimulator (inducible T cell co-stimulator, ICOS, CD278); Inducible T cell co-stimulator ligand (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 (MHC class I polypeptide-related sequence A, MICA); MHC class I polypeptide peptide-related sequence B (MHC class I polypeptide-related sequence B, MICB); CD274 (CD274, PDL1, PD-L1); Programmed cell death 1 (programmed cell death, 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 , 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, 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 (ABC) family transporter (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 transporter (e.g., , solute carrier family 10 member 1 (SLC10A1; also known as Sodium-taurocholate Co-transporting Polypeptide (NTCP)); peptide (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 (solute carrier organic anion transporter, 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 (transferrin receptor, TFR2, TFRC 2) and binds to a target selected from the group consisting of HBV epitopes (e.g., HBV core 18 - 27; env181 - 193; env335 - 343; pol575 - 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 simplex virus (HSV) glycoprotein B (glycoprotein B, gB), glycoprotein C (glycoprotein C, gC), glycoprotein D (glycoprotein D, gD), and glycoprotein E (glycoprotein E, gE). In some embodiments, the antigen - binding domain is CD19; membrane - spanning 4 - domains A1 (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 (interleukin 3 receptor subunit alpha, IL3RA), prominin 1 (prominin, PROM1; CD133); TNFRSF9 (CD137); syndecan 1 (syndecan 1, SDC1; CD138); CD200 molecule (CD200); alpha fetoprotein (alpha fetoprotein, AFP), BAG cochaperone 6 (BAG6); MET oncogene, receptor tyrosine kinase (MET); KIT oncogene, receptor tyrosine kinase (KIT); C-type lectin domain family 12 member A (C-type lectin domain family 12 member A, CLEC12A; CD371) ; C-type lectin domain containing 9A (C-type lectin domain containing 9A, CLEC9A; CD370); cadherin 3 (cadherin, CDH3); carbonic anhydrase 6 (carbonic anhydrase, CA6); carbonic anhydrase 9 (CA9); cancer fetal antigen-related cell adhesion molecule 3 (carcinoembryonic antigen related cell adhesion molecule, CEACAM3); cancer fetal antigen-related cell adhesion molecule 5 (CEACAM5); cancer fetal antigen-related cell adhesion molecule 6 (CEACAM6); chorionic somatomammotropin hormone 1 (chorionic somatomammotropin hormone, CSH1; coagulation factor III, tissue factor (F3); collectin 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 α (FAP); Fibro blast 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 (ITGB7); Leukocyte immunoglobulin-like rece Receptor B1 (leukocyte immunoglobulin like receptor B, LILRB1; ILT2); Leukocyte immunoglobulin-like receptor B2 (LILRB2; ILT4); LY6 / PLAUR domain containing 3 (LY6 / PLAUR domain containing, LYPD3); Glypican 3 (glypican, GPC3); KRAS 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 (mesothelin, MSLN); Mucin 1 (mucin, MUC1), and its splice variants (including, for example, MUC1 / A, C, D, X, Y, Z, and REP); Mucin 16 (MUC16; CA125); Natural killer cell 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 (promyelocytic leukemia, PML); Protein tyrosine Kinase 7 (inactive) (protein tyrosine kinase, 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 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 Signal Transducer 2 (TACSTD2); Fucosyl GM1; Sialyl Lewis adhesion molecule (sLe); and binds to a target selected from the group consisting of Lewis Y antigen or a tumor associated antigen (TAA). In some embodiments, the antigen binding domain binds to an epitope of a target antigen or tumor associated antigen (TAA) presented by a major histocompatibility complex (MHC) molecule. In some embodiments, the TAA is a cancer testis antigen. In some embodiments, the cancer testis antigen is an acrosin binding protein (AC Retinol binding protein (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), 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 (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), Insulin like growth factor 2 mRNA binding protein 3 (IGF2BP3), leucine zipper protein 4 (LUZP4), lymphocyte antigen 6 family member K (LY6K), 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; alias, MPHOSPH1), NDC80 kinetochore complex NUF2 component (NU F2), kinesin family member 20B (KIF20B; alias, MPHOSPH1), NDC80 kinetochore complex NUF2 component (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 X binding family member A1 (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 (SYCP3), testis expressed 14 cell junction forming factor (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) selected from the group consisting of.

[0008] In a further aspect, provided is a conjugate comprising the above-described and herein-described IL-2v, the above-described and herein-described Fc-IL-2v fusion protein, the above-described and herein-described homodimer, or the above-described and herein-described heterodimer, conjugated to a therapeutic agent. In some embodiments, the therapeutic agent is covalently conjugated to, for example, IL-2v, an Fc-IL-2v fusion protein, a homodimer or a 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 an agonist or activator of a pattern recognition receptor (PRR), such as a Toll-like receptor (TLR), a RIG-I-like receptor (RLR), an N OD-like receptor (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 gene (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 besifloxacin (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 selgantolimod (GS-9688) and NKTR-262 (dual TLR7 / TLR8 agonist).

[0009] In a further aspect, provided is a polynucleotide encoding the IL-2v described above and herein, the Fc-IL-2v fusion protein described above and herein, or the 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 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 a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 175-212. Further provided is a polynucleotide or polynucleotides encoding the heterodimeric Fc-IL-2v fusion protein and a second Fc region 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 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 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 comprises a nucleic acid selected from the group consisting of SEQ ID NOs: 214-215, or a nucleic 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 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. Further 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 the polynucleotide or polynucleotides described above and herein, or an expression cassette. 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 from a viral family selected from the group consisting of Adenoviridae (e.g., adenovirus), Arenaviridae (e.g., lymphocytic choriomeningitis mammarenavirus, Calomys callosus arenavirus (synonymous with Pichinde mammarenavirus)), 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 , NDV)), Rhabdoviridae (e.g., Vesicular stomatitis virus (VSV)), Togaviridae (e.g., alphavirus, Sindbis virus), and Enteroviridae (e.g., echovirus). Further provided is a lipoplex, e.g., a lipid nanoparticle (LNP), comprising the polynucleotide or polynucleotides described above and herein, an expression cassette, or a vector.

[0011] In a further aspect, provided is a cell or cell population comprising the polynucleotide or polynucleotides, expression cassette, or vector described above and herein, wherein the cell or cell population expresses the IL-2v, 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 fetal kidney cell.

[0012] In a further aspect, provided is a method for generating an Fc-IL-2 fusion protein heterodimer. In some embodiments, the method comprises: (a) culturing a cell or cell population described hereinabove and herein, which is transformed with at least one polynucleotide or a plurality of polynucleotides encoding an Fc-IL-2v fusion protein described herein, or an expression cassette or a plurality of expression cassettes described herein, in cell culture under conditions sufficient to express an 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 isolation or purification step comprises protein A chromatography. In some embodiments, the isolation or purification step further comprises in-stream pH neutralization or immediate pH neutralization of the protein A chromatography eluate. In some embodiments, the isolation or purification 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 heterodimer molecules are isolated or purified (e.g., in a monodisperse form). In some embodiments, at least 95%, 96%, 97%, 98%, 99% or more of the Fc-IL-2 fusion protein heterodimer molecules are isolated or purified (e.g., in a monodisperse form). In some embodiments, at least 98%, 99% or more of the Fc-IL-2 fusion protein heterodimer molecules are isolated or purified (e.g., in a monodisperse form). In some embodiments, the cell or cell population is cultured in a culture volume of at least 2 L, such as 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 the IL-2v, Fc-IL-2v fusion protein, homodimer, heterodimer, conjugate, polynucleotide or plurality of 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 comprises IL-2v, Fc-IL-2v fusion protein, homodimer, heterodimer, and / or conjugate at a concentration in the range of 0.05 mg / ml to 50 mg / ml, such as 0.05 mg / ml to 20 mg / ml, such as 0.1 mg / ml to 40 mg / ml, such as 1.0 mg / ml to 30 mg / ml, such as 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, 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 second and third therapeutic agents.

[0014] In a further aspect, it is a method for inducing an immune response against human hepatitis B virus (HBV) in a subject in need thereof. Further provided is a method 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 the IL-2v, Fc-IL-2v fusion protein, homodimer, heterodimer, conjugate, polynucleotide or plurality of polynucleotides, expression cassette, vector, lipoplex (e.g., LNP), or pharmaceutical composition described herein. In some embodiments, the subject is infected with HBV, suspected of being infected with HBV, or at risk of infection 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 experiences one or more conditions 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, visible reticulated dilated blood vessels in the skin, dark (e.g., orange or brown) urine, pale feces, fever, persistent malaise, discomfort, abdominal pain, ascites, loss of appetite, nausea, and vomiting. In some embodiments, the subject is coinfected 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), , Entecavir (ETV), Telbivudine (LdT), Tenofovir disoproxate Xylofumarate (tenofovir disoproxil fumarate, TDF), tenofovir alafen In some embodiments, the method further comprises the administration 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 cytosine 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 RNA ... iRNA, DNA-directed RNA interference (ddRNA) i)) co-administering one or more therapeutic agents selected from the group consisting of a gene editor targeting HBV (e.g., CRISPR-Cas (e.g., Cas9, Cas12, Cascade, Cas13), zinc finger nuclease, homing endonuclease, homing megal nuclease (e.g., ARCUS), synthetic nuclease, TALEN), a covalently closed circular DNA (cccDNA) inhibitor, an HBsAg secretion or assembly inhibitor, an HBV viral invasion inhibitor, and CAR-T and T cell bispecificity (redirected T cells).

[0015] In a further aspect, provided is a method of activating a latent viral reservoir in a subject infected with human immunodeficiency virus (HIV). Further provided is a method of doing so in a subject in need of treating and / or preventing human immunodeficiency virus (HIV). 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 plurality of polynucleotides, expression cassette, vector, lipoplex (e.g., LNP), or pharmaceutical composition described herein. In some embodiments, the method further comprises administering an additional therapeutic agent to the subject. In some embodiments, the method further comprises administering one or more anti-HIV broadly neutralizing antibodies to the subject. 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) a high mannose patch comprising a third variable loop (V3) and / or N332 oligomannose glycan, (ii) a second variable loop (V2) and / or Env trimer apex, (iii) a CD4 binding site (CD4bs), (iv) a gp120 / gp41 interface, or (v) a silent face of gp120.In some embodiments, 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 containing the N332 oligomannose glycan and compete with or comprise the VH and VL regions from antibodies 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, 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, one or more anti-HIV broadly neutralizing antibodies bind to an epitope or region of gp120 in the second variable loop (V2) and / or at the Env trimer apex and compete with or comprise the VH and VL regions from antibodies 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, one or more anti-HIV broadly neutralizing antibodies bind to an epitope or region of gp120 at the CD4 binding site (CD4bs), and compete with or comprise the VH and VL regions from antibodies selected from the group consisting of 3BNC117, GS-9723, 3BNC60, b12, F105, VRC01, VRC07, VRC07-523, VRC03, VRC06, VRC06b01 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, one or more anti-HIV broadly neutralizing antibodies bind to an epitope or region of gp120 at the gp120 / gp41 interface, and compete with or comprise the VH and VL regions from antibodies selected from the group consisting of PGT-151, CAP248-2B, 35O22, 8ANC195, ACS202, VRC34, and VRC34.01. In some embodiments, one or more anti-HIV broadly neutralizing antibodies bind to an epitope or region of the silent face of gp120, and compete with or comprise the VH and VL regions from antibodies selected from VRC-PG05 and SF12. In some embodiments, one or more anti-HIV broadly neutralizing antibodies bind to an epitope or region of gp41 in the membrane proximal region (MPER). In some embodiments, one or more anti-HIV broadly neutralizing 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 antibodies selected from the group consisting of 10E8, 10E8v4, 10E8-5R-100cF, 4E10, DH511.11P, 2F5, 7b2, and LN01. In some embodiments, one or more anti-HIV broadly neutralizing antibodies bind to an epitope or region of the gp41 fusion peptide, and compete with or comprise the VH and VL regions from antibodies selected from the group consisting of VRC34 and ACS202.In some embodiments, the subject has not received antiretroviral therapy (ART) or A. RT is interrupted prior to administration of IL-2v, Fc-IL-2v fusion protein, homodimer, heterodimer, conjugate, polynucleotide or plurality of polynucleotides, expression cassette, vector, lipoplex (e.g., LNP), or pharmaceutical composition. In some embodiments, ART is interrupted after one or more administrations of IL-2v, Fc-IL-2v fusion protein, homodimer, heterodimer, conjugate, polynucleotide or plurality of polynucleotides, expression cassette, vector, lipoplex (e.g., LNP), or pharmaceutical composition. In some embodiments, the method further comprises administering to the subject one or more antiretroviral therapy (ART) agents. In some embodiments, the subject is chronically infected with HIV.

[0016] In a further aspect, provided is a method of doing so in a subject in need of enhancing, improving, and / or increasing a response to vaccine therapy. In some embodiments, the vaccine enhancement method comprises (1) co-administering to the subject an effective amount of an IL-2v, Fc-IL-2v fusion protein, homodimer, heterodimer, conjugate, polynucleotide or plurality of 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 antiviral vaccines, antibacterial vaccines, and anticancer vaccines. In some embodiments, the vaccine is hepatitis A virus (HAV), hepatitis B virus (HBV), human immunodeficiency virus (HIV), cytomegalovirus (CMV), herpes simplex virus (HSV), Epstein-Barr virus (EBV), human ortho 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., beta coronavirus, e.g., severe acute respiratory syndrome-related coronavirus, e.g., SARS-CoV2). In some embodiments, the vaccine includes an antibacterial vaccine against a bacterium selected from the group consisting of mycobacterium tuberculosis, whooping cough, tetanus, diphtheria, meningococcus, pneumococcus, Haemophilus influenza, cholera, typhoid fever, and Bacillus anthracis. In some embodiments, the method includes a prime-boost regimen that includes administering a priming composition at a first time point and one or more boosting compositions at one or more subsequent time points. In some embodiments, the priming composition includes an IL-2v, Fc-IL-2v fusion protein, homodimer, heterodimer, conjugate, polynucleotide or plurality of polynucleotides, expression cassette, vector, lipoplex (e.g., LNP), or pharmaceutical composition as described herein. In some embodiments, one or more boosting compositions include an IL-2v, Fc-IL-2v fusion protein, homodimer, heterodimer, conjugate, polynucleotide or plurality of polynucleotides, expression cassette, vector, lipoplex (e.g., LNP), or pharmaceutical composition as described herein. In some embodiments, the priming composition and the boosting composition are the same. In some embodiments, the priming composition and the boosting composition are different. In some embodiments, the method includes a prime-boost regimen that includes administering a priming composition at a first time point and one or more boosting compositions at one or more subsequent time points. In some embodiments, the priming composition includes an IL-2v, Fc-IL-2v fusion protein, homodimer, heterodimer, conjugate, polynucleotide or plurality of polynucleotides, expression cassette, vector, lipoplex (e.g., LNP), or pharmaceutical composition as described herein. In some embodiments, one or more boosting compositions include an IL-2v, Fc-IL-2v fusion protein, homodimer, heterodimer, conjugate, polynucleotide or plurality of polynucleotides, expression cassette, vector, lipoplex (e.g., LNP), or pharmaceutical composition as described herein. In some embodiments, the priming composition and the boosting composition are the same. In some embodiments, the priming composition and the boosting composition are different.

