Fusion containing a CD8 antigen-binding molecule for modulating immune cell function

Abstract

Providing a fusion having a CD8 antigen-binding molecule for modulating immune cell function. [Solution] This specification provides an anti-CD8 antigen-binding molecule and a fusion polypeptide comprising the CD8 antigen-binding molecule for selectively modulating the function of CD8+ T cells more than the function of other immune cells. In addition, this disclosure also provides polynucleotides encoding the disclosed antigen-binding molecule and fusion polypeptide, as well as vectors and host cells comprising such polynucleotides. This disclosure further provides methods for producing the antigen-binding molecule and fusion polypeptide, pharmaceutical compositions comprising them, and uses thereof.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Application No. 63 / 105,162 filed on 23 October 2020, No. 63 / 121,663 filed on 4 December 2020, and No. 63 / 190,669 filed on 19 May 2021, each of which is incorporated herein by reference in its entirety.

[0002] Submission of sequence listings in ASCII text files. The contents of the following submission in ASCII text file are incorporated herein by reference in their entirety: Sequence listing in computer-readable format (CRF) (filename: 182842000440SEQLIST.TXT, date: October 21, 2021, size: 557,283 bytes).

[0003] This disclosure discloses CD8 antigen-binding molecules and fusion polypeptides comprising said CD8 antigen-binding molecules for selectively modulating the function of CD8+ T cells more than the function of other immune cells. In addition, this disclosure also provides polynucleotides encoding the disclosed antigen-binding molecules and fusion polypeptides, as well as vectors and host cells comprising such polynucleotides. This disclosure further provides methods for producing said antigen-binding molecules and fusion polypeptides, pharmaceutical compositions comprising them, and uses thereof. [Background technology]

[0004] CD8+ T cells, which express alpha-beta T cell receptors, are a large subset of major histocompatibility (MHC) class I-restricted T cells that mediate adaptive immunity against various pathogens and cancers. In addition, they can also be pathogenic and disease-causing in certain autoimmune and inflammatory diseases. Therapeutic benefits can be obtained by modulating the function of CD8+ T cells, either by activating their function in relation to infections and cancers, or by inhibiting their function in relation to certain autoimmune diseases.

[0005] The activation and differentiation of CD8+ T cells are primarily regulated by soluble immunomodulatory proteins such as cytokines. The biological activity of cytokines is mediated by their binding, usually with very high affinity, to their respective cytokine receptors on the cell surface. This results in their ability to potently stimulate downstream signaling of these receptors, inducing various cellular processes that regulate the phenotype and function of immune cells. Cytokines typically have multifaceted effects, triggering multiple downstream cellular events, including activation, proliferation, survival, apoptosis, and the secretion of other immunomodulatory proteins. Furthermore, because their receptors are expressed on multiple immune cell subsets, cytokines act not only on CD8+ T cells but also on other immune and non-immune cells that express their receptors.

[0006] For example, interleukin-2 (IL-2) is a cytokine that regulates numerous lymphocyte subsets, including alpha-beta CD4+ and CD8+ T cells, as well as various innate and innate-like lymphocytes such as NK cells, NK T cells, gamma-delta T cells (Tγδ cells), and innate lymphoid cells (ILC1, ILC2, and ILC3 cells).

[0007] IL-2 can transmit signals by binding with moderate affinity to a receptor complex (IL-2Rβγ, moderate-affinity receptor) consisting of IL-2Rβ and IL-2Rγ subunits (both of which are necessary and sufficient to induce downstream signaling in immune cells). In addition, IL-2 binds with high affinity to a receptor complex (IL-2Rαβγ, high-affinity receptor) consisting of IL-2Rα, IL-2Rβ, and IL-2Rγ subunits (Stauber et al, Proc Natl Acad Sci). US A. 2006 Feb 21;103(8):2788-93). IL-2Rα expression is limited to CD4+ Treg cells, activated T lymphocytes, and ILC2 and ILC3 cells, making these subsets most sensitive to IL-2 signaling. The IL-2Rβ and IL-2Rγ subunits are shared with another related cytokine, IL-15, and the IL-2Rγ subunit is shared among other common gamma-chain cytokines (IL-4, IL-7, IL-9, and IL-21). Most native and native-like lymphocytes, including NK cells, NK T cells, Tγδ cells, and ILC1, ILC2, and ILC3 cells, express high levels of IL-2Rβ (ImmGen consortium; Heng TS et al, Immunological Genome Project Consortium. Nat Immunol. 2008 Oct;9(10):1091-4), which also increases their sensitivity to both IL-2 and IL-15 cytokines.

[0008] The binding of IL-2 to its receptor induces phosphorylation of the receptor-associated Janus kinases JAK3 and JAK1, which in turn promotes phosphorylation of the STAT5 transcription factor (pSTAT5), which regulates the transcription of many genes in lymphocytes. IL-2 signaling in lymphocytes enhances cell survival, proliferation, and effector functions, including inflammatory cytokine secretion and cytotoxicity, and in some cases promotes activation-induced cell death (Ross & Cantrell, Annu Rev Immunol. 2018). (This is outlined in Apr 26;36:411-433).

[0009] CD8+ T cells express CD8, a type I transmembrane glycoprotein found on the cell surface as CD8 alpha (CD8α, CD8a) homodimers and CD8 alpha-CD8 beta (CD8β, CD8b) heterodimers. CD8 dimers interact with MHC class I molecules on target cells, and this interaction ensures that the TCR remains tightly engaged with MHC during CD8+ T cell activation. The cytoplasmic end of CD8α contains a binding site for T cell kinase (Lck), which triggers downstream signaling of the TCR during T cell activation, while the role of CD8β is thought to be to increase the binding activity of CD8 to MHC class I and to influence the specificity of the CD8 / MHC / TCR interaction (Bosselut et al, Immunity. 2000 Apr;12(4):409-18). TCR alpha-beta expressing CD8+ T cells typically express both CD8ab and CD8aa dimers. However, in some innate lymphoid cells, such as NK cells, mucosa-associated invariant T (MAIT) cells, and gamma-delta T cells, high levels of CD8aa dimers may be found instead of CD8ab dimers.

[0010] Therefore, antibodies that selectively bind to CD8ab heterodimers rather than CD8aa homodimers are needed. These antibodies can be used, for example, to target specific immunomodulatory polypeptides, such as IL-2, to CD8ab+ T cells and limit their activity in CD8- immune cells and CD8aa+ immune cells such as NK cells. All references cited herein, including patent applications, patent publications, and UniProtKB / Swiss-Prot accession numbers, are incorporated herein by reference in whole, as if each individual reference were specifically and individually indicated to be incorporated by reference. [Prior art documents] [Non-patent literature]

[0011] [Non-Patent Document 1] Stauber et al, Proc Natl Acad Sci U S A. 2006 Feb 21; 103(8): 2788-93 [Non-Patent Document 2] Heng TS et al, Immunological Genome Project Consortium. Nat Immunol. 2008 Oct; 9(10): 1091-4 [Non-Patent Document 3] Ross & Cantrell, Annu Rev Immunol. 2018 Apr 26; 36: 411-433 [Non-Patent Document 4] Bosselut et al, Immunity. 2000 Apr; 12(4): 409-18 [Summary of the Invention] [Means for Solving the Problems]

[0012] The present disclosure particularly describes human or humanized antibodies, antigen-binding fragments, and fusion proteins that bind to human CD8b. In some embodiments, the human or humanized antibodies, antigen-binding fragments, and fusion proteins preferentially bind to human CD8b or human CD8ab heterodimers rather than to the binding to human CD8aa homodimers. In some embodiments, the human or humanized antibodies, antigen-binding fragments, and fusion proteins bind to CD8b, CD8ab, or both.

[0013] Some aspects of this disclosure relate to human or humanized antibodies or their antigen-binding fragments. In some embodiments, the antibody or fragment specifically binds to human CD8b and / or human CD8ab with an affinity at least 10-fold higher than its binding to human CD8a and / or human CD8aa. In some embodiments, the antibody or fragment specifically binds to the extracellular domain(s) of human CD8b and / or human CD8ab with an affinity at least 10-fold higher than its binding to the extracellular domain(s) of human CD8a and / or human CD8aa. In some embodiments, the antibody or fragment binds to cells expressing human CD8ab heterodimers on their surface with an EC50 of less than 1000 nM. In some embodiments, the antibody or fragment binds to human CD8+ T cells. In some embodiments, the antibody or fragment specifically binds to human CD8b and / or human CD8ab with an affinity at least 10-fold higher than its binding to human CD8a and / or human CD8aa expressed on the surface of natural killer (NK) cells. In some embodiments, the antibody or fragment specifically binds to cells expressing human CD8b and / or human CD8ab on their surface (e.g., T cells) with at least 10 times higher affinity than to cells expressing human CD8a and / or human CD8aa on their surface (e.g., NK cells).

[0014] In some embodiments, the antibody or fragment comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 13, CDR-H2 containing the amino acid sequence of SEQ ID NO: 14, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 15, and the VL domain comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 16, CDR-L2 containing the amino acid sequence of SEQ ID NO: 17, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 18. In some embodiments, the antibody or fragment comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 51, CDR-H2 containing the amino acid sequence of SEQ ID NO: 52, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 15, and the VL domain comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 16, CDR-L2 containing the amino acid sequence of SEQ ID NO: 17, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 18. In some embodiments, the VH domain includes an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 62, and the VL domain includes an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 63. In some embodiments, the antibody or fragment includes a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 19, CDR-H2 containing the amino acid sequence of SEQ ID NO: 20, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 21, and the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 22, CDR-L2 containing the amino acid sequence of SEQ ID NO: 23, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 24.In some embodiments, the antibody or fragment comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 53, CDR-H2 containing the amino acid sequence of SEQ ID NO: 52, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 21, and the VL domain comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 22, CDR-L2 containing the amino acid sequence of SEQ ID NO: 23, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 24. In some embodiments, the VH domain comprises an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 64, and the VL domain comprises an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 65. In some embodiments, the antibody or fragment comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 25, CDR-H2 containing the amino acid sequence of SEQ ID NO: 26, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 27, and the VL domain comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 28, CDR-L2 containing the amino acid sequence of SEQ ID NO: 29, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 30. In some embodiments, the antibody or fragment comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 49, CDR-H2 containing the amino acid sequence of SEQ ID NO: 52, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 27, and the VL domain comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 28, CDR-L2 containing the amino acid sequence of SEQ ID NO: 29, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 30. In some embodiments, the VH domain comprises an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 66, and the VL domain comprises an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 67.In some embodiments, the antibody or fragment comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 31, CDR-H2 containing the amino acid sequence of SEQ ID NO: 32, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 33, and the VL domain comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 34, CDR-L2 containing the amino acid sequence of SEQ ID NO: 35, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 36. In some embodiments, the antibody or fragment comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 54, CDR-H2 containing the amino acid sequence of SEQ ID NO: 52, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 33, and the VL domain comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 34, CDR-L2 containing the amino acid sequence of SEQ ID NO: 35, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 36. In some embodiments, the VH domain comprises an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 68, and the VL domain comprises an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 69. In some embodiments, the antibody or fragment comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 37, CDR-H2 containing the amino acid sequence of SEQ ID NO: 38, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 39, and the VL domain comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 40, CDR-L2 containing the amino acid sequence of SEQ ID NO: 41, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 42.In some embodiments, the antibody or fragment comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 55, CDR-H2 containing the amino acid sequence of SEQ ID NO: 56, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 39, and the VL domain comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 40, CDR-L2 containing the amino acid sequence of SEQ ID NO: 41, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 42. In some embodiments, the VH domain comprises an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 70, and the VL domain comprises an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 71. In some embodiments, the antibody or fragment comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 43, CDR-H2 containing the amino acid sequence of SEQ ID NO: 44, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 45, and the VL domain comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 46, CDR-L2 containing the amino acid sequence of SEQ ID NO: 47, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 48. In some embodiments, the antibody or fragment comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 55, CDR-H2 containing the amino acid sequence of SEQ ID NO: 57, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 45, and the VL domain comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 46, CDR-L2 containing the amino acid sequence of SEQ ID NO: 47, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 48. In some embodiments, the VH domain comprises an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 72, and the VL domain comprises an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 73.In some embodiments, the antibody or fragment comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 1, CDR-H2 containing the amino acid sequence of SEQ ID NO: 2, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 3, and the VL domain comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 4, CDR-L2 containing the amino acid sequence of SEQ ID NO: 5, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 6. In some embodiments, the antibody or fragment comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 49, CDR-H2 containing the amino acid sequence of SEQ ID NO: 50, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 3, and the VL domain comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 4, CDR-L2 containing the amino acid sequence of SEQ ID NO: 5, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 6. In some embodiments, the antibody or fragment comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 177, CDR-H2 containing the amino acid sequence of SEQ ID NO: 178, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 179, and the VL domain comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 180, CDR-L2 containing the amino acid sequence of SEQ ID NO: 181, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 182. In some embodiments, the antibody or fragment comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 183, CDR-H2 containing the amino acid sequence of SEQ ID NO: 184, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 179, and the VL domain comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 180, CDR-L2 containing the amino acid sequence of SEQ ID NO: 181, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 182.In some embodiments, the VH domain comprises an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 185, and the VL domain comprises an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 186.

[0015] In some embodiments, the antibody or fragment comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain, and the VH domain comprises a CDR-H1 having the amino acid sequence of X1X2AIS, where X1 is S, K, G, N, R, D, T, or G, and X2 is Y, L, H, or F (SEQ ID NO: 259), X1X2X3PX4X5X6X7X8X9YX 10 QKFX 11 G, where X1 is G or H, X2 is I or F, X3 is I, N, or M, X4 is G, N, H, S, R, I, or A, X5 is A, N, H, S, T, F, or Y, X6 is A, D, or G, X7 is T, E, K, V, Q, or A, X8 is A or T, X9 is N or K, X 10 is A or N, X 11CDR-H2 comprises the amino acid sequence (SEQ ID NO: 260) in which X1 is Q or T, and CDR-H3 comprises the amino acid sequence (SEQ ID NO: 261) in which X1X2X3GX4X5LFX6X7, where X1 is D or A, X2 is A, G, E, R, Y, K, N, Q, L, or F, X3 is A, L, P, or Y, X4 is I or L, X5 is R, A, Q, or S, X6 is A or D, and X7 is D, E, A, or S, and the VL domain comprises the amino acid sequence X1X2SX3X4IX5GX6LN, where X1 is R or G, X2 is A or T, X3 is Q or E, X4 is E, N, T, S, A, K, D, G, R, or Q, and X5 is Y. CDR-L1 includes the amino acid sequence (SEQ ID NO: 262) in which X1 is S and X6 is A or V, CDR-L2 includes the amino acid sequence (SEQ ID NO: 263) in which X1 is A or S, X2 is T, S, E, Q, or D, X3 is N, R, A, E, or H, X4 is Q or A, and X5 is S or D, and CDR-L3 includes the amino acid sequence (SEQ ID NO: 264) in which X1 is S, N, D, Q, A, or E, X2 is T, I, or S, X3 is Y, L, or F, X4 is D, G, T, E, Q, A, or Y, and X5 is A, T, R, S, K, or Y. In some embodiments, the antibody or fragment comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 225, CDR-H2 containing the amino acid sequence of SEQ ID NO: 226, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 227, and the VL domain comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 16, CDR-L2 containing the amino acid sequence of SEQ ID NO: 17, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 228.In some embodiments, the VH domain includes an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 245, and the VL domain includes an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 246. In some embodiments, the antibody or fragment includes a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 225, CDR-H2 containing the amino acid sequence of SEQ ID NO: 232, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 233, and the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 234, CDR-L2 containing the amino acid sequence of SEQ ID NO: 235, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 236. In some embodiments, the VH domain includes an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 251, and the VL domain includes an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 252. In some embodiments, the VH domain includes the amino acid sequence of SEQ ID NO: 251, and the VL domain includes the amino acid sequence of SEQ ID NO: 252. In some embodiments, the antibody or fragment includes a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 225, CDR-H2 containing the amino acid sequence of SEQ ID NO: 232, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 233, and the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 16, CDR-L2 containing the amino acid sequence of SEQ ID NO: 17, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 228. In some embodiments, the VH domain comprises an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 253, and the VL domain comprises an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 254.In some embodiments, the VH domain further comprises FW-1 containing the sequence QVQLVQSGAEVKKPGSSVKVSCKASGGTFS (SEQ ID NO: 274), FW-2 containing the sequence WVRQAPGQGLEWMG (SEQ ID NO: 275), FW-3 containing the sequence RVTITADESTSTAYMELSSLRSEDTAVYYCAR (SEQ ID NO: 276), and / or FW-4 containing the sequence WGQGTLVTVSS (SEQ ID NO: 277). In some embodiments, the VL domain further comprises FW-1 containing the sequence DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 289), FW-2 containing the sequence WYQQKPGKAPKLLIY (SEQ ID NO: 290), FW-3 containing the sequence GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 291), and / or FW-4 containing the sequence FGGGTKVEIK (SEQ ID NO: 292).

[0016] In some embodiments, the antibody or fragment comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain comprises the amino acid sequence GX1X2FX3X4X5, where X1 is G, Y, S, or A, X2 is T, S, G, R, N, or H, X3 is S, T, R, H, Y, G, or P, X4 is S, K, G, N, R, D, T, or G, and X5 is Y, L, H, or F, comprising the amino acid sequence CDR-H1,X1PX2X3X4X5 (SEQ ID NO: 265), CDR-H2 and the amino acid sequence X1X2X3GX4X5LFX6X7, where X1 is D or A, X2 is A, G, E, R, Y, K, N, Q, L, or F, X3 is A, L, P, or Y, X4 is I or L, X5 is R, A, Q, or S, X CDR-H3 comprises the amino acid sequence (SEQ ID NO: 267) in which 6 is A or D and X7 is D, E, A, or S, and the VL domain comprises the amino acid sequence X1X2SX3X4IX5GX6LN in which X1 is R or G, X2 is A or T, X3 is Q or E, X4 is E, N, T, S, A, K, D, G, R, or Q, X5 is Y or S and X6 is A or V, and CDR-L1 comprises the amino acid sequence (SEQ ID NO: 262) in which X1 is A or CDR-L2 comprises the amino acid sequence (SEQ ID NO: 263) in which X2 is S, X3 is T, S, E, Q, or D, X4 is N, R, A, E, or H, X4 is Q or A, and X5 is S or D, and CDR-L3 comprises the amino acid sequence of QX1X2X3X4X5PWT, in which X1 is S, N, D, Q, A, or E, X2 is T, I, or S, X3 is Y, L, or F, X4 is D, G, T, E, Q, A, or Y, and X5 is A, T, R, S, K, or Y.In some embodiments, the VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 238, CDR-H2 containing the amino acid sequence of SEQ ID NO: 239, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 233; the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 16, CDR-L2 containing the amino acid sequence of SEQ ID NO: 17, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 228. In some embodiments, the VH domain includes an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 245; and the VL domain includes an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 246. In some embodiments, the VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 238, CDR-H2 containing the amino acid sequence of SEQ ID NO: 243, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 233; the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 234, CDR-L2 containing the amino acid sequence of SEQ ID NO: 235, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 236. In some embodiments, the VH domain includes an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 251; and the VL domain includes an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 252. In some embodiments, the VH domain includes the amino acid sequence of SEQ ID NO: 251, and the VL domain includes the amino acid sequence of SEQ ID NO: 252. In some embodiments, the VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 238, CDR-H2 containing the amino acid sequence of SEQ ID NO: 243, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 233, and the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 16, CDR-L2 containing the amino acid sequence of SEQ ID NO: 17, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 228.In some embodiments, the VH domain comprises an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 253, and the VL domain comprises an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 254. In some embodiments, the VH domain further comprises FW-1 comprising the sequence QVQLVQSGAEVKKPGSSVKVSCKAS (SEQ ID NO: 278), FW-2 comprising the sequence AISWVRQAPGQGLEWMGGI (SEQ ID NO: 279), FW-3 comprising the sequence ANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAR (SEQ ID NO: 280), and / or FW-4 comprising the sequence WGQGTLVTVSS (SEQ ID NO: 277). In some embodiments, the VL domain further comprises FW-1 containing the sequence DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 289), FW-2 containing the sequence WYQQKPGKAPKLLIY (SEQ ID NO: 290), FW-3 containing the sequence GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 291), and / or FW-4 containing the sequence FGGGTKVEIK (SEQ ID NO: 292).

[0017] In some embodiments, the antibody or fragment comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain comprises CDR-H1, which includes the amino acid sequence (SEQ ID NO: 268) X1YX2MS, where X1 is S, D, E, A, or Q, and X2 is A, G, or T; CDR-H2, which includes the amino acid sequence (SEQ ID NO: 269) DIX1X2X3GX4X5TX6YADSVKG, where X1 is T, N, S, Q, E, H, R, or A, X2 is Y, W, F, or H, X3 is A, S, Q, E, or T, X4 is G or E, X5 is S or I, and X6 is A or G; and X1X2 The CDR-H3 comprises the amino acid sequence (SEQ ID NO: 270) X3YX4WX5X6AX7DX8, where X1 is S or A, X2 is N, H, A, D, L, Q, Y, or R, X3 is A, N, S, or G, X4 is A, V, R, E, or S, X5 is D or S, X6 is D, N, Q, E, S, T, or L, X7 is L, F, or M, and X8 is I, Y, or V. The VL domain comprises CDR-L1 containing the amino acid sequence RASQSVSSNLA (SEQ ID NO: 40), CDR-L2 containing the amino acid sequence GASSRAT (SEQ ID NO: 41), and CDR-L3 containing the amino acid sequence QQYGSSPPVT (SEQ ID NO: 42). In some embodiments, the antibody or fragment comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 229, CDR-H2 containing the amino acid sequence of SEQ ID NO: 230, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 231, and the VL domain comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 40, CDR-L2 containing the amino acid sequence of SEQ ID NO: 41, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 42.In some embodiments, the VH domain contains an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 247, and the VL domain contains an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 248. In some embodiments, the VH domain contains an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 249, and the VL domain contains an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 250. In some embodiments, the VH domain contains the amino acid sequence of SEQ ID NO: 249, and the VL domain contains the amino acid sequence of SEQ ID NO: 250. In some embodiments, the antibody or fragment comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 229, CDR-H2 containing the amino acid sequence of SEQ ID NO: 237, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 231, and the VL domain comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 40, CDR-L2 containing the amino acid sequence of SEQ ID NO: 41, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 42. In some embodiments, the VH domain comprises an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 255, and the VL domain comprises an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 256. In some embodiments, the VH domain includes an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 257, and the VL domain includes an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 258.In some embodiments, the VH domain further comprises FW-1 containing the sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFS (SEQ ID NO: 281), FW-2 containing the sequence WVRQAPGKGLEWVS (SEQ ID NO: 282), FW-3 containing the sequence RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 283), and / or FW-4 containing the sequence WGQGTMVTVSS (SEQ ID NO: 284) or WGQGTLVTVSS (SEQ ID NO: 285). In some embodiments, the VL domain further comprises FW-1 containing the sequence EIVLTQSPGTLSLSPGERATLSC (SEQ ID NO: 293), FW-2 containing the sequence WYQQKPGQAPRLLIY (SEQ ID NO: 294), FW-3 containing the sequence GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC (SEQ ID NO: 295), and / or FW-4 containing the sequence FGQGTKVEIK (SEQ ID NO: 296).

[0018] In some embodiments, the antibody or fragment comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain comprises CDR-H1, which includes the amino acid sequence GFTFX1X2Y (SEQ ID NO: 271), where X1 is S, D, E, Q, S, or A, and X2 is S, D, E, A, or Q; CDR-H2, which includes the amino acid sequence X1X2X3GX4X5 (SEQ ID NO: 272), where X1 is T, N, S, Q, E, H, R, or A, X2 is Y, W, F, or H, X3 is A, S, Q, E, or T, X4 is G or E, and X5 is S or I; and X1X2X3YX4WX5 CDR-H3 comprises the amino acid sequence (SEQ ID NO: 273) X6AX7DX8, where X1 is S or A, X2 is N, H, A, D, L, Q, Y, or R, X3 is A, N, S, or G, X4 is A, V, R, E, or S, X5 is D or S, X6 is D, N, Q, E, S, T, or L, X7 is L, F, or M, and X8 is I, Y, or V. The VL domain comprises CDR-L1 containing the amino acid sequence RASQSVSSNLA (SEQ ID NO: 40), CDR-L2 containing the amino acid sequence GASSRAT (SEQ ID NO: 41), and CDR-L3 containing the amino acid sequence QQYGSSPPVT (SEQ ID NO: 42). In some embodiments, the VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 240, CDR-H2 containing the amino acid sequence of SEQ ID NO: 241, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 242; and the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 40, CDR-L2 containing the amino acid sequence of SEQ ID NO: 41, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 42. In some embodiments, the VH domain includes an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 247; and the VL domain includes an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 248.In some embodiments, the VH domain includes an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 249, and the VL domain includes an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 250. In some embodiments, the VH domain includes the amino acid sequence of SEQ ID NO: 249, and the VL domain includes the amino acid sequence of SEQ ID NO: 250. In some embodiments, the VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 240, CDR-H2 containing the amino acid sequence of SEQ ID NO: 244, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 242, and the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 40, CDR-L2 containing the amino acid sequence of SEQ ID NO: 41, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 42. In some embodiments, the VH domain includes an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 255, and the VL domain includes an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 256. In some embodiments, the VH domain includes an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 257, and the VL domain includes an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 258. In some embodiments, the VH domain further comprises FW-1 containing the sequence EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 286), FW-2 containing the sequence AMSWVRQAPGKGLEWVSDI (SEQ ID NO: 287), FW-3 containing the sequence TAYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 288), and / or FW-4 containing the sequence WGQGTMVTVSS (SEQ ID NO: 284) or WGQGTLVTVSS (SEQ ID NO: 285).In some embodiments, the VL domain further comprises FW-1 containing the sequence EIVLTQSPGTLSLSPGERATLSC (SEQ ID NO: 293), FW-2 containing the sequence WYQQKPGQAPRLLIY (SEQ ID NO: 294), FW-3 containing the sequence GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC (SEQ ID NO: 295), and FW-4 containing the sequence FGQGTKVEIK (SEQ ID NO: 296).

[0019] In some embodiments according to any of the embodiments described herein, the antibody is a multispecific antibody (e.g., a bispecific antibody).

[0020] This specification further provides a fusion protein comprising a first portion containing an antibody or fragment from any of the embodiments described above, and a second portion containing a cytokine, chemokine, or growth factor. In some embodiments, the first portion is fused to the second portion directly or via a linker. In some embodiments, the first part comprises a human or humanized antibody or its antigen-binding fragment that specifically binds to CD8b and / or CD8ab, wherein the antibody or fragment comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain (where the VH domain comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 13, CDR-H2 containing the amino acid sequence of SEQ ID NO: 14, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 15; the VL domain comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 16, CDR-L2 containing the amino acid sequence of SEQ ID NO: 17, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 18; the VH domain comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 19, CDR-H2 containing the amino acid sequence of SEQ ID NO: 20, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 21; and the VL domain comprises the amino acid sequence of SEQ ID NO: 22 The VH domain includes CDR-L1 containing the sequence, CDR-L2 containing the amino acid sequence of SEQ ID NO: 23, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 24; the VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 25, CDR-H2 containing the amino acid sequence of SEQ ID NO: 26, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 27; the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 28, CDR-L2 containing the amino acid sequence of SEQ ID NO: 29, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 30; the VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 31, CDR-H2 containing the amino acid sequence of SEQ ID NO: 32, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 33; the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 34, CDR-L2 containing the amino acid sequence of SEQ ID NO: 35, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 36;The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 37, CDR-H2 containing the amino acid sequence of SEQ ID NO: 38, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 39; the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 40, CDR-L2 containing the amino acid sequence of SEQ ID NO: 41, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 42; the VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 43, CDR-H2 containing the amino acid sequence of SEQ ID NO: 44, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 45; the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 46, CDR-L2 containing the amino acid sequence of SEQ ID NO: 47, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 48; the VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 1, CDR-H2 containing the amino acid sequence of SEQ ID NO: 2, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 3; the VL domain The in includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 4, CDR-L2 containing the amino acid sequence of SEQ ID NO: 5, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 6; the VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 177, CDR-H2 containing the amino acid sequence of SEQ ID NO: 178, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 179, and the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 180, CDR-L2 containing the amino acid sequence of SEQ ID NO: 181, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 182; the VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 225, CDR-H2 containing the amino acid sequence of SEQ ID NO: 226, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 227, and the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 16, CDR-L2 containing the amino acid sequence of SEQ ID NO: 17, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 228;The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 229, CDR-H2 containing the amino acid sequence of SEQ ID NO: 230, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 231; the VL domain includes CDR-L1 containing the amino acid sequence RASQSVSSNLA (SEQ ID NO: 40), CDR-L2 containing the amino acid sequence GASSRAT (SEQ ID NO: 41), and CDR-L3 containing the amino acid sequence QQYGSSPPVT (SEQ ID NO: 42); the VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 225, CDR-H2 containing the amino acid sequence of SEQ ID NO: 232, and the amino acid sequence of SEQ ID NO: 233 The VL domain includes CDR-H3 containing the sequence, and the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 234, CDR-L2 containing the amino acid sequence of SEQ ID NO: 235, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 236; the VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 225, CDR-H2 containing the amino acid sequence of SEQ ID NO: 232, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 233, and the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 16, CDR-L2 containing the amino acid sequence of SEQ ID NO: 17, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 228;Alternatively, the VH domain may include CDR-H1 containing the amino acid sequence of SEQ ID NO: 229, CDR-H2 containing the amino acid sequence of SEQ ID NO: 237, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 231; and the VL domain may include CDR-L1 containing the amino acid sequence RASQSVSSNLA (SEQ ID NO: 40), CDR-L2 containing the amino acid sequence GASSRAT (SEQ ID NO: 41), and CDR-L3 containing the amino acid sequence QQYGSSPPVT (SEQ ID NO: 42). In some embodiments, the first part comprises a human or humanized antibody or its antigen-binding fragment that specifically binds to CD8b and / or CD8ab, wherein the antibody or fragment comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain (where the VH domain comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 51, CDR-H2 containing the amino acid sequence of SEQ ID NO: 52, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 15; the VL domain comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 16, CDR-L2 containing the amino acid sequence of SEQ ID NO: 17, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 18; the VH domain comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 53, CDR-H2 containing the amino acid sequence of SEQ ID NO: 52, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 21; and the VL domain comprises the amino acid sequence of SEQ ID NO: 22 The VH domain includes CDR-L1 containing the sequence, CDR-L2 containing the amino acid sequence of SEQ ID NO: 23, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 24; the VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 49, CDR-H2 containing the amino acid sequence of SEQ ID NO: 52, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 27; the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 28, CDR-L2 containing the amino acid sequence of SEQ ID NO: 29, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 30; the VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 54, CDR-H2 containing the amino acid sequence of SEQ ID NO: 52, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 33; the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 34, CDR-L2 containing the amino acid sequence of SEQ ID NO: 35, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 36;The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 55, CDR-H2 containing the amino acid sequence of SEQ ID NO: 56, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 39; the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 40, CDR-L2 containing the amino acid sequence of SEQ ID NO: 41, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 42; the VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 55, CDR-H2 containing the amino acid sequence of SEQ ID NO: 57, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 45; the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 46, CDR-L2 containing the amino acid sequence of SEQ ID NO: 47, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 48; the VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 49, CDR-H2 containing the amino acid sequence of SEQ ID NO: 50, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 3; the VL domain The main domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 4, CDR-L2 containing the amino acid sequence of SEQ ID NO: 5, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 6; the VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 183, CDR-H2 containing the amino acid sequence of SEQ ID NO: 184, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 179; the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 180, CDR-L2 containing the amino acid sequence of SEQ ID NO: 181, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 182; the VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 238, CDR-H2 containing the amino acid sequence of SEQ ID NO: 239, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 233; the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 16, CDR-L2 containing the amino acid sequence of SEQ ID NO: 17, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 228;The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 240, CDR-H2 containing the amino acid sequence of SEQ ID NO: 241, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 242; the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 40, CDR-L2 containing the amino acid sequence of SEQ ID NO: 41, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 42; the VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 238, CDR-H2 containing the amino acid sequence of SEQ ID NO: 243, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 233; the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 234, CDR-L2 containing the amino acid sequence of SEQ ID NO: 235, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 236; the The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 238, CDR-H2 containing the amino acid sequence of SEQ ID NO: 243, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 233; the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 16, CDR-L2 containing the amino acid sequence of SEQ ID NO: 17, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 228; or the VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 240, CDR-H2 containing the amino acid sequence of SEQ ID NO: 244, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 242; the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 40, CDR-L2 containing the amino acid sequence of SEQ ID NO: 41, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 42).

