Fusions of mutated interleukin-10 polypeptides with antigen-binding molecules for modulating immune cell function

Abstract

Provided herein are mutant interleukin-10 polypeptides and fusion polypeptides comprising mutant interleukin-10 polypeptides and antigen-binding molecules. The present disclosure provides methods for modulating immune cell function by contacting immune cells with fusion polypeptides of the present disclosure. Furthermore, the present disclosure also provides polynucleotides encoding the fusion molecules of the present disclosure, as well as vectors and host cells comprising such polynucleotides. The present disclosure further provides methods for producing the fusion molecules, pharmaceutical compositions comprising the same, and uses thereof.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 123,387, filed December 9, 2020, and U.S. Provisional Patent Application No. 63 / 169,604, filed April 1, 2021, each of which is incorporated by reference in its entirety.

[0002] Submitting a sequence listing as an ASCII text file The contents of the following submission in an ASCII text file are incorporated herein by reference in their entirety: Sequence Listing in Computer Readable Form (CRF) (Filename: 182842000540SEQLIST.TXT, Recorded on: December 8, 2021, Size: 565,560 bytes).

[0003] Field The present disclosure provides mutant interleukin-10 polypeptides, as well as fusion polypeptides comprising the mutant interleukin-10 polypeptides and antigen-binding molecules. The present disclosure provides methods for modulating immune cell function by contacting immune cells with fusion polypeptides of the present disclosure. Furthermore, the present disclosure also provides polynucleotides encoding the fusion molecules of the present disclosure, as well as vectors and host cells comprising such polynucleotides. The present disclosure further provides methods for producing the fusion molecules, pharmaceutical compositions comprising the same, and uses thereof. [Background technology]

[0004] Interleukin-10 (IL-10) is a cytokine that regulates many immune cell subsets, including monocytes, macrophages, dendritic cells, B cells, T cells, and NK cells. IL-10 binds to a heterodimeric receptor (IL-10 receptor, IL-10R), which consists of two subunits: IL-10RA, which is specific for IL-10 and expressed primarily on immune cells, and IL-10RB, which is shared with other cytokines and more widely expressed. Binding of IL-10 to its receptor induces phosphorylation of the receptor-associated Janus kinase JAK1 and the tyrosine kinase TYK2, which promotes phosphorylation of the STAT3 transcription factor (pSTAT3), which regulates the transcription of numerous genes in lymphocytes.

[0005] IL-10 signaling induces diverse effects depending on the target cell (reviewed in Geginat et al., Cytokine Growth Factor Rev. 2016 Aug;30:87-93). IL-10 is considered an immunosuppressive cytokine because its binding to antigen-presenting cells, such as macrophages and dendritic cells, inhibits their production of proinflammatory cytokines and their ability to stimulate T cells. For example, mice and patients with genetic defects in the IL-10 / IL-10R pathway spontaneously develop colitis, suggesting that IL-10 is required to promote intestinal immune cell homeostasis and prevent autoimmunity. However, IL-10 has also been implicated in the development of autoimmune diseases such as systemic lupus erythematosus by acting as a growth and differentiation factor for B cells. Furthermore, IL-10 can promote CD8+ T cell function, and this immunostimulatory activity of IL-10 (Chan et al., J Interferon Cytokine Res. 2015 Dec;35(12):948-55; Nizzoli et al., Eur J Immunol. 2016 Jul;46(7):1622-32) may be related to its ability to induce potent antitumor immune responses in mice (Mumm et al., Cancer Cell. 2011 Dec 13;20(6):781-96; Emmerich et al., Cancer Res. 2012 Jul 15;72(14):3570-81) and to activate CD8+ T cells in cancer patients (Naing et al., Cancer Cell. 2018 Nov 12;34(5):775-791).

[0006] Due to its pleiotropic effects in regulating immune responses, the IL-10 cytokine has been used as a therapeutic agent for both autoimmunity and cancer. However, despite its potent immunosuppressive effects in preclinical models, the clinical benefit of IL-10 administration in Crohn's disease, psoriasis, and rheumatoid arthritis has been limited (O'Garra A, Immunol Rev. 2008;223:114-131). Similarly, the therapeutic effects of IL-10 have been evaluated across multiple advanced solid tumors, and although clinical activity has been demonstrated, clinical benefit has been modest, with the most promising results limited to a few indications (Autio et al., Curr Oncol Rep. 2019 Feb 21;21(2):19).

[0007] These seemingly contradictory effects of IL-10 can be explained by the presence of its receptors on immune cells, which can either suppress or activate immune responses in a given situation. For example, in the setting of cancer, stimulation of macrophages, dendritic cells, and regulatory T cells (Tregs) with IL-10 would result in immunosuppression, whereas stimulation of CD8+ T cells with IL-10 would result in immunoactivation. This suggests that restricting IL-10 activity to specific immune cell subsets may be beneficial for enhancing the therapeutic efficacy of cancer. Furthermore, IL-10 therapy has been associated with severe anemia and hyperferritinemia, which may require blood transfusions in certain patients (Tilg et al., J Immunol. 2002 Aug 15;169(4):2204-9). IL-10 has been shown to directly stimulate ferritin translation in activated mononuclear cells (Tilg et al., J Immunol. 2002 Aug 15;169(4):2204-9), which may lead to sequestration of iron required for erythropoiesis. In addition to inducing ferritin in monocytes, IL-10 may also directly suppress erythropoiesis (Oehler et al., Exp Hematol. 1999 Feb;27(2):217-23; Mullarky et al., Infect Immun. 2007 May;75(5):2630-3).