[0017] In a further aspect, provided is a method of doing so in a subject in need of preventing, reducing, and / or inhibiting the recurrence, growth, proliferation, migration, and / or metastasis of cancer cells or a cancer cell population. In some embodiments, the anti-cancer method comprises administering to the subject a therapeutically effective amount of the IL-2v, Fc-IL-2v fusion protein, homodimer, heterodimer, conjugate, polynucleotide or plurality of 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 agents or chemotherapeutic agents. In some embodiments, the one or more anti-neoplastic agents 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, dicycloplatin, eptaplatin, lobaplatin, miltiplatin), dihydrofolate reductase (DHFR) inhibitors (e.g., methotrexate, trimethoprim, pemetrexed), topoisomerase inhibitors (e.g., doxorubicin, daunorubicin, dactinomycin, eniposide, epirubicin, etoposide, idarubicin, irinotecan, mit platin, dicycloplatin, eptaplatin, lobaplatin, miltiplatin), dihydrofolate reductase (DHFR) inhibitors (e.g., methotrexate, trimethoprim, pemetrexed), topoisomerase inhibitors (e.g., doxorubicin, daunorubicin, dactinomycin, eniposide, epirubicin, etoposide, idarubicin, irinotecan, mit oxantrone, mitomycin C, topotecan, teniposide, amsacrine), alkylating agents (e.g., cyclophosphamide, ifosfamide, melphalan, chlorambucil, busulfan, thiotepa, carmustine, lomustine, semustine, streptozocin, dacarbazine, procarbazine, temozolomide), anti-metabolites (e.g., methotrexate, fluorouracil, gemcitabine, cytarabine, cladribine, pentostatin, fludarabine, pemetrexed), vinca alkalides (e.g., vincristine, vinblastine, vinorelbine, vindesine), corticosteroids (e.g., prednisone, prednisolone, dexamethasone, methylprednisolone), monoclonal antibodies (e.g., rituximab, trastuzumab, bevacizumab, cetuximab, panitumumab), tyrosine kinase inhibitors (e.g., imatinib, gefitinib, erlotinib, lapatinib, sunitinib, sorafenito Xanthron, pixantrone, sobuzoxane, topotecan, irinotecan, MM-398 (liposomal irinotecan), bosaloxicin and GPX-150, aldoxorubicin, AR-67, mavacertinib, AST-2818, avitinib (ACEA-0010), irofulven (MGI-114)), alkylating agents (e.g., nitrogen mustard (e.g., cyclophosphamide, chlorambucil, uramustine or uracil mustard, melphalan, chlorambucil, ifosfamide, bendamustine, temozolomide, carmustine), nitrosourea (e.g., carmustine, lomustine, streptozocin), alkyl sulfonate (e.g., busulfan)), and mixtures thereof. In some embodiments, IL-2v, Fc-IL-2v fusion protein, homodimer, heterodimer, conjugate, polynucleotide, vector, lipoplex (e.g., LNP), and / or pharmaceutical composition are co-administered with FOLFOX regimen, FOLFIRI regimen, FOLFOXIRI regimen, or FOLFIRINOX regimen. In some embodiments, IL-2v, Fc-IL-2v fusion protein, homodimer, heterodimer, conjugate, polynucleotide, vector, lipoplex (e.g., LNP), and / or pharmaceutical composition are 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); alpha-fetoprotein (AFP), BAG cochaperone 6 (BAG6); MET oncogene, receptor tyrosine kinase (MET);KIT oncogene, receptor tyrosine kinase (KIT); C-type lectin domain family member 12A (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 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 (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 beta7 (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 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 (including, for example, MUC1 / A, C, D, X, Y, Z, and REP); mucin 16 (MUC16; CA125); natural killer cell 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); suppression 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);One or more antibodies or their antigen-binding antibody fragments or their antibody-drug conjugates, CD3-targeted bispecific molecules, NK cell-activating receptor-targeted bispecific molecules, or non-immunoglobulin antigen-binding domains, or antibody mimetic proteins directed against one or more targets or tumor-associated antigens (TAAs) selected from the group consisting of triggering receptor expressed on myeloid cells 2 (TREM2); trophoblast glycoprotein (TPBG); trophinin (TRO); tumor-associated calcium signal transducer 2 (TACSTD2); fucosyl GM1; sialyl Lewis adhesion molecule (sLe); and Lewis Y antigen, are co-administered with an immunotherapy. In some embodiments, the one or more antibodies or their antigen-binding antibody fragments or their antibody-drug conjugates, CD3-targeted bispecific molecules, NK cell-activating receptor-targeted bispecific molecules, or non-immunoglobulin antigen-binding domains, or antibody mimetic proteins bind to epitopes of targets or tumor-associated antigens (TAAs) presented by major histocompatibility complex (MHC) molecules. 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, IL-2v, Fc-IL-2v fusion protein, homodimer, heterodimer, conjugate, polynucleotide, vector, lipoplex (e.g., LNP), and / or pharmaceutical composition is natural killer (NK) cells, NK-T cells, T cells, cytokine-induced killer (CIK) cells, macrophage (MAC) cells, tumor infiltrating lymphocyte (TIL), and; one or more cell therapies selected from the group consisting of dendritic cells (DC) are the same It is administered at the right time. In some embodiments, one or more cell therapies include 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, one or more cell therapies include an NK cell therapy, for example, including NK-92 cells. In some embodiments, one or more cell therapies include cells that are autologous, syngeneic, or allogeneic to the subject. In some embodiments, one or more cell therapies include cells that include a chimeric antigen receptor (CAR). In some embodiments, the cells in the cell therapy are 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); Alpha-fetoprotein (AFP), BAG chaperone 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 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; 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 beta-7 (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 (including, for example, MUC1 / A, C, D, X, Y, Z, and REP); Mucin 16 (MUC16; CA125); Natural killer cell cytotoxicity receptor 3 ligand 1 (NCR3LG1; B7-H6); Necudin, 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); Suppression 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 signal transducer 2 (TACSTD2); Fucosyl GM1; Sialyl Lewis adhesion molecule (sLe); and Lewis Y antigen, binds to a target or tumor-associated antigen (TAA) selected from the group consisting of. In some embodiments, the cells of the cell therapy bind to an epitope of a target or tumor-associated antigen (TAA) presented by 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);Selected from the group consisting of 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), NDC80 kinetochore complex NUF2 component (NUF2), nuclear RNA transport factor 2 (NXF2), PAS domain-containing repressor 1 (PASD1), PDZ-binding kinase (PBK), piwi-like RNA-mediated gene silencing 2 (PIWIL-2), melanoma-prioritized antigen (PRAME), sperm-associated antigen 9 (SPAG9), nuclear X-binding 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 structure 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, 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, IL-2v, Fc-IL-2v fusion protein, homodimer, heterodimer, conjugate, polynucleotide, vector, lipoplex (e.g., LNP), and / or pharmaceutical composition is 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 (vascular endothelial growth factor, 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), cyclin-dependent kinase 4 (cyclin dependent kinase, CDK4), cyclin-dependent kinase 6 (CDK6), TNF receptor superfamily member 14 (TNFRSF14, HVEM, CD270), C-C motif chemokine receptor 2 (C-C motif chemokine receptor, CCR2, CD192), C-C motif chemokine receptor 5 (C CR5, CD195), C-C motif chemokine receptor 8 (CCR8, CDw198), C-C motif chemokine receptor 2 (CXCR2, CD182), C-C motif chemokine receptor 3 (CXCR3, CD182, CD183), C-C motif chemokine receptor 4 (CXCR4, CD184), arginase (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, PTGS1, COX-1), prostaglandin-endoperoxide synthase 2 (PTGS2, COX-2), secreted phospholipase A2, prostaglandin E synthase (prostaglandin E synthase, PTGES, PGES), arachidonate 5-lipoxygenase (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 (hypoxia inducible factor 1 subunit alpha, HIF1A), a ngiop oietin 1 (angiopoietin, ANGPT1), endothelial TEK tyrosine kinase (TIE-2, TEK), Janus kinase 1 (Janus kinase, JAK1), catenin beta 1 (catenin It is co-administered with one or more additional therapeutic agents comprising an inhibitor or antagonist of 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 an 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, IL-2v, an Fc-IL-2v fusion protein, a homodimer, a heterodimer, a conjugate, a polynucleotide, a vector, a lipoplex (e.g., LNP), and / or a pharmaceutical composition is co-administered with an oncolytic virus vector.In some embodiments, the oncolytic virus vector comprises a DNA virus or an RNA virus. In some embodiments, the virus vector is from a viral family selected from the group consisting of Adenoviridae (e.g., adenovirus), Arenaviridae (e.g., lymphocytic choriomeningitis mammarenavirus, Calomys callosus arenavirus (also known as Pichinde mammarenavirus)), 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 from cancer.In some embodiments, the subject is a blood cancer, such as 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), undifferentiated 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 (DLBCL), marginal zone lymphoma (MZL), Waldenstrom's macroglobulinemia (WM)), and / or myeloma (e.g., multiple myeloma (M has M). In some embodiments, the subject has a solid tumor. In some embodiments, the tumor is a malignant tumor. In some embodiments, the cancer is a metastatic tumor. In some embodiments, the subject has a cancer selected from the group consisting of epithelial tumors (e.g., carcinoma, squamous cell carcinoma, basal cell carcinoma, squamous intraepithelial neoplasia), ductal tumors (e.g., adenocarcinoma, adenoma, adenomyoma), mesenchymal or soft tissue tumors (e.g., sarcoma, rhabdomyosarcoma, leiomyosarcoma, liposarcoma, fibrosarcoma, dermatofibrosarcoma, neurofibrosarcoma, fibrous histiocytoma, angiosarcoma, angioleiomyoma, leiomyoma, chondroma, chondrosarcoma, cystic soft part sarcoma, epitheloid hemangioendothelioma, Spitz tumor, synovial sarcoma), and lymphomas. In some embodiments, the subject has a cancer of bone (e.g., ameloblastic carcinoma, aneurysmal bone cyst, angiosarcoma, chondroblastoma, chondroma, chondromyxofibroma, chondrosarcoma, chordoma, dedifferentiated chondrosarcoma, enchondroma, epitheloid hemangioendothelioma, fibrous dysplasia, giant cell tumor of bone, hemangioma and related lesions, osteoblastoma, osteochondroma, osteosarcoma, osteoid osteoma, osteoma, perichondroma, tenosynovial giant cell tumor, Ewing sarcoma); lip and oral cavity (e.g., odontogenic ameloblastic carcinoma, oral leukoplakia, oral squamous cell carcinoma, primary oral mucosal melanoma); salivary gland (e.g., pleomorphic adenoma of salivary gland, adenoid cystic carcinoma of salivary gland, mucoepidermoid carcinoma of salivary gland, Warthin tumor of salivary gland); esophagus (e.g., Barrett esophagus, dysplasia, and adenocarcinoma); digestive tract (stomach (e.g., gastric adenoma, primary gastric lymphoma, gastrointestinal stromal tumor (GIST), metastatic deposit, gastric carcinoid, gastric sarcoma, neuroendocrine cancer, primary squamous cell carcinoma of the stomach, 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)-related serous cystic neoplasms, serous cystadenocarcinoma); mucinous cystic neoplasms (MCN), intraductal papillary mucinous neoplasm: IPMN), intraductal oncocytic papillary neoplasm (IOPN), intraductal tubular tumor, cystic glandular papillary neoplasm ( including acinar cell cystadenoma, acinar cell cystadenocarcinoma, pancreatic adenocarcinoma), invasive ductal adenocarcinoma (including tubular adenocarcinoma, adenoid squamous cell 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, gastrin-producing tumor, glucagon-producing tumor, serotonin-producing NET, somatostatin-producing tumor, VIP-producing tumor, solid-pseudopapillary neoplasm (SPN), pancreatoblastoma); gallbladder (For example, cancers of the gallbladder and extrahepatic bile ducts, intrahepatic bile duct cancer); neuroendocrine (for example, adrenocortical cancer, carcinoid tumor, pheochromocytoma, pituitary adenoma); thyroid (for example, poorly differentiated (undifferentiated) cancer tumor, medullary cancer, oncocytic tumor, papillary cancer, adenocarcinoma); liver (for example, adenoma, mixed hepatocellular and cholangiocarcinoma, fibrolamellar cancer tumor, hepatoblastoma, hepatocellular carcinoma, mesenchymal tumor, nested stromal epithelial tumor, undifferentiated cancer; hepatocellular carcinoma, intrahepatic bile duct cancer, cholangiocystadenocarcinoma, epithelioid hemangioendothelioma, angiosarcoma, fetal sarcoma, rhabdomyosarcoma, solitary fibrous tumor, teratoma, yolk sac tumor, carcinosarcoma, rhabdoid tumor); kidney (for example, ALK-rearranged renal cell carcinoma, chromophobe renal cell carcinoma, clear cell renal carcinoma, clear cell sarcoma, metanephric adenoma, metanephric adenofibroma, mucinous tubular and spindle cell carcinoma, renal tumor, nephroblastoma (Wilms tumor), papillary adenoma, papillary renal cell carcinoma, renal oncocytoma, renal cell carcinoma, succinate dehydrogenase-deficient renal cell carcinoma, collecting duct carcinoma); breast (for example, invasive ductal carcinoma (including lobular cell carcinoma, adenoid cystic carcinoma, apocrine adenocarcinoma, cribriform carcinoma, glycogen-rich / clear cell, inflammatory carcinoma, lipid-rich cancer tumor, medullary carcinoma, metaplastic carcinoma, micropapillary carcinoma, mucinous carcinoma, neuroendocrine carcinoma, malignant phyllodes tumor, papillary carcinoma, sebaceous carcinoma, secretory carcinoma, tubular carcinoma, but not limited to these); lobular carcinoma (pleomorphic cell carcinoma, signet ring cell carcinoma, peritoneum (for example, mesothelioma; primary peritoneal cancer);Female genital tissues (ovary (e.g., choriocarcinoma, epithelial tumor, germ cell tumor, sex cord-stromal tumor), fallopian tube (e.g., serous adenocarcinoma, mucinous carcinoma, endometrioid adenocarcinoma, clear cell adenocarcinoma, transitional cell carcinoma, squamous cell carcinoma, undifferentiated carcinoma, müllerian tumor, adenosarcoma, leiomyosarcoma, teratoma, germ cell tumor, choriocarcinoma, chorioma), uterus (e.g., cervical cancer, endometrial polyp, endometrial hyperplasia, intraepithelial carcinoma (EIC), endometrial cancer (e.g., endometrioid cancer, serous cancer, clear cell cancer, mucinous cancer, squamous cell cancer, transitional cancer, small cell cancer, undifferentiated cancer, mesenchymal neoplasm), leiomyoma (e.g., endometrial stromal nodule, leiomyosarcoma, endometrial stromal sarcoma (ESS), mesenchymal tumor), mixed epithelial and mesenchymal tumor (e.g., adenofibroma; fibroma, carcinofibroma, adenosarcoma, carcinosarcoma (malignant mixed mesodermal sarcoma (MMMT)), endometrial stromal tumor, endometrial malignancy müllerian duct mixed tumor, gestational chorioma (partial hydatiform mole, complete hydatiform mole, invasive hydatiform mole, placental site tumor)), vulva, 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 (non-invasive, low-grade, poorly differentiated, glioblastoma; oligodendroglioma, ependymoma included), meningioma, ganglioglioma, schwannoma (neurilemmoma), craniopharyngioma, chordoma, non-Hodgkin lymphoma (NHL), indolent non-Hodgkin's lymphoma (iNHL), refractory iNHL, pituitary tumor); eye (e.g., retinocytoma, retinoblastoma, intraocular melanoma, posterior uveal melanoma, iris nevus); head and neck (e.g., nasopharyngeal carcinoma, endolymphatic sac tumor (ELST), epidermoid carcinoma, laryngeal carcinoma (squamous cell carcinoma (SCC) (e.g., glottic carcinoma, supraglottic carcinoma, subglottic carcinoma, combined laryngeal carcinoma), in situ carcinoma, verrucous, spindle cell, and basaloid SCC, undifferentiated carcinoma, laryngeal adenocarcinoma, adenoid cystic carcinoma, neuroendocrine carcinoma, laryngeal sarcoma) included), paraganglioma of head and neck (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 carcinoma (NSCLC) (squamous cell carcinoma (SCC) SCC), adenocarcinoma, and large cell carcinoma, carcinoid (typical or atypical), carcinosarcoma, pulmonary blastoma, giant cell carcinoma, spindle cell carcinoma, pleuropulmonary blastoma); lymph (e.g., lymphoma (including Hodgkin lymphoma, non-Hodgkin lymphoma (NHL), indolent non-Hodgkin lymphoma (iNHL), refractory iNHL), Epstein-Barr virus (EBV)-associated lymphoproliferative disorders (B cell lymphoma and T cell lymphoma (e.g., including Burkitt lymphoma); large cell type B cell lymphoma, diffuse large cell type B cell lymphoma (DLBCL), mantle cell lymphoma, chronic B cell lymphoma, low-grade B cell lymphoma, fibrin-related diffuse large cell type lymphoma; primary humoral 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 (CNS) (e.g., glioma (astrocytoma (e.g., pilocytic astrocytoma, pilomyxoid astrocytoma, subependymal giant cell astrocytoma, pleomorphic xanthoastrocytoma, diffuse astrocytoma, fibrous astrocytoma, large round cell astrocytoma, protoplasmic astrocytoma, anaplastic astrocytoma, glioblastoma (e.g., giant cell glioblastoma, gliosarcoma, multiform glioblastoma), and cerebral gliomatosis), oligodendroglioma (e.g., oligodendroglioma, anaplastic oligodendroglioma), oligodendroglial astrocytic tumors (e.g., oligodendroglial astrocytoma, anaplastic oligodendroglial astrocytoma), ependymal tumor (e.g., subependymal tumor, myxopapillary ependymoma, ependymoma (e.g., cellular, papillary, clear cell, tanycytic ependymoma), anaplastic ependymoma), optic nerve glioma, and non-glioma (e.g., choroid plexus tumor, neuronal and mixed neuronal-glial tumors, pineal region tumor, germinoma, medulloblastoma, meningioma, primary CNS lymphoma, embryonal cell tumor, pituitary adenoma, cranial and paraspinal nerve tumors, stellar region tumors)); neurofibroma, meningioma, peripheral nerve sheath tumor, neuroblastoma Group tumors (including, but not limited to, neuroblastoma, ganglioneuroblastoma, ganglioneuroma); trisomy 19-related tumors; neuroendocrine tissues (e.g., preganglionic system (including adrenal medulla (pheochromocytoma), and extra-adrenal paraganglia (extra-adrenal paraganglioma)); skin (e.g., clear cell hidradenoma, cutaneous benign fibrous histiocytoma, cylindroma, hidradenoma, melanoma (cutaneous melanoma, mucosal melanoma), trichoblastoma, Spitz tumor); and soft tissues (e.g., aggressive angiomyxoma, alveolar rhabdomyosarcoma, cellular soft tissue sarcoma, angiofibroma, angiomatoid fibrous histiocytoma, synovial sarcoma, biphasic synovial sarcoma, clear cell sarcoma, dermatofibrosarcoma protuberans, desmoid-type fibromatosis, small round cell tumor, fibromatosis with fibroblastic small round cells, elastofibroma, fetal rhabdomyosarcoma, Ewing sarcoma / primitive neurectodermal tumor (PNET), extraskeletal myxoid chondrosarcoma, extraskeletal osteosarcoma, paravertebral sarcoma, inflammatory myofibroblastic tumor, lipoblastoma, lipoma, chondroid lipoma, liposarcoma / malignant lipomatous tumor, liposarcoma, myxoid liposarcoma, fibromyxoid sarcoma, lymphangioleiomyoma, malignant myoepithelioma, soft tissue malignant melanoma, myoepithelial carcinoma, myoepithelioma, myxoinflammatory fibroblastic sarcoma, undifferentiated sarcoma, perivascular cell tumor, rhabdomyosarcoma, non-rhabdomyosarcoma soft tissue sarcoma (NRSTS), soft tissue leiomyosarcoma, undifferentiated sarcoma, well-differentiated type soft tissue sarcoma:NRSTS), soft tissue leiomyosarcoma, undifferentiated sarcoma, well-differentiated type Has a solid tumor in a tissue or organ selected from the group consisting of liposarcoma, or a solid tumor arising therefrom. 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 lymphoma; and multiple myeloma (MM); or a blood cancer selected from the group consisting of melanoma; head and neck; ovarian mesothelioma; endometrium; prostate; sarcoma; neuroblastoma; liver; lung; breast; esophagus, stomach, and pancreas. In some embodiments, 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. In some embodiments, the subject is naive to chemotherapy or has not received chemotherapy. In some embodiments, the subject has received a lymphodepleting chemotherapy regimen. In some embodiments the subject has or does not deplete bone marrow cells.