[0021] In some embodiments of any of the embodiments described herein (e.g., the fusion protein of this disclosure), the second portion induces the activation of CD8+ T cells. In some embodiments, the fusion protein induces the activation of cells expressing human CD8ab heterodimer with at least 10 times higher potency than the activation of cells expressing human CD8aa homodimer. In some embodiments, the fusion protein induces the activation of CD8+ T cells with at least 10 times higher potency than the activation of NK cells. In some embodiments, the potency of activation is measured by EC50, which is assessed by cell proliferation. In some embodiments, the first portion comprises two antibody heavy chain polypeptides having a structure according to formula [I] from the N-terminus to the C-terminus: VH-CH1-Hinge-CH2-CH3 [I] and two antibody light chain polypeptides containing the structure of formula [II] from the N-terminus to the C-terminus: VL-CL [II] The first part comprises (wherein VH is the antibody heavy chain variable (VH) domain, CH1 is the antibody CH1 domain, hinge is the antibody hinge domain, CH2-CH3 is the antibody Fc domain, VL is the antibody light chain variable (VL) domain, and CL is the antibody constant light chain domain), where the N-terminus of the second part is fused to the C-terminus of one of the two CH3 domains (e.g., via the linker of this disclosure). In some embodiments, the first part comprises a first antibody heavy chain polypeptide having a structure according to formula [I] from the N-terminus to the C-terminus: VH-CH1-Hinge-CH2-CH3 [I], Antibody light chain polypeptides containing the structure shown by formula [II] from the N-terminus to the C-terminus: VL-CL [II] and a second antibody heavy chain polypeptide containing the structure of formula [III] from the N-terminus to the C-terminus: Hinge-CH2-CH3 [III], The first antibody heavy chain polypeptide comprises (wherein VH is the antibody heavy chain variable (VH) domain, CH1 is the antibody CH1 domain, Hinge is the antibody hinge domain, CH2-CH3 is the antibody Fc domain, VL is the antibody light chain variable (VL) domain, and CL is the antibody constant light chain domain), where the N-terminus of the second portion is fused to the C-terminus of the CH3 domain of the second antibody heavy chain polypeptide (e.g., via the linker of this disclosure). In some embodiments, the N-terminus of the second portion is fused to the C-terminus of the CH3 domain of the first antibody heavy chain polypeptide (e.g., via the linker of this disclosure). In some embodiments, the first portion comprises a first antibody heavy chain polypeptide having a structure according to formula [I] from the N-terminus to the C-terminus: VH-CH1-Hinge-CH2-CH3 [I], Antibody light chain polypeptides containing the structure shown by formula [II] from the N-terminus to the C-terminus: VL-CL [II] and a second antibody heavy chain polypeptide containing the structure of formula [III] from the N-terminus to the C-terminus: Hinge-CH2-CH3 [III], The first part comprises (wherein VH is the antibody heavy chain variable (VH) domain, CH1 is the antibody CH1 domain, Hinge is the antibody hinge domain, CH2-CH3 is the antibody Fc domain, VL is the antibody light chain variable (VL) domain, and CL is the antibody constant light chain domain), where the C-terminus of the second part is fused to the N-terminus of the hinge domain of the second antibody heavy chain polypeptide (e.g., via the linker of this disclosure). In some embodiments, the first part comprises one or two antibody heavy chain polypeptides and one or two antibody light chain polypeptides. In some embodiments, the first part comprises a single-chain antibody or a single-chain variable fragment (scFv). In some embodiments, the first part comprises a VHH antibody. In some embodiments according to any of the embodiments described herein (e.g., the fusion protein described above), VH and VL form an antigen-binding site (e.g., specifically binding to CD8b and / or CD8ab). In some embodiments, the first part comprises a first antibody heavy chain polypeptide having a structure according to formula [I] from the N-terminus to the C-terminus: VH-CH1-Hinge-CH2-CH3 [I], Antibody light chain polypeptides containing the structure shown by formula [II] from the N-terminus to the C-terminus: VL-CL [II] and a second antibody heavy chain polypeptide containing the structure of formula [III] from the N-terminus to the C-terminus: Hinge-CH2-CH3 [III], The formula includes (wherein VH is the VH domain, CH1 is the antibody CH1 domain, hinge is the antibody hinge domain, CH2-CH3 is the antibody Fc domain, VL is the VL domain, and CL is the antibody constant light chain domain), where the N-terminus of the second part is fused to the C-terminus of the CH3 domain of the first antibody heavy chain polypeptide. In some embodiments, the VH domain of both antibody heavy chain polypeptides comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 13, CDR-H2 containing the amino acid sequence of SEQ ID NO: 14, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 15; the VL domain of both antibody light chain polypeptides comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 16, CDR-L2 containing the amino acid sequence of SEQ ID NO: 17, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 18; the VH domain of both antibody heavy chain polypeptides comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 19, CDR-H2 containing the amino acid sequence of SEQ ID NO: 20, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 21; the VL domain of both antibody light chain polypeptides comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 22, CDR-L2 containing the amino acid sequence of SEQ ID NO: 23, and CDR containing the amino acid sequence of SEQ ID NO: 24 -Includes L3; The VH domains of both antibody heavy chain polypeptides include CDR-H1 containing the amino acid sequence of SEQ ID NO: 25, CDR-H2 containing the amino acid sequence of SEQ ID NO: 26, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 27; The VL domains of both antibody light chain polypeptides include CDR-L1 containing the amino acid sequence of SEQ ID NO: 28, CDR-L2 containing the amino acid sequence of SEQ ID NO: 29, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 30; The VH domains of both antibody heavy chain polypeptides include CDR-H1 containing the amino acid sequence of SEQ ID NO: 31, CDR-H2 containing the amino acid sequence of SEQ ID NO: 32, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 33; The VL domains of both antibody light chain polypeptides include CDR-L1 containing the amino acid sequence of SEQ ID NO: 34, CDR-L2 containing the amino acid sequence of SEQ ID NO: 35, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 36;The VH domains of both antibody heavy chain polypeptides include CDR-H1 containing the amino acid sequence of SEQ ID NO: 37, CDR-H2 containing the amino acid sequence of SEQ ID NO: 38, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 39; the VL domains of both antibody light chain polypeptides include CDR-L1 containing the amino acid sequence of SEQ ID NO: 40, CDR-L2 containing the amino acid sequence of SEQ ID NO: 41, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 42; the VH domains of both antibody heavy chain polypeptides include CDR-H1 containing the amino acid sequence of SEQ ID NO: 43, and SEQ ID NO: 4 The antibody light chain polypeptides include CDR-H2 containing the amino acid sequence of 4, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 45, and the VL domains of both antibody light chain polypeptides include CDR-L1 containing the amino acid sequence of SEQ ID NO: 46, CDR-L2 containing the amino acid sequence of SEQ ID NO: 47, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 48; the VH domains of both antibody heavy chain polypeptides include CDR-H1 containing the amino acid sequence of SEQ ID NO: 1, CDR-H2 containing the amino acid sequence of SEQ ID NO: 2, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 3, and both antibody light chains The VL domain of the polypeptide contains CDR-L1 containing the amino acid sequence of SEQ ID NO: 4, CDR-L2 containing the amino acid sequence of SEQ ID NO: 5, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 6; the VH domain of both antibody heavy chain polypeptides contains CDR-H1 containing the amino acid sequence of SEQ ID NO: 177, CDR-H2 containing the amino acid sequence of SEQ ID NO: 178, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 179; and the VL domain of both antibody light chain polypeptides contains CDR-L1 containing the amino acid sequence of SEQ ID NO: 180, and SEQ ID NO: 181 The antibody heavy chain polypeptides include CDR-L2 containing the amino acid sequence and CDR-L3 containing the amino acid sequence of SEQ ID NO: 182; the VH domains of both antibody heavy chain polypeptides include CDR-H1 containing the amino acid sequence of SEQ ID NO: 225, CDR-H2 containing the amino acid sequence of SEQ ID NO: 226, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 227; and the VL domains of both antibody light chain polypeptides include CDR-L1 containing the amino acid sequence of SEQ ID NO: 16, CDR-L2 containing the amino acid sequence of SEQ ID NO: 17, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 228;The VH domains of both antibody heavy chain polypeptides include CDR-H1 containing the amino acid sequence of SEQ ID NO: 229, CDR-H2 containing the amino acid sequence of SEQ ID NO: 230, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 231; the VL domains of both antibody light chain polypeptides include CDR-L1 containing the amino acid sequence of SEQ ID NO: 40, CDR-L2 containing the amino acid sequence of SEQ ID NO: 41, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 42; the VH domains of both antibody heavy chain polypeptides include CDR-H1 containing the amino acid sequence of SEQ ID NO: 225, CDR-H2 containing the amino acid sequence of SEQ ID NO: 232, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 233; the VL domains of both antibody light chain polypeptides include CDR-L1 containing the amino acid sequence of SEQ ID NO: 234, CDR-L2 containing the amino acid sequence of SEQ ID NO: 235, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 236; The VH domains of both antibody heavy chain polypeptides include CDR-H1 containing the amino acid sequence of SEQ ID NO: 225, CDR-H2 containing the amino acid sequence of SEQ ID NO: 232, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 233; and the VL domains of both antibody light chain polypeptides include CDR-L1 containing the amino acid sequence of SEQ ID NO: 16, CDR-L2 containing the amino acid sequence of SEQ ID NO: 17, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 228; or the VH domains of both antibody heavy chain polypeptides include CDR-H1 containing the amino acid sequence of SEQ ID NO: 229, CDR-H2 containing the amino acid sequence of SEQ ID NO: 237, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 231; and the VL domains of both antibody light chain polypeptides include CDR-L1 containing the amino acid sequence of SEQ ID NO: 40, CDR-L2 containing the amino acid sequence of SEQ ID NO: 41, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 42. In some embodiments, the VH domains of both antibody heavy chain polypeptides include CDR-H1 containing the amino acid sequence of SEQ ID NO: 51, CDR-H2 containing the amino acid sequence of SEQ ID NO: 52, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 15; and the VL domains of both antibody light chain polypeptides include CDR-L1 containing the amino acid sequence of SEQ ID NO: 16, CDR-L2 containing the amino acid sequence of SEQ ID NO: 17, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 18;The VH domains of both antibody heavy chain polypeptides include CDR-H1 containing the amino acid sequence of SEQ ID NO: 53, CDR-H2 containing the amino acid sequence of SEQ ID NO: 52, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 21; the VL domains of both antibody light chain polypeptides include CDR-L1 containing the amino acid sequence of SEQ ID NO: 22, CDR-L2 containing the amino acid sequence of SEQ ID NO: 23, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 24; the VH domains of both antibody heavy chain polypeptides include CDR-H1 containing the amino acid sequence of SEQ ID NO: 49, SEQ ID NO: The antibody light chain polypeptides include CDR-H2 containing the amino acid sequence of 52, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 27; the VL domains of both antibody light chain polypeptides include CDR-L1 containing the amino acid sequence of SEQ ID NO: 28, CDR-L2 containing the amino acid sequence of SEQ ID NO: 29, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 30; the VH domains of both antibody heavy chain polypeptides include CDR-H1 containing the amino acid sequence of SEQ ID NO: 54, CDR-H2 containing the amino acid sequence of SEQ ID NO: 52, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 33. The VL domains of both antibody light chain polypeptides include CDR-L1 containing the amino acid sequence of SEQ ID NO: 34, CDR-L2 containing the amino acid sequence of SEQ ID NO: 35, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 36; the VH domains of both antibody heavy chain polypeptides include CDR-H1 containing the amino acid sequence of SEQ ID NO: 55, CDR-H2 containing the amino acid sequence of SEQ ID NO: 56, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 39; and the VL domains of both antibody light chain polypeptides include CDR-L1 containing the amino acid sequence of SEQ ID NO: 40, SEQ ID NO: The antibody heavy chain polypeptides include CDR-L2 containing the amino acid sequence of 41, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 42; the VH domains of both antibody heavy chain polypeptides include CDR-H1 containing the amino acid sequence of SEQ ID NO: 55, CDR-H2 containing the amino acid sequence of SEQ ID NO: 57, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 45; and the VL domains of both antibody light chain polypeptides include CDR-L1 containing the amino acid sequence of SEQ ID NO: 46, CDR-L2 containing the amino acid sequence of SEQ ID NO: 47, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 48;The VH domains of both antibody heavy chain polypeptides include CDR-H1 containing the amino acid sequence of SEQ ID NO: 49, CDR-H2 containing the amino acid sequence of SEQ ID NO: 50, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 3; the VL domains of both antibody light chain polypeptides include CDR-L1 containing the amino acid sequence of SEQ ID NO: 4, CDR-L2 containing the amino acid sequence of SEQ ID NO: 5, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 6; the VH domains of both antibody heavy chain polypeptides include CDR-H1 containing the amino acid sequence of SEQ ID NO: 183, and SEQ ID NO: 184 The antibody light chain polypeptides include CDR-H2 containing an amino acid sequence and CDR-H3 containing the amino acid sequence of SEQ ID NO. 179, and the VL domains of both antibody light chain polypeptides include CDR-L1 containing the amino acid sequence of SEQ ID NO. 180, CDR-L2 containing the amino acid sequence of SEQ ID NO. 181, and CDR-L3 containing the amino acid sequence of SEQ ID NO. 182; the VH domains of both antibody heavy chain polypeptides include CDR-H1 containing the amino acid sequence of SEQ ID NO. 238, CDR-H2 containing the amino acid sequence of SEQ ID NO. 239, and CDR-H3 containing the amino acid sequence of SEQ ID NO. 233, and both antibody The VL domain of the body light chain polypeptide includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 16, CDR-L2 containing the amino acid sequence of SEQ ID NO: 17, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 228; the VH domain of both antibody heavy chain polypeptides includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 240, CDR-H2 containing the amino acid sequence of SEQ ID NO: 241, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 242; and the VL domain of both antibody light chain polypeptides includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 40, and SEQ ID NO: 41 The antibody heavy chain polypeptides include CDR-L2 containing the amino acid sequence of SEQ ID NO: 42, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 42; the VH domains of both antibody heavy chain polypeptides include CDR-H1 containing the amino acid sequence of SEQ ID NO: 238, CDR-H2 containing the amino acid sequence of SEQ ID NO: 243, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 233; and the VL domains of both antibody light chain polypeptides include CDR-L1 containing the amino acid sequence of SEQ ID NO: 234, CDR-L2 containing the amino acid sequence of SEQ ID NO: 235, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 236;The VH domains of both antibody heavy chain polypeptides contain CDR-H1 containing the amino acid sequence of SEQ ID NO: 238, CDR-H2 containing the amino acid sequence of SEQ ID NO: 243, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 233; and the VL domains of both antibody light chain polypeptides contain CDR-L1 containing the amino acid sequence of SEQ ID NO: 16, CDR-L2 containing the amino acid sequence of SEQ ID NO: 17, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 228; or the VH domains of both antibody heavy chain polypeptides contain CDR-H1 containing the amino acid sequence of SEQ ID NO: 240, CDR-H2 containing the amino acid sequence of SEQ ID NO: 244, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 242; and the VL domains of both antibody light chain polypeptides contain CDR-L1 containing the amino acid sequence of SEQ ID NO: 40, CDR-L2 containing the amino acid sequence of SEQ ID NO: 41, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 42. In the application form, the VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 13, CDR-H2 containing the amino acid sequence of SEQ ID NO: 14, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 15; the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 16, CDR-L2 containing the amino acid sequence of SEQ ID NO: 17, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 18; the VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 19, CDR-H2 containing the amino acid sequence of SEQ ID NO: 20, and CDR- The H3 domain contains CDR-L1 containing the amino acid sequence of SEQ ID NO: 22, CDR-L2 containing the amino acid sequence of SEQ ID NO: 23, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 24; the VH domain contains CDR-H1 containing the amino acid sequence of SEQ ID NO: 25, CDR-H2 containing the amino acid sequence of SEQ ID NO: 26, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 27; the VL domain contains CDR-L1 containing the amino acid sequence of SEQ ID NO: 28, CDR-L2 containing the amino acid sequence of SEQ ID NO: 29, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 30 The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 31, CDR-H2 containing the amino acid sequence of SEQ ID NO: 32, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 33; the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 34, CDR-L2 containing the amino acid sequence of SEQ ID NO: 35, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 36; the VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 37, CDR-H2 containing the amino acid sequence of SEQ ID NO: 38, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 39. Furthermore, the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 40, CDR-L2 containing the amino acid sequence of SEQ ID NO: 41, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 42; the VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 43, CDR-H2 containing the amino acid sequence of SEQ ID NO: 44, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 45; and the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 46, CDR-L2 containing the amino acid sequence of SEQ ID NO: 47, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 48;The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 1, CDR-H2 containing the amino acid sequence of SEQ ID NO: 2, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 3; the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 4, CDR-L2 containing the amino acid sequence of SEQ ID NO: 5, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 6; the VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 177, CDR-H2 containing the amino acid sequence of SEQ ID NO: 178, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 179; the VL domain is The VH domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 180, CDR-L2 containing the amino acid sequence of SEQ ID NO: 181, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 182; the VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 225, CDR-H2 containing the amino acid sequence of SEQ ID NO: 226, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 227; the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 16, CDR-L2 containing the amino acid sequence of SEQ ID NO: 17, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 228; VH domain The VL domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 229, CDR-H2 containing the amino acid sequence of SEQ ID NO: 230, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 231; the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 40, CDR-L2 containing the amino acid sequence of SEQ ID NO: 41, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 42; the VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 225, CDR-H2 containing the amino acid sequence of SEQ ID NO: 232, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 233; VL The domains include CDR-L1 containing the amino acid sequence of SEQ ID NO: 234, CDR-L2 containing the amino acid sequence of SEQ ID NO: 235, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 236; the VH domains include CDR-H1 containing the amino acid sequence of SEQ ID NO: 225, CDR-H2 containing the amino acid sequence of SEQ ID NO: 232, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 233; and the VL domains include CDR-L1 containing the amino acid sequence of SEQ ID NO: 16, CDR-L2 containing the amino acid sequence of SEQ ID NO: 17, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 228;Alternatively, the VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 229, CDR-H2 containing the amino acid sequence of SEQ ID NO: 237, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 231, and the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 40, CDR-L2 containing the amino acid sequence of SEQ ID NO: 41, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 42. In some embodiments, the VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 51, CDR-H2 containing the amino acid sequence of SEQ ID NO: 52, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 15; the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 16, CDR-L2 containing the amino acid sequence of SEQ ID NO: 17, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 18; the VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 53, CDR-H2 containing the amino acid sequence of SEQ ID NO: 52, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 21; the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 22, CDR-L2 containing the amino acid sequence of SEQ ID NO: 23, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 24 -Includes L3; The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 49, CDR-H2 containing the amino acid sequence of SEQ ID NO: 52, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 27; The VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 28, CDR-L2 containing the amino acid sequence of SEQ ID NO: 29, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 30; The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 54, CDR-H2 containing the amino acid sequence of SEQ ID NO: 52, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 33; The VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 34, CDR-L2 containing the amino acid sequence of SEQ ID NO: 35, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 36;The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 55, CDR-H2 containing the amino acid sequence of SEQ ID NO: 56, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 39; the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 40, CDR-L2 containing the amino acid sequence of SEQ ID NO: 41, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 42; the VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 55, CDR-H2 containing the amino acid sequence of SEQ ID NO: 57, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 45, and VL The domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 46, CDR-L2 containing the amino acid sequence of SEQ ID NO: 47, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 48; the VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 49, CDR-H2 containing the amino acid sequence of SEQ ID NO: 50, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 3; the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 4, CDR-L2 containing the amino acid sequence of SEQ ID NO: 5, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 6; the VH domain is The VL domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 183, CDR-H2 containing the amino acid sequence of SEQ ID NO: 184, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 179; the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 180, CDR-L2 containing the amino acid sequence of SEQ ID NO: 181, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 182; the VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 238, CDR-H2 containing the amino acid sequence of SEQ ID NO: 239, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 233; V The L domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 16, CDR-L2 containing the amino acid sequence of SEQ ID NO: 17, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 228; the VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 240, CDR-H2 containing the amino acid sequence of SEQ ID NO: 241, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 242; and the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 40, CDR-L2 containing the amino acid sequence of SEQ ID NO: 41, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 42;The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 238, CDR-H2 containing the amino acid sequence of SEQ ID NO: 243, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 233; the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 234, CDR-L2 containing the amino acid sequence of SEQ ID NO: 235, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 236; the VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 238, CDR-H2 containing the amino acid sequence of SEQ ID NO: 243, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 233. The VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 16, CDR-L2 containing the amino acid sequence of SEQ ID NO: 17, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 228; or the VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 240, CDR-H2 containing the amino acid sequence of SEQ ID NO: 244, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 242, and the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 40, CDR-L2 containing the amino acid sequence of SEQ ID NO: 41, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 42. In some embodiments, the VH domain of both antibody heavy chain polypeptides contains an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 62; the VL domain of both antibody light chain polypeptides contains an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 63; the VH domain of both antibody heavy chain polypeptides contains an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 64. The acid sequence is included, and the VL domain of both antibody light chain polypeptides contains an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 65; the VH domain of both antibody heavy chain polypeptides contains an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 66, and the VL domain of both antibody light chain polypeptides contains an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 67;The VH domain of both antibody heavy chain polypeptides contains an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 68, and the VL domain of both antibody light chain polypeptides contains an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 69; the VH domain of both antibody heavy chain polypeptides contains an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 70, and the VL domain of both antibody light chain polypeptides contains an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 71; The VH domain of both antibody heavy chain polypeptides contains an amino acid sequence that is 0% identical; the VH domain of both antibody heavy chain polypeptides contains an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 72, and the VL domain of both antibody light chain polypeptides contains an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 73; the VH domain of both antibody heavy chain polypeptides contains an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 185, and the VL domain of both antibody light chain polypeptides contains an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 186; the VH domain of both antibody heavy chain polypeptides contains an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 245, and the VL domain of both antibody light chain polypeptides The VH domain of both antibody heavy chain polypeptides contains an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 246; the VH domain of both antibody heavy chain polypeptides contains an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 251, and the VL domain of both antibody light chain polypeptides contains an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 252; the VH domain contains the amino acid sequence of SEQ ID NO: 251, and the VL domain contains the amino acid sequence of SEQ ID NO: 252; the VH domain of both antibody heavy chain polypeptides contains an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 253, and the VL domain of both antibody light chain polypeptides contains an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 254;The VH domain of both antibody heavy chain polypeptides contains an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 247, and the VL domain of both antibody light chain polypeptides contains an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 248; the VH domain of both antibody heavy chain polypeptides contains an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 249, and the VL domain of both antibody light chain polypeptides contains an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 250; the VH domain contains the amino acid sequence of SEQ ID NO: 249, and the VL The domain contains the amino acid sequence of SEQ ID NO: 250; the VH domain of both antibody heavy chain polypeptides contains an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 255, and the VL domain of both antibody light chain polypeptides contains an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 256; or the VH domain of both antibody heavy chain polypeptides contains an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 257, and the VL domain of both antibody light chain polypeptides contains an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 258. In some embodiments, the VH domain includes an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 62, and the VL domain includes an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 63; the VH domain includes an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 64, and the VL domain includes an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 65;The VH domain contains an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 66, and the VL domain contains an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 67; the VH domain contains an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 68, and the VL domain contains an amino acid sequence that is at least 90%, at least 95%, at least 99%, or The VH domain contains an amino acid sequence that is 100% identical; the VL domain contains an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 70, and the VL domain contains an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 71; the VH domain contains an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 72, and the VL domain contains an amino acid sequence that is at least 90%, at least 93% identical to the sequence of SEQ ID NO: 73 The VH domain contains an amino acid sequence that is at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 185, and the VL domain contains an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 186; the VH domain contains an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 245, and the VL domain is The VH domain contains an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 246; the VH domain contains an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 251, and the VL domain contains an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 252; the VH domain contains the amino acid sequence of SEQ ID NO: 251, and the VL domain contains the amino acid sequence of SEQ ID NO: 252;The VH domain contains an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 253, and the VL domain contains an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 254; the VH domain contains an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 247, and the VL domain contains an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 248; the VH domain contains an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 249, and the VL domain contains an amino acid sequence that is at least 90%, The amino acid sequence is at least 95%, at least 99%, or 100% identical; the VH domain contains the amino acid sequence of SEQ ID NO: 249, and the VL domain contains the amino acid sequence of SEQ ID NO: 250; the VH domain contains the amino acid sequence is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 255, and the VL domain contains the amino acid sequence is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 256; or the VH domain contains the amino acid sequence is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 257, and the VL domain contains the amino acid sequence is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 258. In some embodiments, one or both of the antibody heavy chain polypeptides include the following amino acid substitutions: L234A, L235A, and G237A (numbered according to the EU index). In some embodiments, the first antibody heavy chain polypeptide includes the amino acid substitutions Y349C and T366W, and the second antibody heavy chain polypeptide includes the amino acid substitutions S354C, T366S, L368A, and Y407V (numbered according to the EU index).