[0008] CD8+ T cells have been shown to mediate the effects of immunotherapeutic agents, including IL-10, in many preclinical cancer models (Mumm et al., Cancer Cell. 2011 Dec 13;20(6):781-96; Emmerich et al., Cancer Res. 2012 Jul 15;72(14):3570-81), and they also correlate with response to immunotherapy in patients (Sade-Feldman et al., Cell. 2018 Nov 1;175(4):998-1013). CD8+ T cells express CD8, a type I transmembrane glycoprotein found on the cell surface as CD8 alpha (CD8a) homodimers and CD8 alpha-CD8 beta (CD8b) heterodimers. Alpha-beta CD8+ T cells can express both CD8aa and CD8ab dimers, and at the same time, CD8aa homodimers can also be expressed at lower levels on some innate lymphoid cells, such as NK, NK T, and intraepithelial Tγδ cells (Baume et al, Cell Immunol. 1990 Dec;131(2):352-65; Kadivar et al, J Immunol 2016;197:4584-4592; Mayassi & Jabri, Mucosal Immunology 11,1281-1289, 2018). CD8 dimers interact with major histocompatibility (MHC) class I molecules on target cells, and this interaction results in CD8 + During T cell activation, it maintains the TCR tightly bound to MHC. The cytoplasmic tail of CD8a contains a binding site for T cell kinase (Lck), which initiates signaling downstream of the TCR during T cell activation, while CD8b enhances the avidity of CD8 for MHC class I binding and is thought to influence the specificity of the CD8 / MHC / TCR interaction (Bosselut et al., Immunity. 2000 Apr;12(4):409-18).

[0009] There is a need to reduce the toxicity and improve the efficacy of IL-10 by increasing its activity in T cells, including CD8+ T cells, which have been associated with efficacy in preclinical cancer models and cancer patients, and by reducing its activity in other cells, including monocytes, macrophages, dendritic cells, and Tregs, which have been associated with IL-10 toxicity and undesirable effects. All references cited herein, including patent applications, patent publications, and UniProtKB / Swiss-Prot accession numbers, are incorporated by reference in their entirety, as if each individual reference was specifically and individually indicated to be incorporated by reference. [Prior art documents] [Non-patent literature]

[0010] [Non-Patent Document 1] Geginat et al,cytokine Growth Factor Rev.2016 Aug;30:87-93 [Non-patent document 2] Chan et al,J Interferon Cytokine Res.2015 Dec;35(12):948-55 [Non-patent document 3] Nizzoli et al,Eur J Immunol.2016 Jul;46(7):1622-32 [Non-patent document 4] Mumm et al, Cancer Cell.2011 Dec 13;20(6):781-96 [Non-Patent Document 5] Emmerich et al, Cancer Res.2012 Jul 15;72(14):3570-81 [Non-patent document 6] Naing et al, Cancer Cell.2018 Nov 12;34(5):775-791 [Non-Patent Document 7] Autio et al, Curr Oncol Rep. 2019 Feb 21; 21(2): 19

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Summary of the Invention

[0012] In some embodiments, provided herein are mutant IL-10 polypeptides, wherein the mutant IL-10 polypeptides comprise an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence of wild-type mature IL-10 shown in Figure 1A, and the mutant IL-10 polypeptides exhibit reduced binding affinity to an IL-10RA polypeptide having the amino acid sequence shown in Figure 1B. The reduced affinity for IL-10RA is obtained by introducing amino acid substitutions into the sequence of the wild-type IL-10 polypeptide to generate the mutant IL-10 polypeptides of the present disclosure as shown in Figures 2 and 3. The mutant IL-10 polypeptides of the disclosure have one or more amino acid substitutions selected from the group consisting of P20, L23, R24, R27, D28, K34, T35, Q38, M39, D41, L43, D44, N45, L46, K49, 187, V91, L94, L98, K138, S141, E142, D144, N148, E151, and 1158 relative to the amino acid sequence of a wild-type IL-10 polypeptide shown in Figure 1A. In some embodiments, the mutant IL-10 polypeptides of the disclosure exhibit binding affinity that is reduced by 50% or more, reduced by 150% or more, reduced by 2-fold or more, or reduced by 10-fold or more relative to an IL-10RA polypeptide having the amino acid sequence shown in Figure 1B. In some embodiments, the one or more amino acid substitutions are at a position(s) selected from the group consisting of R24, R27, K34, Q38, D44, I87, K138, E142, D144, N148, and E151.In some embodiments, the one or more amino acid substitutions are R24A, R27A, K34A, K34D, K34E, K34S, K34P, K34G, K34T, K34H, K34L, K34N, K34F, K34R, K34Q, K34V, K34Y, Q38A, Q38D, Q38P, Q38G, Q38H, Q38I, Q38L, Q38R, Q38Q ... 8K, Q38N, Q38F, Q38T, Q38E, Q38S, Q38V, Q38Y, D44A, D44E, D44S, D44V, D44G, D44H, D44I, D44K , D44P, D44L, D44N, D44F, D44T, D44R, D44Q, I87A, K138A, E142A, E142G, E142N, E142L, E142F, E142I, E142V, E142K, E142R, E142P, E142Q, E142T, E142S, E142Y, D144A, D144E, D144G, D144H , D144R, D144I, D144K, D144N, D144Q, D144P, D144S, D144L, D144T, D144V, D144Y, N148G, N148 P, N148S, N148D, N148T, N148K, N148V, N148I, N148E, N148F, E151A, E151G, E151H, E151I, E151N, E151F, E151L, E151V, E151R, E151K, E151P, E151Q, E151S, E151T, and E151Y.In some embodiments, the one or more amino acid substitutions are R24A, R27A, K34A, K34D, K34E, K34S, K34P, K34G, K34T, K34H, K34L, K34N, K34F, K34V, K34Y, Q38A, Q38D, Q38P, Q38G, Q38I, Q38L, Q38R, Q38K, Q38F, Q38T, Q38E, Q38S, Q38V, Q38Y, I87A, K138A, E142A, E142G, E142N, E142L, E142F, E142I, E142V, E142K, E142R, E1 42P, E142Q, E142T, E142S, E142Y, D144A, D144E, D144G, D144H, D144R, D144I, D144K, D144N, D144Q, D144P, D144S, D144L, D144T, D144V, D144Y, N148P, N148D, N148I, E151A, E151G, E151H, E151I, E151N, E151F, E151L, E151V, E151R, E151K, E151P, E151Q, E151S, E151T, and E151Y. In some embodiments, the mutant IL-10 polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 87-89, 188-201, and 310-318. In some embodiments, the mutant IL-10 polypeptide comprises an amino acid sequence set forth in Table 4A, Table 8, Table 11, or Table 13.