[0018] Regarding antiviral, anti-cancer, and vaccine-enhancing combination therapy methods, in some embodiments, IL-2v, Fc-IL-2v fusion protein, homodimer, heterodimer, conjugate, polynucleotide, vector, lipoplex (e.g., LNP), and / or pharmaceutical composition are co-administered with one or more additional therapeutic agents. In some embodiments, the one or more additional therapeutic agents include 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 TLR2 agonist, TLR3 agonist, TLR4 agonist, TLR5 agonist, TLR7 agonist, TLR8 agonist, and 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 selgantolimod (GS-9688), R848 (resiquimod), and NKTR-262 (dual TLR7 / TLR8 agonist). In some embodiments, cytokine or chemokine therapy includes co-administering 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, one or more immunostimulatory cytokines or chemokines are 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 / Mig (monokine induced by interferon-γ), CXCL10 / IP10 (interferon-γ inducible 10 kDa protein), and CXCL11 / I-TAC (interferon-inducible 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 regulated on activation normal T expressed and secreted protein (RANTES). RANTES), and selected from the group consisting of RANTES). In some embodiments, the one or more additional therapeutic agents include one or more interleukin receptor agonists of interleukin receptors 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 one or more additional therapeutic agents include one or more cytokines 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), IFN-α, IFN-β, pegylated interferons (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 include one or more innate immune activators. In some embodiments, the one or more innate immune activators are fms-related tyrosine kinase 3 (FLT3, also known as CD135, FLK-2, FLK2, STK1), interferon gene stimulator (STING) receptor, DExD / H-box helicase 58 (DDX58;It comprises an agonist of a receptor selected from the group consisting of retinoic acid-inducible gene I (RIG-I), NLR family pyrin domain containing 3 (NLRP3), and nucleotide binding oligomerization domain containing 2 (NOD2). In some embodiments, one or more innate immune activators comprise one or both of GS-3583 and GS-9992. In some embodiments, one or more additional therapeutic agents include immunotherapy, immunostimulation therapy, cytokine therapy, chemokine therapy, cell therapy, gene therapy, and combinations thereof. In some embodiments, immunotherapy comprises co-administering 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, one or more immune checkpoint proteins or receptors are CD27, CD70; CD40, CD40LG; CD47, CD48 (SLAMF2), transmembrane domain 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 (VTCN1, B7H4); 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-related 2 (HHLA2, B7H7); inducible T cell co-stimulator (ICOS, CD278); inducible T cell co-stimulator 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 immunoreceptor with Ig and ITIM domains (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;Selected from the group consisting of 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 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-administering one or more blocking factors 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 T cell activation inhibitor 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-administering 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 CD27, CD70; CD40, CD40LG; inducible T cell co-stimulator (ICOS, CD278); inducible T cell co-stimulator ligand (ICOSLG, B7H2); TNF receptor; Selected from the group consisting of tumor necrosis factor receptor superfamily member 4 (TNFRSF4, OX40); tumor necrosis factor 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 (Poliovirus receptor: PVR) cell adhesion molecule (PVR, CD155). In some embodiments, the immunotherapy comprises co-administering one or more blocking agents or inhibitors of one or more NK cell inhibitory immune checkpoint proteins or receptors. In some embodiments, the NK cell inhibitory immune checkpoint proteins or receptors are 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-stimulating immune checkpoint protein or receptor is 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 include proteinaceous (e.g., antibody) inhibitors 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, the proteinaceous (e.g., antibody) inhibitor of 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, 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) are selected from the group consisting of 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), dostarlimab (TSR-042, WBP-285), lambrolizumab (MK-3475); sasanlimab (PF-06801591), cetrelimab (JNJ-63723283), serplulimab (HLX-10), retifanlimab (MGA-012), balstilimab (AGEN2034), prorgolimab (BCD 100), budigalimab (ABBV-181), boptatlimab (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-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, proteinaceous (e.g., antibody) inhibitors of the CD274 molecule (NCBI Gene ID: Gene ID: 29126; B7-H, B7H1, PD-L1) include atezolizumab (TECENTRIQ®), avelumab (BAVENCIO®; MSB0010718C), enobafolimab (ASC22), durvalumab (IMFINZI®; MEDI-4736), BMS-936559 (MDX1105), cosibelimab (CK-301), rodaplimab (LY3300054), balstilimab (garivulimab). (BGB A333), enobafolimab (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, one or more immune checkpoint inhibitors include small molecule inhibitors 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 includes BPI-002. In some embodiments, the immunotherapy 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 an antibody or antigen-binding fragment thereof that selectively binds to a cell surface receptor 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, PD-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 alpha E (ITGAE; CD103), interleukin 7 receptor (IL7R; CD127), CD40 ligand (CD40LG; CD154), folate receptor alpha (FOLR1), folate receptor beta (FOLR2), leucine-rich repeat containing 32 (LRRC32; GARP), IKAROS family zinc finger 2 (IKAROS family zinc finger, IKZF2; HELIOS), inducible T cell co-stimulator (ICOS; CD278), lymphocyte activation gene 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 includes co-administering one or more agents that selectively deplete suppressive myeloid cells. In some embodiments, the suppressive myeloid cells are selected from tumor-associated macrophages (TAM) and myeloid derived suppressor cells (MDSC).In some embodiments, the one or more agents that selectively deplete suppressive myeloid cells are colony stimulating factor 1 receptor (CSF1R), C-C motif chemokine receptor 2 (CCR2), C- C motif chemokine ligand 2 (CCL2), triggering receptor expressed on myeloid cells 2 (TREM2), complement C5a receptor 1 (C5AR1), and antibodies or antigen-binding fragments thereof that selectively bind to cell surface receptors selected from the group consisting of mixtures thereof. In some embodiments, the one or more agents that selectively deplete suppressive myeloid cells are agents that inhibit 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 mitogen-activated protein kinase kinase kinase kinase 1 (MAP4K1) (also called 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, or DGK-alpha), T cell immunoreceptor with Ig and ITIM domains (TIGIT), X-linked inhibitor of apoptosis (XIAP), In some embodiments, the one or more agents that selectively deplete suppressive myeloid cells are antibodies or antigen-binding fragments thereof that selectively bind to cell surface receptors selected from the group consisting of colony stimulating factor 1 receptor (CSF1R), C-C motif chemokine receptor 2 (CCR2), C-C motif chemokine ligand 2 (CCL2), triggering 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 suppressive myeloid cells include agents that inhibit 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 mitogen-activated protein kinase kinase kinase kinase 1 (MAP4K1) (also called 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, or DGK-alpha), T cell immunoreceptor with Ig and ITIM domains (TIGIT), X-linked inhibitor of apoptosis (XIAP), 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 mitogen-activated protein kinase kinase kinase kinase 1 (MAP4K1) (also called 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, or DGK-alpha), T cell immunoreceptor with Ig and ITIM domains (TIGIT), X-linked inhibitor of apoptosis (XIAP), subfamily 1 group A member 2 (NR1H2; LXRB). In some embodiments, the one or more additional therapeutic agents are mitogen-activated protein kinase kinase kinase kinase 1 (MAP4K1) (also called 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, or DGK-alpha), T cell immunoreceptor with Ig and ITIM domains (TIGIT), X-linked inhibitor of apoptosis (XIAP), kinase, MAP4K1) (also called 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, or DGK-alpha), T cell immunoreceptor with Ig and ITIM domains (TIGIT), X-linked inhibitor of apoptosis (XIAP), 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), ha, DGKA, DAGK, DAGK1, or DGK-alpha), T cell immunoreceptor with Ig and ITIM domains (TIGIT), X-linked inhibitor of apoptosis (XIAP), It comprises an inhibitor or antagonist of 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). Some examples In some embodiments, the one or more additional therapeutic agents include an activator or agonist of a toll-like receptor (TLR); an interferon gene stimulator (STING) receptor; an inducible T cell co-stimulator (ICOS, CD278); and / or a member of the TNF receptor superfamily (TNFRSF). In some embodiments, the member of the TNF receptor superfamily (TNFRSF) is selected from the group consisting of 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 includes 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 includes urelumab, BMS-663513, utomilumab (PF-05082566), CTX-471, MP-0310, ADG-106, ATOR-1017, AGEN2373, or QL1806; and / or the TNFRSF18 (GITR or CD357) activator or agonist includes 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 includes co-administering 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 method includes co-administering a molecule selected from the group consisting of AGEN1884 (zalifrelimab), AGEN1181, AGEN2034 (bavstilimab), AGEN1307, AGEN1327, AGEN1777, AGEN2373, AGEN1223, and GS-1423.