[0022] In some embodiments according to any of the embodiments described herein, the second portion comprises an IL-2 polypeptide. In some embodiments, the IL-2 polypeptide is an IL-2 polypeptide variant comprising one or more mutations from a human IL-2 polypeptide comprising the sequence of SEQ ID NO: 81. In some embodiments, the IL-2 polypeptide variant has a binding affinity to IL-2Rα that is 50% or less lower compared to the binding affinity to IL-2Rα of the wild-type IL-2 polypeptide comprising the sequence of SEQ ID NO: 81. In some embodiments, the IL-2 polypeptide variant has a binding affinity to IL-2Rβ that is 50% or less lower than the binding affinity to IL-2Rβ of the wild-type IL-2 polypeptide containing the sequence of SEQ ID NO: 81; and / or the IL-2 polypeptide variant has a binding affinity to IL-2Rγ that is 50% or less lower than the binding affinity to IL-2Rγ of the wild-type IL-2 polypeptide containing the sequence of SEQ ID NO: 81. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 having one, two, three, four, or five amino acid substitutions relative to SEQ ID NO: 81, wherein the one, two, three, four, or five substitutions(s) include substitutions(s) at the position of SEQ ID NO: 81 selected from the group consisting of: Q11, H16, L18, L19, D20, Q22, R38, F42, K43, Y45, E62, P65, E68, V69, L72, D84, S87, N88, V91, I92, T123, Q126, S127, I129, and S130. In some embodiments, the IL-2 polypeptide is (relative to the sequence of SEQ ID NO: 81) the following: R38E and F42A; R38D and F42A; F42A and E62Q; R38A and F42K; R38E, F42A, and N88S; R38E, F42A, and N88A; R38E, F42A, and N88G; R38E, F42A, and N88R; R38E, F42A, and N88T; R38E, F42A, and N88D;R38E, F42A, and V91E; R38E, F42A, and D84H; R38E, F42A, and D84K; R38E, F42A, and D84R; H16D, R38E, and F42A; H16E, R38E, and F42A; R38E, F42A, and Q126S; R38D, F42A, and N88S; R38D, F42A, and N88A; R38D, F42A, and N88G; R38D, F42A, and N88R; R38D, F42A, and N88T; R38D , F42A and N88D; R38D, F42A and V91E; R38D, F42A, and D84H; R38D, F42A, and D84K; R38D, F42A, and D84R; H16D, R38D and F42A; H16E, R38D and F42A; R38D, F42A and Q126S; R38A, F42K, and N88S; R38A, F42K, and N88A; R38A, F42K, and N88G; R38A, F42K, and N88R; R38A, F42K, and N88T; R38A, F42K, and N88D; R38A, F42K, and V91E; R38A, F42K, and D84H; R38A, F42K, and D84K; R38A, F42K, and D84R; H16D, R38A, and F42K; H16E, R38A, and F42K; R38A, F42K, and Q126S; F42A, E62Q, and N88S; F42A, E62Q, and N88A; F42A, E62Q, and N88G; F The sequence of SEQ ID NO: 81 includes one of the amino acid substitution sets of 42A, E62Q, and N88R; F42A, E62Q, and N88T; F42A, E62Q, and N88D; F42A, E62Q, and V91E; F42A, E62Q, and D84H; F42A, E62Q, and D84K; F42A, E62Q, and D84R; H16D, F42A, and E62Q; H16E, F42A, and E62Q; F42A, E62Q, and Q126S. In some embodiments, the IL-2 polypeptide includes the sequence of SEQ ID NO: 81 having a further amino acid substitution at position C125 relative to SEQ ID NO: 81. In some embodiments, the IL-2 polypeptide is (relative to the sequence of SEQ ID NO: 81) R38E, F42A, and C125A; R38D, F42A, and C125A; F42A, E62Q, and C125A; R38A, F42K, and C125A;R38E, F42A, N88S, and C125A; R38E, F42A, N88A, and C125A; R38E, F42A, N88G, and C125A; R38E, F42A, N88R, and C125A; R38E, F42A, N88T, and C125A; R38E, F42A, N88D, and C125A; R38E, F42A, V91E, and C125A; R38E, F42A, D84H, and C125A; R38E, F42A, D84K, and C125A; R38E, F42A, D84R, and C125A; H16D, R38E, F42A, and C125A;H16E, R38E, F42A, and C125A;R38E, F42A, C125A and Q126S;R38D, F42A, N88S, and C125A;R38D, F42A, N88A, and C125A;R38D, F42A, N88G, and C125A;R38D, F42A, N88R, and C125A;R38D, F42A, N88T, and C125A;R38D, F42A, N88D, and C125A;R38D, F42A, V91E, and C125A;R38D, F42A, D84H, and C125A;R38D, F42A, D8 4K, and C125A; R38D, F42A, D84R, and C125A; H16D, R38D, F42A, and C125A; H16E, R38D, F42A, and C125A; R38D, F42A, C125A, and Q126S; R38A, F42K, N88S, and C125A; R38A, F42K, N88G, and C125A; R38A, F42K, N88R, and C125A; R38A, F42K, N88T, and C125A; R38A, F42K, N88D, and C125A; R38A, F42K, N88A, and C125A; R38A, F42K, V91E, and C125A; R38A, F42K, D84H, and C125A; R38A, F42K, D84K, and C125A; R38A, F42K, D84R, and C125A; H16D, R38A, F42K, and C125A; H16E, R38A, F42K, and C125A; R38A, F42K, C125A, and Q126S; F42A, E62Q, N88S, and C125A; F42A, E62Q, N88A, and C125A; F42A, E62Q, N88G, and C125A; F42A, E62Q, N88R, and C125A;F42A, E62Q, N88T, and C125A; F42A, E62Q, N88D, and C125A; F42A, E62Q, V91E, and C125A; F42A, E62Q, and D84H, and C125A; F42A, E62Q, and D84K, and C125A; F42A, E62Q, and D84R, and C125A; H16D, F42A, and E62Q, and C125A; H16E, F42A, E62Q, and C125A; F42A, E62Q, C125A, and Q126S; F42A, N88S, and C125A; F42A, N88A, and C125A; F42A, N88G, and C125A; F42A, N88R, and C125A; F42A, N88T, and C125A; F42A, N88D, and C125A; F42A, V91E, and C125A; F42A, D84H, and C125A; F42A, D84K, and C125A; F42A, D84R, and C125A; H16D, F42A, and C125A; H16E, F42A, and C125A;The sequence also includes the sequence of SEQ ID NO: 81, which has one of the amino acid substitution sets of F42A, C125A, and Q126S. In some embodiments, the IL-2 polypeptide includes the sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISAINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 80). In some embodiments, the IL-2 polypeptide includes the sequence APTSSSTKKTQLQLEELLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 297). In some embodiments, the IL-2 polypeptide comprises a sequence selected from the group consisting of SEQ ID NOs. 85-155 and 190-216. In some embodiments, the IL-2 polypeptide comprises a sequence selected from the group consisting of SEQ ID NOs. 80, 85-155, 190-216, 297, and 354-383. In some embodiments, the second portion comprises a polypeptide that induces signal transduction via IL2Rβγ. In some embodiments, the second portion comprises an IL-21 polypeptide.

[0023] In some embodiments according to any of the embodiments described herein (e.g., the fusion proteins of this disclosure), one or both of the antibody Fc domains include a human IgG1 Fc domain having the following amino acid substitutions: L234A, L235A, G237A, and K322A (numbered according to the EU index). In some embodiments, one or both of the antibody Fc domains lack C-terminal lysine. In some embodiments, one or both of the antibody Fc domains include a human IgG1 Fc domain having the following amino acid substitutions: L234A, L235A, and G237A (numbered according to the EU index). In some embodiments, one or both of the antibody Fc domains lack C-terminal lysine. In some embodiments, the first of the two Fc domains comprises a human IgG1 Fc domain having amino acid substitutions Y349C and T366W, and the second of the two Fc domains comprises a human IgG1 Fc domain having amino acid substitutions S354C, T366S, L368A and Y407V (numbering according to the EU index). In some embodiments, one or both of the antibody Fc domains lack a C-terminal lysine. In some embodiments (e.g., the fusion proteins of this disclosure), the linker comprises the sequence (GGGS)xGn (SEQ ID NO: 74), (GGGGS)xGn (SEQ ID NO: 75), or (GGGGGS)xGn (SEQ ID NO: 76), S(GGGS)xGn (SEQ ID NO: 386), S(GGGGS)xGn (SEQ ID NO: 387), or S(GGGGGS)xGn (SEQ ID NO: 388), where x = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, and n = 0, 1, 2, or 3. In some embodiments, the linker comprises the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 79) or SGGGGSGGGGSGGGGS (SEQ ID NO: 389).In some embodiments (for example, in the fusion protein of the present disclosure), a linker connects a first portion of the present disclosure (e.g., a human or humanized antibody or its antigen-binding fragment that specifically binds to CD8b and / or CD8ab) to a second portion of the present disclosure (e.g., the IL-2 polypeptide of the present disclosure, the IL-21 polypeptide of the present disclosure, or a polypeptide that induces signaling via IL2Rβγ of the present disclosure).

[0024] In some embodiments, the fusion protein of the Disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO: 156, a heavy chain containing the amino acid sequence of SEQ ID NO: 157, and a heavy chain containing the amino acid sequence of SEQ ID NO: 158. In some embodiments, the fusion protein of the Disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO: 156, a heavy chain containing the amino acid sequence of SEQ ID NO: 157, and a heavy chain containing the amino acid sequence of SEQ ID NO: 217. In some embodiments, the fusion protein of the Disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO: 159, a heavy chain containing the amino acid sequence of SEQ ID NO: 160, and a heavy chain containing the amino acid sequence of SEQ ID NO: 161. In some embodiments, the fusion protein of the Disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO: 159, a heavy chain containing the amino acid sequence of SEQ ID NO: 160, and a heavy chain containing the amino acid sequence of SEQ ID NO: 218. In some embodiments, the fusion protein of the Disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO: 162, a heavy chain containing the amino acid sequence of SEQ ID NO: 163, and a heavy chain containing the amino acid sequence of SEQ ID NO: 164. In some embodiments, the fusion protein of the Disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO: 162, a heavy chain containing the amino acid sequence of SEQ ID NO: 163, and a heavy chain containing the amino acid sequence of SEQ ID NO: 219. In some embodiments, the fusion protein of the Disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO: 165, a heavy chain containing the amino acid sequence of SEQ ID NO: 166, and a heavy chain containing the amino acid sequence of SEQ ID NO: 167. In some embodiments, the fusion protein of the Disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO: 165, a heavy chain containing the amino acid sequence of SEQ ID NO: 166, and a heavy chain containing the amino acid sequence of SEQ ID NO: 220. In some embodiments, the fusion protein of the Disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO: 168, a heavy chain containing the amino acid sequence of SEQ ID NO: 169, and a heavy chain containing the amino acid sequence of SEQ ID NO: 170. In some embodiments, the fusion protein of the Disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO: 168, a heavy chain containing the amino acid sequence of SEQ ID NO: 169, and a heavy chain containing the amino acid sequence of SEQ ID NO: 221.In some embodiments, the fusion protein of the Disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO: 171, a heavy chain containing the amino acid sequence of SEQ ID NO: 172, and a heavy chain containing the amino acid sequence of SEQ ID NO: 173. In some embodiments, the fusion protein of the Disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO: 171, a heavy chain containing the amino acid sequence of SEQ ID NO: 172, and a heavy chain containing the amino acid sequence of SEQ ID NO: 222. In some embodiments, the fusion protein of the Disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO: 174, a heavy chain containing the amino acid sequence of SEQ ID NO: 175, and a heavy chain containing the amino acid sequence of SEQ ID NO: 176. In some embodiments, the fusion protein of the Disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO: 174, a heavy chain containing the amino acid sequence of SEQ ID NO: 175, and a heavy chain containing the amino acid sequence of SEQ ID NO: 223. In some embodiments, the fusion protein of the Disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO: 187, a heavy chain containing the amino acid sequence of SEQ ID NO: 188, and a heavy chain containing the amino acid sequence of SEQ ID NO: 189. In some embodiments, the fusion protein of the Disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO: 187, a heavy chain containing the amino acid sequence of SEQ ID NO: 188, and a heavy chain containing the amino acid sequence of SEQ ID NO: 224. In some embodiments, the fusion protein of the Disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO: 298, a heavy chain containing the amino acid sequence of SEQ ID NO: 299, and a heavy chain containing the amino acid sequence of SEQ ID NO: 300. In some embodiments, the fusion protein of the Disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO: 298, a heavy chain containing the amino acid sequence of SEQ ID NO: 299, and a heavy chain containing the amino acid sequence of SEQ ID NO: 301. In some embodiments, the fusion protein of the Disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO: 302, a heavy chain containing the amino acid sequence of SEQ ID NO: 303, and a heavy chain containing the amino acid sequence of SEQ ID NO: 304. In some embodiments, the fusion protein of the Disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO: 302, a heavy chain containing the amino acid sequence of SEQ ID NO: 303, and a heavy chain containing the amino acid sequence of SEQ ID NO: 305.In some embodiments, the fusion protein of the Disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO: 306, a heavy chain containing the amino acid sequence of SEQ ID NO: 307, and a heavy chain containing the amino acid sequence of SEQ ID NO: 308. In some embodiments, the fusion protein of the Disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO: 306, a heavy chain containing the amino acid sequence of SEQ ID NO: 307, and a heavy chain containing the amino acid sequence of SEQ ID NO: 309. In some embodiments, the fusion protein of the Disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO: 310, a heavy chain containing the amino acid sequence of SEQ ID NO: 311, and a heavy chain containing the amino acid sequence of SEQ ID NO: 312. In some embodiments, the fusion protein of the Disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO: 310, a heavy chain containing the amino acid sequence of SEQ ID NO: 311, and a heavy chain containing the amino acid sequence of SEQ ID NO: 313. In some embodiments, the fusion protein of the Disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO: 314, a heavy chain containing the amino acid sequence of SEQ ID NO: 315, and a heavy chain containing the amino acid sequence of SEQ ID NO: 316. In some embodiments, the fusion protein of the Disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO: 314, a heavy chain containing the amino acid sequence of SEQ ID NO: 315, and a heavy chain containing the amino acid sequence of SEQ ID NO: 317. In some embodiments, the fusion protein of the Disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO: 318, a heavy chain containing the amino acid sequence of SEQ ID NO: 319, and a heavy chain containing the amino acid sequence of SEQ ID NO: 320. In some embodiments, the fusion protein of the Disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO: 318, a heavy chain containing the amino acid sequence of SEQ ID NO: 319, and a heavy chain containing the amino acid sequence of SEQ ID NO: 321. In some embodiments, the fusion protein of the Disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO: 322, a heavy chain containing the amino acid sequence of SEQ ID NO: 323, and a heavy chain containing the amino acid sequence of SEQ ID NO: 324. In some embodiments, the fusion protein of the Disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO: 322, a heavy chain containing the amino acid sequence of SEQ ID NO: 323, and a heavy chain containing the amino acid sequence of SEQ ID NO: 325.In some embodiments, the fusion protein of the Disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO: 326, a heavy chain containing the amino acid sequence of SEQ ID NO: 327, and a heavy chain containing the amino acid sequence of SEQ ID NO: 328. In some embodiments, the fusion protein of the Disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO: 326, a heavy chain containing the amino acid sequence of SEQ ID NO: 327, and a heavy chain containing the amino acid sequence of SEQ ID NO: 329. In some embodiments, the fusion protein of the Disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO: 330, a heavy chain containing the amino acid sequence of SEQ ID NO: 331, and a heavy chain containing the amino acid sequence of SEQ ID NO: 332. In some embodiments, the fusion protein of the Disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO: 330, a heavy chain containing the amino acid sequence of SEQ ID NO: 331, and a heavy chain containing the amino acid sequence of SEQ ID NO: 333. In some embodiments, the fusion protein of the Disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO: 334, a heavy chain containing the amino acid sequence of SEQ ID NO: 335, and a heavy chain containing the amino acid sequence of SEQ ID NO: 336. In some embodiments, the fusion protein of the Disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO: 334, a heavy chain containing the amino acid sequence of SEQ ID NO: 335, and a heavy chain containing the amino acid sequence of SEQ ID NO: 337. In some embodiments, the fusion protein of the Disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO: 338, a heavy chain containing the amino acid sequence of SEQ ID NO: 339, and a heavy chain containing the amino acid sequence of SEQ ID NO: 340. In some embodiments, the fusion protein of the Disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO: 338, a heavy chain containing the amino acid sequence of SEQ ID NO: 339, and a heavy chain containing the amino acid sequence of SEQ ID NO: 341. In some embodiments, the fusion protein of the Disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO: 342, a heavy chain containing the amino acid sequence of SEQ ID NO: 343, and a heavy chain containing the amino acid sequence of SEQ ID NO: 344. In some embodiments, the fusion protein of the Disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO: 342, a heavy chain containing the amino acid sequence of SEQ ID NO: 343, and a heavy chain containing the amino acid sequence of SEQ ID NO: 345.In some embodiments, the fusion protein of the Disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO: 346, a heavy chain containing the amino acid sequence of SEQ ID NO: 347, and a heavy chain containing the amino acid sequence of SEQ ID NO: 348. In some embodiments, the fusion protein of the Disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO: 346, a heavy chain containing the amino acid sequence of SEQ ID NO: 347, and a heavy chain containing the amino acid sequence of SEQ ID NO: 349. In some embodiments, the fusion protein of the Disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO: 350, a heavy chain containing the amino acid sequence of SEQ ID NO: 351, and a heavy chain containing the amino acid sequence of SEQ ID NO: 352. In some embodiments, the fusion protein of the Disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO: 156, a heavy chain containing the amino acid sequence of SEQ ID NO: 157, and a heavy chain containing the amino acid sequence of SEQ ID NO: 217. In some embodiments, the fusion protein of the Disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO: 159, a heavy chain containing the amino acid sequence of SEQ ID NO: 160, and a heavy chain containing the amino acid sequence of SEQ ID NO: 218. In some embodiments, the fusion protein of the Disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO: 162, a heavy chain containing the amino acid sequence of SEQ ID NO: 163, and a heavy chain containing the amino acid sequence of SEQ ID NO: 219. In some embodiments, the fusion protein of the Disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO: 165, a heavy chain containing the amino acid sequence of SEQ ID NO: 166, and a heavy chain containing the amino acid sequence of SEQ ID NO: 220. In some embodiments, the fusion protein of the Disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO: 168, a heavy chain containing the amino acid sequence of SEQ ID NO: 169, and a heavy chain containing the amino acid sequence of SEQ ID NO: 221. In some embodiments, the fusion protein of the Disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO: 171, a heavy chain containing the amino acid sequence of SEQ ID NO: 172, and a heavy chain containing the amino acid sequence of SEQ ID NO: 222. In some embodiments, the fusion protein of the Disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO: 174, a heavy chain containing the amino acid sequence of SEQ ID NO: 175, and a heavy chain containing the amino acid sequence of SEQ ID NO: 223.In some embodiments, the fusion protein of the present disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO: 187, a heavy chain containing the amino acid sequence of SEQ ID NO: 188, and a heavy chain containing the amino acid sequence of SEQ ID NO: 224. In some embodiments, the fusion of the present disclosure. The protein comprises one or two light chains containing the amino acid sequence of SEQ ID NO: 298, a heavy chain containing the amino acid sequence of SEQ ID NO: 299, and a heavy chain containing the amino acid sequence of SEQ ID NO: 301. In some embodiments, the fusion protein of the Disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO: 302, a heavy chain containing the amino acid sequence of SEQ ID NO: 303, and a heavy chain containing the amino acid sequence of SEQ ID NO: 305. In some embodiments, the fusion protein of the Disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO: 306, a heavy chain containing the amino acid sequence of SEQ ID NO: 307, and a heavy chain containing the amino acid sequence of SEQ ID NO: 309. In some embodiments, the fusion protein of the Disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO: 310, a heavy chain containing the amino acid sequence of SEQ ID NO: 311, and a heavy chain containing the amino acid sequence of SEQ ID NO: 313. In some embodiments, the fusion protein of the Disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO: 314, a heavy chain containing the amino acid sequence of SEQ ID NO: 315, and a heavy chain containing the amino acid sequence of SEQ ID NO: 317. In some embodiments, the fusion protein of the Disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO: 318, a heavy chain containing the amino acid sequence of SEQ ID NO: 319, and a heavy chain containing the amino acid sequence of SEQ ID NO: 321. In some embodiments, the fusion protein of the Disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO: 322, a heavy chain containing the amino acid sequence of SEQ ID NO: 323, and a heavy chain containing the amino acid sequence of SEQ ID NO: 325. In some embodiments, the fusion protein of the Disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO: 326, a heavy chain containing the amino acid sequence of SEQ ID NO: 327, and a heavy chain containing the amino acid sequence of SEQ ID NO: 329. In some embodiments, the fusion protein of the Disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO: 330, a heavy chain containing the amino acid sequence of SEQ ID NO: 331, and a heavy chain containing the amino acid sequence of SEQ ID NO: 333. In some embodiments, the fusion protein of the present disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO: 334, a heavy chain containing the amino acid sequence of SEQ ID NO: 335, and a heavy chain containing the amino acid sequence of SEQ ID NO: 337.In some embodiments, the fusion protein of the Disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO: 338, a heavy chain containing the amino acid sequence of SEQ ID NO: 339, and a heavy chain containing the amino acid sequence of SEQ ID NO: 341. In some embodiments, the fusion protein of the Disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO: 342, a heavy chain containing the amino acid sequence of SEQ ID NO: 343, and a heavy chain containing the amino acid sequence of SEQ ID NO: 345. In some embodiments, the fusion protein of the Disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO: 346, a heavy chain containing the amino acid sequence of SEQ ID NO: 347, and a heavy chain containing the amino acid sequence of SEQ ID NO: 349. In some embodiments, the fusion protein of the Disclosure comprises one or two light chains containing the amino acid sequence of SEQ ID NO: 350, a heavy chain containing the amino acid sequence of SEQ ID NO: 351, and a heavy chain containing the amino acid sequence of SEQ ID NO: 353. In some embodiments, the fusion protein comprises one or two antigen-binding sites, each antigen-binding site comprising a VL domain from one of the light chains and a VH domain from one of the heavy chains (for example, the fusion protein has two antigen-binding sites: one comprising a VL domain from one of two light chains and a VH domain from one of the heavy chains, and the other comprising a VL domain from the other light chain and a VH domain from the other heavy chain).

[0025] In some embodiments, the fusion protein of the Disclosure comprises one or two polypeptides comprising the amino acid sequence of SEQ ID NO: 156, a polypeptide comprising the amino acid sequence of SEQ ID NO: 157, and a polypeptide comprising the amino acid sequence of SEQ ID NO: 158. In some embodiments, the fusion protein of the Disclosure comprises one or two polypeptides comprising the amino acid sequence of SEQ ID NO: 156, a polypeptide comprising the amino acid sequence of SEQ ID NO: 157, and a polypeptide comprising the amino acid sequence of SEQ ID NO: 217. In some embodiments, the fusion protein of the Disclosure comprises one or two polypeptides comprising the amino acid sequence of SEQ ID NO: 159, a polypeptide comprising the amino acid sequence of SEQ ID NO: 160, and a polypeptide comprising the amino acid sequence of SEQ ID NO: 161. In some embodiments, the fusion protein of the Disclosure comprises one or two polypeptides comprising the amino acid sequence of SEQ ID NO: 159, a polypeptide comprising the amino acid sequence of SEQ ID NO: 160, and a polypeptide comprising the amino acid sequence of SEQ ID NO: 218. In some embodiments, the fusion protein of the Disclosure comprises one or two polypeptides comprising the amino acid sequence of SEQ ID NO: 162, a polypeptide comprising the amino acid sequence of SEQ ID NO: 163, and a polypeptide comprising the amino acid sequence of SEQ ID NO: 164. In some embodiments, the fusion protein of the Disclosure comprises one or two polypeptides comprising the amino acid sequence of SEQ ID NO: 162, a polypeptide comprising the amino acid sequence of SEQ ID NO: 163, and a polypeptide comprising the amino acid sequence of SEQ ID NO: 219. In some embodiments, the fusion protein of the Disclosure comprises one or two polypeptides comprising the amino acid sequence of SEQ ID NO: 165, a polypeptide comprising the amino acid sequence of SEQ ID NO: 166, and a polypeptide comprising the amino acid sequence of SEQ ID NO: 167. In some embodiments, the fusion protein of the Disclosure comprises one or two polypeptides comprising the amino acid sequence of SEQ ID NO: 165, a polypeptide comprising the amino acid sequence of SEQ ID NO: 166, and a polypeptide comprising the amino acid sequence of SEQ ID NO: 220.In some embodiments, the fusion protein of the Disclosure comprises one or two polypeptides containing the amino acid sequence of SEQ ID NO: 168, a polypeptide containing the amino acid sequence of SEQ ID NO: 169, and a polypeptide containing the amino acid sequence of SEQ ID NO: 170. In some embodiments, the fusion protein of the Disclosure comprises one or two polypeptides containing the amino acid sequence of SEQ ID NO: 168, a polypeptide containing the amino acid sequence of SEQ ID NO: 169, and a polypeptide containing the amino acid sequence of SEQ ID NO: 221. In some embodiments, the fusion protein of the Disclosure comprises one or two polypeptides containing the amino acid sequence of SEQ ID NO: 171, a polypeptide containing the amino acid sequence of SEQ ID NO: 172, and a polypeptide containing the amino acid sequence of SEQ ID NO: 173. In some embodiments, the fusion protein of the Disclosure comprises one or two polypeptides containing the amino acid sequence of SEQ ID NO: 171, a polypeptide containing the amino acid sequence of SEQ ID NO: 172, and a polypeptide containing the amino acid sequence of SEQ ID NO: 222. In some embodiments, the fusion protein of the Disclosure comprises one or two polypeptides containing the amino acid sequence of SEQ ID NO: 174, a polypeptide containing the amino acid sequence of SEQ ID NO: 175, and a polypeptide containing the amino acid sequence of SEQ ID NO: 176. In some embodiments, the fusion protein of the Disclosure comprises one or two polypeptides comprising the amino acid sequence of SEQ ID NO: 174, a polypeptide comprising the amino acid sequence of SEQ ID NO: 175, and a polypeptide comprising the amino acid sequence of SEQ ID NO: 223. In some embodiments, the fusion protein of the Disclosure comprises one or two polypeptides comprising the amino acid sequence of SEQ ID NO: 187, a polypeptide comprising the amino acid sequence of SEQ ID NO: 188, and a polypeptide comprising the amino acid sequence of SEQ ID NO: 189. In some embodiments, the fusion protein of the Disclosure comprises one or two polypeptides comprising the amino acid sequence of SEQ ID NO: 187, a polypeptide comprising the amino acid sequence of SEQ ID NO: 188, and a polypeptide comprising the amino acid sequence of SEQ ID NO: 224.In some embodiments, the fusion protein of the Disclosure comprises one or two polypeptides containing the amino acid sequence of SEQ ID NO: 298, a polypeptide containing the amino acid sequence of SEQ ID NO: 299, and a polypeptide containing the amino acid sequence of SEQ ID NO: 300. In some embodiments, the fusion protein of the Disclosure comprises one or two polypeptides containing the amino acid sequence of SEQ ID NO: 298, a polypeptide containing the amino acid sequence of SEQ ID NO: 299, and a polypeptide containing the amino acid sequence of SEQ ID NO: 301. In some embodiments, the fusion protein of the Disclosure comprises one or two polypeptides containing the amino acid sequence of SEQ ID NO: 302, a polypeptide containing the amino acid sequence of SEQ ID NO: 303, and a polypeptide containing the amino acid sequence of SEQ ID NO: 304. In some embodiments, the fusion protein of the Disclosure comprises one or two polypeptides containing the amino acid sequence of SEQ ID NO: 302, a polypeptide containing the amino acid sequence of SEQ ID NO: 303, and a polypeptide containing the amino acid sequence of SEQ ID NO: 305. In some embodiments, the fusion protein of the Disclosure comprises one or two polypeptides containing the amino acid sequence of SEQ ID NO: 306, a polypeptide containing the amino acid sequence of SEQ ID NO: 307, and a polypeptide containing the amino acid sequence of SEQ ID NO: 308. In some embodiments, the fusion protein of the Disclosure comprises one or two polypeptides comprising the amino acid sequence of SEQ ID NO: 306, a polypeptide comprising the amino acid sequence of SEQ ID NO: 307, and a polypeptide comprising the amino acid sequence of SEQ ID NO: 309. In some embodiments, the fusion protein of the Disclosure comprises one or two polypeptides comprising the amino acid sequence of SEQ ID NO: 310, a polypeptide comprising the amino acid sequence of SEQ ID NO: 311, and a polypeptide comprising the amino acid sequence of SEQ ID NO: 312. In some embodiments, the fusion protein of the Disclosure comprises one or two polypeptides comprising the amino acid sequence of SEQ ID NO: 310, a polypeptide comprising the amino acid sequence of SEQ ID NO: 311, and a polypeptide comprising the amino acid sequence of SEQ ID NO: 313.In some embodiments, the fusion protein of the Disclosure comprises one or two polypeptides containing the amino acid sequence of SEQ ID NO: 314, a polypeptide containing the amino acid sequence of SEQ ID NO: 315, and a polypeptide containing the amino acid sequence of SEQ ID NO: 316. In some embodiments, the fusion protein of the Disclosure comprises one or two polypeptides containing the amino acid sequence of SEQ ID NO: 314, a polypeptide containing the amino acid sequence of SEQ ID NO: 315, and a polypeptide containing the amino acid sequence of SEQ ID NO: 317. In some embodiments, the fusion protein of the Disclosure comprises one or two polypeptides containing the amino acid sequence of SEQ ID NO: 318, a polypeptide containing the amino acid sequence of SEQ ID NO: 319, and a polypeptide containing the amino acid sequence of SEQ ID NO: 320. In some embodiments, the fusion protein of the Disclosure comprises one or two polypeptides containing the amino acid sequence of SEQ ID NO: 318, a polypeptide containing the amino acid sequence of SEQ ID NO: 319, and a polypeptide containing the amino acid sequence of SEQ ID NO: 321. In some embodiments, the fusion protein of the Disclosure comprises one or two polypeptides containing the amino acid sequence of SEQ ID NO: 322, a polypeptide containing the amino acid sequence of SEQ ID NO: 323, and a polypeptide containing the amino acid sequence of SEQ ID NO: 324. In some embodiments, the fusion protein of the Disclosure comprises one or two polypeptides comprising the amino acid sequence of SEQ ID NO: 322, a polypeptide comprising the amino acid sequence of SEQ ID NO: 323, and a polypeptide comprising the amino acid sequence of SEQ ID NO: 325. In some embodiments, the fusion protein of the Disclosure comprises one or two polypeptides comprising the amino acid sequence of SEQ ID NO: 326, a polypeptide comprising the amino acid sequence of SEQ ID NO: 327, and a polypeptide comprising the amino acid sequence of SEQ ID NO: 328. In some embodiments, the fusion protein of the Disclosure comprises one or two polypeptides comprising the amino acid sequence of SEQ ID NO: 326, a polypeptide comprising the amino acid sequence of SEQ ID NO: 327, and a polypeptide comprising the amino acid sequence of SEQ ID NO: 329.In some embodiments, the fusion protein of the Disclosure comprises one or two polypeptides containing the amino acid sequence of SEQ ID NO: 330, a polypeptide containing the amino acid sequence of SEQ ID NO: 331, and a polypeptide containing the amino acid sequence of SEQ ID NO: 332. In some embodiments, the fusion protein of the Disclosure comprises one or two polypeptides containing the amino acid sequence of SEQ ID NO: 330, a polypeptide containing the amino acid sequence of SEQ ID NO: 331, and a polypeptide containing the amino acid sequence of SEQ ID NO: 333. In some embodiments, the fusion protein of the Disclosure comprises one or two polypeptides containing the amino acid sequence of SEQ ID NO: 334, a polypeptide containing the amino acid sequence of SEQ ID NO: 335, and a polypeptide containing the amino acid sequence of SEQ ID NO: 336. In some embodiments, the fusion protein of the Disclosure comprises one or two polypeptides containing the amino acid sequence of SEQ ID NO: 334, a polypeptide containing the amino acid sequence of SEQ ID NO: 335, and a polypeptide containing the amino acid sequence of SEQ ID NO: 337. In some embodiments, the fusion protein of the Disclosure comprises one or two polypeptides containing the amino acid sequence of SEQ ID NO: 338, a polypeptide containing the amino acid sequence of SEQ ID NO: 339, and a polypeptide containing the amino acid sequence of SEQ ID NO: 340. In some embodiments, the fusion protein of the Disclosure comprises one or two polypeptides comprising the amino acid sequence of SEQ ID NO: 338, a polypeptide comprising the amino acid sequence of SEQ ID NO: 339, and a polypeptide comprising the amino acid sequence of SEQ ID NO: 341. In some embodiments, the fusion protein of the Disclosure comprises one or two polypeptides comprising the amino acid sequence of SEQ ID NO: 342, a polypeptide comprising the amino acid sequence of SEQ ID NO: 343, and a polypeptide comprising the amino acid sequence of SEQ ID NO: 344. In some embodiments, the fusion protein of the Disclosure comprises one or two polypeptides comprising the amino acid sequence of SEQ ID NO: 342, a polypeptide comprising the amino acid sequence of SEQ ID NO: 343, and a polypeptide comprising the amino acid sequence of SEQ ID NO: 345.In some embodiments, the fusion protein of the Disclosure comprises one or two polypeptides comprising the amino acid sequence of SEQ ID NO: 346, a polypeptide comprising the amino acid sequence of SEQ ID NO: 347, and a polypeptide comprising the amino acid sequence of SEQ ID NO: 348. In some embodiments, the fusion protein of the Disclosure comprises one or two polypeptides comprising the amino acid sequence of SEQ ID NO: 346, a polypeptide comprising the amino acid sequence of SEQ ID NO: 347, and a polypeptide comprising the amino acid sequence of SEQ ID NO: 349. In some embodiments, the fusion protein of the Disclosure comprises one or two polypeptides comprising the amino acid sequence of SEQ ID NO: 350, a polypeptide comprising the amino acid sequence of SEQ ID NO: 351, and a polypeptide comprising the amino acid sequence of SEQ ID NO: 352. In some embodiments, the fusion protein of the Disclosure comprises one or two polypeptides comprising the amino acid sequence of SEQ ID NO: 350, a polypeptide comprising the amino acid sequence of SEQ ID NO: 351, and a polypeptide comprising the amino acid sequence of SEQ ID NO: 353.