[0013] In other embodiments, the mutant IL-10 polypeptides of the present disclosure also i) exhibit increased binding affinity to an IL-10RB polypeptide having the amino acid sequence depicted in Figure 1C, and ii) may have one or more amino acid substitutions selected from the group consisting of N18, N21, M22, R24, D25, D28, S31, R32, D55, M68, I69, L73, E74, M77, P78, Q79, E81, N82, K88, A89, H90, N92, S93, G95, E96, N97, K99, T100, L101, L103, R104, R107, R110, and F111 relative to the amino acid sequence of the wild-type mature IL-10 polypeptide depicted in Figure 1A. In yet another embodiment, the mutant IL-10 polypeptide exhibits increased binding affinity of 150% or more to an IL-10RB polypeptide having the amino acid sequence depicted in Figure 1C. In some embodiments, the one or more amino acid substitutions are at a position(s) selected from the group consisting of N18, D28, N92, K99, and L103, numbered according to SEQ ID NO: 1. In some embodiments, the one or more amino acid substitutions are selected from the group consisting of N18F, N18L, N18Y, D28Q, D28R, N92F, N92H, N92I, N92K, N92L, N92R, N92S, N92T, N92V, N92Y, K99N, L103N, and L103Q, numbered according to SEQ ID NO: 1.