[0019] Regarding antiviral, anticancer, and vaccine enhancement methods, in some embodiments, IL-2v, Fc-IL-2v fusion protein, homodimer, heterodimer, conjugate, polynucleotide, vector, lipoplex (e.g., LNP), and / or pharmaceutical composition are administered systemically or locally via a route selected from, for example, intravenous, subcutaneous, intramuscular, intradermal, intratumoral, and mucosal (e.g., oral, intranasal, rectal, vaginal). In some embodiments, IL-2v, Fc-IL-2v fusion protein, homodimer, heterodimer, conjugate, polynucleotide, vector, lipoplex (e.g., LNP), and / or pharmaceutical composition, and one or more additional therapeutic agents are administered by the same or different administration routes. In various embodiments, IL-2v, Fc-IL-2v fusion protein, homodimer, heterodimer, conjugate, polynucleotide, vector, lipoplex (e.g., LNP), and / or pharmaceutical composition, and one or more additional therapeutic agents are co-administered according to the same schedule (e.g., co-administered simultaneously at the same time intervals) or different schedules (e.g., co-administered simultaneously at different time intervals).In some embodiments, IL-2v, Fc-IL-2v fusion protein, homodimer, heterodimer, conjugate, polynucleotide, vector, lipoplex (e.g., LNP), and / or pharmaceutical composition are administered at a dose in the range of 0.5 μg / kg to 1000 μg / kg, such as in the range of 1 μg / kg to 500 μg / kg, such as in the range of 10 μg / kg to 300 μg / kg, such as in the range of 30 μg / kg to 600 μg / kg, such as at least 0.5 μg / kg per dose to a maximum of 0.2 μg / kg, 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, 15 μg / kg, 20 μg / kg, 25 μg / kg, 30 μg / kg, 40 μg / kg, 50 μg / kg, 60 μg / kg, 70 μg / kg, 80 μg / kg, 90 μg / kg, 100 μg / kg, 110 μg / kg, 120 μg / kg, 130 μg / kg, 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. In some embodiments, IL-2v, Fc-IL-2v fusion protein, homodimer, heterodimer, conjugate, polynucleotide, vector, lipoplex (e.g., LNP), and / or pharmaceutical composition are administered at a dose in the range of 0.02 mg to 100 mg, such as 0.04 mg to 80 mg, such as at least 0.02 mg per dose to a maximum of 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.In various embodiments, the method optionally includes administering IL-2v, an Fc-IL-2v fusion protein, a homodimer, a heterodimer, a conjugate, a polynucleotide, a vector, a lipoplex (e.g., LNP), and / or a pharmaceutical composition multiple times at predetermined intervals, optionally with one or more additional therapeutic agents. In various embodiments, the method optionally includes administering IL-2v, an Fc-IL-2v fusion protein, a homodimer, a heterodimer, a conjugate, a polynucleotide, a vector, a lipoplex (e.g., LNP), and / or a pharmaceutical composition multiple times 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, optionally with one or more additional therapeutic agents. In some embodiments, the method optionally includes administering IL-2v, an Fc-IL-2v fusion protein, a homodimer, a heterodimer, a conjugate, a polynucleotide, a vector, a lipoplex (e.g., LNP), and / or a pharmaceutical composition one or more times at predetermined intervals that are at least 1 week and up to at least 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months apart, optionally with one or more additional therapeutic agents. In some embodiments, IL-2v, an Fc-IL-2v fusion protein, a homodimer, a heterodimer, a conjugate, a polynucleotide, a vector, a lipoplex (e.g., LNP), and / or a pharmaceutical composition is administered once a week (i.e., QW), once every other week (i.e., once every 1 week or once every 2 weeks, i.e., Q2W), once every 3 weeks (i.e., once every 3 weeks, i.e., Q3W), once a month (i.e., QM), or once every other month (i.e., once every 1 month or once every 2 months, i.e., Q2M), once every 3 months (Q3M), once every 4 months (Q4M), once every 5 months (Q5M), once every 6 months (Q6M), or at a lower frequency.In some embodiments, IL-2v, Fc-IL-2v fusion protein, homodimer, heterodimer, conjugate, polynucleotide, vector, lipoplex (e.g., LNP), and / or pharmaceutical composition are administered subcutaneously two or more times at one or more intervals between once every other week (i.e., once every week or once every two weeks, i.e., Q2W) to once every three weeks (i.e., once every three weeks, i.e., Q3W). In some embodiments, IL-2v, Fc-IL-2v fusion protein, homodimer, heterodimer, or conjugate has a serum half-life of at least 6, 9, 12, 15, 18, 21, 24 hours, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days or more in humans. In some embodiments, the subject or mammal is human.

[0020] In another aspect, provided is a kit. In various embodiments, the kit comprises one or more unit doses of the IL-2v, Fc-IL-2v fusion protein, homodimer, heterodimer, conjugate, polynucleotide, vector, lipoplex (e.g., LNP), and / or pharmaceutical composition 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 kit comprises one or more containers selected from the group consisting of vials, ampoules, and pre-filled syringes. In some embodiments, the kit comprises one or more containers containing the fusion protein and / or homodimer in an aqueous solution. In some embodiments, the aqueous solution has a concentration in the range of 0.05 mg / ml to 50 mg / ml, such as 0.05 mg / ml to 20 mg / ml, such as 0.1 mg / ml to 40 mg / ml, such as 1.0 mg / ml to 30 mg / ml, such as 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, 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 and contains the IL-2v, Fc-IL-2v fusion protein, homodimer, heterodimer, conjugate, polynucleotide, vector, lipoplex (e.g., LNP), and / or pharmaceutical composition. In various embodiments, the one or more unit doses are the same or different. In some embodiments, each unit dose is in the range of 0.5 μg / kg to 1000 μg / kg, such as 1 μg / kg to 500 μg / kg, such asIn 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, 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, 15 μg / kg, 20 μg / kg, 25 μg / kg, 30 μg / kg, 40 μg / kg, 50 μg / kg, 60 μg / kg, 70 μg / kg, 80 μg / kg, 90 μg / kg, 100 μg / kg, 110 μg / kg, 120 μg / kg, 130 μg / kg, 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. In some embodiments, each unit dose is in the range of 0.02 mg to 100 mg, for example, 0.04 mg to 80 mg, for example, at least 0.02 mg per dose to a maximum of 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 additional therapeutic agents in one or more unit doses. In some embodiments, the kit comprises one or more antiviral agents for, for example, HBV, HIV, HSV, or coronavirus in one or more unit doses. 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 TLR2 agonist, TLR3 agonist, TLR4 agonist, TLR5 agonist, TLR7 agonist, TLR8 agonist, and TLR9 agonist. In some embodiments, the TLR7 agonist is GS9620 (vesatolimod),Selected from the group consisting of 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 selgantolimod (GS-9688), R848 (resiquimod), and NKTR-262 (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 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 / Mig (monokine induced by interferon-γ), CXCL10 / IP10 (interferon-γ-inducible 10 kDa protein), and CXCL11 / I-TAC (interferon-inducible 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 regulated on activation, normal T cell expressed and secreted (RANTES), and are selected from the group consisting of. In some embodiments, the kit comprises one or more interleukin receptor agonists of interleukin receptors 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 kit comprises 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 interferon (e.g.,It 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), interferon gene stimulator (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 domain 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 (VTCN1, B7H4); V-set immune regulatory receptor (VSIR, B7H5, VISTA); immunoglobulin superfamily member 11 (IGSF11, VSIG3); natural killer cell cytotoxicity receptor 3 ligand 1 (NCR3LG1, B7H6); HERV-H LTR-related 2 (HHLA2, B7H7); inducible T cell co-stimulator (ICOS, CD278); inducible T cell co-stimulator 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 immunoreceptor with Ig and ITIM domains (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, Selected from the group consisting of 4 (KLRC4, NKG2F); 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 immunoglobulin-like receptor, three Ig domains, and a 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 blocking factors 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 T cell activation inhibitor 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 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-stimulating immune checkpoint protein or receptor is selected from the group consisting of CD27, CD70; CD40, CD40LG; inducible T cell co-stimulator (ICOS, CD278); inducible T cell co-stimulator 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 (Poliovirus receptor: PVR) cell adhesion molecule (PVR, CD155). In some embodiments, the kit further comprises one or more blocking factors 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 kit comprises one or more agonists or activators of one or more NK cell-stimulating immune checkpoint proteins or receptors.In some embodiments, the NK cell-stimulating immune checkpoint protein or receptor is CD16, CD226 (DNAM-1), 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., an antibody) inhibitor of PD-L1 (CD274), PD-1 (PDCD1), or CTLA4. In some embodiments, the kit comprises a proteinaceous (e.g., an antibody) inhibitor of PD-L1 (CD274) or PD-1 (PDCD1). In some embodiments, the proteinaceous (e.g., an antibody) inhibitor of 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, 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) are selected from the group consisting of 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), dostarlimab (TSR-042, WBP-285), lambrolizumab (MK-3475); sasanelimab (PF-06801591), cetrelimab (JNJ-63723283), serplulimab (HLX-10), retifanlimab (MGA-012), balstilimab (AGEN2034), prorgolimab (BCD 100), budigalimab (ABBV-181), boptatlimab (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-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, proteinaceous (e.g., antibody) inhibitors of the CD274 molecule (NCBI Gene ID: Gene ID: 29126; B7-H, B7H1, PD-L1) are selected from the group consisting of atezolizumab (TECENTRIQ®), avelumab (BAVENCIO®; MSB0010718C), enobafolimab (ASC22), durvalumab (IMFINZI®; MEDI-4736), BMS-936559 (MDX1105), cosibelimab (CK-301), rodapolimab (LY3300054), valbribumab (BGB A333), enobafolimab (KN035), opdivolimab (HLX20), manelimumab (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 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 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), 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 alpha E (ITGAE; CD103), interleukin 7 receptor (IL7R; CD127), CD40 ligand (CD40LG; CD154), folate receptor alpha (FOLR1), folate receptor beta (FOLR2), leucine rich repeat containing. Comprising an antibody or an antigen-binding fragment thereof that selectively binds to a cell surface receptor selected from the group consisting of 32 (LRRC32; GARP), IKAROS family zinc finger 2 (IKZF2; HELIOS), inducible T cell co-stimulator (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 comprises mitogen-activated protein kinase kinase kinase kinase kinase 1 (MAP4K1) (also known as hematopoietic progenitor kinase 1 (HPK1)), phosphatidylinositol-4,5-bisphosphate 3-kinase 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), inhibitor of apoptosis X-linked inhibitor (XIAP, BIRC4, IAP-3), baculovirus IAP repeat-containing 2 (BIRC2, cIAP1), baculovirus IAP repeat-containing 3 (BIRC3, cIAP2), baculovirus IAP repeat-containing 5 (BIRC5, survivin), or cytokine-inducible SH2-containing protein (CISH) inhibitor or antagonist, one or more additional therapeutic agents. In some embodiments, the kit comprises one or more additional therapeutic agents comprising an activator or agonist of toll-like receptor (TLR); interferon gene stimulator (STING) receptor; inducible T cell co-stimulator (ICOS, CD278); and / or TNF receptor superfamily (TNFRSF) member.In some embodiments, the TNF receptor superfamily (TNFRSF) member is selected from the group consisting of 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 comprises a TNFRSF4 (OX40 or CD134) activator or agonist selected from INCAGN1949, tabalumab (MEDI0562), pogalizumab (MOXR0916 / RG7888), MEDI6469, BMS-986178, PF-04518600, GSK3174998, IBI101, ATOR-1015, ABBV-368, or SL-279252, a TNFRSF9 (4-1BB or CD137) activator or agonist selected from urelumab, BMS-663513, utomilumab (PF-05082566), CTX-471, MP-0310, ADG-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 binds simultaneously 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 (zarifrelimab), AGEN1181, AGEN2034 (baltirlimab), AGEN1307, AGEN2373, AGEN1223, and GS-1423.

[0021] Regarding kits comprising therapeutic agents for anti-HBV combination therapy, in some embodiments, 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 + 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 single-unit doses of one or more therapeutic agents selected from the group consisting of HBV antigen inhibitors (e.g., HBV core antigen (HBcAg) inhibitors, HBV surface antigen (HBsAg) inhibitors, HBx inhibitors, HBV E antigen inhibitors), anti-HBV antigen antibodies, inhibitory nucleic acids targeting HBV (e.g., antisense oligonucleotides, small interfering RNA (siRNA), DNA-directed RNA interference (ddRNAi)), gene editors targeting HBV (e.g., CRISPR-Cas (e.g., Cas9, Cas12, Cascade, Cas13), zinc finger nucleases, homing endonucleases, homing megonucleases (e.g., ARCUS), synthetic nucleases, TALEN), covalently closed circular DNA (cccDNA) inhibitors, HBsAg secretion or assembly inhibitors, HBV virus entry inhibitors, and CAR-T and T cell bispecifics (redirected T cells).

[0022] Regarding kits containing therapeutic agents for anti-HBV combination therapy, in some embodiments, the kit contains one or more anti-retroviral agents in one or more unit doses. In some embodiments, the kit contains 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 containing the N332 oligomannose glycan, (ii) the second variable loop (V2) and / or the Env trimer apex, (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 high-mannose patch containing the third variable loop (V3) and / or the N332 oligomannose glycan, and compete with or contain the VH and VL regions from antibodies 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, 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, one or more anti-HIV broadly neutralizing antibodies bind to an epitope or region of gp120 at the second variable loop (V2) and / or the Env trimer apex, and compete with or comprise VH and VL regions from antibodies 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, one or more anti-HIV broadly neutralizing antibodies bind to an epitope or region of gp120 at the CD4 binding site (CD4bs), including 3BNC117, GS-9723, 3BNC60, b12, F105, VRC01, VRC07, VRC07-523, VRC03, VRC06, VRC06b01. The VH and VL regions that compete with, or comprise, 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, one or more anti-HIV broadly neutralizing antibodies bind to an epitope or region of gp120 at the gp120 / gp41 interface and compete with, or comprise, the 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, one or more anti-HIV broadly neutralizing antibodies bind to an epitope or region of the silent face of gp120 and compete with, or comprise, the VH and VL regions from an antibody selected from VRC-PG05 and SF12. In some embodiments, one or more anti-HIV broadly neutralizing antibodies bind to an epitope or region of gp41 in the membrane-proximal region (MPER). In some embodiments, one or more anti-HIV broadly neutralizing 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, one or more anti-HIV broadly neutralizing antibodies bind to an epitope or region of the 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 a kit comprising a therapeutic agent for vaccine augmentation 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 is 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 coronavirus (e.g., beta coronavirus, e.g., severe acute respiratory syndrome-related coronavirus, e.g., SARS-CoV2), and comprises an antiviral vaccine. In some embodiments, the vaccine is an antibacterial vaccine against a bacterium selected from the group consisting of mycobacterium tuberculosis, whooping cough, tetanus, diphtheria, meningococcus, pneumococcus, Haemophilus influenza, cholera, typhoid fever, and Bacillus anthracis.