[0026] This specification further provides a fusion protein comprising a first portion that binds to human CD8b and a second portion that comprises an IL2 polypeptide, the fusion protein comprising four polypeptide chains: (1) the first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 334, the second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 335, the third polypeptide chain comprising the amino acid sequence of SEQ ID NO: 336, and the fourth polypeptide chain comprising the amino acid sequence of SEQ ID NO: 334; (2) the first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 334, the second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 335, and the third polypeptide chain comprising SEQ ID NO: 337 (3) The first polypeptide chain includes the amino acid sequence of SEQ ID NO: 338, the second polypeptide chain includes the amino acid sequence of SEQ ID NO: 339, the third polypeptide chain includes the amino acid sequence of SEQ ID NO: 340, and the fourth polypeptide chain includes the amino acid sequence of SEQ ID NO: 338; or (4) The first polypeptide chain includes the amino acid sequence of SEQ ID NO: 338, the second polypeptide chain includes the amino acid sequence of SEQ ID NO: 339, the third polypeptide chain includes the amino acid sequence of SEQ ID NO: 341, and the fourth polypeptide chain includes the amino acid sequence of SEQ ID NO: 338.

[0027] This specification further provides polynucleotides (e.g., isolated polynucleotides) encoding an antibody or fusion protein according to any one of the embodiments described above. This specification further provides vectors (e.g., expression vectors) comprising polynucleotides (or more) according to any one of the embodiments described above. This specification further provides host cells (e.g., isolated host cells or cell lines) comprising polynucleotides (or more) or vectors (or more) according to any one of the embodiments described above. This specification further provides a method for producing an antibody or fusion protein, comprising culturing host cells according to any one of the embodiments described above under conditions suitable for the production of the antibody or fusion protein. In some embodiments, the method further includes recovering the antibody or fusion protein from the host cells.

[0028] This specification further provides a pharmaceutical composition comprising an antibody or fusion protein and a pharmaceutically acceptable carrier according to any one of the embodiments described above.

[0029] This specification further provides the use of an antibody, fusion protein, or composition according to any one of the above embodiments as a pharmaceutical. This specification further provides the use of an antibody, fusion protein, or composition according to any one of the above embodiments in a method for treating cancer or an infectious disease (e.g., a chronic infectious disease). This specification further provides the use of an antibody, fusion protein, or composition according to any one of the above embodiments toward the manufacture of a pharmaceutical for treating cancer or an infectious disease (e.g., a chronic infectious disease). This specification further provides a method for treating cancer, comprising administering an effective amount of an antibody, fusion protein, or composition according to any one of the above embodiments to an individual having cancer. In some embodiments, the method further comprises administering a T-cell therapy, a cancer vaccine, a chemotherapeutic agent, or an immune checkpoint inhibitor (ICI) to the individual. This specification further provides a method for treating an infectious disease (e.g., a chronic infectious disease), comprising administering an effective amount of an antibody, fusion protein, or composition according to any one of the above embodiments to an individual having an infectious disease. This specification further provides a method (e.g., ex vivo) for increasing T cells, comprising contacting one or more T cells (e.g., tumor-infiltrating lymphocytes) with an effective amount of an antibody, fusion protein, or composition according to any one of the above embodiments, ex vivo.

[0030] It should be understood that one, some, or all of the characteristics of the various embodiments described herein may be combined to form other embodiments of the Disclosure. These and other embodiments of the Disclosure will be apparent to those skilled in the art. These and other embodiments of the Disclosure will be further described by embodiments for carrying out subsequent inventions. [Brief explanation of the drawing]

[0031] [Figure 1] This diagram illustrates three types of CD8ab antibodies that can be identified according to their binding priority to CD8a, CD8b, and CD8ab heterodimers. A represents an antibody that binds to the CD8a epitope, B represents an antibody that binds to an epitope spanning both CD8a and CD8b, and C represents an antibody that binds to the CD8b epitope. The binding priority for each antibody type to CD8a, CD8b, and CD8ab heterodimers is also shown. [Figure 2-1] Figure 1A-C shows the results of ELISA assays used to distinguish between the three types of CD8ab antibodies. Binding to recombinant CD8a (black square), CD8b (black triangle), CD8ab heterodimer (black circle), and the unrelated antigen obalbumin (white triangle) was measured. xhCD8a1 (clone OKT8) and xhCD8a2 antibodies (clone SK1) are as previously described. [Figure 2-2] Figure 1A-C shows the results of ELISA assays used to distinguish between the three types of CD8ab antibodies. Binding to recombinant CD8a (black square), CD8b (black triangle), CD8ab heterodimer (black circle), and the unrelated antigen obalbumin (white triangle) was measured. [Figure 2-3] Figure 1A-C shows the results of ELISA assays used to distinguish between the three types of CD8ab antibodies. Binding to recombinant CD8a (black square), CD8b (black triangle), CD8ab heterodimer (black circle), and the unrelated antigen obalbumin (white triangle) was measured. [Figure 3-1] Figures 2A-2C illustrate the binding preference of the three types of anti-CD8ab antibodies to various CD8+ immune cell types. The antibody against the CD8a epitope, as shown in Figure 1A, binds to both CD8ab+ T cells and CD8aa+ NK cells, as shown in Figure 3A. [Figure 3-2]Figures 2A-2C illustrate the binding preference of the three types of anti-CD8ab antibodies to various CD8+ immune cell types. The antibody targeting an epitope spanning both CD8a and CD8b, as illustrated in Figure 1B, preferentially binds to CD8ab+ T cells rather than CD8aa+ NK cells, as illustrated in Figure 3B. [Figure 3-3] Figures 2A-2C illustrate the binding preference of the three types of anti-CD8ab antibodies to various CD8+ immune cell types. The antibody against the CD8b epitope, as shown in Figure 1C, preferentially binds to CD8ab+ T cells rather than CD8aa+ NK cells, as shown in Figure 3C. [Figure 4] Figure 1A-C shows the results of a flow cytometry assay to detect the binding of CD8ab antibodies to human PBMCs and to distinguish between the three types of CD8ab antibodies depicted. Binding of CD8ab antibodies from hPBMCs to T cells was detected by staining with anti-human Fc antibody conjugated to APC. Anti-hFc was used to measure the binding of hFc-containing CD8 antibodies. The mean fluorescence intensity (MFI) of anti-hFc staining was used as an indicator of binding. Anti-hFc binding was measured in CD3+CD8a+CD8b+ cells (CD8+ T cells). [Figure 5] Figure 1A-C shows the results of a flow cytometry assay to detect the binding of CD8ab antibodies to human PBMCs and to distinguish between the three types of CD8ab antibodies depicted. Binding of CD8ab antibodies from hPBMCs to NK cells was detected by staining with anti-human Fc antibody conjugated to APC. Anti-hFc was used to measure the binding of hFc-containing CD8 antibodies. The mean fluorescence intensity (MFI) of anti-hFc staining was used as an indicator of binding. Anti-hFc binding was measured in CD3-CD56+CD8a+ cells (CD8+NK cells). [Figure 6] This shows the results of a flow cytometry assay measuring the binding of CD8ab antibodies xhCD8v1~xhCD8v5, which bind to the CD8b epitope, to CD8+ T cells. Negative control antibodies that bind to the HA antigen are also included. [Figure 7] Four different formats (Formats A, B, C, and D) of the fusion protein containing the CD8ab antibody of this disclosure are depicted according to several embodiments. [Figure 8-1] Figures 1A-C and 3A-3C depict fusion proteins containing three types of CD8ab antibodies and IL-2Rbg-binding polypeptides. These proteins demonstrate preferential activation of CD8ab+ T cells over CD8aa+ NK cells. [Figure 8-2] Figures 1A-C and 3A-3C depict fusion proteins containing three types of CD8ab antibodies and IL-2Rbg-binding polypeptides. These proteins demonstrate preferential activation of CD8ab+ T cells over CD8aa+ NK cells. [Figure 8-3] Figures 1A-C and 3A-3C depict fusion proteins containing three types of CD8ab antibodies and IL-2Rbg-binding polypeptides. These proteins demonstrate preferential activation of CD8ab+ T cells over CD8aa+ NK cells. [Figure 9]These are the results of an assay to determine the selectivity of a fusion protein containing the CD8ab antibody and the IL-2Rbg-binding polypeptide IL2m1. The expression of the proliferation marker Ki-67 in CD8+ T cells and NK cells derived from hPBMC donors was measured by flow cytometry after co-culturing with the following fusion proteins for 5 days: control Ab-IL2v (top of figure) containing the control antibody and a previously published IL-2 variant (Klein et al, Oncoimmunol. 2017; 6(3); e1277306); xhCD8a1-IL-2m1 (bottom left of figure) containing xhCD8a1, a CD8 antibody targeting the CD8a epitope, and IL2m1, the IL-2 polypeptide variant of this disclosure; and xhCD8v1-IL2m1 (bottom right of figure) containing xhCD8v1, the CD8ab antibody of this disclosure, and IL2m1. xhCD8a1-IL2m1 was in format C, while the control Ab-IL2v and xhCD8v1-IL2m1 were in format A. Ki-67 expression was measured in CD8+ T cells (black circles) and NK cells (black squares). NK cells were defined as CD3-CD56+. CD8 expression was measured by staining with the CD8 antibody SK1. [Figure 10-1] The expression of the Ki-67 proliferation marker after co-culturing hPBMCs from three different donors with the indicated fusion protein for 5 days is shown. All fusion proteins contained the IL-2 polypeptide variant IL2m1 and the CD8ab antibodies of this disclosure, xhCD8v1 to xhCD8v7, and were in format A. Ki-67 expression was measured in CD8+ T cells (solid line) and NK cells (dashed line). [Figure 10-2] The expression of the Ki-67 proliferation marker after co-culturing hPBMCs from three different donors with the indicated fusion protein for 5 days is shown. All fusion proteins contained the IL-2 polypeptide variant IL2m1 and the CD8ab antibodies of this disclosure, xhCD8v1 to xhCD8v7, and were in format A. Ki-67 expression was measured in CD8+ T cells (solid line) and NK cells (dashed line). [Figure 10-3]The expression of the Ki-67 proliferation marker after co-culturing hPBMCs from three different donors with the indicated fusion protein for 5 days is shown. All fusion proteins contained the IL-2 polypeptide variant IL2m1 and the CD8ab antibodies of this disclosure, xhCD8v1 to xhCD8v7, and were in format A. Ki-67 expression was measured in CD8+ T cells (solid line) and NK cells (dashed line). [Figure 11-1] The results of a phosphorylated STAT5 assay using hPBMCs from one donor cultured with the indicated fusion proteins (all in format C) are shown. The pSTAT5-expressing cell percentage is represented in the following hPBMC subset: CD8+ T cells. [Figure 11-2] The results of a phosphorylated STAT5 assay using hPBMCs from one donor cultured with the indicated fusion proteins (all in format C) are shown. pSTAT5-expressing cell percentages are represented in the following hPBMC subset: NK cells. NK cells were identified as CD3-perforin+. [Figure 11-3] The results of a phosphorylated STAT5 assay using hPBMCs from one donor cultured with the indicated fusion proteins (all in format C) are shown. pSTAT5-expressing cell percentages are represented in the following hPBMC subset: Treg cells. Treg cells were identified as CD4+Foxp3+CD25+. [Figure 11-4] The results of a phosphorylated STAT5 assay using hPBMCs from one donor cultured with the indicated fusion proteins (all in format C) are shown. The pSTAT5-expressing cell percentage is represented in the following hPBMC subset: CD4+Foxp3-T cells. [Figure 12]The expression of the Ki-67 proliferation marker after co-culturing hPBMCs from one donor with the indicated fusion protein for 5 days is shown. All fusion proteins contained xhCD8v1, the CD8ab antibody of this disclosure, and various IL-2 polypeptide variants of this disclosure, IL2m1 to IL2m5, and were in format A. Ki-67 expression was measured in CD8+ T cells (left) and NK cells (right). [Figure 13-1] The results of a phosphorylated STAT5 assay using hPBMCs from one donor cultured with the indicated fusion protein are shown. pSTAT5-expressing cell percentages are expressed in the following hPBMC subsets: CD8+ T cells and NK cells. All fusion proteins were in format A and contained xhCD8v1, the CD8ab antibody of this disclosure, as well as various IL-2 polypeptide variants of this disclosure, IL2m1, IL2m2, and IL2m6-IL2m10. NK cells were identified as CD3-perforin+. [Figure 13-2] The results of a phosphorylated STAT5 assay using hPBMCs from one donor cultured with the indicated fusion protein are shown. pSTAT5-expressing cell percentages are expressed in the following hPBMC subsets: CD8+ T cells and NK cells. The fusion protein contained the CD8ab antibodies xhCD8v11 and xhCD8v12 of this disclosure, as well as various IL-2 polypeptide variants of this disclosure, IL2m11, IL2m12, and IL2m13, and was in format A. NK cells were identified as CD3-perforin+. [Figure 14-1]The expression of the Ki-67 proliferation marker after co-culturing hPBMCs from one donor with xhCD8v1-IL2m1 (left) and xhCD8v6-IL2m1 (right) for 5 days is shown. Three different fusion protein formats were tested as depicted in Figure 7: Format A, Format B, and Format C. Solid lines represent Ki-67 expression in CD8+ T cells, and dashed lines represent Ki-67 expression in NK cells. In Format A, activity of both CD8ab antibodies tested was significantly increased in CD8+ T cells compared to NK cells. This suggests that Format A is a preferred format for fusion proteins containing CD8ab antibodies targeting an epitope between CD8a and CD8b (Figure 8B) or a CD8b epitope (Figure 8C). [Figure 14-2] Figure 7 shows the binding of xhCD8v6-IL2m1 to CD8+ T cells from a single donor (left) and the induction of Ki67 (right). Four different fusion protein formats were tested, as shown in Figure 7: Format A, Format B, Format C, and Format D. In cell binding, Format A was approximately 5 times stronger than the other formats, and in Ki67 induction, Format A was 20 to 40 times stronger than the other formats. [Figure 14-3] This figure shows the results for nine fusion molecules in formats A and D when evaluating binding to CD8+ T cells and Ki67 induction in CD8+ T cells. An example of the binding and Ki67 curves for xhCD8v12-IL2m4 is shown on the left. On the right, the EC50 ratio of format D to format A for a given conjugate-mutant protein pair is shown for binding and Ki67. For all molecules tested, format A showed approximately a 10-fold increase in potency in binding EC50 compared to format D. However, when evaluating the activation of the downstream proliferation marker Ki67, format A showed approximately a 1000-fold increase in potency in Ki67 induction compared to format D. [Figure 15]The expression of the Ki-67 proliferation marker after co-culturing hPBMCs for 5 days with xhCD8v8-IL2m1, a fusion protein of format A containing the IL-2 polypeptide variant IL2m1 and the CD8ab antibody xhCD8v8 of this disclosure. Ki-67 expression was measured in CD8+ T cells (square) and NK cells (triangle). [Figure 16-1] This figure shows Ki-67 expression in tumor-infiltrating lymphocytes (TILs) gated with CD8+ T cells (left) and NK cells (right). Prior to analysis, TILs were incubated with rhIL-2 or the expressed fusion protein (Format A) for 5 days. [Figure 16-2] This shows the number of CD8+ T cells, NK cells, and CD4+ T cells per well for each of the displayed conditions. [Figure 17-1] The expression of the Ki-67 proliferation marker after co-culturing hPBMCs for 5 days with a fusion protein of format A containing an IL-2 polypeptide variant (IL2m1 or IL2m4) and the CD8ab antibody of this disclosure is shown. CD8ab antibodies xhCD8v9~14 were tested. Ki-67 expression was measured in CD8+ T cells (square) and NK cells (triangle). [Figure 17-2] The expression of the Ki-67 proliferation marker after co-culturing hPBMCs for 5 days with a fusion protein of format A containing an IL-2 polypeptide variant (IL2m1 or IL2m4) and the CD8ab antibody of this disclosure is shown. CD8ab antibodies xhCD8v9~14 were tested. Ki-67 expression was measured in CD8+ T cells (square) and NK cells (triangle). [Figure 17-3] The expression of the Ki-67 proliferation marker after co-culturing hPBMCs for 5 days with a fusion protein of format A containing an IL-2 polypeptide variant (IL2m1 or IL2m4) and the CD8ab antibody of this disclosure is shown. CD8ab antibodies xhCD8v9~14 were tested. Ki-67 expression was measured in CD8+ T cells (square) and NK cells (triangle). [Figure 17-4]The expression of the Ki-67 proliferation marker after co-culturing hPBMCs for 5 days with a fusion protein of format A containing an IL-2 polypeptide variant (IL2m1 or IL2m4) and the CD8ab antibody of this disclosure is shown. CD8ab antibodies xhCD8v9~14 were tested. Ki-67 expression was measured in CD8+ T cells (square) and NK cells (triangle). [Figure 17-5] The expression of the Ki-67 proliferation marker after co-culturing hPBMCs for 5 days with a fusion protein of format A containing an IL-2 polypeptide variant (IL2m1 or IL2m4) and the CD8ab antibody of this disclosure is shown. CD8ab antibodies xhCD8v9~14 were tested. Ki-67 expression was measured in CD8+ T cells (square) and NK cells (triangle). [Figure 18-1] This shows the number of amino acid defects (e.g., putative N-linked glycosylation, deamination, or acid cleavage sites) in the displayed anti-CD8ab antibody, as well as the number of amino acid mismatches compared to human germline cells. [Figure 18-2] This alignment illustrates how the VH (top) and VK (bottom) CDRs (italicized) of xhCD8v1 were transplanted onto the VH1-69 and VK1-39 human germline frameworks, respectively. The VH domain sequences (top) correspond to SEQ ID NOs. 58, 384, and 60, respectively (from top to bottom). The VL domain sequences (bottom) correspond to SEQ ID NOs. 59, 385, and 61, respectively (from top to bottom). Underlined residues represent human germline framework residues. [Figure 18-3] This shows the alignment of the VH domains of the anti-CD8ab antibody of this disclosure. The VH domain sequences correspond to SEQ ID NOs. 60, 62, 64, 66, 68, 245, 251, and 253, respectively (from top to bottom). [Figure 18-4] This shows the alignment of the VL domains of the anti-CD8ab antibody of this disclosure. The VL domain sequences correspond to SEQ ID NOs. 61, 63, 65, 67, 69, 246, 252, and 254, respectively (from top to bottom). [Figure 19]The results of an ELISA assay used to confirm glycosylation (removed by glycosidase treatment (black dashed line, black square)) of the parent xhCD8v6 (solid black line, black square) are shown. Glycosylation is not detected in xhCD8v11 (gray line, white triangle). Fetuin is shown as a positive control. [Modes for carrying out the invention]

[0032] definition As used herein and in the appended claims, unless otherwise clearly indicated by the context, the singular forms "a," "an," and "the" refer to multiple objects. For example, a reference to "a molecule" may optionally include combinations of two or more such molecules.

[0033] It should be understood that the aspects and embodiments of this disclosure include the terms "includes," "consist of," and "essentially consist of" the aspects and embodiments.

[0034] As used herein, the term “about” refers to the normal margin of error for each value, as readily understood by those skilled in the art. References to values ​​or parameters “about” herein include (and are described) embodiments for which that value or parameter is the primary focus.

[0035] As used herein, “immune cells” are cells of the immune system that react to organisms or other entities that are considered foreign to the host’s immune system. They protect the host from foreign pathogens, organisms, and diseases. Immune cells, also known as leukocytes, are involved in both innate and adaptive immune responses to fight pathogens. The innate immune response occurs immediately upon exposure to a pathogen without further antigenic stimulation or learning processes. The adaptive immune process requires initial antigenic stimulation followed by memory formation, resulting in enhanced responsiveness in subsequent encounters with the same pathogen. Examples of innate immune cells include, but are not limited to, monocytes, macrophages, dendritic cells, innate lymphoid cells (ILCs) including natural killer (NK) cells, neutrophils, megakaryocytes, eosinophils, and basophils. Examples of adaptive immune cells include B and T lymphocytes / cells. T cell subsets include, but are not limited to, alpha-beta CD4+ T cells (naive CD4+, memory CD4+, effector memory CD4+, effector CD4+, regulatory CD4+) and alpha-beta CD8+ T cells (naive CD8+, memory CD8+, effector memory CD8+, effector CD8+). B cell subsets include, but are not limited to, naive B cells, memory B cells, and plasma cells. NK T cells and T gamma-delta (Tγδ) cells exhibit characteristics of both innate and acquired lymphocytes.

[0036] T cells, or T lymphocytes, are immune cells that play a crucial role in organizing immune responses in health and disease. There are two major T cell subsets with distinct functions and characteristics: T cells that express the CD8 antigen (CD8 + T cells are cytotoxic or killer T cells that can lyse target cells using cytotoxic proteins such as granzymes and perforins; and T cells that express the CD4 antigen (CD4 + T cells are CD8 + Helper T cells are capable of controlling the functions of many other immune cell types, including T cells, B cells, and macrophages. Furthermore, CD4 +T cells are further subdivided into several subsets, such as regulatory T (Treg) cells that can suppress the immune response, and helper T1 (Th1), helper T2 (Th2), and helper T17 (Th17) cells that control different types of immune responses by secreting immunomodulatory proteins such as cytokines. T cells recognize their targets via an alpha-beta T cell receptor that binds to a specific antigenic motif, and this recognition mechanism is generally required to induce their cytotoxic and cytokine-secreting functions. "Innate lymphocytes" can also exhibit the characteristics of CD8 + and CD4 + T cells. Some of these innate lymphocyte subsets include NK cells and ILC1, ILC2, and ILC3 cells; innate-like T cells such as Tγδ cells; and NK T cells. Typically, these cells can respond rapidly to inflammatory stimuli from infected or damaged tissues, such as immunomodulatory cytokines, but, unlike alpha-beta T cells, they can respond without the need to recognize an antigen-specific pattern.

[0037] Cytokines are a form of immunomodulatory polypeptide that mediates crosstalk between progenitor / primary cells and target / effector cells. Cytokines can function as a soluble form or by associating with the cell surface and binding to "cytokine receptors" on target immune cells to activate signaling. As used herein, a "cytokine receptor" is a cell surface polypeptide that activates intracellular signaling as soon as the cytokine binds to the extracellular surface of the cell. Examples of cytokines include, but are not limited to, chemokines, interferons, interleukins, lymphokines, and tumor necrosis factor. Cytokines are produced by a wide range of cells, including immune cells, endothelial cells, fibroblasts, and stromal cells. A given cytokine may be produced by more than one cell type. Cytokines are multifaceted, and their receptors are expressed on multiple immune cell subsets, so one cytokine can activate multiple intracellular signaling pathways. However, depending on the cell type, cytokine signaling events can result in different downstream cellular events, such as activation, proliferation, survival, apoptosis, effector function, and secretion of other immunomodulatory proteins.

[0038] As used herein, “amino acids” refers to naturally occurring carboxy-α-amino acids, including alanine (3-letter code: ala, 1-letter code: A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine ​​(cys, C), glutamine (gln, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y), and valine (val, V).

[0039] As used herein, “polypeptide” or “protein” refers to a molecule in which monomers (amino acids) are linearly linked to one another by peptide bonds (also known as amide bonds). The term “polypeptide” refers to any chain of two or more amino acids and does not refer to a specific length of the product. Thus, peptide, dipeptide, tripeptide, oligopeptide, “protein,” “amino acid chain,” or any other term used to refer to a chain of two or more amino acids are included in the definition of “polypeptide,” and the term “polypeptide” may be used in place of or synonymously with any of these terms. The term “polypeptide” is also intended to refer to a polypeptide product that may be derived from natural biosources or produced by recombinant technology, but is not necessarily translated from a specified nucleic acid sequence. Polypeptides can be produced in any manner, including by chemical synthesis. Polypeptides usually have a defined three-dimensional structure, but are not necessarily required to have such a structure. Polypeptides of this disclosure may have sizes of approximately 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1,000 or more, or 2,000 or more amino acids. Polypeptides having a defined three-dimensional structure are referred to as folded, while polypeptides that do not have a defined three-dimensional structure and can rather take on many different conformations are referred to as unfolded. Polypeptides may further form multimers, such as dimers, trimers, and higher low polymers, i.e., multimers consisting of more than one polypeptide molecule. The polypeptide molecules forming such dimers, trimers, etc., may or may not be identical. Therefore, the corresponding higher-order structures of such multimers are called homodimers or heterodimers, homodimers or heterotrimers, etc. The terms “polypeptide” and “protein” also refer to modified polypeptides / proteins, including, but not limited to, post-expression modifications such as glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or modification with amino acids that do not exist naturally.

[0040] As used herein, “residue” means the position within a protein and the corresponding amino acid itself. For example, Leu 234 (also known as Leu234 or L234) is the residue at position 234 in the human antibody IgG1.

[0041] In this specification, "wild-type" means an amino acid or nucleotide sequence found in nature, including allelic mutations. Wild-type proteins have an amino acid or nucleotide sequence that has not been intentionally modified.

[0042] "Substitution" or "mutation" refers to a change in the polypeptide backbone in which a naturally occurring amino acid in the wild-type sequence of a polypeptide is replaced by another amino acid that is not naturally occurring at the same position within the polypeptide. Preferably, mutations are introduced to alter the polypeptide's affinity for its receptor, thereby changing the polypeptide's activity to differ from that of the wild-type homologous polypeptide. Mutations can also improve the polypeptide's biophysical properties. Amino acid mutations can be induced using genetic or chemical methods well known in the art. Genetic methods may include site-directed mutagenesis, PCR, gene synthesis, and similar methods. Methods other than genetic manipulation, such as chemical modification, to alter the side chain groups of amino acids may also be useful.

[0043] As used herein, “CD8” refers to any natural human CD8. Unless otherwise indicated explicitly or by context, references to “CD8” refer to CD8aa and / or CD8ab. The amino acid sequence of human CD8b, the exemplary beta chain of human CD8, is listed under UniProt P10966(CD8B_HUMAN). “CD8a” refers to the alpha chain of human CD8 (e.g., listed under UniProt P01732(CD8A_HUMAN)). “CD8aa” refers to the homodimer of CD8a. “CD8ab” refers to the heterodimer of CD8a and CD8b. “CD8,” “CD8a,” “CD8b,” “CD8aa,” and “CD8ab” encompass both the unprocessed form and the mature form resulting from intracellular processing. Furthermore, "CD8," "CD8a," "CD8b," "CD8aa," and "CD8ab" include, but are not limited to, naturally occurring variants, such as alleles or splice variants or variants.