[0014] In some aspects, provided herein are mutant IL-10 polypeptides that exhibit increased binding affinity to an IL-10RB polypeptide, e.g., comprising the amino acid sequence of SEQ ID NO: 3 or having the amino acid sequence shown in Figure 1C. In some embodiments, the mutant IL-10 polypeptide comprises one or more amino acid substitutions relative to the amino acid sequence of a wild-type mature IL-10 polypeptide according to SEQ ID NO: 1, wherein the one or more amino acid substitutions are at a position(s) selected from the group consisting of N18, N21, M22, R24, D25, D28, S31, R32, D55, M68, I69, L73, E74, M77, P78, Q79, E81, N82, K88, A89, H90, N92, S93, G95, E96, N97, K99, T100, L101, L103, R104, R107, R110, and F111. In some embodiments, the one or more amino acid substitutions are at position(s) selected from the group consisting of N18, D28, N92, K99, and L103, numbered according to SEQ ID NO: 1. In some embodiments, the one or more amino acid substitutions are selected from the group consisting of N18F, N18L, N18Y, D28Q, D28R, N92F, N92H, N92I, N92K, N92L, N92R, N92S, N92T, N92V, N92Y, K99N, L103N, and L103Q, numbered according to SEQ ID NO: 1. In some embodiments, the mutant IL-10 polypeptide exhibits a binding affinity for an IL-10RB polypeptide that is increased by 50% or more, 100% or more, or 150% or more, e.g., comprises the amino acid sequence of SEQ ID NO: 3 or has the amino acid sequence shown in Figure 1C.In some embodiments, the mutant IL-10 polypeptide comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence of wild-type mature IL-10 shown in FIG. 1A, and the mutant IL-10 polypeptide exhibits reduced binding affinity to an IL-10RA polypeptide, e.g., comprises the amino acid sequence of SEQ ID NO:2 or has the amino acid sequence shown in FIG. 1B. In some embodiments, the mutant IL-10 polypeptide further comprises one or more amino acid substitutions relative to the amino acid sequence of a wild-type IL-10 polypeptide according to SEQ ID NO: 1, wherein the one or more amino acid substitutions are at a position(s) selected from the group consisting of P20, L23, R24, R27, D28, K34, T35, Q38, M39, D41, L43, D44, N45, L46, K49, 187, V91, L94, L98, K138, S141, E142, D144, N148, E151, and 1158. In some embodiments, the one or more amino acid substitutions are at a position(s) selected from the group consisting of R24, R27, K34, Q38, D44, 187, K138, E142, D144, N148, and E151.In some embodiments, the one or more amino acid substitutions are R24A, R27A, K34A, K34D, K34E, K34S, K34P, K34G, K34T, K34H, K34L, K34N, K34F, K34R, K34Q, K34V, K34Y, Q38A, Q38D, Q38P, Q38G, Q38H, Q38I, Q38L, Q38R, Q38Q ... 8K, Q38N, Q38F, Q38T, Q38E, Q38S, Q38V, Q38Y, D44A, D44E, D44S, D44V, D44G, D44H, D44I, D44K , D44P, D44L, D44N, D44F, D44T, D44R, D44Q, I87A, K138A, E142A, E142G, E142N, E142L, E142F, E142I, E142V, E142K, E142R, E142P, E142Q, E142T, E142S, E142Y, D144A, D144E, D144G, D144H , D144R, D144I, D144K, D144N, D144Q, D144P, D144S, D144L, D144T, D144V, D144Y, N148G, N148 P, N148S, N148D, N148T, N148K, N148V, N148I, N148E, N148F, E151A, E151G, E151H, E151I, E151N, E151F, E151L, E151V, E151R, E151K, E151P, E151Q, E151S, E151T, and E151Y.In some embodiments, the one or more amino acid substitutions are R24A, R27A, K34A, K34D, K34E, K34S, K34P, K34G, K34T, K34H, K34L, K34N, K34F, K34V, K34Y, Q38A, Q38D, Q38P, Q38G, Q38I, Q38L, Q38R, Q38K, Q38F, Q38T, Q38E, Q38S, Q38V, Q38Y, I87A, K138A, E142A, E142G, E142N, E142L, E142F, E142I, E142V, E142K, E142R, E1 42P, E142Q, E142T, E142S, E142Y, D144A, D144E, D144G, D144H, D144R, D144I, D144K, D144N, D144Q, D144P, D144S, D144L, D144T, D144V, D144Y, N148P, N148D, N148I, E151A, E151G, E151H, E151I, E151N, E151F, E151L, E151V, E151R, E151K, E151P, E151Q, E151S, E151T, and E151Y. In some embodiments, the mutant IL-10 polypeptide exhibits a binding affinity that is at least 50% reduced, at least 100% reduced, at least 150% reduced, at least 2-fold reduced, or at least 10-fold reduced relative to an IL-10RA polypeptide, e.g., comprising the amino acid sequence of SEQ ID NO:2 or having the amino acid sequence shown in FIG. 1B.