[0024] Regarding kits containing therapeutic agents for cancer combination therapy, in some embodiments, the kit comprises one or more antineoplastic agents or chemotherapeutic agents in one or more unit doses. In some embodiments, the kit comprises 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, dicroplatin, eptaplatin, lobaplatin, miloplatin), dihydrofolate reductase (DHFR) inhibitors (e.g., methotrexate, trimethoprim, pemetrexed), topoisomerase inhibitors (e.g., doxorubicin, daunorubicin, dactinomycin, etoposide, epirubicin, etoposide, idarubicin, irinotecan, mitoxantrone, pixantrone, sobuzoxane, topotecan, irinotecan, MM-398 (liposomal irinotecan), bosaroxacin and GPX-150, aldoxorubicin, AR-67, maberlatinib, AST-2818, abitinib (ACEA-0010), irofulven (MGI-114)), alkylating agents (e.g., nitrogen mustards (e.g., cyclophosphamide, chlorambucil, uramustine or uracil mustard, melphalan, chlorambucil, ifosfamide, bendamustine, temozolomide, carmustine), nitrosoureas (e.g., carmustine, lomustine, streptozocin), alkyl sulfonates (e.g., busulfan)), and mixtures thereof, one or more antineoplastic agents or chemotherapeutic agents in one or more unit doses selected from the group consisting of. 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); 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-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; 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; osteactivin); 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, group G (HLA-G); MHC class I polypeptide-related sequence A (MICA); MHC class I polypeptide-related sequence B (MICB); integrin subunit beta-7 (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 cell cytotoxicity receptor 3 ligand 1 (NCR3LG1; B7-H6); nectin, 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); suppression 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 signal transducer 2 (TACSTD2); fucosyl GM1; sialyl Lewis adhesion molecule (sLe);One or more single unit doses of one or more antibodies or their antigen-binding antibody fragments or their antibody-drug conjugates, CD3-targeted bispecific molecules, NK cell activating receptor-targeted bispecific molecules, or non-immunoglobulin antigen-binding domains, or antibody mimetic proteins, directed against one or more targets or tumor-associated antigens (TAAs) selected from the group consisting of Lewis Y antigen. In some embodiments, the kit comprises one or more antibodies or their antigen-binding antibody fragments or their antibody-drug conjugates, CD3-targeted bispecific molecules, NK cell activating receptor-targeted bispecific molecules, or non-immunoglobulin antigen-binding domains, or antibody mimetic proteins, that bind to epitopes of targets or tumor-associated antigens (TAAs) presented by major histocompatibility complex (MHC) molecules. 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 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 an 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, the one or more CARs comprise 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); Alpha-Fetoprotein (AFP), BAG Chaperone 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 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; 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); Folic Acid Hydrolase 1 (FOLH1); Folic Acid Receptor 1 (FOLR1); GD2 Ganglioside; Glycoprotein NMB (GPNMB; Osteoactivin); Guanylate Cyclase 2C (GUCY2C); Human Papillomavirus; S (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 beta7 (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 (including, for example, MUC1 / A, C, D, X, Y, Z, and REP); mucin 16 (MUC16; CA125); natural killer cell cytotoxicity receptor 3 ligand 1 (NCR3LG1; B7-H6); nectin,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); suppression 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); trofinin (TRO); tumor-associated calcium signal transducer 2 (TACSTD2); fucosyl GM1; sialyl Lewis adhesion molecule (sLe); and Lewis Y antigen binds to a target or tumor-associated antigen (TAA) selected from the group consisting of. In some embodiments, one or more CARs bind to an epitope of a target or tumor-associated antigen (TAA) presented by 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 D2 (MAGED2), kinesin family member 20B (KIF20B; also known as MPHOSPH1), NDC80 kinetochore complex NUF2 component (NUF2), 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), melanoma-prioritized expressed antigen (PRAME), sperm-associated antigen 9 (SPAG9), nuclear X-binding family member A1-related sperm protein (SPANXA1), SPANX family member A2 (SPANXA2), SPANX family member C (SPANXC),Selected from the group consisting of SPANX family member D (SPANXD), SSX family member 1 (SSX1), SSX family member 2 (SSX2), synaptonemal complex protein 3 (SYCP3), testis-expressed 14 cell junction formation 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 comprises one or more unit doses of 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 (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), C-C motif chemokine receptor 2 (CCR2, CD192), C-C motif chemokine receptor 5 (CCR5, CD195), C-C motif chemokine receptor 8 (CCR8, CDw198), C-C motif chemokine receptor 2 (CXCR2, CD182), C-C motif chemokine receptor 3 (CXCR3, CD182, CD183), C-C motif chemokine receptor 4 (CXCR4,Inhibitors or antagonists of 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), secreted phospholipase A2, prostaglandin E synthase (PTGES, PGES), arachidonic acid-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, inhibitors of 5'-nucleotidase ecto (NT5E or CD73) are 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, inhibitors of CCR2 and / or CCR5 are selected from the group consisting of BMS-813160, PF-04136309, and CCX-872. In some embodiments, inhibitors of MCL1 are 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, inhibitors of PTPN11 or SHP2 are TNO155 (SHP-099), RMC-4550, JAB-3068,and selected from the group consisting of 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. Methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, but 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 not intended to be limiting.

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

Brief Description of the Drawings

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Figure 22

Mode for Carrying Out the Invention

[0049] 1. Introduction Provided is a fusion protein comprising a serum half-life extending polypeptide operably linked to a variant or mutant or non-naturally occurring IL-2 (IL-2v). In various embodiments, the IL-2v of the fusion protein has at least 5 amino acids cleaved at the N-terminus compared to wild-type IL-2 (i.e., does not contain the 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 have several structural features that significantly reduce the dosing frequency while enhancing their safety and therapeutic efficacy. In addition, these features contribute to improved manufacturability through high-level production of soluble products using expression and purification platforms typically used for monoclonal antibody production. For example, the Fc-IL-2v fusion proteins described herein were engineered to have very low affinity for IL-2Rα, which is highly expressed on Treg cells, by introduction of point mutations into IL-2 at the IL-2 / IL-2Rα interface. In addition, the use of a heterodimeric Fc enabled fusion of a single copy of IL-2v to the dimeric Fc. This fusion design mimics the monovalent nature of native IL-2 and circumvents 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 that disrupts Protein A binding and avoids co-purification of the corresponding homodimeric contaminant with the desired heterodimeric product.The Fc derived from IgG4, which naturally lacks the ability to activate complement and has reduced Fc gamma receptor (FcγR) binding compared to IgG1, is used in combination with additional mutations to further minimize FcγR binding, thereby eliminating the potential for antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), or complement-dependent cytotoxicity (CDC). These Fc modifications. are combined with additional changes in the IL-2v protein to further enhance manufacturability, i.e., by substituting unpaired cysteine residues with serine to prevent unwanted aggregation or modification that can occur with these unpaired cysteine residues. The unpaired cysteines within the Fc domain can be substituted, for example, with serine (e.g., at a position corresponding to position 136 of any one of SEQ ID NOs: 45-56 or 141-143). Further, deletion of the first five residues of the mature native IL-2 sequence eliminates a potential O-glycosylation site on threonine (T3), improving manufacturing control. Deletion of the first five residues of mature native IL-2 does not require 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 a safer, more effective, less frequently dosed IL-2-based therapeutic agent, which can be applied to the treatment of a more diverse range of diseases than is currently the case.

[0050] In particular, the present disclosure identifies the cause of the problem of determining the minimal combination of amino acid substitutions in wild-type (wt) IL-2 that can abrogate binding to IL-2Rα to such an extent that it is sufficient to minimize the stimulation of immunosuppressive Treg cells over effector CD4+ and CD8+ T cells and NK cells. The various literature reports describing IL-2 substitution studies do not provide a comprehensive analysis of the contribution of individual amino acid side chains to IL-2α binding, either due to the limited set of IL-2 residues evaluated in any single study or, for example, due to the drastic nature of the substitutions reported in such studies when substituting an amino acid with a charged side chain with an amino acid with a side chain carrying the opposite charge. The present disclosure provides, in particular, particularly useful and effective constructs comprising variant IL-2 moieties. The present disclosure provides an IL-2v construct that combines the minimal number of substitutions capable of eliciting the desired biology (i.e., limiting substitutions to either alanine or glycine while minimizing the stimulation of immunosuppressive Treg cells over effector immune cells). For example, the present disclosure provides a heterodimeric Fc-IL-2v fusion protein comprising the amino acid substitutions R38G, F42A, and E62A in IL-2. In some embodiments, the provided heterodimeric Fc-IL-2v fusion protein may further comprise (i) the amino acid substitution C125S and / or (ii) the deletion of the first five residues of the mature native IL-2 sequence. Without 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, without 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 may be useful and / or effective in improving manufacturing control and reducing the risk of sequence-dependent immunogenicity in some embodiments.

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

[0052] IL-2 variants have been the subject of intensive research for over 20 years, and much effort has been focused on developing variants with reduced affinity for IL-2Rα as part of the trimeric IL 2Rαβγ (e.g., compared to wild-type IL-2 as set forth in SEQ ID NO: 216). Particular positions of focus include R38, F42, K43, Y45, E61, and E62, which were determined in studies of analogs using binding assays for IL-2Rα (see, e.g., Ju, et al, 1990, The Biology and Clinical Applications of Interleukin-2, ed. Rees, R.C. (Oxford Univ. Press, Oxford), pp. 7-14). Various reports have described substitutions at one or more of these positions and evaluated their effects on IL-2Rα binding. However, these studies have often utilized drastic substitutions to the native side chains, such as substituting a native amino acid with a charged side chain for an amino acid with a side chain carrying the opposite charge, or, for example, substituting a native amino acid with an uncharged side chain for an amino acid with a charged side chain (see Sauve et al., Proc. Natl. Acad. Sci. U.S.A., 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). While such drastic changes may seem favorable with respect to binding disruption, they can 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 is advantageous to reduce the risk of immunogenicity by incorporating fewer amino acid substitutions into the IL-2 molecule. Additionally, there have been no studies that globally compare the effects of a large panel of single point substitutions and / or their combinations, and it is difficult to design minimally substituted IL-2 variants that achieve the dual goal of restricting the stimulation of immunosuppressive Treg cells preferentially over effector CD4+ and CD8+ T cells and NK cells while simultaneously restricting the total number and nature of the substitutions to reduce the likelihood that the variant is immunogenic in humans.

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

[0054] For example, the evaluation of IL-2 variants was performed by making combinations of substitutions including A at positions 38, 42, 45, and 62 (Carmenate et al., J Immunol. 190(12)6230-6238, 2013), and determining its ability to stimulate the proliferation of CD8+ T cells and NK cells, reduce Treg proliferation, and reduce IL-2Rα binding. The results showed that the IL-2 variant containing R38A, F42A, Y45A, and E62A significantly reduced Treg proliferation and IL-2Rα binding while stimulating CD8+ T cell and NK cell proliferation as well as wtIL-2. Thus, at least four substitutions including the substitution of 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 includes G at position 38, A at position 42, and A at position E62 and does not include the substitution of A at position 45.

[0055] In another example, the above-described IL-2 variant containing the combination of A at positions 38, 42, 45, and 62 (Carmenate, et al., 2013, supra) was further evaluated to assess the contribution of each substitution to the impairment of IL-2Rα binding (Rojas, et al., J Mol Recognit. 28(4):261-8, 2015). The results of the binding assay showed that the single IL-2 substitutions of R38A and Y45A still bound to IL-2Rα (34% and 4% of wtIL-2, respectively), while the single IL-2 substitutions of F42A and E62A resulted in very little IL-2Rα binding (less than 1% of wtIL-2 each). The results of the proliferation assay showed that the single IL-2 substitutions of R38A and Y45A still caused proliferation in IL-2Rα-expressing CTLL-2 cells (48.2% and 7.1% of wt, respectively), while the single IL-2 substitutions of F42A and E62A caused very little 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) resulted in undetectable CTLL-2 cell proliferation. These results indicate that (i) at least four mutations are necessary 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 helpful for the most residue binding to IL-2Rα and the highest proliferation in CTLL-2 cells. Different from the study of Carmenate et al, the present disclosure demonstrates that R38G is a more effective single mutation than R38A in reducing IL-2Rα binding, and thus, an IL-2 variant having only three substitutions including R38G combined with F42A and E62A attenuates STAT5 activation in CTLL-2 cells by more than 30,000-fold compared to an equivalent molecule having a native IL-2Rα binding interface, and induces the desired biology for primary human immune cells by minimizing the stimulation of immunosuppressive Treg cells preferentially over effector T cells and NK cells.

[0056] In addition, research by Roche (see, for example, WO 2012 / 107417) describes the development of IL-2 variants related to heterodimeric IgG fusion molecules having antigen-specific antigen-binding domains, including, for example, tumor antigens. This research shows, inter alia, (i) that the substitution of G for L72 in IL-2 is necessary for the desired loss of IL-2Rα binding and reduction of human Treg activation associated with the substitutions F42A and Y45A, (ii) that the desired IgG IL-2 fusion has the T3A substitution in IL-2 to eliminate the O-glycosylation site, and (iii) that the desired IgG IL-2 fusion has the C125A substitution to avoid intermolecular disulfide bridging. The present disclosure demonstrates that an IL-2 variant containing substitutions at the positions of R38G, F42A, and E62A reduces STAT5 activation in CTLL-2 cells by 30,000-fold, while an IL-2 variant having substitutions at F42A, Y45A, and L72G reduces STAT5 activation in CTLL-2 cells by only 3,800-fold compared to an equivalent molecule having a 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 triple substitutions of R38G / F42A / E62A or F42A / Y45A / L72G had comparable activities in the activation of CD8+ T cells. However, while both of these triple substitution variants significantly reduced activity against Treg cells, the R38G / F42A / E62A-containing variant was superior in that it exhibited a more modest difference in 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 for eliminating the O-glycosylation site because it avoids any mutagenesis of the native IL-2 sequence for this purpose that could increase the risk of potential immunogenicity in humans.