[0044] As used herein, “interleukin-2” or “IL-2” refers to any natural human IL-2 unless otherwise indicated. “IL-2” includes unprocessed IL-2, as well as “mature IL-2,” which is a form of IL-2 resulting from intracellular processing. The sequence of “mature IL-2” is shown in Figure 1A. One exemplary form of unprocessed human IL-2 consists of an additional N-terminal amino acid signal peptide bound to mature IL-2. “IL-2” includes, but is not limited to, naturally occurring variants of IL-2, such as alleles or splice variants or variants. The amino acid sequence of an exemplary human IL-2 is listed under UniProt P60568(IL2_HUMAN).

[0045] "Affinity" or "binding affinity" refers to the total strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects the 1:1 interaction between members of a binding pair (e.g., between an antibody and an antigen). Affinity is the ratio of the dissociation rate constant to the association rate constant (koff and kon, respectively). D Affinity can generally be expressed by the following: Therefore, equivalent affinity may include different rate constants, provided that the ratio of rate constants remains the same. Affinity can be measured by common methods known in the art, such as enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR) (e.g., BIAcore), biolayer interference (BLI) (e.g., Octet), and other conventional binding assays (Heeley, Endocr Res 28, 217-229 (2002)).

[0046] As used herein, “binding” or “specific binding” refers to the ability of a polypeptide or antigen-binding molecule to selectively interact with the receptor of that polypeptide or target antigen, respectively, and this specific interaction may be distinguished from untargeted interactions, undesirable interactions, or nonspecific interactions. Examples of specific binding include, but are not limited to, the binding of an IL-2 cytokine to its specific receptor (e.g., IL-2Rα, IL-2Rβ, and IL-2Rγ) and the binding of an antigen-binding molecule to a specific antigen (e.g., CD8 or PD-1).

[0047] An "IL-2 polypeptide variant" refers to an IL-2 polypeptide in which its affinity for its receptor is reduced, and this reduction in affinity results in a reduction in the biological activity of the variant. The reduction in affinity and the resulting reduction in activity can be achieved by introducing a small number of amino acid mutations or substitutions. IL-2 polypeptide variants may also have other modifications to the peptide backbone to produce a final construct with desired properties, such as reduced affinity for IL-2Rβγ. Such modifications include, but are not limited to, amino acid deletion, rearrangement, cyclization, disulfide bonding or post-translational modification (e.g., glycosylation or carbohydrate modification) of the polypeptide, chemical or enzymatic modification of the polypeptide (e.g., PEG attachment to the polypeptide backbone), addition of peptide tags or labels, or fusion to a protein or protein domain. Desired activity may also include improved biophysical properties compared to the wild-type IL-2 polypeptide. Multiple modifications may be combined to achieve desired activity modifications, such as reduced affinity or improved biophysical properties. As a non-limiting example, an amino acid sequence for consensus N-linked glycosylation can be incorporated into the polypeptide to enable glycosylation. Another non-limiting example is the incorporation of lysine onto the polypeptide to enable PEGylation. Preferably, mutations are introduced into the polypeptide to modify its activity.

[0048] The terms “antibody” and “immunoglobulin” are used synonymously and are used herein in the broadest sense, encompassing a wide range of antibody structures, including, but not limited to, monoclonal antibodies (e.g., full-length or intact monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), antibody fragments, and single-domain antibodies (as described in more detail herein), insofar as they exhibit the desired antigen-binding activity.

[0049] Antibodies (immunoglobulins) refer to proteins that have a structure substantially similar to that of natural antibodies. "Natural antibodies" refer to naturally occurring immunoglobulin molecules with a variety of structures. For example, the IgG class of natural immunoglobulins is a heterotetrameric glycoprotein with approximately 150,000 daltons, composed of two disulfide-linked light chains and two heavy chains. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called a variable heavy chain domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3), also called heavy chain constant domains. Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called a variable light chain domain or light chain variable domain, followed by a constant light chain (CL) domain, also called a light chain constant domain. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known and are generally described, for example, in Abbas et al., 2000, Cellular and Mol, and Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., page 91 (2007). Antibodies (immunoglobulins) are assigned to different classes depending on the amino acid sequence of the heavy chain constant domain. There are five main classes of antibodies: α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), some of which can be further divided into subtypes, e.g., γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2). The light chain of immunoglobulins can be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain. Immunoglobulins essentially consist of two Fab molecules and an Fc domain, linked via an immunoglobulin hinge region.

[0050] As used herein, “Fc” or “Fc region” or “Fc domain” refers to the C-terminal region of an antibody heavy chain containing at least a portion of the constant region. This term includes both native sequence Fc regions and variant Fc regions. Fc may refer to the last two constant region immunoglobulin domains of IgA, IgD, and IgG (e.g., CH2 and CH3), the last three constant region immunoglobulin domains of IgE and IgM, and optionally, all or a portion of the N-terminal flexible hinges of these domains. For IgA and IgM, Fc may include the J chain. The IgG Fc region includes the IgG CH2 and IgG CH3 domains, possibly including the hinges. Unless otherwise specified herein, the numbering of amino acid residues within the Fc region or constant region is as defined by Kabat et al. This is based on the EU numbering system, also known as the EU index, as described in al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991. The “hinge” region typically extends from approximately position 216 to approximately position 230. In this specification, the hinge region may be a natural hinge domain or a variant hinge domain. The “CH2 domain” of the human IgG Fc region typically extends from approximately position 231 to approximately position 340. In this specification, the CH2 domain may be a natural sequence CH2 domain or a variant CH2 domain. The “CH3 domain” includes a stretch of residues from the C-terminus of the Fc region to the CH2 domain (from approximately position 341 to approximately position 447 of IgG). In this specification, a CH3 region may be a native sequence CH3 domain or a variant CH3 domain (for example, a CH3 domain in which a “protrusion” (“knob”) is introduced on one strand and a “cavity” (“hole”) is introduced on the other strand; see U.S. Patent No. 5,821,333, which is more clearly incorporated herein by reference). Thus, the definition of “Fc domain” includes both amino acids 231-447 (CH2-CH3) or 216-447 (hinge-CH2-CH3), or fragments thereof. In this context, an “Fc fragment” may contain fewer amino acids from either or both of the N-terminus and / or C-terminus, but still retains the ability to form dimers with other Fc domains or Fc fragments, so that they can be detected using standard methods (e.g., non-denaturing chromatography, size exclusion chromatography, etc.) that are generally size-based. Human IgG Fc domains are particularly useful in this disclosure and may be Fc domains from human IgG1, IgG2, or IgG4.

[0051] A "variant Fc domain," "Fc variant," or "variant Fc" has amino acid modifications (e.g., substitutions, additions, and deletions) compared to the parent Fc domain. This term also includes naturally occurring allelic variants of the Fc region of immunoglobulins. Generally, a variant Fc domain has at least about 80, 85, 90, 95, 97, 98, or 99 percent identity with the corresponding parent human IgG Fc domain (using the identity algorithm discussed below, or using default parameters in one embodiment utilizing the BLAST algorithm as is known in the art). Alternatively, a variant Fc domain may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications compared to the parent Fc domain. For example, one or more amino acids can be deleted from the N-terminus or C-terminus of the Fc region of an immunoglobulin without significant loss of biological function. In addition, as discussed herein, the variant Fc domains herein still retain the ability to form dimers with other Fc domains, as measured using known techniques described herein, such as non-denaturing gel electrophoresis.

[0052] As used herein, “Fc gamma receptor,” “FcγR,” or “Fc gamma R” means any member of the family of proteins that bind to the Fc region of an IgG antibody and are encoded by the FcγR gene. In humans, this family includes, but is not limited to, any undiscovered human FcγR or FcγR isoform or allotype, as well as FcγRI(CD64), including isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII(CD32), including isoforms FcγRIIa (including allotypes H131 and R131), FcγRIIb (including allotypes FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII(CD16), including isoforms FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIb-NA1 and FcγRIIb-NA2) (the entire family is incorporated by reference, Jefferis et al., 2002, Immunol Lett 82:57-65). FcγR may originate from any organism, including but not limited to humans, mice, rats, rabbits, and monkeys. Mouse FcγR includes, but is not limited to, FcγRI(CD64), FcγRII(CD32), FcγRIII(CD16), and FcγRIII-2(CD16-2), as well as any undiscovered mouse FcγR or FcγR isoform or allotype.

[0053] As used herein, “effector function” refers to a biochemical event resulting from the interaction between an antibody Fc region and an Fc receptor or ligand, which varies depending on the antibody isotype. Effector functions include, but are not limited to, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), complement-dependent cell-mediated cytotoxicity (CDC), cytokine secretion, immune complex-mediated antigen uptake by antigen-presenting cells, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation. “Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to a cell-mediated reaction in which nonspecific cytotoxic cells expressing FcR (such as natural killer (NK) cells, neutrophils, and macrophages) recognize an antibody bound to a target cell, subsequently causing lysis of that target cell. ADCC correlates with binding to FcγRIIIa, and increased binding to FcγRIIIa leads to increased ADCC activity. To evaluate the ADCC activity of a target molecule, an in vitro ADCC assay, such as those described in U.S. Patent No. 5,500,362 or No. 5,821,337, can be performed. As used herein, “ADCP” or antibody-dependent cell-mediated phagocytosis means a cell-mediated response in which nonspecific cytotoxic cells expressing FcγR recognize an antibody bound to a target cell, and subsequently induce phagocytosis of that target cell.

[0054] The terms "Fc null" and "Fc null variant" are used synonymously and are used herein to describe modified Fc having reduced or absent effector function. Such Fc nulls or Fc null variants are reduced or absent with respect to FcγR and / or complement receptors. Preferably, such Fc nulls or Fc null variants have absent effector function. Exemplary methods for modification include, but are not limited to, chemical changes, amino acid residue substitutions, insertions, and deletions. At exemplary amino acid positions on the Fc molecule, one or more modifications have been introduced to reduce the effector function of the resulting variants at positions i) IgG1: C220, C226, C229, E233, L234, L235, G237, P238, S239, D265, S267, N297, L328, P331, K322, A327 and P329, ii) IgG2: V234, G237, D265, H268, N297, V309, A330, A331, K322, and iii) IgG4: L235, G237, D265 and E318 (numbering is based on the EU numbering scheme).Examples of Fc molecules with reduced effector function include those having one or more of the following substitutions: i) IgG1:N297A, N297Q, N297G, D265A / N297A, D265A / N297Q, C220S / C226S / C229S / P238S, S267E / L328F, C226S / C229S / E233P / L234V / L235 A, L234F / L235E / P331S, L234A / L235A, L234A / L235A / G237A, L234A / L235A / G237A / K322A, L234A / L235 A / G237A / A330S / A331S, L234A / L235A / P329G, E233P / L234V / L235A / G236 deletion / S239K, E233P / L234V / L23 5A / G236 deletion / S267K, E233P / L234V / L235A / G236 deletion / S239K / A327G, E233P / L234V / L235A / G236 deletion / S267K / A327G and E233P / L234V / L235A / G236 deletion, L234A / L235A / G237 deletion; ii) IgG2: A330S / A331S, V234A / G237A, V234A / G237A / D265A, D265A / A330S / A331S, V234A / G237A / D265A / A330S / A331S, and H268Q / V309L / A330S / A331S;iii)IgG4:L235A / G237A / E318A, D265A, L235A / G237A / D265A and L235A / G237A / D265A / E318A.

[0055] As used herein, “epitope” refers to a determinant that can specifically bind to the variable region of an antibody molecule, known as a paratope. An epitope is a collection of molecules, such as amino acids or sugar side chains, and typically possesses specific structural and charge properties. A single antigen may have more than one epitope. An epitope may include amino acid residues directly involved in binding and other amino acid residues not directly involved in binding, such as amino acid residues effectively blocked by the antigen-binding peptide (in other words, these amino acid residues are within the footprint of the antigen-binding peptide). An epitope may be either a conformational epitope or a linear epitope. An epitope typically contains at least three, usually more, at least five or eight to ten amino acids. Antibodies that recognize the same epitope can be validated by a simple immunoassay (e.g., “binning”) that reveals the ability of one antibody to block the binding of another antibody to its target antigen.

[0056] As used herein, “linker” refers to a molecule that connects two polypeptide chains. The linker may be a polypeptide linker or a synthetic chemical linker (see, for example, Protein Engineering, 9(3), 299-305, 1996). The length and sequence of the polypeptide linker are not particularly limited and may be selected according to the purpose by those skilled in the art. The polypeptide linker contains one or more amino acids. In some embodiments, the polypeptide linker is a peptide having a length of at least 5 amino acids, preferably 5 to 100, more preferably 10 to 50 amino acids. In one embodiment, the peptide linker is G, S, GS, SG, SGG, GGS, and GSG (G = glycine and S = serine). In another embodiment, the peptide linker is (GGGS)xGn (SEQ ID NO: 74), (GGGGS)xGn (SEQ ID NO: 75), (GGGGGS)xGn (SEQ ID NO: 76), S(GGGS)xGn (SEQ ID NO: 386), S(GGGGS)xGn (SEQ ID NO: 387), or S(GGGGGS)xGn (SEQ ID NO: 388) (x=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, and n=0, 1, 2, or 3). Preferably, the linker is (GGGGS)xGn (x=2, 3, or 4, and n=0) (SEQ ID NO: 77), and more preferably, the linker is (GGGGS)xGn (x=3, and n=0) (SEQ ID NO: 78). In some embodiments, the linker includes the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 79) or SGGGGSGGGGSGGGGS (SEQ ID NO: 389).The synthetic chemical linkers include crosslinking agents routinely used to crosslink peptides, such as N-hydroxysuccinimide (NHS), disuccinimidyl suberate (DSS), bis(succinimidyl) suberate (BS3), dithiobis(succinimidyl propionate) (DSP), dithiobis(succinimidyl propionate) (DTSSP), ethylene glycol bis(succinimidyl succinate) (EGS), ethylene glycol bis(sulfosuccinimidyl succinate) (sulfo-EGS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST), bis[2-(succinimideoxycarbonyloxy)ethyl]sulfone (BSOCOES), and bis[2-(succinimideoxycarbonyloxy)ethyl]sulfone (sulfo-BSOCOES).

[0057] The term “polynucleotide” refers to an isolated nucleic acid molecule or construct, e.g., messenger RNA (mRNA), viral RNA, or plasmid DNA (pDNA), encoding the polypeptide of the Disclosure. Polynucleotides may contain conventional phosphodiester bonds or non-conventional bonds (e.g., amide bonds, such as those found in peptide nucleic acids (PNAs)). The term “nucleic acid molecule” refers to any one or more nucleic acid segments present in a polynucleotide, e.g., DNA or RNA fragments. In some embodiments, one or more vectors (in particular, expression vectors) containing such nucleic acids are provided. In one embodiment, a method for producing the polypeptide of the Disclosure is provided, comprising culturing a host cell containing the nucleic acid encoding the polypeptide under conditions suitable for polypeptide expression, and recovering the polypeptide from the host cell. “Recombinant” means a protein produced in exogenous host cells using recombinant nucleic acid techniques. Recombinant-produced proteins expressed in host cells are considered isolated in the spirit of the Disclosure, and native or recombinant proteins isolated, fractionated, or partially or substantially purified by any preferred technique are also considered isolated.

[0058] When used herein to describe the various polypeptides disclosed herein, “isolated” means a polypeptide identified, separated, and / or recovered from cells or cell cultures expressing that polypeptide. Typically, an isolated polypeptide will be purified by at least one purification step. There is no requirement for a specific level of purification; “purified” or “purified” refers to an increase in the concentration of the target protein relative to the concentration of impurities in the composition compared to the starting material. As used herein, “isolated protein” refers to a target protein that is substantially free from other proteins with different binding specificities.

[0059] The term "cancer" refers to a physiological condition in mammals typically characterized by uncontrolled and abnormal cell proliferation that can invade or spread to other parts of the body. Examples of cancer include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemias. More specific examples of such cancers include lung cancer, small cell lung cancer, non-small cell lung (NSCL) cancer, bronchioloalviolar cell lung cancer, squamous cell carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, head and neck cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, skin or intraocular melanoma, thyroid cancer, uterine cancer, gastrointestinal cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, and gastric cancer. This includes, but is not limited to, cancers such as colon cancer, breast cancer, endometrial cancer, uterine cancer, fallopian tube cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine cancers, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, bladder cancer, kidney or ureteral cancer, renal cell carcinoma, renal pelvis cancer, mesothelioma, bladder cancer, liver cancer, hepatoma, hepatocellular carcinoma, cervical cancer, salivary gland cancer, biliary tract cancer, central nervous system (CNS) tumors, spinal axis tumors, brainstem gliomas, glioblastoma multiforme, astrocytoma, schwannoma, ependymoma, medulloblastoma, meningioma, squamous cell carcinoma, pituitary adenoma, and Ewing's sarcoma, refractory versions of any of the above cancers, or combinations of one or more of the above cancers.

[0060] Antibody and antigen binding domains Certain aspects of this disclosure relate to antibodies, antibody fragments, and antigen-binding domains that specifically bind to human CD8b and / or human CD8ab. Any of the anti-CD8 antibodies of this disclosure (e.g., those that specifically bind to human CD8b and / or human CD8ab) may be used in the fusion proteins, methods, and uses disclosed herein.

[0061] In some embodiments, the anti-CD8 antibodies of this disclosure specifically bind to human CD8b and / or human CD8ab with an affinity at least 10 times, at least 20 times, at least 30 times, at least 40 times, at least 50 times, at least 60 times, at least 70 times, at least 80 times, at least 90 times, at least 100 times, or at least 200 times higher than binding to human CD8a and / or human CD8aa, for example, expressed on natural killer (NK) cells (e.g., human NK cells). In some embodiments, the anti-CD8 antibodies of this disclosure specifically bind to human CD8b and / or human CD8ab with an affinity at least 10 times higher than binding to human CD8a and / or human CD8aa, for example, expressed on natural killer (NK) cells. In some embodiments, human CD8b and / or human CD8ab are expressed on the surface of human cells, for example, human T cells.

[0062] In some embodiments, the anti-CD8 antibody of this disclosure specifically binds to cells expressing the human CD8ab heterodimer on its surface (e.g., human T cells) with an EC50 of less than 1000 nM. In some embodiments, the anti-CD8 antibody of this disclosure specifically binds to human CD8+ T cells.

[0063] In some embodiments, the anti-CD8 antibody of this disclosure is a human antibody or antibody fragment. In some embodiments, the human antibody or antibody fragment includes a human-derived CDR and framework sequence in a variable domain, for example, isolated from a human or generated using a library having a human antibody sequence (e.g., a CDR sequence). In some embodiments, the anti-CD8 antibody of this disclosure is a humanized antibody or antibody fragment. In some embodiments, the humanized antibody or antibody fragment includes a non-human-derived CDR (e.g., derived from mouse, rabbit, goat, etc.) and a human-derived framework sequence in a variable domain. In some embodiments, the human or humanized antibody further includes a human Fc region. In some embodiments, the human Fc region further includes one or more Fc mutations, for example, as disclosed herein. Intact antibodies have five main classes: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), for example, IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant domains corresponding to different classes of antibodies are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

[0064] Several definitions of CDR sequences of antibody variable domains are known in the art. Unless otherwise specified, CDR sequences are described herein according to Kabat's definition (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3). However, other definitions are known and intended for use. For example, in some embodiments, CDR sequences can be described according to Chothia's definition (see, e.g., Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). Depending on the specific CDR definition used, the exact framework sequences may also differ, but as is well known in the art, the first framework sequence (FW-1) refers to the sequence from the N-terminus of the VH or VL domain to the beginning of CDR-H1 / -L1, the second framework sequence (FW-2) refers to the sequence from the end of CDR-H1 / -L1 to the beginning of CDR-H2 / -L2, the third framework sequence (FW-3) refers to the sequence from the end of CDR-H2 / -L2 to the beginning of CDR-H3 / -L3, and the fourth framework sequence (FW-4) refers to the sequence from the end of CDR-H3 / -L3 to the C-terminal boundary of the VH or VL domain.

[0065] In some embodiments, the anti-CD8 antibody of this disclosure comprises a VH domain comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3, and a VL domain comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6. In some embodiments, the anti-CD8 antibody of this disclosure comprises one, two, or three heavy chain CDRs of antibody xhCD8v1 (e.g., those shown in Tables 1-3) and / or one, two, or three light chain CDRs of antibody xhCD8v1 (e.g., those shown in Tables 1-3). In some embodiments, the antibody is humanized. In some embodiments, the anti-CD8 antibody of the present disclosure comprises a VH domain containing CDR-H1, CDR-H2, and CDR-H3 of the sequence of SEQ ID NO: 58, and a VL domain containing CDR-L1, CDR-L2, and CDR-L3 of the sequence of SEQ ID NO: 59.

[0066] In some embodiments, the anti-CD8 antibody of this disclosure comprises a VH domain comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 177, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 178, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 179, and a VL domain comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 180, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 181, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 182. In some embodiments, the anti-CD8 antibody of this disclosure comprises one, two, or three heavy chain CDRs of antibody xhCD8v8 (e.g., those shown in Tables 1-3) and / or one, two, or three light chain CDRs of antibody xhCD8v8 (e.g., those shown in Tables 1-3). In some embodiments, the antibody is humanized. In some embodiments, the anti-CD8 antibody of this disclosure comprises a VH domain containing CDR-H1, CDR-H2, and CDR-H3 of the sequence of SEQ ID NO: 185, and a VL domain containing CDR-L1, CDR-L2, and CDR-L3 of the sequence of SEQ ID NO: 186.

[0067] In some embodiments, the anti-CD8 antibody of the present disclosure comprises a VH domain comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 13, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 14, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 15, and a VL domain comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 16, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 17, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 18. In some embodiments, the VH domain comprises an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 62, and / or the VL domain comprises an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 63. In some embodiments, the anti-CD8 antibody of this disclosure comprises one, two, or three heavy chain CDRs of antibody xhCD8v2 (e.g., those shown in Tables 1-3) and / or one, two, or three light chain CDRs of antibody xhCD8v2 (e.g., those shown in Tables 1-3). In some embodiments, the antibody is humanized. In some embodiments, the anti-CD8 antibody of this disclosure comprises a VH domain containing CDR-H1, CDR-H2, and CDR-H3 of the sequence of SEQ ID NO: 62, and a VL domain containing CDR-L1, CDR-L2, and CDR-L3 of the sequence of SEQ ID NO: 63.

[0068] In some embodiments, the anti-CD8 antibody of the present disclosure comprises a VH domain comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 19, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 20, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 21, and a VL domain comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 22, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 23, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 24. In some embodiments, the VH domain comprises an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 64, and / or the VL domain comprises an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 65. In some embodiments, the anti-CD8 antibody of this disclosure comprises one, two, or three heavy chain CDRs of antibody xhCD8v3 (e.g., those shown in Tables 1-3) and / or one, two, or three light chain CDRs of antibody xhCD8v3 (e.g., those shown in Tables 1-3). In some embodiments, the antibody is humanized. In some embodiments, the anti-CD8 antibody of this disclosure comprises a VH domain containing CDR-H1, CDR-H2, and CDR-H3 of the sequence of SEQ ID NO: 64, and a VL domain containing CDR-L1, CDR-L2, and CDR-L3 of the sequence of SEQ ID NO: 65.

[0069] In some embodiments, the anti-CD8 antibody of the present disclosure comprises a VH domain comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 25, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 26, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 27, and a VL domain comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 28, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 29, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 30. In some embodiments, the VH domain comprises an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 66, and / or the VL domain comprises an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 67. In some embodiments, the anti-CD8 antibody of this disclosure comprises one, two, or three heavy chain CDRs of antibody xhCD8v4 (e.g., those shown in Tables 1-3) and / or one, two, or three light chain CDRs of antibody xhCD8v4 (e.g., those shown in Tables 1-3). In some embodiments, the antibody is humanized. In some embodiments, the anti-CD8 antibody of this disclosure comprises a VH domain containing CDR-H1, CDR-H2, and CDR-H3 of the sequence of SEQ ID NO: 66, and a VL domain containing CDR-L1, CDR-L2, and CDR-L3 of the sequence of SEQ ID NO: 67.

[0070] In some embodiments, the anti-CD8 antibody of the present disclosure comprises a VH domain comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 31, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 32, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 33, and a VL domain comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 34, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 35, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 36. In some embodiments, the VH domain comprises an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 68, and / or the VL domain comprises an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 69. In some embodiments, the anti-CD8 antibody of this disclosure comprises one, two, or three heavy chain CDRs of antibody xhCD8v5 (e.g., those shown in Tables 1-3) and / or one, two, or three light chain CDRs of antibody xhCD8v5 (e.g., those shown in Tables 1-3). In some embodiments, the antibody is humanized. In some embodiments, the anti-CD8 antibody of this disclosure comprises a VH domain containing CDR-H1, CDR-H2, and CDR-H3 of the sequence of SEQ ID NO: 68, and a VL domain containing CDR-L1, CDR-L2, and CDR-L3 of the sequence of SEQ ID NO: 69.

[0071] In some embodiments, the anti-CD8 antibody of the present disclosure comprises a VH domain comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 37, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 38, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 39, and a VL domain comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 40, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 41, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 42. In some embodiments, the VH domain comprises an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 70, and / or the VL domain comprises an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 71. In some embodiments, the anti-CD8 antibody of this disclosure comprises one, two, or three heavy chain CDRs of antibody xhCD8v6 (e.g., those shown in Tables 1-3) and / or one, two, or three light chain CDRs of antibody xhCD8v6 (e.g., those shown in Tables 1-3). In some embodiments, the antibody is human. In some embodiments, the anti-CD8 antibody of this disclosure comprises a VH domain containing CDR-H1, CDR-H2, and CDR-H3 of the sequence of SEQ ID NO: 70, and a VL domain containing CDR-L1, CDR-L2, and CDR-L3 of the sequence of SEQ ID NO: 71.

[0072] In some embodiments, the anti-CD8 antibody of the present disclosure comprises a VH domain comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 43, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 44, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 45, and a VL domain comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 46, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 47, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 48. In some embodiments, the VH domain comprises an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 72, and / or the VL domain comprises an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 73. In some embodiments, the anti-CD8 antibody of this disclosure comprises one, two, or three heavy chain CDRs of antibody xhCD8v7 (e.g., those shown in Tables 1-3) and / or one, two, or three light chain CDRs of antibody xhCD8v7 (e.g., those shown in Tables 1-3). In some embodiments, the antibody is human. In some embodiments, the anti-CD8 antibody of this disclosure comprises a VH domain containing CDR-H1, CDR-H2, and CDR-H3 of the sequence of SEQ ID NO: 72, and a VL domain containing CDR-L1, CDR-L2, and CDR-L3 of the sequence of SEQ ID NO: 73.

[0073] In some embodiments, the anti-CD8 antibody of the present disclosure is CDR-H1, X1X2X3PX4X5X6X7X8X9YX, comprising the amino acid sequence X1X2AIS (SEQ ID NO: 259), where X1 is S, K, G, N, R, D, T, or G, and X2 is Y, L, H, or F. 10 QKFX 11 The amino acid sequence of G, where X1 is G or H, X2 is I or F, X3 is I, N, or M, X4 is G, N, H, S, R, I, or A, X5 is A, N, H, S, T, F, or Y, X6 is A, D, or G, X7 is T, E, K, V, Q, or A, X8 is A or T, X9 is N or K, X 10 is A or N, X11CDR-H2 comprising the amino acid sequence (SEQ ID NO: 260) where X1 is Q or T, and CDR-H3 comprising the amino acid sequence (SEQ ID NO: 261) where X1 is D or A, X2 is A, G, E, R, Y, K, N, Q, L, or F, X3 is A, L, P, or Y, X4 is I or L, X5 is R, A, Q, or S, X6 is A or D, X7 is D, E, A, or S, and the amino acid sequence X1 is X2, X3, X4, X5, G, X6, LN comprising the amino acid sequence (SEQ ID NO: 261) where X1 is R or G, X2 is A or T, X3 is Q or E, X4 is E, N, T, S, A, K, D, G, R, or Q, and X5 is Y or S. Yes, CDR-L1 includes the amino acid sequence (SEQ ID NO: 262) in which X6 is A or V, CDR-L2 includes the amino acid sequence (SEQ ID NO: 263) in which X1 is A or S, X2 is T, S, E, Q, or D, X3 is N, R, A, E, or H, X4 is Q or A, and X5 is S or D, and CDR-L3 includes the amino acid sequence (SEQ ID NO: 264) in which X1 is S, N, D, Q, A, or E, X2 is T, I, or S, X3 is Y, L, or F, X4 is D, G, T, E, Q, A, or Y, and X5 is A, T, R, S, K, or Y, and includes a VL domain including CDR-L3. In some embodiments, the VH domain further comprises FW-1 containing the sequence QVQLVQSGAEVKKPGSSVKVSCKASGGTFS (SEQ ID NO: 274), FW-2 containing the sequence WVRQAPGQGLEWMG (SEQ ID NO: 275), FW-3 containing the sequence RVTITADESTSTAYMELSSLRSEDTAVYYCAR (SEQ ID NO: 276), and / or FW-4 containing the sequence WGQGTLVTVSS (SEQ ID NO: 277).In some embodiments, the VL domain further comprises FW-1 containing the sequence DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 289), FW-2 containing the sequence WYQQKPGKAPKLLIY (SEQ ID NO: 290), FW-3 containing the sequence GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 291), and / or FW-4 containing the sequence FGGGTKVEIK (SEQ ID NO: 292).