[0015] In some embodiments, the amino acid sequence comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence of SEQ ID NO:1. Provided herein are mutant IL-10 polypeptides having one or more amino acid substitutions relative to the amino acid sequence of sequence number 1, wherein the one or more amino acid substitutions are at a position(s) selected from the group consisting of N18, N21, M22, R24, D25, D28, S31, R32, D55, M68, 169, L73, E74, M77, P78, Q79, E81, N82, K88, A89, H90, N92, S93, G95, E96, N97, K99, T100, L101, L103, R104, R107, R110, and F111. In some embodiments, the one or more amino acid substitutions are at a position(s) selected from the group consisting of N18, D28, N92, K99, and L103, numbered according to SEQ ID NO: 1. In some embodiments, the one or more amino acid substitutions are selected from the group consisting of N18F, N18L, N18Y, D28Q, D28R, N92F, N92H, N92I, N92K, N92L, N92R, N92S, N92T, N92V, N92Y, K99N, L103N, and L103Q, numbered according to SEQ ID NO: 1. In some embodiments, the mutant IL-10 polypeptide exhibits increased binding affinity for an IL-10RB polypeptide, e.g., comprising the amino acid sequence of SEQ ID NO: 3 (e.g., compared to the binding affinity of a wild-type mature IL-10 polypeptide comprising the amino acid sequence of SEQ ID NO: 1 for an IL-10RB polypeptide comprising the amino acid sequence of SEQ ID NO: 3).In some embodiments, the mutant IL-10 polypeptide exhibits a binding affinity that is increased by 50% or more, 100% or more, or 150% or more for an IL-10RB polypeptide, e.g., comprising the amino acid sequence of SEQ ID NO: 3 (e.g., compared to the binding affinity of a wild-type mature IL-10 polypeptide comprising the amino acid sequence of SEQ ID NO: 1 for an IL-10RB polypeptide comprising the amino acid sequence of SEQ ID NO: 3). In some embodiments, the mutant IL-10 polypeptide comprises one or more additional amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 1, wherein the one or more additional amino acid substitutions are at a position(s) selected from the group consisting of P20, L23, R24, R27, D28, K34, T35, Q38, M39, D41, L43, D44, N45, L46, K49, 187, V91, L94, L98, K138, S141, E142, D144, N148, E151, and 1158, numbered according to SEQ ID NO: 1. In some embodiments, the mutant IL-10 polypeptide comprises one or more additional amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 1, wherein the one or more additional amino acid substitutions are at a position(s) selected from the group consisting of R24, R27, K34, Q38, D44, I87, K138, E142, D144, N148, and E151.In some embodiments, the one or more additional amino acid substitutions are R24A, R27A, K34A, K34D, K34E, K34S, K34P, K34G, K34T, K34H, K34L, K34N, K34F, K34R, K34Q, K34V, K34Y, Q38A, Q38D, Q38P, Q38G, Q38H, Q38I, Q38L, Q38R , Q38K, Q38N, Q38F, Q38T, Q38E, Q38S, Q38V, Q38Y, D44A, D44E, D44S, D44V, D44G, D44H, D44I, D 44K, D44P, D44L, D44N, D44F, D44T, D44R, D44Q, I87A, K138A, E142A, E142G, E142N, E142L, E142 F, E142I, E142V, E142K, E142R, E142P, E142Q, E142T, E142S, E142Y, D144A, D144E, D144G, D14 4H, D144R, D144I, D144K, D144N, D144Q, D144P, D144S, D144L, D144T, D144V, D144Y, N148G, N14 8P, N148S, N148D, N148T, N148K, N148V, N148I, N148E, N148F, E151A, E151G, E151H, E151I, E151N, E151F, E151L, E151V, E151R, E151K, E151P, E151Q, E151S, E151T, and E151Y.In some embodiments, the one or more additional amino acid substitutions are R24A, R27A, K34A, K34D, K34E, K34S, K34P, K34G, K34T, K34H, K34L, K34N, K34F, K34V, K34Y, Q38A, Q38D, Q38P, Q38G, Q38I, Q38L, Q38R, Q38K, Q38F, Q38T, Q38E, Q38S, Q38V, Q38Y, I87A, K138A, E142A, E142G, E142N, E142L, E142F, E142I, E142V, E142K, E142R, E142P, E142Q, E142T, E142S, E142Y, D144A, D144E, D144G, D144H, D144R, D144I, D144K, D144N, D144Q, D144P, D144S, D144L, D144T, D144V, D144Y, N148P, N148D, N148I, E151A, E151G, E151H, E151I, E151N, E151F, E151L, E151V, E151R, E151K, E151P, E151Q, E151S, E151T, and E151Y. In some embodiments, the mutant IL-10 polypeptide exhibits reduced binding affinity for, e.g., an IL-10RA polypeptide comprising the amino acid sequence of SEQ ID NO: 2 (e.g., compared to the binding affinity of a wild-type mature IL-10 polypeptide comprising the amino acid sequence of SEQ ID NO: 1 to an IL-10RA polypeptide comprising the amino acid sequence of SEQ ID NO: 2). In some embodiments, the mutant IL-10 polypeptide exhibits 50% or more reduced, 100% or more reduced, 150% or more reduced, 2-fold or more reduced, or 10-fold or more reduced binding affinity for, e.g., an IL-10RA polypeptide comprising the amino acid sequence of SEQ ID NO: 2 (e.g., compared to the binding affinity of a wild-type mature IL-10 polypeptide comprising the amino acid sequence of SEQ ID NO: 1 to an IL-10RA polypeptide comprising the amino acid sequence of SEQ ID NO: 2).In some embodiments according to any of the embodiments described herein, a mutant IL-10 polypeptide comprising one or more amino acid substitutions (e.g., comprising the amino acid sequence of SEQ ID NO: 3, e.g., resulting in increased binding affinity for an IL-10RB polypeptide) comprises one, two, three, four, five, or more amino acid substitutions. In some embodiments according to any of the embodiments described herein, a mutant IL-10 polypeptide comprising one or more amino acid substitutions (e.g., comprising the amino acid sequence of SEQ ID NO: 2, e.g., resulting in decreased binding affinity for an IL-10RA polypeptide) comprises one, two, three, four, or five or more amino acid substitutions. In some embodiments according to any of the embodiments described herein, a mutant IL-10 polypeptide comprises one or more amino acid substitutions (e.g., one or two amino acid substitutions) associated with increased binding affinity for an IL-10RB polypeptide and one or more amino acid substitutions (e.g., one or two amino acid substitutions) associated with decreased binding affinity for an IL-10RA polypeptide.