[0057] Furthermore, recent studies reported by Cugene (see, e.g., WO 2020 / 252418) describe the development of IL-2 variants related to bivalent IL-2 homodimer Fc fusions, and in particular, (i) at position R38, none of the A, F, or G substitutions dramatically improved specificity for IL-2Rβγ compared to equivalently fused IL-2Rαβγ, (ii) among the substitutions tested, R38A (EC50 of 3.23) was more useful than any other in such improvement of specificity (EC50 of 2.0 and 0.42 for R38G and R38F, respectively), (iii) at position E62, all of the F, H, L, and A substitutions improved specificity for IL-2Rβγ compared to equivalently fused IL-2Rαβγ, (iv) among the substitutions tested, E62F (EC50 of 151) was more useful than any other in such improvement of specificity (EC50 of 2.57, 2.38, and 60.5 for E26H, E62L, and E62A, respectively), and (iii) that a desirable IL-2-Fc fusion does not have an S residue at position 125. Indeed, the reference IL-2 used in Cugene's study clearly naturally contains S125, and Cugene recommends substituting away from S (in particular, using the S125I substitution), whereas the present disclosure substitutes to S (in particular, using the C125S substitution). In contrast to this recent study by Cugene, the present disclosure provides an IL-2 Fc fusion protein variant comprising 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 available teachings prior to the present disclosure, whether taken individually or together, have led to the development of IL-2 variants different from those described herein, particularly with respect to Fc fusions. 2. Variant Interleukin-2 (IL-2v) Protein a. Variant IL-2 (IL-2v) with Reduced Binding Affinity for Interleukin-2 Receptor Alpha Subunit (IL-2RA)

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

[0060] In various embodiments, for example, the variant IL-2 (IL-2v) domain of the fusion protein described herein promotes or induces equal or greater proliferation of CD8+ T cells compared to wt IL-2, which is any one of the IL-2v of SEQ ID NOs: 43 and 44. Additionally, in some embodiments, for example, the IL-2v of the fusion protein described herein induces 50% of the maximum signal transduction and transcriptional activation of signal transducer and activator of transcription 5 (STAT5) in regulatory T (Treg) cells at a concentration (EC 50 ) that is the EC for STAT5 activation or signal transduction of wt IL-2 or any one of the IL-2v of SEQ ID NOs: 43 and 44 50It is at least 1000 times higher, for example, at least 1500 times higher, for example, at least 1700 times higher, for example, at least 2000 times higher, for example, at least 2500 times higher compared to. See, for example, 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 "suppressor T cells", are immunosuppressive and are generally a subpopulation of T cells that suppress or downregulate the induction and proliferation of effector T cells. Treg express the surface biomarkers CD4 and CD25 (IL-2 receptor α chain) and the intracellular DNA-binding biomarker FOXP3. Human Foxp3+CD4+ T cells are 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, while the Foxp3loCD45RA-CD25lo fraction marks activated T effector (Teff) cells without suppressive activity. In addition, in healthy subjects Compared to that of, Treg cells in cancer patients are typically characterized by different 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, for example, 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, the IL-2v of the fusion protein described herein is 50% of the maximum IL-2Rαβγ-mediated STAT5 activation or signal transduction (measured as STAT5 activation in, for example, CTLL2 cells) (EC 50) The concentration that induces 50 is at least 2500-fold, for example, at least 5000-fold, for example, at least 7500-fold, for example, at least 10,000-fold, for example, at least 15,000-fold, for example, at least 20,000-fold higher compared to the EC for STAT5 activation or signal transduction of wt IL-2 or any one of IL-2v of SEQ ID NOs: 43 and 44. 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. 50 For some embodiments, for example, the IL-2v of the fusion protein described herein has an EC 50 that induces 50% of the maximum proliferation of natural killer (NK) cells, which is, for example, at least 10-fold, for example, at least 12-fold, for example, at least 15-fold, for example, at least 16-fold, for example, at least 18-fold, for example, at least 20-fold higher compared to the EC 50 for the proliferation of wt IL-2 or any one of IL-2v of SEQ ID NOs: 43 and 44, as measured using, for example, the cell line KHYG-1. See, for example, Suck, et al., Exp Hematol (2005) Oct;33(10):1160-71, Yagita, et al., Leukemia (2000) 14(5):922-30; the cell line KHYG-1 has DSMZ number: ACC725; ExPASy cynomolgus KHYG-1 (CVCL_2976); CellBank Australia CODE: JCRB0156.

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

[0063] As used herein, the term "polypeptide variant" refers to a polypeptide specifically disclosed herein, which is typically a different polypeptide with one or more substitutions, deletions, additions, and / or insertions. Such variants may occur naturally or, for example, by modifying one or more of the polypeptide sequences described herein, by evaluating one or more biological activities of the polypeptides described herein, and / or by using any of several techniques well known in the art, may be synthetically generated.

[0064] The term "variant" may also refer to any naturally occurring or genetically engineered molecule that contains 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 the same B cell lineage or are derived from the same B cell lineage. Genetically engineered variants may include all single or combinatorial mutations made to an antibody.

[0065] With respect to structural attributes, generally, for example, the IL-2v domain of the fusion protein described herein does not include the first five amino acid residues corresponding to amino acid positions 1 to 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 the position of any given polymer component (e.g., an amino acid, nucleotide, generally also referred to as a "residue") not being the actual numerical position of the component in the given polymer, but rather by reference to the same or equivalent position in a selected amino acid or nucleic acid polymer (e.g., based on optimal alignment or consensus sequence), the numbering of the selected or referenced amino acid polymer or nucleic acid polymer, "corresponds to", "corresponding to", or "relative to" ". 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 are cleaved. For fusion proteins based on human IL-2, the IL-2 position numbering is done with reference to the mature human IL-2 (NCBI Gene ID: 3558) shown below, or with reference to SEQ ID NO: 44 (IL-2v having serine at position 125 (C125S)): APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 216).

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

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

[0068] In some embodiments, IL-2v comprises at least two or at least three substitutions (e.g., to glycine or alanine) at position 125 serine (C125) and at amino acid positions selected from the group consisting of R38, F42, Y45, E61, E62, and L72. In some embodiments, IL-2v comprises at least two or at least three, three or less, two or less substitutions (e.g., to glycine or alanine) at position 125 serine (C125) and at amino acid positions selected from the group consisting of R38, F42, Y45, E61, and E62. In some embodiments, IL-2v comprises at least two or at least three, three or less, two or less substitutions (e.g., to glycine or alanine) at position 125 serine (C125) and at amino acid positions selected from the group consisting of R38, F42, Y45, and E62. In some embodiments, IL-2v comprises at least two or at least three, three or less, two or less substitutions (e.g., to glycine or alanine) at position 125 serine (C125) and at amino acid positions selected from the group consisting of R38, F42, and E62. In some embodiments, IL-2v comprises at least two or at least three, three or less, two or less substitutions at position 125 serine (C125) and at amino acid positions selected from the group consisting of R38G, F42A, Y45G, E61A, E62A, and L72G. In some embodiments, IL-2v comprises at least two or at least three, three or less, two or less substitutions at position 125 serine (C125) and at amino acid positions selected from the group consisting of R38G, F42A, Y45G, E61A, and E62A. In some embodiments, IL-2v comprises at least two or at least three, three or less, two or less substitutions at position 125 serine (C125) and at amino acid positions selected from the group consisting of R38G, F42A, Y45G, and E62A. In some embodiments, IL-2v comprises at least two or at least three, three or less, two or less substitutions at position 125 serine (C125) and at amino acid positions selected from the group consisting of R38G, F42A, and E62A. The foregoing position numbers relate to IL-2v of SEQ ID NO: 44.In some embodiments, IL-2v does not contain amino acid substitutions at one or more or all of the positions selected from the group consisting of D20, Y45, E61, E68, V69, L72, A73, L80, R81, L85, L86, I87, I92, and Q126. In some embodiments, IL-2v does not contain amino acid substitutions at one or more or all of the 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 to the IL-2v described herein, as well as to the structures of IL-2v fusion polynucleotides and polypeptides, and functional molecules encoding variant or derivative polypeptides having desirable characteristics can also be obtained. When it is desired to alter the amino acid sequence of a polypeptide to create an equivalent or even improved variant or portion of a polypeptide described herein, one of ordinary skill in the art will typically change one or more of the codons of the encoding DNA sequence.

[0071] For example, a particular amino acid can be substituted with another amino acid in the protein structure without apparently losing its ability to bind to other polypeptides (e.g., antigens) or cells. Since this is the binding ability and property of the protein that defines its biological functional activity, certain amino acid sequence substitutions can be made in the protein sequence and, of course, in the underlying DNA coding sequence, and yet a protein with similar properties can be obtained. Therefore, it is contemplated that various changes can be made to the polypeptide sequences of the disclosed antibodies and their antigen-binding fragments, or to the corresponding DNA sequences encoding such polypeptides, without apparently losing their biological utility or activity.

[0072] Often, polypeptide variants contain one or more conservative substitutions. A "conservative substitution" is one in which an amino acid is replaced with another amino acid having similar properties, such that one of ordinary skill in peptide chemistry would expect that the secondary structure and hydropathicity properties of the polypeptide would not substantially change.

[0073] As used herein, "identity" means the percentage of identical nucleotides or amino acid residues at corresponding positions in two or more sequences when the sequences are aligned, i.e., to maximize sequence matching taking into account gaps and insertions. Sequences are generally aligned for maximum correspondence over a specified 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 can extend to the full length of the reference polypeptide or polynucleotide sequence. For sequence comparison, typically one sequence functions as the reference sequence and the test sequence is compared to that sequence. When using a sequence comparison algorithm, the test and reference sequences are input into a computer program, subsequence coordinates are designated as 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 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 the sequences of nucleotides or amino acids in the two sequences are the same when the two sequences are aligned for maximum correspondence as described below. The 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 a segment of at least 20 contiguous positions, usually 30 to 75 contiguous positions, 40 to 50 contiguous positions, or the full length of the sequence, and the sequences within the comparison window can be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.

[0075] Optimal alignment of the arrays 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, "M.O." (1978) A model of evolutionary change in proteins-Matrices for detecting distant relationships.In Dayhoff, M.O. (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, D.G. and Sharp, P.M. (1989) CABIOS 5:151-153; Myers, E.W. and Muller W. (1988) CABIOS 4:11-17; Robinson, E.D. (1971) Comb. Theor 77:105; Santou, N. Nes, M. (1987) Mol. Biol. Evol. 4:406-425; Sneath, P.H.A. and Sokal, R.R. (1973) Numerical Taxonomy-the Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco, CA; Wilbur, W.J. and Lipman, D.J. (1983) Proc. Natl. Acad., Sci. USA80:726-730。

[0076] Alternatively, the optimal alignment of sequences for comparison may be performed by the local identity algorithm of Smith and Waterman (1981), "Add. APL. Math" Vol. 2: p. 482, by the identity alignment algorithm of Needleman and Wunsch (1970), "J. Mol. Biol." Vol. 48: p. 443, by the similarity search of Pearson and Lipman (1988) "Proc. Natl. Acad. Sci. USA" Vol. 85: p. 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] An example of an algorithm suitable for determining percent sequence identity and sequence similarity is 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 the percent sequence identity of the polynucleotides and polypeptides described herein. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (blast.ncbi.nlm.nih.gov / Blast.cgi).

[0078] In an exemplary case, for a nucleotide sequence, the cumulative score can be calculated using parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for a mismatched residue; always <0). The extension of the word hit in each direction stops when: the cumulative alignment score drops by component X from its maximum achieved value; the cumulative score becomes zero or less due to the accumulation of alignments of one or more negative-score 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 a word length (W) of 11 and an expectation value (E) of 10 as default, and BLOSUM62 is used as the scoring matrix (see the following: Henikoff and Henikoff (1989) "Proc. Natl. Acad. Sci. USA" Vol. 89: p. 10915) for alignment, (B) 50, expectation value (E) 10, M = 5, N = -4, and comparison of both strands as default.

[0079] For an amino acid sequence, the cumulative score can be calculated using a scoring matrix. The extension of the word hit in each direction stops when: the cumulative alignment score drops by component X from its maximum achieved value; the cumulative score becomes zero or less due to the accumulation of alignments of one or more negative-score 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, the "percentage of sequence identity" is determined by comparing two sequences optimally aligned over a comparison window of at least 20 positions. The portion of the polynucleotide or polypeptide sequence in the comparison window may include additions or deletions (i.e., gaps) of 20 percent or less, usually 5 - 15 percent, or 10 - 12 percent, as compared to a reference sequence (excluding additions or deletions) for optimal alignment of the two sequences. This percentage is calculated by determining the number of positions at which the identical nucleic acid bases or amino acid residues occur in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the reference sequence (i.e., window size), and multiplying the result by 100 to obtain the percentage of sequence identity.

[0081] In some embodiments, IL-2v, serum half-life extended IL-2v, e.g., Fc-IL-2v fusion protein, and their homodimers and heterodimers do not contain a signal peptide. In some embodiments, IL-2v, serum half-life extended IL-2v, e.g., Fc-IL-2v fusion protein, and their homodimers and heterodimers contain an N-terminal signal peptide. The signal peptide can be from 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 serum proteins, immunoglobulins, cytokines, chemokines, chaperone proteins, invariant proteins, and proteins that direct proteins to the lysosomal compartment. In various embodiments, the signal peptide or leader sequence is colony stimulating factor 2 (CSF2, GM-CSF), tissue-type plas nogen activator (PLAT, t-PA), C-C motif chemokine ligand 7 (CCL7, MCP-3), C-X-C motif chemokine ligand 10 (CXCL10, IP-10), CD74 (p33; DHLAG; HLA DG; immunoglobulin kappa; Ia-GAMMA, invariant chain), serum albumin (ALB), SPARC (osteonectin), cwcv and kazal-like domain proteoglycan 1 (SPOCK1); SPARC (osteonectin), cwcv and kazal-like domain proteoglycan 2 (SPOCK2); polyubiquitin B / C (UBB / UBC), calreticulin (CALR), and vesicular stomatitis virus G protein (VSV-G). 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 the 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 to 231, or a sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 218 to 231. Exemplary signal sequences that can be used in this IL-2v, serum half-life extended IL-2v, e.g., Fc-IL-2v fusion protein, and their homodimers and heterodimers are provided in Table B.