[0074] In some embodiments, the anti-CD8 antibody of the present disclosure comprises a VH domain comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 225, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 226, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 227, and a VL domain comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 16, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 17, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 228. In some embodiments, the VH domain comprises an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 245, and / or the VL domain comprises an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 246. In some embodiments, the anti-CD8 antibody of this disclosure comprises one, two, or three heavy chain CDRs of antibody xhCD8v9 (e.g., those shown in Tables 1-3) and / or one, two, or three light chain CDRs of antibody xhCD8v9 (e.g., those shown in Tables 1-3). In some embodiments, the antibody is humanized. In some embodiments, the anti-CD8 antibody of this disclosure comprises a VH domain containing CDR-H1, CDR-H2, and CDR-H3 of the sequence of SEQ ID NO: 245, and a VL domain containing CDR-L1, CDR-L2, and CDR-L3 of the sequence of SEQ ID NO: 246. In some embodiments, the VH domain further comprises FW-1 containing the sequence QVQLVQSGAEVKKPGSSVKVSCKASGGTFS (SEQ ID NO: 274), FW-2 containing the sequence WVRQAPGQGLEWMG (SEQ ID NO: 275), FW-3 containing the sequence RVTITADESTSTAYMELSSLRSEDTAVYYCAR (SEQ ID NO: 276), and / or FW-4 containing the sequence WGQGTLVTVSS (SEQ ID NO: 277).In some embodiments, the VL domain further comprises FW-1 containing the sequence DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 289), FW-2 containing the sequence WYQQKPGKAPKLLIY (SEQ ID NO: 290), FW-3 containing the sequence GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 291), and / or FW-4 containing the sequence FGGGTKVEIK (SEQ ID NO: 292).

[0075] In some embodiments, the anti-CD8 antibody of the present disclosure comprises a VH domain comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 225, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 232, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 233, and a VL domain comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 234, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 235, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 236. In some embodiments, the VH domain comprises an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 251, and / or the VL domain comprises an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 252. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO: 251, and the VL domain comprises the amino acid sequence of SEQ ID NO: 252. In some embodiments, the anti-CD8 antibody of this disclosure comprises one, two, or three heavy chain CDRs of antibody xhCD8v12 (e.g., those shown in Tables 1-3) and / or one, two, or three light chain CDRs of antibody xhCD8v12 (e.g., those shown in Tables 1-3). In some embodiments, the antibody is humanized. In some embodiments, the anti-CD8 antibody of this disclosure comprises a VH domain containing CDR-H1, CDR-H2, and CDR-H3 of the sequence of SEQ ID NO: 251, and a VL domain containing CDR-L1, CDR-L2, and CDR-L3 of the sequence of SEQ ID NO: 252. In some embodiments, the VH domain further comprises FW-1 containing the sequence QVQLVQSGAEVKKPGSSVKVSCKASGGTFS (SEQ ID NO: 274), FW-2 containing the sequence WVRQAPGQGLEWMG (SEQ ID NO: 275), FW-3 containing the sequence RVTITADESTSTAYMELSSLRSEDTAVYYCAR (SEQ ID NO: 276), and / or FW-4 containing the sequence WGQGTLVTVSS (SEQ ID NO: 277).In some embodiments, the VL domain further comprises FW-1 containing the sequence DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 289), FW-2 containing the sequence WYQQKPGKAPKLLIY (SEQ ID NO: 290), FW-3 containing the sequence GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 291), and / or FW-4 containing the sequence FGGGTKVEIK (SEQ ID NO: 292).

[0076] In some embodiments, the anti-CD8 antibody of the present disclosure comprises a VH domain comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 225, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 232, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 233, and a VL domain comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 16, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 17, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 228. In some embodiments, the VH domain comprises an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 253, and / or the VL domain comprises an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 254. In some embodiments, the anti-CD8 antibody of this disclosure comprises one, two, or three heavy chain CDRs of antibody xhCD8v13 (e.g., those shown in Tables 1-3) and / or one, two, or three light chain CDRs of antibody xhCD8v13 (e.g., those shown in Tables 1-3). In some embodiments, the antibody is humanized. In some embodiments, the anti-CD8 antibody of this disclosure comprises a VH domain containing CDR-H1, CDR-H2, and CDR-H3 of the sequence of SEQ ID NO: 253, and a VL domain containing CDR-L1, CDR-L2, and CDR-L3 of the sequence of SEQ ID NO: 254. In some embodiments, the VH domain further comprises FW-1 containing the sequence QVQLVQSGAEVKKPGSSVKVSCKASGGTFS (SEQ ID NO: 274), FW-2 containing the sequence WVRQAPGQGLEWMG (SEQ ID NO: 275), FW-3 containing the sequence RVTITADESTSTAYMELSSLRSEDTAVYYCAR (SEQ ID NO: 276), and / or FW-4 containing the sequence WGQGTLVTVSS (SEQ ID NO: 277).In some embodiments, the VL domain further comprises FW-1 containing the sequence DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 289), FW-2 containing the sequence WYQQKPGKAPKLLIY (SEQ ID NO: 290), FW-3 containing the sequence GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 291), and / or FW-4 containing the sequence FGGGTKVEIK (SEQ ID NO: 292).

[0077] In some embodiments, the anti-CD8 antibodies of the present disclosure include CDR-H1 comprising the amino acid sequence X1YX2MS, where X1 is S, D, E, A, or Q, and X2 is A, G, or T (Sequence ID 268); CDR-H2 comprising the amino acid sequence DIX1X2X3GX4X5TX6YADSVKG, where X1 is T, N, S, Q, E, H, R, or A, X2 is Y, W, F, or H, X3 is A, S, Q, E, or T, X4 is G or E, X5 is S or I, and X6 is A or G (Sequence ID 269); and the amino acid sequence X1X2X3YX4WX5X6AX7DX8. Here, X1 is S or A, X2 is N, H, A, D, L, Q, Y, or R, X3 is A, N, S, or G, X4 is A, V, R, E, or S, X5 is D or S, X6 is D, N, Q, E, S, T, or L, X7 is L, F, or M, and X8 is I, Y, or V, comprising a VH domain including CDR-H3 containing the amino acid sequence (SEQ ID NO: 270), and a VL domain including CDR-L1 containing the amino acid sequence of RASQSVSSNLA (SEQ ID NO: 40), CDR-L2 containing the amino acid sequence of GASSRAT (SEQ ID NO: 41), and CDR-L3 containing the amino acid sequence of QQYGSSPPVT (SEQ ID NO: 42). In some embodiments, the VH domain further comprises FW-1 containing the sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFS (SEQ ID NO: 281), FW-2 containing the sequence WVRQAPGKGLEWVS (SEQ ID NO: 282), FW-3 containing the sequence RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 283), and / or FW-4 containing the sequence WGQGTMVTVSS (SEQ ID NO: 284) or WGQGTLVTVSS (SEQ ID NO: 285).In some embodiments, the VL domain further comprises FW-1 containing the sequence EIVLTQSPGTLSLSPGERATLSC (SEQ ID NO: 293), FW-2 containing the sequence WYQQKPGQAPRLLIY (SEQ ID NO: 294), FW-3 containing the sequence GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC (SEQ ID NO: 295), and / or FW-4 containing the sequence FGQGTKVEIK (SEQ ID NO: 296).

[0078] In some embodiments, the anti-CD8 antibody of the present disclosure comprises a VH domain comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 229, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 230, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 231, and a VL domain comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 40, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 41, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 42. In some embodiments, the VH domain comprises an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 247, and / or the VL domain comprises an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 248. In some embodiments, the anti-CD8 antibody of this disclosure comprises one, two, or three heavy chain CDRs of antibody xhCD8v10 (e.g., those shown in Tables 1-3) and / or one, two, or three light chain CDRs of antibody xhCD8v10 (e.g., those shown in Tables 1-3). In some embodiments, the antibody is humanized. In some embodiments, the anti-CD8 antibody of this disclosure comprises a VH domain containing CDR-H1, CDR-H2, and CDR-H3 of the sequence of SEQ ID NO: 247, and a VL domain containing CDR-L1, CDR-L2, and CDR-L3 of the sequence of SEQ ID NO: 248. In some embodiments, the VH domain further comprises FW-1 containing the sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFS (SEQ ID NO: 281), FW-2 containing the sequence WVRQAPGKGLEWVS (SEQ ID NO: 282), FW-3 containing the sequence RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 283), and / or FW-4 containing the sequence WGQGTMVTVSS (SEQ ID NO: 284) or WGQGTLVTVSS (SEQ ID NO: 285).In some embodiments, the VL domain further comprises FW-1 containing the sequence EIVLTQSPGTLSLSPGERATLSC (SEQ ID NO: 293), FW-2 containing the sequence WYQQKPGQAPRLLIY (SEQ ID NO: 294), FW-3 containing the sequence GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC (SEQ ID NO: 295), and / or FW-4 containing the sequence FGQGTKVEIK (SEQ ID NO: 296).

[0079] In some embodiments, the anti-CD8 antibody of the present disclosure comprises a VH domain comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 229, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 230, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 231, and a VL domain comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 40, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 41, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 42. In some embodiments, the VH domain comprises an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 249, and / or the VL domain comprises an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 250. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO: 249, and the VL domain comprises the amino acid sequence of SEQ ID NO: 250. In some embodiments, the anti-CD8 antibody of this disclosure comprises one, two, or three heavy chain CDRs of antibody xhCD8v11 (e.g., those shown in Tables 1-3) and / or one, two, or three light chain CDRs of antibody xhCD8v11 (e.g., those shown in Tables 1-3). In some embodiments, the antibody is humanized. In some embodiments, the anti-CD8 antibody of this disclosure comprises a VH domain containing CDR-H1, CDR-H2, and CDR-H3 of the sequence of SEQ ID NO: 249, and a VL domain containing CDR-L1, CDR-L2, and CDR-L3 of the sequence of SEQ ID NO: 250. In some embodiments, the VH domain further comprises FW-1 containing the sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFS (SEQ ID NO: 281), FW-2 containing the sequence WVRQAPGKGLEWVS (SEQ ID NO: 282), FW-3 containing the sequence RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 283), and / or FW-4 containing the sequence WGQGTMVTVSS (SEQ ID NO: 284) or WGQGTLVTVSS (SEQ ID NO: 285).In some embodiments, the VL domain further comprises FW-1 containing the sequence EIVLTQSPGTLSLSPGERATLSC (SEQ ID NO: 293), FW-2 containing the sequence WYQQKPGQAPRLLIY (SEQ ID NO: 294), FW-3 containing the sequence GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC (SEQ ID NO: 295), and / or FW-4 containing the sequence FGQGTKVEIK (SEQ ID NO: 296).

[0080] In some embodiments, the anti-CD8 antibody of the present disclosure comprises a VH domain comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 229, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 237, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 231, and a VL domain comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 40, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 41, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 42. In some embodiments, the VH domain comprises an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 255, and / or the VL domain comprises an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 256. In some embodiments, the anti-CD8 antibody of this disclosure comprises one, two, or three heavy chain CDRs of antibody xhCD8v14 (e.g., those shown in Tables 1-3) and / or one, two, or three light chain CDRs of antibody xhCD8v14 (e.g., those shown in Tables 1-3). In some embodiments, the antibody is humanized. In some embodiments, the anti-CD8 antibody of this disclosure comprises a VH domain containing CDR-H1, CDR-H2, and CDR-H3 of the sequence of SEQ ID NO: 255, and a VL domain containing CDR-L1, CDR-L2, and CDR-L3 of the sequence of SEQ ID NO: 256. In some embodiments, the VH domain further comprises FW-1 containing the sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFS (SEQ ID NO: 281), FW-2 containing the sequence WVRQAPGKGLEWVS (SEQ ID NO: 282), FW-3 containing the sequence RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 283), and / or FW-4 containing the sequence WGQGTMVTVSS (SEQ ID NO: 284) or WGQGTLVTVSS (SEQ ID NO: 285).In some embodiments, the VL domain further comprises FW-1 containing the sequence EIVLTQSPGTLSLSPGERATLSC (SEQ ID NO: 293), FW-2 containing the sequence WYQQKPGQAPRLLIY (SEQ ID NO: 294), FW-3 containing the sequence GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC (SEQ ID NO: 295), and / or FW-4 containing the sequence FGQGTKVEIK (SEQ ID NO: 296).

[0081] In some embodiments, the anti-CD8 antibody of the present disclosure comprises a VH domain comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 229, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 237, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 231, and a VL domain comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 40, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 41, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 42. In some embodiments, the VH domain comprises an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 257, and / or the VL domain comprises an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 258. In some embodiments, the anti-CD8 antibody of this disclosure comprises one, two, or three heavy chain CDRs of antibody xhCD8v15 (e.g., those shown in Tables 1-3) and / or one, two, or three light chain CDRs of antibody xhCD8v15 (e.g., those shown in Tables 1-3). In some embodiments, the antibody is humanized. In some embodiments, the anti-CD8 antibody of this disclosure comprises a VH domain containing CDR-H1, CDR-H2, and CDR-H3 of the sequence of SEQ ID NO: 257, and a VL domain containing CDR-L1, CDR-L2, and CDR-L3 of the sequence of SEQ ID NO: 258. In some embodiments, the VH domain further comprises FW-1 containing the sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFS (SEQ ID NO: 281), FW-2 containing the sequence WVRQAPGKGLEWVS (SEQ ID NO: 282), FW-3 containing the sequence RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 283), and / or FW-4 containing the sequence WGQGTMVTVSS (SEQ ID NO: 284) or WGQGTLVTVSS (SEQ ID NO: 285).In some embodiments, the VL domain further comprises FW-1 containing the sequence EIVLTQSPGTLSLSPGERATLSC (SEQ ID NO: 293), FW-2 containing the sequence WYQQKPGQAPRLLIY (SEQ ID NO: 294), FW-3 containing the sequence GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC (SEQ ID NO: 295), and / or FW-4 containing the sequence FGQGTKVEIK (SEQ ID NO: 296).

[0082] In some embodiments, the anti-CD8 antibody of the present disclosure comprises a VH domain comprising CDR-H1 containing the amino acid sequence of SEQ ID NO: 49, CDR-H2 containing the amino acid sequence of SEQ ID NO: 50, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 3, and a VL domain comprising CDR-L1 containing the amino acid sequence of SEQ ID NO: 4, CDR-L2 containing the amino acid sequence of SEQ ID NO: 5, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 6. In some embodiments, the anti-CD8 antibody of the present disclosure comprises a VH domain comprising CDR-H1 containing the amino acid sequence of SEQ ID NO: 51, CDR-H2 containing the amino acid sequence of SEQ ID NO: 52, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 15, and a VL domain comprising CDR-L1 containing the amino acid sequence of SEQ ID NO: 16, CDR-L2 containing the amino acid sequence of SEQ ID NO: 17, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 18. In some embodiments, the anti-CD8 antibody of the present disclosure comprises a VH domain comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 53, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 52, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 21, and a VL domain comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 22, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 23, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 24. In some embodiments, the anti-CD8 antibody of the present disclosure comprises a VH domain comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 49, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 52, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 27, and a VL domain comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 28, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 29, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 30. In some embodiments, the anti-CD8 antibody of the present disclosure comprises a VH domain comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 54, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 52, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 33, and a VL domain comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 34, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 35, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 36.In some embodiments, the anti-CD8 antibody of the present disclosure comprises a VH domain comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 55, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 56, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 39, and a VL domain comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 40, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 41, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 42. In some embodiments, the anti-CD8 antibody of the present disclosure comprises a VH domain comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 55, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 57, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 45, and a VL domain comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 46, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 47, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 48. In some embodiments, the anti-CD8 antibody of the present disclosure comprises a VH domain comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 183, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 184, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 179, and a VL domain comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 180, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 181, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 182.

[0083] In some embodiments, the anti-CD8 antibody of the present disclosure is an amino acid sequence of CDR-H1,X1PX2X3X4X5 comprising the amino acid sequence (SEQ ID NO: 265) GX1X2FX3X4X5, where X1 is G, Y, S, or A, X2 is T, S, G, R, N, or H, X3 is S, T, R, H, Y, G, or P, X4 is S, K, G, N, R, D, T, or G, and X5 is Y, L, H, or F, where X1 is I, N, or M, X2 is G, N, H, S, R, I, CDR-H2 comprises the amino acid sequence (SEQ ID NO: 266) in which X1 is A, X3 is A, N, H, S, T, F, or Y, X4 is A, D, or G, and X5 is T, E, K, V, Q, or A, and the amino acid sequence X1X2X3GX4X5LFX6X7, where X1 is D or A, X2 is A, G, E, R, Y, K, N, Q, L, or F, X3 is A, L, P, or Y, X4 is I or L, X5 is R, A, Q, or S, X6 is A or D, and X7 is D, E, A, or S. A VH domain containing CDR-H3 containing a certain amino acid sequence (SEQ ID NO: 267), and an amino acid sequence of X1X2SX3X4IX5GX6LN containing CDR-L1, GX1X2X3LX4X5, where X1 is R or G, X2 is A or T, X3 is Q or E, X4 is E, N, T, S, A, K, D, G, R, or Q, X5 is Y or S, and X6 is A or V (SEQ ID NO: 262). CDR-L2 comprises the amino acid sequence (SEQ ID NO: 263) in which X1 is D, X3 is N, R, A, E, or H, X4 is Q or A, and X5 is S or D, and includes a VL domain comprising CDR-L3 comprising the amino acid sequence (SEQ ID NO: 264) of QX1X2X3X4X5PWT, where X1 is S, N, D, Q, A, or E, X2 is T, I, or S, X3 is Y, L, or F, X4 is D, G, T, E, Q, A, or Y, and X5 is A, T, R, S, K, or Y.In some embodiments, the VH domain further comprises FW-1 containing the sequence QVQLVQSGAEVKKPGSSVKVSCKAS (SEQ ID NO: 278), FW-2 containing the sequence AISWVRQAPGQGLEWMGGI (SEQ ID NO: 279), FW-3 containing the sequence ANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAR (SEQ ID NO: 280), and / or FW-4 containing the sequence WGQGTLVTVSS (SEQ ID NO: 277). In some embodiments, the VL domain further comprises FW-1 containing the sequence DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 289), FW-2 containing the sequence WYQQKPGKAPKLLIY (SEQ ID NO: 290), FW-3 containing the sequence GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 291), and / or FW-4 containing the sequence FGGGTKVEIK (SEQ ID NO: 292).

[0084] In some embodiments, the anti-CD8 antibody of the present disclosure comprises a VH domain comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 238, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 239, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 233, and a VL domain comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 16, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 17, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 228. In some embodiments, the VH domain further comprises FW-1 comprising the sequence QVQLVQSGAEVKKPGSSVKVSCKAS (SEQ ID NO: 278), FW-2 comprising the sequence AISWVRQAPGQGLEWMGGI (SEQ ID NO: 279), FW-3 comprising the sequence ANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAR (SEQ ID NO: 280), and / or FW-4 comprising the sequence WGQGTLVTVSS (SEQ ID NO: 277). In some embodiments, the VL domain further comprises FW-1 containing the sequence DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 289), FW-2 containing the sequence WYQQKPGKAPKLLIY (SEQ ID NO: 290), FW-3 containing the sequence GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 291), and / or FW-4 containing the sequence FGGGTKVEIK (SEQ ID NO: 292).

[0085] In some embodiments, the anti-CD8 antibody of the present disclosure comprises a VH domain comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 238, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 243, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 233, and a VL domain comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 234, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 235, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 236. In some embodiments, the VH domain further comprises FW-1 comprising the sequence QVQLVQSGAEVKKPGSSVKVSCKAS (SEQ ID NO: 278), FW-2 comprising the sequence AISWVRQAPGQGLEWMGGI (SEQ ID NO: 279), FW-3 comprising the sequence ANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAR (SEQ ID NO: 280), and / or FW-4 comprising the sequence WGQGTLVTVSS (SEQ ID NO: 277). In some embodiments, the VL domain further comprises FW-1 containing the sequence DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 289), FW-2 containing the sequence WYQQKPGKAPKLLIY (SEQ ID NO: 290), FW-3 containing the sequence GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 291), and / or FW-4 containing the sequence FGGGTKVEIK (SEQ ID NO: 292).

[0086] In some embodiments, the anti-CD8 antibody of the present disclosure comprises a VH domain comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 238, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 243, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 233, and a VL domain comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 16, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 17, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 228. In some embodiments, the VH domain further comprises FW-1 comprising the sequence QVQLVQSGAEVKKPGSSVKVSCKAS (SEQ ID NO: 278), FW-2 comprising the sequence AISWVRQAPGQGLEWMGGI (SEQ ID NO: 279), FW-3 comprising the sequence ANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAR (SEQ ID NO: 280), and / or FW-4 comprising the sequence WGQGTLVTVSS (SEQ ID NO: 277). In some embodiments, the VL domain further comprises FW-1 containing the sequence DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 289), FW-2 containing the sequence WYQQKPGKAPKLLIY (SEQ ID NO: 290), FW-3 containing the sequence GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 291), and / or FW-4 containing the sequence FGGGTKVEIK (SEQ ID NO: 292).

[0087] In some embodiments, the anti-CD8 antibody of the present disclosure is CDR-H1 comprising the amino acid sequence GFTFX1X2Y, where X1 is S, D, E, Q, S, or A, and X2 is S, D, E, A, or Q (Sequence ID 271); CDR-H2 comprising the amino acid sequence X1X2X3GX4X5, where X1 is T, N, S, Q, E, H, R, or A, X2 is Y, W, F, or H, X3 is A, S, Q, E, or T, X4 is G or E, and X5 is S or I (Sequence ID 272); and the amino acid sequence X1X2X3YX4WX5X6AX7DX8, where X1 The VH domain includes CDR-H3 containing the amino acid sequence (SEQ ID NO: 273) in which X2 is S or A, X3 is N, H, A, D, L, Q, Y, or R, X4 is A, N, S, or G, X5 is D or S, X6 is D, N, Q, E, S, T, or L, X7 is L, F, or M, and X8 is I, Y, or V; and the VL domain includes CDR-L1 containing the amino acid sequence of RASQSVSSNLA (SEQ ID NO: 40), CDR-L2 containing the amino acid sequence of GASSRAT (SEQ ID NO: 41), and CDR-L3 containing the amino acid sequence of QQYGSSPPVT (SEQ ID NO: 42). In some embodiments, the VH domain further comprises FW-1 containing the sequence EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 286), FW-2 containing the sequence AMSWVRQAPGKGLEWVSDI (SEQ ID NO: 287), FW-3 containing the sequence TAYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 288), and / or FW-4 containing the sequence WGQGTMVTVSS (SEQ ID NO: 284) or WGQGTLVTVSS (SEQ ID NO: 285). In some embodiments, the VL domain further comprises FW-1 containing the sequence EIVLTQSPGTLSLSPGERATLSC (SEQ ID NO: 293), FW-2 containing the sequence WYQQKPGQAPRLLIY (SEQ ID NO: 294), FW-3 containing the sequence GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC (SEQ ID NO: 295), and / or FW-4 containing the sequence FGQGTKVEIK (SEQ ID NO: 296).

[0088] In some embodiments, the anti-CD8 antibody of the present disclosure comprises a VH domain comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 240, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 241, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 242, and a VL domain comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 40, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 41, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 42. In some embodiments, the VH domain further comprises FW-1 comprising the sequence EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 286), FW-2 comprising the sequence AMSWVRQAPGKGLEWVSDI (SEQ ID NO: 287), FW-3 comprising the sequence TAYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 288), and / or FW-4 comprising the sequence WGQGTMVTVSS (SEQ ID NO: 284) or WGQGTLVTVSS (SEQ ID NO: 285). In some embodiments, the VL domain further comprises FW-1 containing the sequence EIVLTQSPGTLSLSPGERATLSC (SEQ ID NO: 293), FW-2 containing the sequence WYQQKPGQAPRLLIY (SEQ ID NO: 294), FW-3 containing the sequence GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC (SEQ ID NO: 295), and / or FW-4 containing the sequence FGQGTKVEIK (SEQ ID NO: 296).

[0089] In some embodiments, the anti-CD8 antibody of the present disclosure comprises a VH domain comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 240, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 244, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 242, and a VL domain comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 40, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 41, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 42. In some embodiments, the VH domain further comprises FW-1 comprising the sequence EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 286), FW-2 comprising the sequence AMSWVRQAPGKGLEWVSDI (SEQ ID NO: 287), FW-3 comprising the sequence TAYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 288), and / or FW-4 comprising the sequence WGQGTMVTVSS (SEQ ID NO: 284) or WGQGTLVTVSS (SEQ ID NO: 285). In some embodiments, the VL domain further comprises FW-1 containing the sequence EIVLTQSPGTLSLSPGERATLSC (SEQ ID NO: 293), FW-2 containing the sequence WYQQKPGQAPRLLIY (SEQ ID NO: 294), FW-3 containing the sequence GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC (SEQ ID NO: 295), and / or FW-4 containing the sequence FGQGTKVEIK (SEQ ID NO: 296). In some embodiments, the disclosure provides anti-CD8 antibodies comprising VH domains containing the CDR-H1, CDR-H2, and CDR-H3 sequences of a single antibody listed in Table 1, and VL domains containing the CDR-L1, CDR-L2, and CDR-L3 sequences of a single antibody listed in Table 1. For example, anti-CD8 antibodies include the antibodies xhCD8v1, xhCD8v1.1, xhCD8v2, xhCD8v3, xhCD8v4, xhCD8v5, xhCD8v6, xhCD8v7, xhCD8v8, xhCD8v9, xhCD8v10, xhCD8v11, xhCD8v12, xhCD8v13, xhCD8v14, ​​xhCD8v15, the V9 family, or the six CDRs of the V11 family, as shown in Table 1.In some embodiments, the Disclosure provides anti-CD8 antibodies comprising a VH domain containing the CDR-H1, CDR-H2, and CDR-H3 sequences of a single antibody listed in Table 2, and a VL domain containing the CDR-L1, CDR-L2, and CDR-L3 sequences of a single antibody listed in Table 2. For example, an anti-CD8 antibody comprises six CDRs from the antibodies xhCD8v1, xhCD8v1.1, xhCD8v2, xhCD8v3, xhCD8v4, xhCD8v5, xhCD8v6, xhCD8v7, xhCD8v8, xhCD8v9, xhCD8v10, xhCD8v11, xhCD8v12, xhCD8v13, xhCD8v14, ​​xhCD8v15, the V9 family, or the V11 family, as shown in Table 2. In some embodiments, the Disclosure provides a fusion protein comprising an anti-CD8 antibody comprising a VH domain containing the CDR-H1, CDR-H2, and CDR-H3 sequences of a single antibody listed in Table 1, and a VL domain containing the CDR-L1, CDR-L2, and CDR-L3 sequences of a single antibody listed in Table 1. For example, the anti-CD8 antibody of the fusion protein comprises six CDRs from the antibodies xhCD8v1, xhCD8v1.1, xhCD8v2, xhCD8v3, xhCD8v4, xhCD8v5, xhCD8v6, xhCD8v7, xhCD8v8, xhCD8v9, xhCD8v10, xhCD8v11, xhCD8v12, xhCD8v13, xhCD8v14, ​​xhCD8v15, the V9 family, or the V11 family, as shown in Table 1. In some embodiments, the disclosure provides a fusion protein comprising an anti-CD8 antibody comprising a VH domain containing the CDR-H1, CDR-H2, and CDR-H3 sequences of a single antibody listed in Table 2, and a VL domain containing the CDR-L1, CDR-L2, and CDR-L3 sequences of a single antibody listed in Table 2.For example, the anti-CD8 antibodies for the fusion protein include the antibodies xhCD8v1, xhCD8v1.1, xhCD8v2, xhCD8v3, xhCD8v4, xhCD8v5, xhCD8v6, xhCD8v7, xhCD8v8, xhCD8v9, xhCD8v10, xhCD8v11, xhCD8v12, xhCD8v13, xhCD8v14, ​​xhCD8v15, the V9 family, or the six CDRs of the V11 family, as shown in Table 2. In some embodiments, the Disclosure provides anti-CD8 antibodies comprising a VH domain containing the CDR-H1, CDR-H2, and CDR-H3 sequences of the VH domain listed in Table 3, and a VL domain containing the CDR-L1, CDR-L2, and CDR-L3 sequences of the VL domain listed in Table 3 (in some embodiments, the VH and VL domains are derived from the same single antibody listed in Table 3). For example, an anti-CD8 antibody comprising the VH and VL of the antibodies xhCD8v1, xhCD8v1.1, xhCD8v2, xhCD8v3, xhCD8v4, xhCD8v5, xhCD8v6, xhCD8v7, xhCD8v8, xhCD8v9, xhCD8v10, xhCD8v11, xhCD8v12, xhCD8v13, xhCD8v14, ​​or xhCD8v15 shown in Table 3. In some embodiments, the Disclosure provides a fusion protein comprising an anti-CD8 antibody comprising a VH domain containing the CDR-H1, CDR-H2, and CDR-H3 sequences of the VH domain listed in Table 3, and a VL domain containing the CDR-L1, CDR-L2, and CDR-L3 sequences of the VL domain listed in Table 3 (in some embodiments, the VH and VL domains are derived from the same single antibody listed in Table 3). In some embodiments, the Disclosure provides an anti-CD8 antibody comprising the VH domain sequence and the VL domain sequence of a single antibody as listed in Table 3. In some embodiments, the Disclosure provides a fusion protein comprising an anti-CD8 antibody comprising the VH domain sequence and the VL domain sequence of a single antibody as listed in Table 3.For example, the anti-CD8 antibodies for the fusion protein include the VH and VL versions of the antibodies xhCD8v1, xhCD8v1.1, xhCD8v2, xhCD8v3, xhCD8v4, xhCD8v5, xhCD8v6, xhCD8v7, xhCD8v8, xhCD8v9, xhCD8v10, xhCD8v11, xhCD8v12, xhCD8v13, xhCD8v14, ​​or xhCD8v15 shown in Table 3. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4]

[0090] Fusion protein This specification further provides fusion proteins comprising any one of the anti-CD8 antibodies, antigen-binding domains, or antibody fragments disclosed herein. In some embodiments, the fusion proteins of this disclosure comprise a first portion comprising a human or humanized antibody or its antigen-binding fragment (e.g., any one of the anti-CD8 antibodies described above) that specifically binds to CD8b and / or CD8ab, and a second portion comprising a cytokine, chemokine, or growth factor. In some embodiments, the first portion is directly fused to the second portion. In some embodiments, the first portion is fused to the second portion via a linker. Exemplary and non-limiting diagrams of the fusion proteins of this disclosure are shown in Figure 7.