[0016] In some embodiments, the mutated IL-10 polypeptide comprises an amino acid substitution at position R24, e.g., R24A, relative to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutated IL-10 polypeptide comprises an amino acid substitution at position R27, e.g., R27A, relative to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutated IL-10 polypeptide comprises an amino acid substitution at position K34, e.g., K34A, K34D, K34E, K34S, K34P, K34G, K34T, K34H, K34L, K34N, K34F, K34R, K34Q, K34V, or K34Y, relative to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutated IL-10 polypeptide comprises an amino acid substitution at position Q38 relative to the amino acid sequence of SEQ ID NO: 1, e.g., Q38A, Q38D, Q38P, Q38G, Q38H, Q38I, Q38L, Q38R, Q38K, Q38N, Q38F, Q38T, Q38E, Q38S, Q38V, or Q38Y. In some embodiments, the mutated IL-10 polypeptide comprises an amino acid substitution at position D44 relative to the amino acid sequence of SEQ ID NO: 1, e.g., D44A, D44E, D44S, D44V, D44G, D44H, D44I, D44K, D44P, D44L, D44N, D44F, D44T, D44R, or D44Q. In some embodiments, the mutated IL-10 polypeptide comprises an amino acid substitution at position 187 relative to the amino acid sequence of SEQ ID NO: 1, e.g., I87A. In some embodiments, the mutated IL-10 polypeptide comprises an amino acid substitution at position K138 relative to the amino acid sequence of SEQ ID NO: 1, e.g., K138A. In some embodiments, the mutated IL-10 polypeptide comprises an amino acid substitution at position E142 relative to the amino acid sequence of SEQ ID NO: 1, e.g., E142A, E142G, E142N, E142L, E142F, E142I, E142V, E142K, E142R, E142P, E142Q, E142T, E142S, or E142Y.In some embodiments, the mutant IL-10 polypeptide comprises an amino acid substitution at position D144 relative to the amino acid sequence of SEQ ID NO: 1, e.g., D144A, D144E, D144G, D144H, D144R, D144I, D144K, D144N, D144Q, D144P, D144S, D144L, D144T, D144V, or D144Y. In some embodiments, the mutant IL-10 polypeptide comprises an amino acid substitution at position N148 relative to the amino acid sequence of SEQ ID NO: 1, e.g., N148G, N148P, N148S, N148D, N148T, N148K, N148V, N148I, N148E, or N148F. In some embodiments, the mutated IL-10 polypeptide comprises an amino acid substitution at position E151 relative to the amino acid sequence of SEQ ID NO: 1, e.g., E151A, E151G, E151H, E151I, E151N, E151F, E151L, E151V, E151R, E151K, E151P, E151Q, E151S, E151T, or E151Y. In some embodiments, the mutated IL-10 polypeptide comprises an amino acid substitution at position N18 relative to the amino acid sequence of SEQ ID NO: 1, e.g., N18F, N18L, or N18Y. In some embodiments, the mutated IL-10 polypeptide comprises an amino acid substitution at position D28 relative to the amino acid sequence of SEQ ID NO: 1, e.g., D28Q or D28R. In some embodiments, the mutated IL-10 polypeptide comprises an amino acid substitution at position N92 relative to the amino acid sequence of SEQ ID NO: 1, e.g., N92F, N92H, N92I, N92K, N92L, N92R, N92S, N92T, N92V, or N92Y. In some embodiments, the mutated IL-10 polypeptide comprises an amino acid substitution at position K99 relative to the amino acid sequence of SEQ ID NO: 1, e.g., K99N. In some embodiments, the mutated IL-10 polypeptide comprises an amino acid substitution at position L103 relative to the amino acid sequence of SEQ ID NO: 1, e.g., L103N or L103Q.