Table 2

[0082] Signal peptides can be designed, for example, to be cleaved after secretion from a cell to form a mature fusion protein. A modified human serum albumin signal peptide for secreting a protein in a cell in which use in the expression of this fusion protein can be found is described, for example, in Attallah, et al., Protein Expr Purif. (2017) 132:27-33. Additional signal peptide sequences for use in the expression of 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 murine or rat IL-2 sequences. Mus musculus IL-2 is identified as NCBI Gene ID 16183. For fusion proteins based on murine IL-2, the numbering of the IL-2 positions refers to the mature murine IL-2 shown below: APTSSSTSSSTAEAQQQQQQQQQQQQHLEQLLMDLQELLSRMENYRNLKLPRMLTFKFYLPKQATELKDLQCLEDELGPLRHVLDLTQSKSFQLEDAENFISNIRVTVVKLKGSDNTFECQFDDESATVVDFLRRWIAFCQSIISTSPQ (SEQ ID NO: 234).

[0084] Exemplary IL-2v domains based on or derived from wild-type murine IL-2 with reduced binding to murine IL-2RA (also known as il2ra, CD25; Il2r; Ly-43; NCBI Gene ID: 16184) are provided in Table D. Generally, the IL-2v domains of the fusion proteins described herein do not include the first 23 amino acid residues of the mature wild-type murine IL-2 sequence (e.g., do not include the amino acids APTSSSTSSSTAEAQQQQQQQQQ (SEQ ID NO: 235)). In some embodiments, murine IL-2v includes 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, L86, where the position numbers are with respect to the sequence of mature murine IL-2 represented by SEQ ID NO: 234. In some embodiments, murine IL-2v includes alanine at position 140 (C140) and at least one, two, or at least three substitutions to alanine or glycine at amino acid positions selected from the group consisting of R52, F56, Y59, E76, L86, where the position numbers are with respect to the sequence of mature murine IL-2 represented by SEQ ID NO: 234. In some embodiments, murine IL-2v includes 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 the 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] Optionally or if desired, the IL-2v polypeptides described herein may or may not be PEGylated. b. Serum half-life extended polypeptide

[0086] The fusion proteins described herein include variant IL-2 that binds to IL-2RA with reduced binding affinity and a polypeptide with an extended serum half-life. In some embodiments, the fusion protein includes a polypeptide with an extended serum half-life (e.g., an Fc region) and IL-2v in order from the N-terminus to the C-terminus. In some embodiments, the fusion protein includes IL-2v and a polypeptide with an extended serum half-life (e.g., an Fc region) in order from the N-terminus to the C-terminus. For example, polypeptides that can be used to extend the serum half-life of another polypeptide via binding or fusion are well-known in the art and can be used in the present fusion proteins. Exemplary polypeptides with an extended serum half-life that can bind or fuse with IL-2v described herein include, but are not limited to, the crystallizable fragment of an immunoglobulin (Fc region), one or more serum albumin moieties, albumin-binding proteins or peptides, IgG, XTEN polypeptides, proline / alanine / serine-rich (PAS) polypeptides, elastin-like polypeptides. IL-2v bound 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., need not 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 bind or fuse to either or both the N-terminus and the C-terminus of IL-2v.Exemplary XTEN protein polymers that can be used in the present IL-2v fusion protein 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, International Publication No. 2010 / 091122, International Publication No. 2011 / 123813, International Publication No. 2013 / 130683, International Publication No. 2016 / 077505, and International Publication No. 2017 / 197048. Exemplary proline / alanine / serine-rich (PAS) polypeptides that can be used in the present IL-2v fusion protein 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, International Publication No. 2016122806 and International Publication No. 2016130451. The foregoing references are hereby incorporated 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 (e.g., human, dog, cat, mouse, or monkey) as the IL-2 main. 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 modification 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, these particular modifications decrease antibody effector function and increase the half-life of the antibody. In some embodiments, the Fc-IL-2v fusion proteins described herein include two or more, three or more, four or more, five or more, six or more, six or less, five or less, four or less, three or less, two or less, 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 a hinge region. This is wholly or partially cleaved or deleted. The structural hinge regions of human IgG1, IgG2, and IgG4 antibodies are peptide linkers of 19 - 23 amino acids containing 2 - 4 cysteine residues, genetically encoded on the hinge exon together with the 5' end of the CH2 exon, and enabling disulfide bridging 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 includes or is derived from the human IgG4 isotype and does not include the amino acid sequence ESKYGPPCPPCP (SEQ ID NO: 236). In some embodiments, the Fc region includes or is derived from the human IgG1 isotype and does not include 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 amino acid modifications that promote 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 substitution of methionine to tyrosine at position 252 (EU numbering), a substitution of serine to threonine at position 254 (EU numbering), and a substitution of threonine to glutamic acid at position 256 (EU numbering). See, for example, U.S. Patent No. 7,658,921. This type of variant, named the "YTE variant," exhibits a 4-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., Antimicrob Agents Chemotherap., 57(12):6147-6153 (2013)). In certain embodiments, the Fc-IL-2v fusion protein described herein comprises one, two, three or more amino acid substitutions among the amino acid residues at positions 251-257, 285-290, 308-314, 385-389, and 428-436 (EU numbering), and comprises an IgG constant domain. Alternatively, the M428L and N434S ("LS") substitutions can increase the pharmacokinetic half-life of the fusion protein. In other embodiments, the Fc-IL-2v fusion protein described herein comprises the M428L and N434S substitutions (EU numbering). In other embodiments, the Fc-IL-2v fusion protein described herein comprises the T250Q and M428L (EU numbering) mutations. In other embodiments, the Fc-IL-2v fusion protein described herein comprises the H433K and N434F (EU numbering) mutations. Fc mutations that reduce or eliminate effector activity

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

[0092] In some embodiments, the Fc region is altered by substituting at least one amino acid residue with a different amino acid residue to reduce or eliminate the effector function of the antibody. 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 different amino acid residues such that the fusion protein has a reduced affinity for effector ligands. Effector ligands whose affinity is altered can be, for example, Fc receptors (e.g., residue positions 234, 235, 236, 237, 297 (EU numbering)), or C1 complement components (e.g., residue positions 297, 318, 320, 322 (EU numbering)). U.S. Patent Nos. 5,624,821 and 5,648,260 are both by Winter et al.

[0093] Fc modifications that reduce or eliminate effector functions include, for example, substitutions, insertions, and deletions at one or more positions including 234, 235, 236, 237, 267, 269, 325, and 328, such as 234G, 235G, 236R, 237K, 267R, 269R, 325L, and 328R (EU numbering). Further, the Fc variant may include 236R / 328R. Other modifications for reducing FcγR and complement interactions 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 functions (both ADCC and complement activation) can be reduced by mutating IgG residues at one or more positions between positions 233 - 236 and 327 - 331 (in IgG1, E233P, L234V, L235A, optionally G236A, A327G, A330S, and P331S; in IgG4, E233P, F234V, L235A, optionally G236A; in IgG2, A330S and P331S (EU numbering), etc.) while maintaining neonatal FcR binding (while maintaining half-life). See Armour et al. (1999) Eur. J. Immunol. 29:2613; WO 99 / 58572. Other mutations for reducing effector functions include L234A and L235A in IgG1 (Alegre et al. (1994) Transplantation 57:1537); V234A and G237A in IgG2 (Cole et al. (1997) J. Immunol. 159:3613 (see also U.S. Patent No. 5,834,597), and the S228P and L235E of 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. Generally, see Labrijn et al. (2008) Curr. Op. Immunol. 20:479. Additional mutations found to reduce effector function in relation to the Fc(IgG1) fusion protein (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 with respect to the ADCC site of 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 with reduced binding to FcγR includes the amino acid substitutions L234A, L235E, and G237A. Gross, et al., (2001) Immunity 15:289. Modifications in the IgG Fc region for reducing binding to FcγRI to reduce ADCC identified in International Publication No. 88 / 007089 (e.g., 234A; 235E; 236A; G237A) can be used in the fusion proteins of the present 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 (consideration of the effects of these mutations on FcγRIII 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 antibody to bind C1q and activate complement (Idusogie et al., (2000) J. Immunol. 164:4178, International Publication No. 99 / 51642. Modification at position 331 of IgG1 (e.g., P331S) has been shown to reduce complement binding (Tao 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 acids 231-239 are altered, thereby reducing 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 present 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 are disclosed in Glaesner et 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. (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 is essentially effector - function - free. For example, the Fc reduces or eliminates binding to FcγR and reduces or eliminates complement fixation, e.g., it is effectorless. Exemplary effectorless IgG1 Fc contains the following five mutations: L234A, L235E, G237A, A330S, and P331S (EU numbering) (Gross et al. (2001) Immunity 15:289). Combining these five substitutions with N297A may 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 outlined in Ha, et al., Front. Immunol. (2016) 7:394. In some embodiments, the first Fc domain and the second Fc domain each contain the following amino acid substitutions (EU numbering) (or the reverse): T366W and T366S / L368A / Y407V; T366W / S354C and T366S / L368A / Y407V / Y349C; S364H / F405A and Y349T / T394F; T350V / L351Y / F405A / Y407V and T350V / T366L / K392L / 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, the Fc region heterodimerization of two different heavy-chain-containing species can be facilitated 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. Additionally, the C220S mutation can be incorporated into the IgG1 hinge region of the scFv-containing arm to eliminate the free cysteine that otherwise forms a disulfide bond with the corresponding cysteine in the light chain of wild-type IgG1. Co-transfection of such constructs results in the preferential formation of heterodimeric Fc with a low level of homodimeric contaminants. Additionally, the incorporation of the S354C mutation can be incorporated into the Fc containing the "knob" mutation, and optionally the incorporation of the Y349C mutation into the Fc containing the "hole" mutation can be used to generate a covalent bond between the two halves of the heterodimeric Fc when additional thermodynamic stability is desired (Merchant et al. 1998. Nat. Biotechnol. 16:677-81).

[0101] To facilitate the purification of heterodimeric molecules from contaminating homodimeric products, the H435R or H435R+Y436F mutation for reducing or eliminating Protein A binding can be introduced into only one of the two Fc-containing chains (Jendeberg, L. et al. 1997 J. Immunol. Methods 201:25-34). This reduces or eliminates the Protein A binding of homodimeric contaminants containing those mutations, and the purification of the desired heterodimer from the remaining homodimeric contaminants via additional chromatographic steps (e.g., ion exchange, e.g., anion exchange) is significantly simplified. In embodiments where the H435R (or H435R+Y436F) mutation is incorporated into the first or second Fc region of the heavy chain, if the VH region of the same heavy chain is derived from the VH3 family variable region, this VH region can also contain the amino acid substitutions described herein to reduce or eliminate the 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, CH2 regions of IgG4 and the CH3 region of IgG1).

[0103] IgG1 antibodies exist in various allotypes and isotypes. In certain embodiments, the Fc-IL-2v fusion proteins described herein comprise an IgG1 heavy chain having the allotype of G1m1;nG1m2;G1m3;G1m17,1;G1m17,1,2;G1m3,1; or G1m17. Each of these allotypes or isotypes 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 directly linked or linked via a intervening amino acid sequence (e.g., a G-S linker) to the wild-type IgG1m3 sequence or a fragment thereof provided below. EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:74). For example, in various embodiments, the IgG1m3 fragment has the first 5 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 the 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 the wild-type Fc region of the same isotype). In some embodiments, one or more amino acid substitutions are N297A, N297Q (Bolt Set al. (1993) Eur J Immunol 23:403-411), D265A, L234A, L235A (McEarchem et al., (2007) Blood, 109:1185-1192), C226S, C229S (McEarchem et al., (2007) Blood. 109:1185 - 1192), P238S (Davis et al., (2007) J Rheumatol, 34:2204 - 2210), E233P, L234V (McEarchern et 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., (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, wherein the amino acid positions are according to the EU numbering convention. In certain embodiments, the Fc region further comprises an amino acid deletion at the 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 the human IgG1 isotype and contains one or more amino acid substitutions in the Fc region at residue positions 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, T394D, A330L, M252Y, S254T, T256E, M428L, N434S, T366W, T366S, L368A, Y407V, H435R, Y436F, and any combination thereof, wherein the residue numbering follows EU numbering. In some embodiments, the Fc region comprises the human IgG1 isotype and contains 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, wherein the residue numbering follows EU numbering. IgG4 isotype Fc

[0108] In cases where effector function should be completely avoided, for example, where antigen binding alone is sufficient to produce the desired therapeutic benefit and the effector function only results in (or increases the risk of) unwanted side effects, an IgG4 antibody may be used, or an antibody or fragment lacking the Fc region or a substantial portion thereof may be devised, or Fc may be mutated to completely eliminate glycosylation (e.g., N297A). Alternatively, a hybrid construct of human IgG2 (CH1 domain and hinge region) and human IgG4 (CH2 domain and CH3 domain) has been generated that lacks effector function, lacks the ability to bind to FcγR (like IgG2), and cannot 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 for reducing 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 includes 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 the 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, and the amino acid positions are according to the EU numbering convention. See, for example, 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 includes the human IgG4 isotype and contains 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, and the residue numbering is according to EU numbering.

[0110] In some embodiments, the IgG4 variants of the present disclosure can be combined with the S228P mutation according to the EU numbering convention (Angal et al., (1993) Mol Immunol, 30:105-108) and / or one or more mutations described in 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 the human IgG2 constant region. In some embodiments, the human IgG2 constant region includes the 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 the 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, and 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 protein described herein comprises the L234F, L235E, D265A mutations, which are collectively referred to as "FEA". The FEA mutations reduce or abolish effector function. In certain embodiments, the Fc-IL-2v fusion protein described herein comprises the L234F, L235E, D265A, and F405L mutations, which are collectively referred to as "FEAL". In certain embodiments, the Fc-IL-2v fusion protein described herein comprises 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 protein described herein comprises the L234F, L235E, D265A, and K409R mutations, which are collectively referred to as "FEAR". In certain embodiments, FEAL and FEAR constitute the fusion protein described herein. In certain embodiments, the Fc-IL-2v fusion protein described herein further comprises the M428L and N434S mutations, which are collectively referred to as LS. In certain embodiments, the Fc-IL-2v fusion protein described herein comprises the L234F, L235E, D265A, F405L, M428L, and N434S mutations, which are collectively referred to as "FEALLS". In certain embodiments, the Fc-IL-2v fusion protein described herein comprises 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 protein described herein comprises the L234F, L235E, D265A, K409R, M428L, and N434S mutations, which are collectively referred to as "FEARLS".In certain embodiments, FEALLS and FEARLS constitute the fusion proteins described herein. By reducing or suppressing effector functions on the Fc domain of the Fc-IL-2v fusion protein, cells bound by the molecule are not killed by natural effector cells such as NK cells and macrophages.