[0091] Fusion protein format In some embodiments, the first portion comprises an antibody (e.g., the anti-CD8 antibody of the Disclosure). In some embodiments, the first portion comprises an antibody fragment (e.g., the anti-CD8 antibody fragment of the Disclosure). In some embodiments, the first portion comprises a single-chain antibody or a single-chain variable fragment (scFv). In some embodiments, the first portion comprises a VHH antibody. In some embodiments, the first portion comprises one or two antibody heavy chain polypeptides and one or two antibody light chain polypeptides (e.g., those of the anti-CD8 antibody of the Disclosure). In some embodiments, the first portion comprises three heavy chain CDRs and / or three light chain CDRs of a single anti-CD8 antibody of the Disclosure (e.g., those shown in Tables 1-3). In some embodiments, the first portion comprises the VH and / or VL domains (or more) of a single anti-CD8 antibody of the Disclosure (e.g., those shown in Table 3). In some embodiments, the first portion further comprises one or two human IgG Fc domains. In some embodiments, one or two human IgG Fc domains are IgG1, IgG2, IgG3, or IgG4 Fc domains. In some embodiments, one or two human IgG Fc domains lack a C-terminal lysine residue. In some embodiments, one or two human IgG Fc domains include amino acid modifications (substitutions, deletions, additions, etc.). In some embodiments, Fc domain modifications promote heterodimer formation (e.g., those shown in Table 4). In some embodiments, one or two Fc domains include Fc gamma-null mutations.

[0092] In some embodiments, the first part comprises two antibody heavy chain polypeptides having a structure according to formula [I] from the N-terminus to the C-terminus: VH-CH1-Hinge-CH2-CH3 [I] and two antibody light chain polypeptides containing the structure of formula [II] from the N-terminus to the C-terminus: VL-CL [II] The formula includes (wherein VH is the VH domain, CH1 is the antibody CH1 domain, hinge is the antibody hinge domain, CH2-CH3 is the antibody Fc domain, VL is the VL domain, and CL is the antibody constant light chain domain). In some embodiments, the N-terminus of the second portion is fused to the C-terminus of one of the two CH3 domains (see, for example, Format A in Figure 7).

[0093] In some embodiments, the first part comprises a first antibody heavy chain polypeptide having a structure according to formula [I] from the N-terminus to the C-terminus: VH-CH1-Hinge-CH2-CH3 [I], Antibody light chain polypeptides containing the structure shown by formula [II] from the N-terminus to the C-terminus: VL-CL [II] and a second antibody heavy chain polypeptide containing the structure of formula [III] from the N-terminus to the C-terminus: Hinge-CH2-CH3 [III], The formula includes (wherein VH is the VH domain, CH1 is the antibody CH1 domain, hinge is the antibody hinge domain, CH2-CH3 is the antibody Fc domain, VL is the VL domain, and CL is the antibody constant light chain domain). In some embodiments, the N-terminus of the second portion is fused to the C-terminus of the CH3 domain of the second antibody heavy chain polypeptide (see, for example, Format B in Figure 7). In some embodiments, the N-terminus of the second portion is fused to the C-terminus of the CH3 domain of the first antibody heavy chain polypeptide (see, for example, Format D in Figure 7).

[0094] In some embodiments, the first part comprises a first antibody heavy chain polypeptide having a structure according to formula [I] from the N-terminus to the C-terminus: VH-CH1-Hinge-CH2-CH3 [I], Antibody light chain polypeptides containing the structure shown by formula [II] from the N-terminus to the C-terminus: VL-CL [II] and a second antibody heavy chain polypeptide containing the structure of formula [III] from the N-terminus to the C-terminus: Hinge-CH2-CH3 [III], The formula includes (wherein VH is the VH domain, CH1 is the antibody CH1 domain, hinge is the antibody hinge domain, CH2-CH3 is the antibody Fc domain, VL is the VL domain, and CL is the antibody constant light chain domain). In some embodiments, the C-terminus of the second portion is fused to the N-terminus of the hinge domain of the second antibody heavy chain polypeptide (see, for example, Format C in Figure 7).

[0095] In some embodiments, the anti-CD8 antibodies of this disclosure are multispecific (e.g., bispecific) antibodies or antibody fragments. For example, in some embodiments, a multispecific antibody (e.g., a bispecific antibody) comprises a first antigen-binding domain (e.g., those described above) that binds to CD8 and a second antigen-binding domain that binds to a target of interest. In some embodiments, a bispecific antibody may be produced by fusing an additional binding site to either the heavy or light chain of an immunoglobulin. Examples of additional binding sites include, but are not limited to, variable regions, scFv, Fab, VHH, and peptides.

[0096] In some embodiments, the recombinant bispecific antibodies disclosed herein can be broadly classified into two categories: i) formats resulting from a combination of only variable regions, and ii) formats combining a variable region and an Fc domain. Representatives of the first category include tandem scFv (taFv), diabody (Db), DART, single-chain diabody (scDbs), Fab-Fc, tandem Fab, bivariate-region Fab, and tandem dAb / VHH. The two variable regions may be linked to each other via covalent or non-covalent interactions.

[0097] Non-covalent interactions may be involved in pairing variable regions using a dock-and-lock method with leucine zippers, cAMP-dependent protein kinase (PKA) regulatory subunits and A kinase anchored protein (AKAP) anchoring domains, or with heterodimerization modules such as knob-into-holes CH3 domains (U.S. Patents 5,731,168, 7,695,936, Ridgway et al., Prot Eng 9,617-621 (1996) and Carter, J Immunol Meth 248,7-15 (2001)).

[0098] In some embodiments, bispecific antibodies are generated on a native immunoglobulin structure containing two pairs of heavy and light chain combinations, each pair having different binding specificities. Homodimerization of the two heavy chains in IgG is mediated by CH3 interactions. To promote heterodimerization, genetic modifications are introduced into each of the two CH3 regions. In this regard, heterodimerizing mutations are often involved in steric repulsion, charge steering interactions, or interchain disulfide bond formation. Exemplary and non-limiting Fc modifications to promote heterodimerization include: [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4]

[0099] In some embodiments, bispecific antibodies can be generated by assembly after the production of semi-antibodies, thereby resolving the issue of heavy-light chain mispairs. These antibodies often contain modifications that support heterodimerization of semi-antibodies. Exemplary systems include, but are not limited to, knob-into-hole, IgG1(EEE-RRR), IgG2(EEE-RRRR) (Strop et al. J Mol Biol (2012)), and DuoBody(F405L-K409R), listed in Table 5. In such cases, semi-antibodies are produced and purified individually in independent cell lines. The purified antibodies are then subjected to mild reduction to obtain semi-antibodies, which are then combined to construct bispecific antibodies. The heterodimer bispecific antibodies are then purified from the mixture using conventional purification methods.

[0100] In some embodiments, bispecific antibody production strategies that do not rely on preferred chain pairing can also be employed. These strategies typically involve introducing genetic modifications to antibodies so that the heterodimer has different biochemical or biophysical properties than the homodimer. Thus, the combined or expressed heterodimer can be selectively purified from the homodimer. For example, H435R / Y436F was introduced into the IgG1 CH3 domain to eliminate Fc binding to protein A resin, and then the H435R / Y436F variant was co-expressed with wild-type Fc. The resulting homodimeric antibody containing two copies of H435R / Y436F could not bind to the protein A column, while the heterodimeric antibody containing one copy of the H435R / Y436F mutation had reduced affinity for protein A compared to the strong interaction of the homodimeric wild-type antibody (Tustian et al Mabs 2016). Other examples include kappa / lambda antibodies (Fischer et al., Nature Communications 2015) and the introduction of charge differences (E357Q, S267K or N208D / Q295E / N384D / Q418E / N421D) into their respective chains (US 2018 / 0142040 A1, (Strop et al. J Mol Biol (2012))).

[0101] In some embodiments, bispecific antibodies can be generated by fusing an additional binding site to either the heavy or light chain of an immunoglobulin. Examples of additional binding sites include, but are not limited to, variable regions, scFv, Fab, VHH, and peptides.

[0102] Fusion protein Fc region In some embodiments, the antibodies or fusion proteins of this disclosure include an Fc region. In some embodiments, the Fc region includes one or more mutations that reduce or eliminate FcγR binding and / or effector function. In some embodiments, the Fc region (e.g., IgG1 Fc region) includes substitutions at one or more of the following positions: C220, C226, C229, E233, L234, L235, G237, P238, S239, D265, S267, N297, L328, P331, K322, A327, and P329. In some embodiments, the Fc region (e.g., IgG2 Fc region) includes substitutions at one or more of the following positions: V234, G237, D265, H268, N297, V309, A330, A331, K322. In some embodiments, the Fc region (e.g., IgG4 Fc region) includes substitutions at one or more of the following positions: L235, G237, D265 and E318. In some embodiments, the Fc region (e.g., IgG1 The Fc region contains one or more of the following mutations or mutant groups: N297A, N297Q, N297G, D265A / N297A, D265A / N297Q, C220S / C226S / C229S / P238S, S267E / L328F, C226S / C229S / E233P / L234V / L235A, L234F / L235E / P331S, L234A / L235A, L234A / L235A / G237A, L234A / L235A / G237A / K322A, L234A / L23 5A / G237A / A330S / A331S, L234A / L235A / P329G, E233P / L234V / L235A / G236 missing / S239K, E233P / L234V / L235A / G236 missing / S267K, E233P / L234V / L235A / G236 missing / S239K / A327G, E233P / L234V / L235A / G236 missing / S267K / A327G and E233P / L234V / L235A / G236 missing, L234A / L235A / G237 missing.In some embodiments, the Fc region (e.g., the IgG2 Fc region) contains one or more of the following mutations or groups of mutations: A330S / A331S, V234A / G237A, V234A / G237A / D265A, D265A / A330S / A331S, V234A / G237A / D265A / A330S / A331S, and H268Q / V309L / A330S / A331S. In some embodiments, the Fc region (e.g., the IgG4 Fc region) contains one or more of the following mutations or groups of mutations: L235A / G237A / E318A, D265A, L235A / G237A / D265A, and L235A / G237A / D265A / E318A. In some embodiments, the Fc region contains one, two, three, or all of the following mutations: L234A, L235A, G237A, and K322A (numbered according to the EU index).

[0103] In some embodiments, the first and second Fc domains of the fusion protein contain one or more of the following Fc mutations according to EU numbering to reduce effector function: L234A, L235A, G237A, and K322A. In some embodiments, the first and second Fc domains of the fusion protein contain the following Fc mutations according to EU numbering to reduce effector function: L234A, L235A, and G237A. In some embodiments, the first and second Fc domains of the fusion protein contain the following Fc mutations according to EU numbering to reduce effector function: L234A, L235A, G237A, and K322A. In some embodiments, the first and second Fc domains of the fusion protein contain the following amino acid substitutions to promote heterodimer formation: Y349C / T366W (knob) and S354C, T366S, L368A and Y407V (hole).

[0104] In some embodiments, heterodimer mutations and / or mutations to alter Fc gamma receptor binding resulted in decreased Fc stability. Therefore, additional mutations(s) were added to the Fc region to improve its stability. For example, one or more pairs of disulfide bonds, such as A287C and L306C, V259C and L306C, R292C and V302C, and V323C and I332C, were introduced into the Fc region. Another example is the introduction of S228P into IgG4-based bispecific antibodies to stabilize the hinge disulfide. Additional examples include the introduction of K338I, A339K, and K340S mutations to enhance Fc stability and agglutination resistance (Gao et al, 2019 Mol Pharm. 2019;16:3647).

[0105] Fusion protein cytokine In some embodiments, the second portion induces CD8+ T cell activation. In some embodiments, the second portion includes a polypeptide that induces IL2Rβγ-mediated signaling. For example, in some embodiments, the second portion includes an IL-15 polypeptide (e.g., human IL-15 polypeptide or a derivative thereof) or neoleukin. In some embodiments, the second portion includes an IL-21 polypeptide (e.g., human IL-21 polypeptide or a derivative thereof).

[0106] In some embodiments, the second portion comprises an IL-2 polypeptide (e.g., a human IL-2 polypeptide or a derivative thereof).

[0107] In some embodiments, the IL-2 polypeptide variant has a binding affinity to IL-2Rα that is 50% or less lower than that of the wild-type IL-2 polypeptide containing the sequence of SEQ ID NO: 81. In some embodiments, the IL-2 polypeptide variant has a binding affinity to IL-2Rβ that is 50% or less lower than that of the wild-type IL-2 polypeptide containing the sequence of SEQ ID NO: 81. In some embodiments, the IL-2 polypeptide variant has a binding affinity to IL-2Rγ that is 50% or less lower than that of the wild-type IL-2 polypeptide containing the sequence of SEQ ID NO: 81. The difference in binding affinity to IL-2Rα and IL-2Rβ between the wild-type and the disclosed polypeptide variants can be measured, for example, by a standard surface plasmon resonance (SPR) assay, which measures the affinity of protein-protein interactions, as is well known to those skilled in the art. The difference in binding affinity to IL-2Rγ between the wild-type and disclosed polypeptide variants cannot be reliably measured by the SPR assay because the affinity of the wild-type IL-2 polypeptide to IL-2Rγ is very low. Instead, their reduced affinity to IL-2Rγ can be estimated by performing an in vitro assay that measures pSTAT5 in IL-2R-expressing cells and compares the activity of IL-2 polypeptides with and without IL-2Rγ affinity-reducing substitutions. Exemplary sequences for IL-2Rα, IL-2Rβ, and IL-2Rγ are shown below. IL-2Rα: MDSYLLMWGLLTFIMVPGCQAELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQASPEGRPESETSCLVTTTDFQIQTEMAATMETSIFTTEYQVAVAGCVFLLISVLLLSGLTWQRRQRKSRRTI (SEQ ID NO: 82) IL-2Rβ: MAAPALSWRLPLLILLLPLATSWASAAVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKDTIPWLGHLLVGLSGAFGFIILVYLLINCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPLQPLSGEDDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLV (SEQ ID NO: 83) IL-2Rγ: MLKPSLPFTSLLFLQLPLLGVGLNTTILTPNGNEDTTADFFLTTMPTDSLSVSTLPLPEVQCFVFNVEYMNCTWNSSSEPQPTNLTLHYWYKNSDNDKVQKCSHYLFSEEITSGCQLQKKEIHLYQTFVVQLQDPREPRRQATQMLKLQNLVIPWAPENLTLHKLSESQLELNWNNRFLNHCLEHLVQYRTDWDHSWTEQSVDYRHKFSLPSVDGQKRYTFRVRSRFNPLCGSAQHWSEWSHPIHWGSNTSKENPFLFALEAVVISVGSMGLIISLLCVYFWLERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIPPKGGALGEGPGASPCNQHSPYWAPPCYTLKPET(Sequence ID 84)

[0108] In some embodiments, the IL-2 polypeptide is an IL-2 polypeptide variant comprising one or more mutations from a human IL-2 polypeptide comprising the sequence of SEQ ID NO: 81. In some embodiments, the IL-2 polypeptide variant of the present disclosure has one or more, two or more, or three or more affinity-decreasing amino acid substitutions from a wild-type mature IL-2 polypeptide (e.g., having the amino acid sequence of SEQ ID NO: 81). In some embodiments, one or more, two or more, or three or more residues to be substituted are selected from the following group: Q11, H16, L18, L19, D20, D84, S87, Q22, R38, F42, K43, Y45, E62, P65, E68, V69, L72, D84, S87, N88, V91, I92, T123, Q126, S127, I129, and S130. The decrease in affinity for IL-2Rα may be obtained by substituting one or more of the following residues in the sequence of the wild-type mature IL-2 polypeptide: R38, F42, K43, Y45, E62, P65, E68, V69, and L72. A decrease in affinity for IL-2Rβ may be obtained by substituting one or more of the following residues: E15, H16, L19, D20, D84, S87, N88, V91, and I92. A decrease in affinity for IL-2Rγ may be obtained by substituting one or more of the following residues in the sequence of the wild-type mature IL-2 polypeptide: Q11, L18, Q22, T123, Q126, S127, I129, and S130.

[0109] In some embodiments, the IL-2 polypeptide variant includes the amino acid substitution F42A or F42K relative to the wild-type mature IL-2 amino acid sequence. In some embodiments, the IL-2 polypeptide variant includes the amino acid substitution F42A or F42K and the amino acid substitution R38A, R38D, R38E, E62Q, E68A, E68Q, E68K, or E68R relative to the wild-type mature IL-2 amino acid sequence. For example, in some embodiments, the IL-2 polypeptide variant includes amino acid substitutions of F42A;R38A and F42A;R38D and F42A;R38E and F42A;F42A and E62Q;F42A and E68A;F42A and E68Q;F42A and E68K;F42A and E68R; or R38A and F42K relative to the wild-type mature IL-2 amino acid sequence (e.g., shown in SEQ ID NO: 81). In some embodiments, the IL-2 polypeptide variant includes amino acid substitutions of R38E and F42A relative to the wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 polypeptide variant includes amino acid substitutions of R38D and F42A relative to the wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 polypeptide variant includes amino acid substitutions of F42A and E62Q relative to the wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 polypeptide variant includes the amino acid substitutions R38A and F42K relative to the wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 polypeptide variant includes the amino acid substitutions R38D and F42A relative to the wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 polypeptide variant includes the amino acid substitutions R38A and F42K relative to the wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 polypeptide variant includes the amino acid substitutions F42A and E62Q relative to the wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 polypeptide variant includes the amino acid substitutions H16E, H16D, D20N, M23A, M23R, M23K, D84L, D84N, D84V, D84H, D84Y, D84R, D84K, S87K, S87A, N88A, N88D,Includes amino acid substitutions of N88G, N88S, N88T, N88R, N88I, V91A, V91T, V91E, I92A, E95S, E95A, E95R, T123A, T123E, T123K, T123Q, Q126A, Q126S, Q126T, Q126E, S127A, S127E, S127K, or S127Q. In some embodiments, IL-2 polypeptide variants are amino acid substitutions (multiple substitutions) of F42A;R38A and F42A;R38D and F42A;R38E and F42A;F42A and E62Q;F42A and E68A;F42A and E68Q;F42A and E68K;F42A and E68R; or R38A and F42K relative to the wild-type mature IL-2 amino acid sequence (e.g., as shown in SEQ ID NO: 81), H16E, H16D, Includes amino acid substitutions of D20N, M23A, M23R, M23K, D84L, D84N, D84V, D84H, D84Y, D84R, D84K, S87K, S87A, N88A, N88D, N88G, N88S, N88T, N88R, N88I, V91A, V91T, V91E, I92A, E95S, E95A, E95R, T123A, T123E, T123K, T123Q, Q126A, Q126S, Q126T, Q126E, S127A, S127E, S127K, or S127Q. For example, in some embodiments, the IL-2 polypeptide variant includes the amino acid substitutions R38E, F42A, and H16E relative to the wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 polypeptide variant includes the amino acid substitutions R38E, F42A, and H16D relative to the wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 polypeptide variant includes the amino acid substitutions R38E, F42A, and D84K relative to the wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 polypeptide variant includes the amino acid substitutions R38E, F42A, and D84R relative to the wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 polypeptide variant includes the amino acid substitutions R38E, F42A, and N88S relative to the wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 polypeptide variant,The IL-2 polypeptide variant includes the amino acid substitutions R38E, F42A, and N88A relative to the wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 polypeptide variant includes the amino acid substitutions R38E, F42A, and N88G relative to the wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 polypeptide variant includes the amino acid substitutions R38E, F42A, and N88R relative to the wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 polypeptide variant includes the amino acid substitutions R38E, F42A, and N88T relative to the wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 polypeptide variant includes the amino acid substitutions R38E, F42A, and N88D relative to the wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 polypeptide variant includes the amino acid substitutions R38E, F42A, and V91E relative to the wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 polypeptide variant includes the amino acid substitutions R38E, F42A, and Q126S relative to the wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 polypeptide variant includes the amino acid sequence of SEQ ID NO: 81 having one of the following sets of amino acid substitutions (relative to the sequence of SEQ ID NO: 81): R38E and F42A; R38D and F42A; F42A and E62Q; R38A and F42K; R38E, F42A, and N88S; R38E, F42A, and N88A; R38E, F42A, and N88G; R38E, F42A, and N88R; R38E, F42A, and N88T; R38E, F42A, and N88D; R38E, F42A, and V91E; R38E, F42A, and D84H; R38E, F42A, and D84K; R38E, F42A, and D84R; H16D, R38E, and F42A; H16E, R38E, and F42A; R38E, F42A, and Q126S; R38D, F42A, and N88S; R38D, F42A, and N88A; R38D, F42A, and N88G; R38D, F42A, and N88R; R38D, F42A, and N88T; R38D, F42A, and N88D; R38D, F42A, and V91E; R38D, F42A, and D84H; R38D, F42A, and D84K; R38D, F42A,and D84R;H16D, R38D and F42A;H16E, R38D and F42A;R38D, F42A and Q126S;R38A, F42K, and N88S;R38A, F42K, and N88A;R38A, F42K, and N88G;R38A, F42K, and N88R;R38A, F42K, and N88T;R38A, F42K, and N88D;R38A, F42K, and V91E;R38A, F42K, and D84H;R38A, F42K, and D84K;R38A, F42K, and D84R;H16D, R38A, and F42K;H16E, R3 8A, and F42K; R38A, F42K, and Q126S; F42A, E62Q, and N88S; F42A, E62Q, and N88A; F42A, E62Q, and N88G; F42A, E62Q, and N88R; F42A, E62Q, and N88T; F42A, E62Q, and N88D; F42A, E62Q, and V91E; F42A, E62Q, and D84H; F42A, E62Q, and D84K; F42A, E62Q, and D84R; H16D, F42A, and E62Q; H16E, F42A, and E62Q; F42A, E62Q, and Q126S; R38E, F42A, and C125A; R38D, F42A, and C125A; F42A, E62Q, and C125A; R38A, F42K, and C125A; R38E, F42A, N88S, and C125A; R38E, F42A, N88A, and C125A; R38E, F42A, N88G, and C125A; R38E, F42A, N88R, and C125A; R38E, F42A, N88T, and C125A; R38E, F42A, N88D, and C125A; R38E, F42A, V91E, and C125A; R38E, F42A, D84H, and C125A;R38E, F42A, D84K, and C125A;R38E, F42A, D84R, and C125A;H16D, R38E, F42A, and C125A;H16E, R38E, F42A, and C125A;R38E, F42A, C125A and Q126S;R38D, F42A, N88S, and C125A;R38D, F42A, N88A, and C125A;R38D, F42A, N88G, and C125A;R38D, F42A, N88R, and C125A;R38D, F42A, N88T, and C125A;R38D, F42A, N88D,and C125A;R38D, F42A, V91E, and C125A;R38D, F42A, D84H, and C125A;R38D, F42A, D84K, and C125A;R38D, F42A, D84R, and C125A;H16D, R38D, F42A, and C125A;H16E, R38D, F42A, and C125A;R38D, F42A, C125A, and Q126S;R38A, F42K, N88S, and C125A;R38A, F42K, N88A, and C125A;R38A, F42K, N88G, and C125A;R38A, F42K, N 88R, and C125A; R38A, F42K, N88T, and C125A; R38A, F42K, N88D, and C125A; R38A, F42K, V91E, and C125A; R38A, F42K, D84H, and C125A; R38A, F42K, D84K, and C125A; R38A, F42K, D84R, and C125A; H16D, R38A, F42K, and C125A; H16E, R38A, F42K, and C125A; R38A, F42K, C125A and Q126S; F42A, E62Q, N88S, and C125A; F42A, E62 Q, N88A, and C125A; F42A, E62Q, N88G, and C125A; F42A, E62Q, N88R, and C125A; F42A, E62Q, N88T, and C125A; F42A, E62Q, N88D, and C125A; F42A, E62Q, V91E, and C125A; F42A, E62Q, and D84H, and C125A; F42A, E62Q, and D84K, and C125A; F42A, E62Q, and D84R, and C125A; H16D, F42A, and E62Q, and C125A; H16E, F42A, E62Q, and C12 5A;F42A, E62Q, C125A and Q126S;F42A, N88S, and C125A;F42A, N88A, and C125A;F42A, N88G, and C125A;F42A, N88R, and C125A;F42A, N88T, and C125A;F42A, N88D, and C125A;F42A, V91E, and C125A;F42A, D84H, and C125A;F42A, D84K, and C125A;F42A, D84R, and C125A;H16D, F42A, and C125A;H16E, F42A, and C125A;and F42A,C125A and Q126S. In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 having 1, 2, 3, 4, or 5 amino acid substitutions relative to SEQ ID NO: 81, where the 1, 2, 3, 4, or 5 substitutions (multiple) are: Q11, H16, L18, L19, D20, Q22, R38, This includes substitutions (multiple substitutions possible) at the position of sequence number 81, selected from the group consisting of F42, K43, Y45, E62, P65, E68, V69, L72, D84, S87, N88, V91, I92, T123, Q126, S127, I129, and S130.In some embodiments, the IL-2 polypeptide is (relative to the sequence of SEQ ID NO: 81) the following: R38E and F42A; R38D and F42A; F42A and E62Q; R38A and F42K; R38E, F42A, and N88S; R38E, F42A, and N88A; R38E, F42A, and N88G; R38E, F42A, and N88R; R38E, F42A, and N88T; R38E, F42A, and N88D; R38E, F42A, and V91E; R38E, F42A, and D84H; R38E, F42A, and D84K;R38E, F42A, and D84R;H16D, R38E and F42A;H16E, R38E and F42A;R38E, F42A and Q126S;R38D, F42A and N88S;R38D, F42A and N88A;R38D, F42A and N88G;R38D, F42A and N88R;R38D, F42A and N88T;R38D, F42A and N88D;R38D, F42A and V91E;R38D, F42A, and D84H;R38D, F42A, and D84K;R38D, F42A, and D84R;H16 D, R38D and F42A; H16E, R38D and F42A; R38D, F42A and Q126S; R38A, F42K, and N88S; R38A, F42K, and N88A; R38A, F42K, and N88G; R38A, F42K, and N88R; R38A, F42K, and N88T; R38A, F42K, and N88D; R38A, F42K, and V91E; R38A, F42K, and D84H; R38A, F42K, and D84K; R38A, F42K, and D84R; H16D, R38A, and F42K; H16E The sequence of Sequence ID No. 81 includes one of the amino acid substitution sets of R38A, and F42K; R38A, F42K, and Q126S; F42A, E62Q, and N88S; F42A, E62Q, and N88A; F42A, E62Q, and N88G; F42A, E62Q, and N88R; F42A, E62Q, and N88T; F42A, E62Q, and N88D; F42A, E62Q, and V91E; F42A, E62Q, and D84H; F42A, E62Q, and D84K; and F42A, E62Q, and D84R.

[0110] In some embodiments, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 having a further amino acid substitution at position C125 relative to SEQ ID NO: 81. In some embodiments, the IL-2 polypeptide comprises the following (relative to the sequence of SEQ ID NO: 81): R38E, F42A, and C125A; R38D, F42A, and C125A; F42A, E62Q, and C125A; R38A, F42K, and C125A; R38E, F42A, N88S, and C125A; R38E, F42A, N88A, and C125A; R38E, F42A, N88G, and C125A; R38E, F42A, N88R, and C125A; R38E, F42A, N88D, and C125A ;R38E, F42A, N88T, and C125A;R38E, F42A, V91E, and C125A;R38E, F42A, D84H, and C125A;R38E, F42A, D84K, and C125A;R38E, F42A, D84R, and C125A;H16D, R38E, F42A, and C125A;H16E, R38E, F42A, and C125A;R38E, F42A, C125A and Q126S;R38D, F42A, N88S, and C125A;R38D, F42A, N88A, and C125A; R38D, F42A, N88G, and C125A; R38D, F42A, N88R, and C125A; R38D, F42A, N88T, and C125A; R38D, F42A, N88D, and C125A; R38D, F42A, V91E, and C125A; R38D, F42A, D84H, and C125A; R38D, F42A, D84K, and C125A; R38D, F42A, D84R, and C125A; H16D, R38D, F42A, and C125A; H16E, R38D, F42A, and C125A; R38D, F42A, C125A, and Q126S; R38A, F42K, N88S, and C125A; R38A, F42K, N88G, and C125A; R38A, F42K, N88R, and C125A; R38A, F42K, N88T, and C125A; R38A, F42K, N88D, and C125A; R38A, F42K, N88A, and C125A; R38A, F42K, V91E, and C125A; R38A, F42K, D84H, and C125A; R38A, F42K, D84K, and C125A;R38A, F42K, D84R, and C125A; H16D, R38A, F42K, and C125A; H16E, R38A, F42K, and C125A; R38A, F42K, C125A and Q126S; F42A, E62Q, N88S, and C125A; F42A, E62Q, N88A, and C125A; F42A, E62Q, N88G, and C125A; F42A, E 62Q, N88R, and C125A; F42A, E62Q, N88T, and C125A; F42A, E62Q, N88D, and C125A; F42A, E62Q, V91E, and C125A; F42A, E62Q, and D84H, and C125A; F42A, E62Q, and D84K, and C125A; F42A, E62Q, and D84R, and C125A; H16D, F 42A, and E62Q, and C125A; H16E, F42A, E62Q, and C125A; F42A, E62Q, C125A and Q126S; F42A, N88S, and C125A; F42A, N88A, and C125A; F42A, N88G, and C125A; F42A, N88R, and C125A; F42A, N88T, and C125A; F42A, N88D, and The sequence of Sequence ID No. 81 includes C125A;F42A, V91E, and C125A;F42A, D84H, and C125A;F42A, D84K, and C125A;F42A, D84R, and C125A;H16D, F42A, and C125A;H16E, F42A, and C125A; and one of the amino acid substitution sets of F42A, C125A, and Q126S.