[0017] In some embodiments, an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence of SEQ ID NO:1. Provided herein is a mutant IL-10 polypeptide comprising the sequence and having one or more amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 1, wherein the one or more amino acid substitutions are at a position(s) selected from the group consisting of P20, L23, R24, R27, D28, K34, T35, Q38, M39, D41, L43, D44, N45, L46, K49, 187, V91, L94, L98, K138, S141, E142, D144, N148, E151, and 1158, numbered according to SEQ ID NO: 1. In some embodiments, the one or more amino acid substitutions are at a position(s) selected from the group consisting of R24, R27, K34, Q38, D44, 187, K138, E142, D144, N148, and E151, numbered according to SEQ ID NO: 1.In some embodiments, the one or more amino acid substitutions are R24A, R27A, K34A, K34D, K34E, K34S, K34P, K34G, K34T, K34H, K34L, K34N, K34F, K34R, K34Q, K34V, K34Y, Q38A, Q38D, Q38P, Q38G, Q38H, Q38I, Q38L, Q38R, Q38Q ... 8K, Q38N, Q38F, Q38T, Q38E, Q38S, Q38V, Q38Y, D44A, D44E, D44S, D44V, D44G, D44H, D44I, D44K , D44P, D44L, D44N, D44F, D44T, D44R, D44Q, I87A, K138A, E142A, E142G, E142N, E142L, E142F, E142I, E142V, E142K, E142R, E142P, E142Q, E142T, E142S, E142Y, D144A, D144E, D144G, D144H , D144R, D144I, D144K, D144N, D144Q, D144P, D144S, D144L, D144T, D144V, D144Y, N148G, N148 P, N148S, N148D, N148T, N148K, N148V, N148I, N148E, N148F, E151A, E151G, E151H, E151I, E151N, E151F, E151L, E151V, E151R, E151K, E151P, E151Q, E151S, E151T, and E151Y.In some embodiments, the one or more amino acid substitutions are R24A, R27A, K34A, K34D, K34E, K34S, K34P, K34G, K34T, K34H, K34L, K34N, K34F, K34V, K34Y, Q38A, Q38D, Q38P, Q38G, Q38I, Q38L, Q38R, Q38K, Q38F, Q38T, Q38E, Q38S, Q38V, Q38Y, I87A, K138A, E142A, E142G, E142N, E142L, E142F, E142I, E142V, E142K, E142R, E1 42P, E142Q, E142T, E142S, E142Y, D144A, D144E, D144G, D144H, D144R, D144I, D144K, D144N, D144Q, D144P, D144S, D144L, D144T, D144V, D144Y, N148P, N148D, N148I, E151A, E151G, E151H, E151I, E151N, E151F, E151L, E151V, E151R, E151K, E151P, E151Q, E151S, E151T, and E151Y. In some embodiments, the one or more amino acid substitutions are selected from the group consisting of E151A and K138A, E151A and D144A, E151A and R27A, Q38A and R27A, R24A and Q38A, R24A and E151A, Q38A and E142A, E138A and E142A, R27A and K138A, R24A and K138A, and R24A and R27A. In some embodiments, the mutant IL-10 polypeptide exhibits reduced binding affinity to an IL-10RA polypeptide, e.g., comprising the amino acid sequence of SEQ ID NO: 2 (e.g., compared to the binding affinity of a wild-type mature IL-10 polypeptide comprising the amino acid sequence of SEQ ID NO: 1 to an IL-10RA polypeptide comprising the amino acid sequence of SEQ ID NO: 2).In some embodiments, the mutant IL-10 polypeptide exhibits a binding affinity that is 50% or more reduced, 100% or more reduced, 150% or more reduced, 2-fold or more reduced, or 10-fold or more reduced to an IL-10RA polypeptide, e.g., comprising the amino acid sequence of SEQ ID NO:2 (e.g., compared to the binding affinity of a wild-type mature IL-10 polypeptide comprising the amino acid sequence of SEQ ID NO:1 to an IL-10RA polypeptide comprising the amino acid sequence of SEQ ID NO:2). In some embodiments, the mutant IL-10 polypeptide comprises one or more additional amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 1, wherein the one or more additional amino acid substitutions are at a position(s) selected from the group consisting of N18, N21, M22, R24, D25, D28, S31, R32, D55, M68, I69, L73, E74, M77, P78, Q79, E81, N82, K88, A89, H90, N92, S93, G95, E96, N97, K99, T100, L101, L103, R104, R107, R110, and F111, numbered according to SEQ ID NO: 1. In some embodiments, the one or more amino acid substitutions are at a position(s) selected from the group consisting of N18, D28, N92, K99, and L103, numbered according to SEQ ID NO: 1. In some embodiments, the one or more amino acid substitutions are selected from the group consisting of N18F, N18L, N18Y, D28Q, D28R, N92F, N92H, N92I, N92K, N92L, N92R, N92S, N92T, N92V, N92Y, K99N, L103N, and L103Q, numbered according to SEQ ID NO: 1. In some embodiments, the mutant IL-10 polypeptide exhibits increased binding affinity for an IL-10RB polypeptide comprising, e.g., the amino acid sequence of SEQ ID NO: 3 (e.g., compared to the binding affinity of a wild-type mature IL-10 polypeptide comprising the amino acid sequence of SEQ ID NO: 1 to an IL-10RB polypeptide comprising the amino acid sequence of SEQ ID NO: 3).In some embodiments, the mutant IL-10 polypeptide exhibits a binding affinity that is increased by 50% or more, 100% or more, or 150% or more for an IL-10RB polypeptide, e.g., comprising the amino acid sequence of SEQ ID NO: 3 (e.g., compared to the binding affinity of a wild-type mature IL-10 polypeptide comprising the amino acid sequence of SEQ ID NO: 1 for an IL-10RB polypeptide comprising the amino acid sequence of SEQ ID NO: 3). In some embodiments, the mutant IL-10 polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 87-89, 188-201, and 310-318.

[0018] In some embodiments, the mutated IL-10 polypeptide further comprises an amino acid substitution at position R107 relative to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutated IL-10 polypeptide further comprises an R107A mutation, numbered according to SEQ ID NO: 1. In some embodiments, the mutated IL-10 monomer polypeptide comprises a sequence selected from the group consisting of SEQ ID NOs: 422-428. In some embodiments, the mutated IL-10 monomer polypeptide comprises the sequence of a mutated monomeric IL-10 polypeptide set forth in Table 11. In some embodiments, the mutated IL-10 monomer polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 87-89, 188-201, 310-318, and 422-428.