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

[0114] Mutations that reduce Fc receptor binding and are found for 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 contain the L234F and L235E mutations. In certain embodiments, the Fc-IL-2v fusion proteins described herein contain the L234F, L235E, and D265A mutations. In certain embodiments, the Fc-IL-2v fusion proteins described herein contain the L234F, L235E, and N297Q mutations. In certain embodiments, the Fc-IL-2v fusion proteins described herein contain the N297A or N297Q mutation. In certain embodiments, the Fc-IL-2v fusion proteins described herein contain 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 into 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 an altered (e.g., reduced) affinity for effector ligands (e.g., Fc receptors or C1 component of complement) while retaining the antigen-binding ability of the parental antibody. In certain embodiments, the Fc-IL-2v fusion proteins described herein contain the E233P, L234V, L235A, and / or G236A mutations (EU numbering). In some embodiments, the Fc-IL-2v fusion proteins described herein contain the A327G, A330S, and / or P331S mutations (EU numbering).In some embodiments, the Fc-IL-2v fusion protein described herein comprises the K322A mutation (EU numbering). In some embodiments, the Fc-IL-2v fusion protein described herein comprises the E318A, K320A, and K322A (EU numbering) mutations. In certain embodiments, the Fc-IL-2v fusion protein described herein comprises the 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, the terminal Fc amino acid residue (e.g., K447) is removed or excluded. 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 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: 45-72. 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 bound, or continuously bound, or adjacent to a serum half-life extending polypeptide (e.g., an Fc domain). In some embodiments, the IL-2v domain or a truncated fragment thereof is operably bound to a serum half-life extending polypeptide (e.g., an Fc domain) via a linker, e.g., the linker is positioned between the serum half-life extending polypeptide (e.g., an Fc domain) and 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 of the GGGS motif (SEQ ID NO: 265), e.g., 1 to 5 repeats or units, e.g., 3 to 5 repeats or units, e.g., 3 or 4 or 5 repeats, e.g., 3 or 4 or 5 repeats of the GGGGS motif (SEQ ID NO: 264), or a mixture thereof ("G-S 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 4 repeats of the GGGGS motif (SEQ ID NO: 246).

[0118] In certain embodiments, the IL-2v domain or a truncated fragment thereof is directly bound to, or bound via an intervening amino acid sequence (e.g., a G-S linker) to, human IgG1 (e.g., the 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] Further provided is an Fc-IL-2v fusion protein comprising the IL-2v domain described above and herein and the Fc domain described above and herein.

[0120] Functionally, in various embodiments, the Fc-IL-2v fusion protein has an equilibrium dissociation constant (K D ) of at least 60 μM (e.g., 60 μM or greater), e.g., at least 70 μM, 80 μM, 90 μM, 100 μM or greater for binding to IL-2RA (indicating a 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 D 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 Fc-IL-2v fusion protein promotes equivalent or greater proliferation of CD8+ T cells compared to any one of the IL-2v of SEQ ID NOs: 43 and 44, a fusion protein comprising Fc operably linked to wt IL-2, or any one of the fusion proteins of SEQ ID NOs: 117, 118, 161, and 162. In some embodiments, the concentration (EC 50 ) at which the IL-2v fusion protein induces 50% of the maximum signal transduction and transcriptional activation of signal transducer and activator of transcription 5 (STAT5) in regulatory T (Treg) 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 compared to the EC 50 for STAT5 activation or signal transduction of wt IL-2, or any one of the IL-2v of SEQ ID NOs: 43 and 44, a fusion protein comprising Fc operably linked to wt IL-2, or any one of the fusion proteins of SEQ ID NOs: 117, 118, 161, or 162. In some embodiments, the EC 50The concentration that induces is the EC for STAT5 activation or signal transduction of wt IL-2, or any one of the IL-2vs of SEQ ID NOs: 43 and 44, a fusion protein containing Fc operably linked to wt IL-2, or any one of the fusion proteins of SEQ ID NOs: 117, 118, 161, or 162 50 is at least 2500-fold, e.g., at least 5000-fold, e.g., at least 7500-fold, e.g., at least 10,000-fold, e.g., at least 15,000-fold, e.g., at least 20,000-fold higher compared to. In some embodiments, the concentration (EC 50 ) that induces 50% of the maximum proliferation of natural killer (NK) cells, e.g., measured using KHYG-1 cells, is the EC for the proliferation of wt IL-2, or any one of the IL-2vs of SEQ ID NOs: 43 and 44, a fusion protein containing Fc operably linked to wt IL-2, or any one of the fusion proteins of SEQ ID NOs: 117, 118, 161, or 162 50 is at least 10-fold, e.g., at least 12-fold, e.g., at least 15-fold, e.g., at least 16-fold, e.g., at least 18-fold, e.g., at least 20-fold 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 mouse wild-type Fc and mouse wild-type IL-2 and comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 166-171, or 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%, 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 mouse wild-type Fc and mouse wild-type IL-2 are provided in Table D. In various embodiments, the mouse Fc-IL-2v fusion protein is, for example, in the form of a heterodimer with an Fc domain that comprises the amino acid sequence of SEQ ID NO: 250, or 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%, 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 to an interferon.

[0124] In some embodiments, the Fc-IL-2v fusion protein is not glycosylated. In some embodiments, the IL-2v in the Fc-IL-2v fusion protein is not glycosylated. In some embodiments, the Fc region or Fc domain of the Fc-IL-2v fusion protein is glycosylated, for example, 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 heterodimer 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 in this specification, or their homodimers or heterodimers, can be conjugated. IL-2v, IL-2v fusion proteins, or their homodimers or heterodimers can be polymers (e.g., polyethylene glycol (PEG), polyethyleneimine (PEI) modified with PEG (PEI-PEG), polyglutamic acid (PGA), N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer), hyaluronic acid, radioactive substances (e.g., 90 Y, 131 I, 125 I, 35 S, 3 H, 121 In, 99It can be conjugated or attached to various molecules (e.g., labels) including, but not limited to, macromolecules such as Tc), 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 drugs (including the antiviral and anticancer drugs described herein).

[0126] The conjugate IL-2v, IL-2v fusion protein, or their homodimers or heterodimers can be prepared, for example, according to known methods for chemically modifying the IL-2v, IL-2v fusion protein, or their homodimers or heterodimers described herein. In certain embodiments, the label moiety or therapeutic moiety is conjugated to the Fc portion of the fusion protein. Methods for modifying the antibody Fc region are well known in the art (e.g., U.S. Patent No. 5,057,313 and U.S. Patent No. 5,156,840).

[0127] In some embodiments, the IL-2v, IL-2v fusion protein, or their homodimers or heterodimers are conjugated to a drug or therapeutic agent. In various embodiments, the drug is a small organic compound or an inhibitory nucleic acid, e.g., short interfering RNA (siRNA), microRNA (miRNA). In some embodiments, the drug or therapeutic agent is an anti-neoplastic agent 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, such as GS-4224 or GS-4416. In some embodiments, the therapeutic agent is an agonist or activator of a pattern recognition receptor (PRR), such as a Toll-like receptor (TLR), RIG-I-like receptor (RLR), NOD-like receptor (NLR), AIM2-like receptor (ALR), C-type lectin receptor (CLR), DNA receptor, or 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 stimulator of interferon genes (STING) receptor. In some embodiments, the TLR agonist or activator is selected from the group consisting of TLR2 agonist, TLR3 agonist, TLR4 agonist, TLR5 agonist, TLR7 agonist, TLR8 agonist, and TLR9 agonist. In some embodiments, the TLR7 agonist is selected from the group consisting of besatollimod (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 selgantolimod (GS-9688), and / or dual TLR7 / TLR8 agonists such as NKTR-262, telratolimod, BDB-001, and CV8102.

[0129] In some embodiments, the drug or therapeutic agent is monomethyl auristatin E, monomethyl auristatin F, calicheamicin, ansamitocin, maytansine or an analog thereof (e.g., mertansine / emtansine (DM1), labtansine / sorlabtansine (DM4)), anthracyline (e.g., doxorubicin, daunorubicin, epirubicin, idarubicin), pyrrolobenzodiazepine , the DNA cross-linking agent SC-DR002 (D6.5), duocarmycin, a microtubule inhibitor (MTI) (e.g., taxane, vinca alkaloid, epothilone), pyrrolobenzodiazepine (PBD) or a dimer thereof, and duocarmycin (A, B1, B2, C1, C2, D, SA, CC-1065). is selected from the group consisting of f. Homodimer

[0130] Also provided is a homodimer. In some embodiments, the homodimer comprises two Fc-IL-2v fusion proteins described above and herein. g. Heterodimer

[0131] Also provided is a heterodimer. In various forms and configurations, the heterodimer 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 heterodimer generally comprises a half-life extension moiety (e.g., an Fc domain, one or more serum albumin moieties, an albumin-binding protein or peptide, IgG, an XTEN polypeptide, a proline / alanine / serine-rich (PAS) polypeptide, an elastin-like polypeptide). In some embodiments, the heterodimer is monovalent with respect to IL-2v, e.g., has one IL-2v domain. or a non-target (IL-2βγ receptor complex-targeted) heterodimer

[0132] In some embodiments, the heterodimer comprises two IL-2v domains. Such a heterodimer comprises (i) a first Fc-IL-2v fusion protein described herein that comprises a first Fc domain, and (ii) a second Fc-IL-2v fusion protein described herein that comprises a second Fc domain. In such non-targeted 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-targeted 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., a single) Fc-IL-2v fusion protein that comprises a first Fc domain, and (ii) a second Fc domain. In such non-targeted embodiments, the second Fc domain does not comprise or is not fused to an antigen-binding domain. In such non-targeted embodiments, the second Fc domain is "empty" but is heterodimerized to the Fc-IL-2v fusion protein (e.g., using Fc substitution) to, for example, promote the stability of the molecule and reduce or prevent the homodimerization or assembly of molecules having two IL-2v domains. In some embodiments, neither the first Fc domain 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 the heterodimer 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). Specific binding of the heterodimer is at least 10 7 、108 , 10 9 , or 10 10 M -1 means affinity. Specific binding is detectably high and distinguishable from non-specific binding that occurs to at least one irrelevant target. Specific binding can be the result of binding between specific functional groups or the formation of a specific spatial fit (e.g., lock and key type), while non-specific 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), and the first Fc domain and the second Fc domain are different. To promote heterodimerization, in some embodiments, the first Fc domain and the second Fc domain each have the following amino acid substitutions (EU numbering): T366W and T366S / L368A / Y407V; T366S / L368A / Y407V and T366W; T366W / S354C and T366S / L368A / Y407V / Y349C; T366S / L368A / 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 / D399V / 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 the extended serum half-life of the heterodimer, in some embodiments, one or both of the first Fc domain and the second Fc domain have 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 promote the purification of the heterodimeric protein A, in some embodiments, either the first Fc domain or the second Fc domain contains, at the indicated positions (EU index numbering), the following amino acids: arginine at position 435 and phenylalanine at position 436. In some embodiments, either the first Fc domain or the second Fc domain contains arginine at position 435, phenylalanine at position 436, and the T366S / L368A / Y407V amino acid substitutions.

[0137] In some embodiments, the effector function of one or both of the 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 contain the human IgG4 isotype and include 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, with residue numbering according to EU numbering. In some embodiments, one or both of the first Fc domain and the second Fc domain contain the human IgG1 isotype and include 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, with residue numbering according to EU numbering.

[0138] In some embodiments, the terminal Fc amino acid residue (e.g., K447) is removed or excluded 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 each comprise the amino acid sequences described below, or each 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 sequences described below: 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 to 116, 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: 75 to 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 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, 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 NO: 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 at least 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 80, 107, and 114, and 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.

[0140] In some embodiments, the heterodimer comprises: (i) the first amino acid sequence described 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 described below; and (ii) the second amino acid sequence described below, or a second Fc region comprising 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 described below, and includes a human IgG4 Fc-IL-2v fusion protein: 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,Array number 114 and array number 46, array number 115 and array number 46, or array number 116 and array number 46. In some embodiments, the heterodimer comprises (i) the first amino acid sequence described 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 described below, and (ii) the second amino acid sequence described below, or a second Fc region comprising 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 described below, and comprises a human IgG4 Fc-IL-2v fusion protein: array number 80 and array number 46, array number 107 and array number 46, or array number 114 and array number 46.,

[0141] In some embodiments, the heterodimer comprises: (i) the 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) the amino acid sequence of SEQ ID NO: 46, or a second Fc region comprising 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: (i) the first amino acid sequence of SEQ ID NO: 114, or an amino acid sequence that contains the amino acid substitutions R38G, F42A, and E62A 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 of SEQ ID NO: 114 is A, and the amino acid at position 305 is A), 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 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) the amino acid sequence of SEQ ID NO: 46, or a second Fc region comprising 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. The heterodimer is included in, or consists of, a human IgG4 Fc-IL-2v fusion protein.In some embodiments, the heterodimer comprises: (i) an amino acid sequence of SEQ ID NO: 114, or an amino acid sequence that contains amino acid substitutions of R38G, F42A, and E62A 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 of SEQ ID NO: 114 is A, and the amino acid at position 305 is A), does not contain residues corresponding to amino acids 1 to 5 of wild-type human IL-2 (e.g., does not contain APTSS (SEQ ID NO: 163)), and is 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 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: (i) the 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 the complex of the Fc receptor or interleukin-2 receptor subunit beta (IL-2RB; CD122) and interleukin-2 receptor subunit gamma (IL-2RG; CD132). In some embodiments, the heterodimer does not contain an immunoglobulin antigen-binding domain (i.e., does not contain the immunoglobulin heavy or light chain variable regions, i.e., does not contain VH or VL). In some embodiments, the IL-2 domain does not contain, or in addition to R38G, F42A, E62A, and C125S, does not contain amino acid substitutions 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 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: 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 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: 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 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: 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 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: 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 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: 124, 151, and 158, and the second Fc region comprises the amino acid sequence of SEQ ID NO: 58, 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: 58.

[0143] In some embodiments, the heterodimer comprises: (i) the first amino acid sequence described 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 described below, and (ii) the second amino acid sequence described below, or a second Fc region comprising 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 described below, and a human IgG4 Fc-IL-2v fusion protein: 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,Array number 157 and array number 58, array number 158 and array number 58, array number 159 and array number 58, or array number 160 and array number 58. In some embodiments, the heterodimer comprises (i) the first amino acid sequence described 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 described below, and (ii) the second amino acid sequence described 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 described below, and a second Fc region comprising the same: 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.,

[0144] Exemplary polypeptide sequences of the 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 polypeptide comprising the first Fc domain and / or the polypeptide comprising the second Fc domain may comprise an N-terminal signal peptide or leader sequence. In embodiments where both the polypeptide comprising the first Fc domain and / or the polypeptide comprising the second Fc domain may comprise an N-terminal signal peptide or leader sequence, the first N-terminal signal peptide or leader sequence and the second first N-terminal signal peptide or leader sequence may be the same or different. Targeted heterodimer

[0146] Also provided is a targeted heterodimer. 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 an 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 ...

Claims

[Claim 1] The invention described in the specification.

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