[0111] In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISAINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 80). In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 85). In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 86). In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 87). In some embodiments, the IL-2 polypeptide variant contains the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 88).In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISSINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 89). In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISAINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 90). In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINEIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 91). In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRHLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 92). In some embodiments, the IL-2 polypeptide variant contains the amino acid sequence APTSSSTKKTQLQLEDLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 93).In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEELLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFFCQSIISTLT (SEQ ID NO: 94). In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCSSIISTLT (SEQ ID NO: 95). In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISSINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 96). In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISAINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 97). In some embodiments, the IL-2 polypeptide variant contains the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINEIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 98).In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRHLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 99). In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEDLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 100). In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEELLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFFCQSIISTLT (SEQ ID NO: 101). In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCSSIISTLT (SEQ ID NO: 102). In some embodiments, the IL-2 polypeptide variant contains the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISSINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 103).In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISAINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 104). In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINEIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 105). In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRHLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 106). In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEDLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 107). In some embodiments, the IL-2 polypeptide variant contains the amino acid sequence APTSSSTKKTQLQLEELLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 108).In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCSSIISTLT (SEQ ID NO: 109). In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISSINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 110). In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISAINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 111). In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKN. The amino acid sequence PKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISNINEIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 112) is included. In some embodiments, the IL-2 polypeptide variant includes the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRHLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 113). In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEDLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 114). In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEELLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 115). In some embodiments, the IL-2 polypeptide variant contains the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCSSIISTLT (SEQ ID NO: 116)In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 117). In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 118). In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 119). In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 120). In some embodiments, the IL-2 polypeptide variant contains the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISSINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 121)In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISAINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 122). In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINEIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 123). In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRHLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 124). In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEDLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 125). In some embodiments, the IL-2 polypeptide variant contains the amino acid sequence APTSSSTKKTQLQLEELLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 126)In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFASSIISTLT (SEQ ID NO: 127). In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISSINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 128). In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISAINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 129). In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINEIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 130). In some embodiments, the IL-2 polypeptide variant contains the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRHLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 131).In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEDLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 132). In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEELLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 133). In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFASSIISTLT (SEQ ID NO: 134). In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISSINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 135). In some embodiments, the IL-2 polypeptide variant contains the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISAINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 136)In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINEIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 137). In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRHLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 138). In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEDLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 139). In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEELLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLI. The IL-2 polypeptide variant contains the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFASSIISTLT (SEQ ID NO: 141). In some embodiments, the IL-2 polypeptide variant contains the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISSINVIVLELKGSETTFMCEYADETATIVEFLNRWITFASSIISTLT (SEQ ID NO: 142). In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISAINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 143). In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISNINEIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 144). In some embodiments, the IL-2 polypeptide variant contains the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRHLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 145)In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEDLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 146). In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEELLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 147). In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFASSIISTLT (SEQ ID NO: 148). In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISSINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 149). In some embodiments, the IL-2 polypeptide variant contains the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISAINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 150)In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINEIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 151). In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRHLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 152). In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEDLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 153). In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEELLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 154). In some embodiments, the IL-2 polypeptide variant contains the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFASSIISTLT (SEQ ID NO: 155)In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISGINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 190). In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRKLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 191). In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRRLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 192). In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISGINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 193). In some embodiments, the IL-2 polypeptide variant contains the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRKLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 194).In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRRLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 195). In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISGINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 196). In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRKLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 197). In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRRLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 198). In some embodiments, the IL-2 polypeptide variant contains the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISGINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 199).In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRKLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 200). In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRRLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 201). In some embodiments, the IL-2 polypeptide variant has the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISGINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 2). 02) is included. In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRKLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 203). In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRRLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 204). In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISGINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 205). In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRKLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 206). In some embodiments, the IL-2 polypeptide variant contains the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRRLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 207).In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISGINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 208). In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRKLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 209). In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRRLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 210). In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISGINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 211). In some embodiments, the IL-2 polypeptide variant contains the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRKLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 212).In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRRLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 213). In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISGINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 214). In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRKLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 215). In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRRLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 216). In some embodiments, the IL-2 polypeptide variant contains the amino acid sequence APTSSSTKKTQLQLEELLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 297)In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 354). In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISTINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 355). In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 356). In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 357). In some embodiments, the IL-2 polypeptide variant contains the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISTINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 358).In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 359). In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 360). In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISTINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 361). In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 362). In some embodiments, the IL-2 polypeptide variant contains the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 363).In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISTINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 364). In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 365). In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 366). In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQ. The amino acid sequence LQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISTINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 367) is included. In some embodiments, the IL-2 polypeptide variant includes the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 368). In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 369). In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISTINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 370). In some embodiments, the IL-2 polypeptide variant contains the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 371).In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 372). In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISTINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 373). In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 374). In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 375). In some embodiments, the IL-2 polypeptide variant contains the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISTINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 376)In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 377). In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 378). In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISTINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 379). In some embodiments, the IL-2 polypeptide variant comprises the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 380). In some embodiments, the IL-2 polypeptide variant contains the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 381).In some embodiments, the IL-2 polypeptide variant contains the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISTINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 382). In some embodiments, the IL-2 polypeptide variant contains the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 383). In some embodiments, the IL-2 polypeptide variant contains the amino acid sequences of the IL-2 polypeptide listed in Table 7. [Table 5-1] [Table 5-2]

[0112] In some embodiments, the IL-2 polypeptide variants of this disclosure also include other modifications, including but not limited to mutations and deletions, that provide additional advantages, such as improved biophysical properties. Improved biophysical properties include, but are not limited to, improved thermal stability, cohesiveness, acid reversibility, viscosity, and production in mammalian, bacterial, or yeast cells. For example, residue C125 may be replaced with a neutral amino acid such as serine, alanine, threonine, or valine, and the N-terminal A1 residue may be deleted (both of which are described in U.S. Patent No. 4,518,584). IL-2 polypeptide variants may also include mutations in residue M104, such as M104A, as described in U.S. Patent No. 5,206,344. Therefore, in certain embodiments, the IL-2 polypeptide variants of this disclosure include the amino acid substitution C125A. In other embodiments, one, two, or three N-terminal residues are deleted.

[0113] Fusion protein linker In some embodiments, the fusion proteins of this disclosure include a linker. In some embodiments, the linker is a chemical linker (see, for example, those disclosed in Protein Engineering, 9(3), 299-305, 1996). The synthetic chemical linkers include crosslinking agents routinely used to crosslink peptides, such as N-hydroxysuccinimide (NHS), disuccinimidyl suberate (DSS), bis(succinimidyl) suberate (BS3), dithiobis(succinimidyl propionate) (DSP), dithiobis(succinimidyl propionate) (DTSSP), ethylene glycol bis(succinimidyl succinate) (EGS), ethylene glycol bis(sulfosuccinimidyl succinate) (sulfo-EGS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST), bis[2-(succinimideoxycarbonyloxy)ethyl]sulfone (BSOCOES), and bis[2-(succinimideoxycarbonyloxy)ethyl]sulfone (sulfo-BSOCOES).

[0114] In some embodiments, the linker is an amino acid-based or peptide-based linker. In some embodiments, the polypeptide linker is a peptide having a length of at least 5 amino acids, preferably 5 to 100, more preferably 10 to 50 amino acids. In one embodiment, the peptide linker is G, S, GS, SG, SGG, GGS, and GSG (G = glycine and S = serine). In some embodiments, the linker includes the sequence (GGGS)xGn (SEQ ID NO: 74), (GGGGS)xGn (SEQ ID NO: 75), (GGGGGS)xGn (SEQ ID NO: 76), S(GGGS)xGn (SEQ ID NO: 386), S(GGGGS)xGn (SEQ ID NO: 387), or S(GGGGGS)xGn (SEQ ID NO: 388), where x = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, and n = 0, 1, 2, or 3. In some embodiments, the linker includes the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 79) or SGGGGSGGGGSGGGGS (SEQ ID NO: 389).

[0115] Fusion protein properties In some embodiments, the fusion protein induces activation of cells expressing human CD8ab heterodimers with at least 2-fold, at least 5-fold, or at least 10-fold higher potency than activation of cells expressing human CD8aa homodimers. In some embodiments, the fusion protein induces activation of CD8+ T cells with at least 2-fold, at least 5-fold, or at least 10-fold higher potency than activation of NK cells. In some embodiments, the potency of activation is measured by EC50, which is assessed by cell proliferation. Exemplary assays are described further below.

[0116] The preferential activity of targeted IL-2 fusion proteins containing IL-2 polypeptide variants against antigen-expressing cells is demonstrated in assays involving antigen-expressing and non-antigen-expressing cells that also express IL-2Rβγ or IL-2Rαβγ. One such assay is an in vitro assay that measures the phosphorylation of STAT5 (pSTAT5) and / or the expression of the proliferation marker Ki-67 in human immune cells, such as human peripheral blood cells and / or tumor-infiltrating immune cells, upon exposure to IL-2 polypeptides. In one form of the assay, the activity of the targeted IL-2 fusion protein is measured in antigen-expressing and non-expressing cells to demonstrate selectivity in antigen-expressing cells. In another form of the assay, the activity of targeted IL-2 fusion proteins containing IL-2 polypeptide variants against antigen-expressing cells is compared to the activity of non-targeted IL-2 fusion proteins containing the same IL-2 polypeptide variant and a control antibody that does not recognize any antigen, to demonstrate the magnitude of the rescue in signaling provided by the IL-2 polypeptide variants when fused to antigen-binding molecules.

[0117] In some embodiments, the fusion protein of this disclosure containing a CD8b antigen-binding molecule activates CD8b+IL-2Rβ+ cells at least 10-fold, at least 50-fold, or at least 100-fold more than CD8b-IL-2Rβ+ cells. In some embodiments, the fusion protein activates CD8b+IL-2Rβ+ cells 50-fold, 100-fold, or more than 200-fold more than a fusion molecule containing the IL-2 polypeptide variant and a control antibody that does not bind to any antigen expressed on the cells. The activation of the cells by the IL-2 fusion protein is determined by measuring the expression of pSTAT5 or the cell proliferation marker Ki67 in the cells after treatment with the IL-2 fusion protein.

[0118] In some embodiments, the fusion proteins of the Disclosure exhibit one or more of the following: they bind to human CD8 and do not block the interaction between CD8 and MHC class I; and they activate CD8+ T cells with at least 10-fold, 25-fold, 50-fold, 100-fold, 250-fold, 500-fold, or 1000-fold greater potency compared to the activation of NK cells. In some embodiments, whether the anti-CD8 antibody or fusion protein of the Disclosure blocks the interaction between CD8 and MHC class I can be analyzed, for example, by quantifying the activation state of CD8+ T cells in or out of the presence of the anti-CD8 antibody or fusion protein (e.g., upon antigen stimulation). In some embodiments, the activation of CD8+ T cells and / or NK cells can be measured by quantifying one or more markers (e.g., the percentage of treated cells representing one or more markers) among, for example, proliferation (e.g., Ki67), IL-2Rβ / γ downstream signaling, and / or STAT5 downstream signaling.

[0119] Exemplary fusion protein In some embodiments, the fusion proteins of this disclosure comprise one, two, or all three polypeptides shown for a single fusion protein in Table 5. In some embodiments, the fusion proteins of this disclosure comprise two light chains, a heavy chain containing an IL-2 fusion, and a heavy chain not containing an IL-2 fusion, for a single fusion protein in Table 5. As is known in the art, the C-terminal lysine of some antibody heavy chain species can be cleaved in some fractions of the molecule. Accordingly, in some embodiments, the fusion proteins of the present disclosure, for a single fusion protein in Table 5, comprise two light chains, a heavy chain containing an IL-2 fusion, and a heavy chain without an IL-2 fusion, the heavy chain without an IL-2 fusion comprising, for each fusion protein, the sequence of SEQ ID NOs: 158, 161, 164, 167, 170, 173, 176, 189, 300, 304, 308, 312, 326, 320, 324, 328, 332, 336, 340, 344, 348, or 352. In some em...

Claims

1. A fusion protein, the following: (a) A first portion comprising a human antibody or antigen-binding fragment specifically bound to CD8b and / or CD8ab, wherein the antibody or fragment comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain: (i) The VH domain comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 229, CDR-H2 containing the amino acid sequence of SEQ ID NO: 230, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 231, and the VL domain comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 40, CDR-L2 containing the amino acid sequence of SEQ ID NO: 41, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 42; or (ii) The VH domain includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 240, CDR-H2 containing the amino acid sequence of SEQ ID NO: 241, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 242, and the VL domain includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 40, CDR-L2 containing the amino acid sequence of SEQ ID NO: 41, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 42, the first portion, and (b) A second portion comprising the IL-2 polypeptide, The fusion protein wherein the first portion fuses with the second portion directly or via a linker.

2. The second portion induces the activation of CD8+ T cells, (a) The fusion protein induces the activation of cells expressing human CD8ab heterodimer with at least 10 times greater potency than the activation of cells expressing human CD8aa homodimer, or (b) The fusion protein induces the activation of CD8+ T cells with at least 10 times greater potency than the activation of NK cells. The fusion protein according to claim 1.

3. The fusion protein according to claim 2, wherein the activating efficacy is measured by EC50, which is evaluated by cell proliferation.

4. The first portion comprises two antibody heavy chain polypeptides having a structure according to formula [I] from the N-terminus to the C-terminus: VH-CH1-Hinge-CH2-CH3 [I] and two antibody light chain polypeptides containing the structure of formula [II] from the N-terminus to the C-terminus: VL-CL [II] The formula includes (wherein VH is the VH domain, CH1 is the antibody CH1 domain, Hinge is the antibody hinge domain, CH2-CH3 is the antibody Fc domain, VL is the VL domain, and CL is the antibody constant light chain domain), The fusion protein according to any one of claims 1 to 3, wherein the N-terminus of the second portion is fused to the C-terminus of one of the two CH3 domains.

5. (a) The first portion comprises a first antibody heavy chain polypeptide having a structure according to formula [I] from the N-terminus to the C-terminus: VH-CH1-Hinge-CH2-CH3 [I], Antibody light chain polypeptides containing the structure of formula [II] from the N-terminus to the C-terminus: VL-CL [II], and a second antibody heavy chain polypeptide comprising the structure of formula [III] from the N-terminus to the C-terminus: Hinge-CH2-CH3 [III] The formula includes (wherein VH is the VH domain, CH1 is the antibody CH1 domain, Hinge is the antibody hinge domain, CH2-CH3 is the antibody Fc domain, VL is the VL domain, and CL is the antibody constant light chain domain), The N-terminus of the second portion is fused to the C-terminus of the CH3 domain of the second antibody heavy chain polypeptide. (b) The first portion is a first antibody heavy chain polypeptide comprising the structure of formula [I] from the N-terminus to the C-terminus: VH-CH1-Hinge-CH2-CH3 [I], Antibody light chain polypeptides containing the structure of formula [II] from the N-terminus to the C-terminus: VL-CL [II], and a second antibody heavy chain polypeptide comprising the structure of formula [III] from the N-terminus to the C-terminus: Hinge-CH2-CH3 [III] The formula includes (wherein VH is the VH domain, CH1 is the antibody CH1 domain, Hinge is the antibody hinge domain, CH2-CH3 is the antibody Fc domain, VL is the VL domain, and CL is the antibody constant light chain domain), The C-terminus of the second portion is fused to the N-terminus of the hinge domain of the second antibody heavy chain polypeptide, or (c) The first portion is a first antibody heavy chain polypeptide comprising the structure of formula [I] from the N-terminus to the C-terminus: VH-CH1-Hinge-CH2-CH3 [I], Antibody light chain polypeptides containing the structure of formula [II] from the N-terminus to the C-terminus: VL-CL [II], and a second antibody heavy chain polypeptide comprising the structure of formula [III] from the N-terminus to the C-terminus: Hinge-CH2-CH3 [III] The formula includes (wherein VH is the VH domain, CH1 is the antibody CH1 domain, Hinge is the antibody hinge domain, CH2-CH3 is the antibody Fc domain, VL is the VL domain, and CL is the antibody constant light chain domain), The fusion protein according to any one of claims 1 to 3, wherein the N-terminus of the second portion is fused to the C-terminus of the CH3 domain of the first antibody heavy chain polypeptide.

6. The VH domain of both antibody heavy chain polypeptides comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 229, CDR-H2 containing the amino acid sequence of SEQ ID NO: 230, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 231, and the VL domain of both antibody light chain polypeptides comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 40, CDR-L2 containing the amino acid sequence of SEQ ID NO: 41, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 42; or The fusion protein according to claim 4, wherein the VH domain of both antibody heavy chain polypeptides comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 240, CDR-H2 containing the amino acid sequence of SEQ ID NO: 241, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 242, and the VL domain of both antibody light chain polypeptides comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 40, CDR-L2 containing the amino acid sequence of SEQ ID NO: 41, and CDR-L3 containing the amino acid sequence of SEQ ID NO:

42.

7. The fusion protein according to claim 4 or claim 6, wherein the VH domain of both antibody heavy chain polypeptides comprises an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 249, and the VL domain of both antibody light chain polypeptides comprises an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO:

250.

8. The fusion protein according to claim 7, wherein the VH domain of both antibody heavy chain polypeptides comprises the amino acid sequence of SEQ ID NO: 251, and the VL domain of both antibody light chain polypeptides comprises the amino acid sequence of SEQ ID NO:

252.

9. The fusion protein according to claim 5, wherein the VH domain comprises an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 249, and the VL domain comprises an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO:

250.

10. The fusion protein according to claim 9, wherein the VH domain comprises the amino acid sequence of SEQ ID NO: 249, and the VL domain comprises the amino acid sequence of SEQ ID NO:

250.

11. One or both of the antibody heavy chain polypeptides comprise the following amino acid substitutions: L234A, L235A, and G237A, numbered according to the EU index, and / or The first antibody heavy chain polypeptide contains amino acid substitutions Y349C and T366W, and the second antibody heavy chain polypeptide contains amino acid substitutions S354C, T366S, L368A and Y407V, with numbering according to the EU index. A fusion protein according to any one of claims 4 to 10.

12. (a) The first portion comprises one or two antibody heavy chain polypeptides and one or two antibody light chain polypeptides, or (b) The first portion includes a single-chain variable fragment (scFv), A fusion protein according to any one of claims 1 to 3.

13. The IL-2 polypeptide is APTSSSSTKKTQLQLEHLLLLDLQMILNGINNYKNPKLTRMMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRRPRDLISNINVIVLEELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (Sequence ID 81) The fusion protein according to any one of claims 1 to 12, which is an IL-2 polypeptide variant comprising one or more mutations in a human IL-2 polypeptide containing the sequence.

14. (a) The IL-2 polypeptide variant has a binding affinity to IL-2Rα that is 50% or less lower than the binding affinity to IL-2Rα of the wild-type IL-2 polypeptide containing the sequence of SEQ ID NO:

81. (b) The IL-2 polypeptide variant has a binding affinity to IL-2Rβ that is 50% or less lower than that of the wild-type IL-2 polypeptide containing the sequence of SEQ ID NO: 81; and / or (c) The fusion protein according to claim 13, wherein the IL-2 polypeptide variant has a binding affinity to IL-2Rγ that is 50% or less lower than that of the wild-type IL-2 polypeptide containing the sequence of SEQ ID NO:

81.

15. The fusion protein according to any one of claims 1 to 14, wherein the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 having one, two, three, four, or five amino acid substitutions relative to SEQ ID NO: 81, and the one, two, three, four, or five substitutions (or more) comprises substitutions (or more) at the position of SEQ ID NO: 81 selected from the group consisting of: Q11, H16, L18, L19, D20, Q22, R38, F42, K43, Y45, E62, P65, E68, V69, L72, D84, S87, N88, V91, I92, T123, Q126, S127, I129, and S130.

16. The IL-2 polypeptide is (relative to the sequence of Sequence ID No. 81) the following: R38E and F42A; R38D and F42A; F42A and E62Q; R38A and F42K; R38E, F42A, and N88S; R38E, F42A, and N88A; R38E, F42A, and N88G; R38E, F42A, and N88R; R38E, F42A, and N88T; R38E, F42A, and N88D; R38E, F42A, and V91E; R38E, F42A, and D84H; R38E, F42A, and D84K; R38E, F42A , and D84R; H16D, R38E and F42A; H16E, R38E and F42A; R38E, F42A and Q126S; R38D, F42A and N88S; R38D, F42A and N88A; R38D, F42A and N88G; R38D, F42A and N88R; R38D, F42A and N88T; R38D, F42A and N88D; R38D, F42A and V91E; R38D, F42A and D84H; R38D, F42A and D84K; R38D, F42A and D84R; H16D, R38D and F42A; H1 6E, R38D and F42A; R38D, F42A and Q126S; R38A, F42K, and N88S; R38A, F42K, and N88A; R38A, F42K, and N88G; R38A, F42K, and N88R; R38A, F42K, and N88T; R38A, F42K, and N88D; R38A, F42K, and V91E; R38A, F42K, and D84H; R38A, F42K, and D84K; R38A, F42K, and D84R; H16D, R38A, and F42K; H16E, R38A, and F42K; R38 The fusion protein according to claim 15, comprising the sequence of Sequence ID No. 81 having one of the amino acid substitution sets of A, F42K, and Q126S; F42A, E62Q, and N88S; F42A, E62Q, and N88A; F42A, E62Q, and N88G; F42A, E62Q, and N88R; F42A, E62Q, and N88T; F42A, E62Q, and N88D; F42A, E62Q, and V91E; F42A, E62Q, and D84H; F42A, E62Q, and D84K; and F42A, E62Q, and D84R.

17. The fusion protein according to claim 15 or claim 16, wherein the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81 having a further amino acid substitution at position C125 relative to SEQ ID NO:

81.

18. The IL-2 polypeptide is (relative to the sequence of SEQ ID NO: 81) the following: R38E, F42A, and C125A; R38D, F42A, and C125A; F42A, E62Q, and C125A; R38A, F42K, and C125A; R38E, F42A, N88S, and C125A; R38E, F42A, N88A, and C125A; R38E, F42A, N88G, and C125A; R38E, F42A, N88R, and C125A; R38E, F42A, N88D, and C125A; R38E, F42A, N88T, and C125A; R3 8E, F42A, V91E, and C125A; R38E, F42A, D84H, and C125A; R38E, F42A, D84K, and C125A; R38E, F42A, D84R, and C125A; H16D, R38E, F42A, and C125A; H16E, R38E, F42A, and C125A; R38E, F42A, C125A and Q126S; R38D, F42A, N88S, and C125A; R38D, F42A, N88A, and C125A; R38D, F42A, N88G, and C125A; R38D, F42A, N88R, and C125A; R38D, F42A, N88T, and C125A; R38D, F42A, N88D, and C125A; R38D, F42A, V91E, and C125A; R38D, F42A, D84H, and C125A; R38D, F42A, D84K, and C125A; R38D, F42A, D84R, and C125A; H16D, R38D, F42A, and C125A; H16E, R38D, F42A, and C125A; R38D, F42A, C125A, and Q126S; R38A, F42K, N88S, and C125A; R38A, F42 K, N88G, and C125A; R38A, F42K, N88R, and C125A; R38A, F42K, N88T, and C125A; R38A, F42K, N88D, and C125A; R38A, F42K, N88A, and C125A; R38A, F42K, V91E, and C125A; R38A, F42K, D84H, and C125A; R38A, F42K, D84K, and C125A; R38A, F42K, D84R, and C125A; H16D, R38A, F42K, and C125A; H16E, R38A, F42K, and C125A;R38A, F42K, C125A and Q126S; F42A, E62Q, N88S and C125A; F42A, E62Q, N88A and C125A; F42A, E62Q, N88G and C125A; F42A, E62Q, N88R and C125A; F42A, E62Q, N88T and C125A; F42A, E62Q, N88D, and and C125A; F42A, E62Q, V91E, and C125A; F42A, E62Q, and D84H, and C125A; F42A, E62Q, and D84K, and C125A; F42A, E62Q, and D84R, and C125A; H16D, F42A, and E62Q, and C125A; H16E, F42A, E62Q, and C125A; F 42A, E62Q, C125A and Q126S; F42A, N88S and C125A; F42A, N88A and C125A; F42A, N88G and C125A; F42A, N88R and C125A; F42A, N88T and C125A; F42A, N88D and C125A; F42A, V91E and C125A; F42A, D The fusion protein according to claim 17, comprising the sequence of Sequence ID No. 81 having one of the amino acid substitution sets of 84H and C125A; F42A, D84K and C125A; F42A, D84R and C125A; H16D, F42A and C125A; H16E, F42A and C125A; and F42A, C125A and Q126S.

19. The IL-2 polypeptide is sequence APTSSSSTKKTQLQLEHLLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLIISAINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (Sequence ID 80), or array APTSSSSTKKTQLQLEELLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRRPRDLISNINVIVLEELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (Sequence ID 297) A fusion protein according to any one of claims 1 to 14, comprising:

20. The fusion protein according to claim 13 or claim 14, wherein the IL-2 polypeptide comprises a sequence selected from the group consisting of SEQ ID NOs: 80, 85-155, 190-216, 297, and 354-383.

21. The fusion protein according to any one of claims 4 to 20, wherein one or both of the antibody Fc domains comprises a human IgG1 Fc domain having the amino acid substitutions L234A, L235A, and G237A, numbered according to the EU index.

22. The one or both of the antibody Fc domains lack a C-terminal lysine, and / or The first of the two Fc domains comprises a human IgG1 Fc domain having amino acid substitutions Y349C and T366W, and the second of the two Fc domains comprises a human IgG1 Fc domain having amino acid substitutions S354C, T366S, L368A and Y407V. It includes FC domains, and the numbering is based on the EU Index. The fusion protein according to claim 21.

23. (a) The linker includes the sequence (GGGS)xGn (SEQ ID NO: 74), (GGGGGS)xGn (SEQ ID NO: 75), or (GGGGGGGS)xGn (SEQ ID NO: 76), S(GGGS)xGn (SEQ ID NO: 386), S(GGGGGS)xGn (SEQ ID NO: 387), or S(GGGGGGGS)xGn (SEQ ID NO: 388), where x = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 and n = 0, 1, 2, or 3, or (b) The linker includes sequence GGGGGSGGGGGSGGGGGS (SEQ ID NO: 79) or SGGGGGGSGGGGGSGGGGGS (SEQ ID NO: 389), A fusion protein according to any one of claims 1 to 22.

24. The fusion protein is as follows: One or two light chains containing the amino acid sequence of SEQ ID NO: 306, a heavy chain containing the amino acid sequence of SEQ ID NO: 307, and a heavy chain containing the amino acid sequence of SEQ ID NO: 308; One or two light chains containing the amino acid sequence of SEQ ID NO: 306, a heavy chain containing the amino acid sequence of SEQ ID NO: 307, and a heavy chain containing the amino acid sequence of SEQ ID NO: 309; One or two light chains containing the amino acid sequence of SEQ ID NO: 334, a heavy chain containing the amino acid sequence of SEQ ID NO: 335, and a heavy chain containing the amino acid sequence of SEQ ID NO: 336; or A fusion protein according to any one of claims 1 to 22, comprising one or two light chains containing the amino acid sequence of SEQ ID NO: 334, a heavy chain containing the amino acid sequence of SEQ ID NO: 335, and a heavy chain containing the amino acid sequence of SEQ ID NO:

337.

25. A fusion protein comprising a first portion that binds to human CD8b and a second portion that comprises an IL2 polypeptide, wherein the fusion protein comprises four polypeptide chains: The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 334, the second polypeptide chain contains the amino acid sequence of SEQ ID NO: 335, the third polypeptide chain contains the amino acid sequence of SEQ ID NO: 336, and the fourth polypeptide chain contains the amino acid sequence of SEQ ID NO: 334; or The fusion protein wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 334, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 335, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 337, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:

334.

26. One or more polynucleotides encoding the fusion protein according to any one of claims 1 to 25.

27. ​​One or more vectors comprising one or more polynucleotides as described in Claim 26.

28. The vector(s)(s)(s)(s)(s)) are expression vectors(s) as described in Claim 27, one or more vectors as described in Claim 27.

29. An isolated host cell comprising one or more polynucleotides as described in claim 26 or one or more vectors as described in claim 27 or claim 28.

30. A method for producing a fusion protein, comprising culturing the host cell described in claim 29 under conditions suitable for the production of the fusion protein.

31. The method according to claim 30, further comprising recovering the fusion protein from the host cell.

32. A pharmaceutical composition comprising a fusion protein according to any one of claims 1 to 25 and a pharmaceutically acceptable carrier.

33. A composition comprising the fusion protein according to any one of claims 1 to 25 for use as a pharmaceutical.

34. A composition for the treatment of cancer, comprising a fusion protein according to any one of claims 1 to 25, characterized in that the composition is administered to an individual having cancer.

35. A composition comprising a fusion protein according to any one of claims 1 to 25 for the treatment of an infectious disease, characterized in that the composition is administered to an individual in need thereof.

36. The composition according to claim 35, wherein the infectious disease is a viral infectious disease.

37. A method for increasing T cells ex vivo, comprising contacting one or more T cells ex vivo with an effective amount of the fusion protein described in any one of claims 1 to 25 or the composition described in claim 32.

38. The method according to claim 37, wherein the one or more T cells are tumor-infiltrating lymphocytes (TILs).

Images