[0019] In some embodiments, the mutant IL-10 polypeptide is a dimer, e.g., a homodimer or a heterodimer. In some embodiments, the mutant IL-10 polypeptide is a monomer, e.g., comprising an amino acid or peptide insertion between N116 and K117 that enables folding and expression as a monomer (e.g., as shown in FIG. 1D ). In some embodiments, the insertion is 1 to 15 amino acids in length. In some embodiments, the insertion is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in length. In certain embodiments, the insertion is 6 amino acids in length. In some embodiments, the mutant IL-10 monomer polypeptide comprises the amino acid sequence of SEQ ID NO: 1 with a single amino acid, or a peptide insertion of between 1 and 15 amino acids, immediately following residues C114, E115, N116, K117, S118, K119, or A120, numbered based on SEQ ID NO: 1. Examples of insertions can include, but are not limited to, G, GG, GGG, GGGG (SEQ ID NO: 80), GGGSG (SEQ ID NO: 81), GGGGG (SEQ ID NO: 82), GGGGGG (SEQ ID NO: 83), and GGGSGG (SEQ ID NO: 84). In some embodiments, the mutant IL-10 monomer polypeptide comprises the amino acid sequence of SEQ ID NO: 187. In some embodiments, a mutant monomeric IL-10 polypeptide of the disclosure has reduced binding affinity for an IL-10RA polypeptide having the amino acid sequence shown in FIG. 1B and has an amino acid substitution selected from the group of P20, L23, R24, R27, D28, K34, T35, Q38, M39, D41, L43, D44, N45, L46, K49, 187, V91, L94, L98, K138, S141, E142, D144, N148, E151, and 1158 (or selected from the group of R24, R27, K34, Q38, D44, 187, K138, E142, D144, N148, and E151), where amino acid numbering refers to the corresponding amino acid in a wild-type IL-10 polypeptide that does not include the 6-linker insertion.In some embodiments, a mutant monomeric IL-10 polypeptide of the present disclosure also has increased binding affinity for an IL-10RB polypeptide having the amino acid sequence shown in Figure 1C and comprises an amino acid substitution selected from the group of N18, N21, M22, R24, D25, D28, S31, R32, D55, M68, I69, L73, E74, M77, P78, Q79, E81, N82, K88, A89, H90, N92, S93, G95, E96, N97, K99, T100, L101, L103, R104, R107, R110, and F111 (or selected from the group consisting of N18, D28, N92, K99, and L103). In some embodiments, the mutant IL-10 monomer polypeptide comprises an amino acid substitution at position N92 based on numbering in accordance with SEQ ID NO:1. In some embodiments, the mutated IL-10 monomer polypeptide comprises the amino acid substitutions N92I, N92A, N92V, N92L, N92M, N92Y, N92F, N92S, N92T, N92H, or N92Q. In some embodiments, the mutated IL-10 monomer polypeptide comprises the amino acid substitutions N92F, N92H, N92K, N92L, N92R, N92S, N92T, N92V, or N92Y. In some embodiments, the mutated IL-10 monomer polypeptide further comprises one or more of the amino acid substitutions N18I, K99N, and F111L, numbered based on SEQ ID NO: 1. In some embodiments, the mutated IL-10 monomer polypeptide further comprises the amino acid substitutions N18I, K99N, and F111L, numbered based on SEQ ID NO: 1. In some embodiments, the mutated IL-10 monomer polypeptide comprises the amino acid substitutions N18I, N92I, K99N, and F111L, numbered based on SEQ ID NO: 1. In some embodiments, the mutated IL-10 monomer polypeptide comprises the sequence of SEQ ID NO: 188. In some embodiments, the mutated IL-10 monomer polypeptide comprises the amino acid substitutions N18I, N92I, K99N, and F111L, and further comprises one or more additional amino acid substitutions at position(s) R24, R27, Q38, 187, K138, E142, D144, and / or E151, numbered based on SEQ ID NO: 1.In some embodiments, the mutant IL-10 monomer polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 87-89, 188-201, 310-318, and 422-428.

[0020] Due to their reduced binding affinity to the IL-10R complex, the mutant IL-10 polypeptides disclosed herein have a reduced ability to stimulate IL-10R-expressing cells, including CD8+ T cells, which have been shown to be associated with the beneficial effects of IL-10 in preclinical cancer models (Mumm et al., Cancer Cell. 2011 Dec 13;20(6):781-96; Emmerich et al., Cancer Res. 2012 Jul 15;72(14):3570-81). To make mutant IL-10 polypeptides safer and potentially more effective therapeutic agents for the treatment of cancer and other immune-related diseases, such as certain infectious diseases, fusion proteins comprising the disclosed mutant IL-10 polypeptides and antigen-binding molecules, e.g., antibodies, against antigens present on CD8+ T cells, such as CD8, have been generated. Such fusion proteins, composed of mutant IL-10 polypeptides and antibodies that bind to a specific antigen, are also referred to as "targeted" fusion proteins, because they bind to the antigen recognized by the antigen-binding molecule of the fusion. This distinguishes them from "non-targeting" fusion proteins that comprise mutant IL-10 polypeptides and control antibodies that do not bind to any particular antigen (i.e., Fc fusions or control antibody fusions with IL-10 polypeptides; Poutahidis et al, Carcinogenesis. 2007 Dec;28(12):2614-23).

[0021] Without wishing to be bound by theory, Figure 4A shows a general mechanism for how an antigen-binding molecule that binds to an antigen on CD8+ T cells may work to increase the binding and / or stimulation of CD8+ T cells by a mutant IL-10 polypeptide in the context of a disclosed targeting fusion protein containing a mutant IL-10 polypeptide. When fused to a mutant IL-10 polypeptide, a particular antigen-binding molecule has the ability to substantially increase the binding and / or activity of the mutant IL-10 polypeptide only on cells that express the antigen for which the fusion antigen-binding molecule is expressed, resulting in preferential activation of antigen-expressing cells over non-antigen-expressing cells (Figure 4A). Unlike targeting fusion proteins, non-targeting fusion proteins containing the same mutant IL-10 polypeptide do not preferentially bind to and / or activate antigen-expressing cells (Figure 4A). Figure 4B shows a general mechanism for a mutant monomeric IL-10 polypeptide.

[0022] Without wishing to be bound by theory, it is believed that differences in activation of antigen-expressing cells versus non-antigen-expressing cells by targeting fusion proteins, and differences in activation of antigen-expressing cells by targeting fusion proteins versus non-targeting fusion proteins, are important to the effectiveness of the targeting fusion protein as a therapeutic. Fusion proteins that are selective for cells associated with an effect, such as CD8+ T cells, over other cells associated with undesirable effects on toxicity or efficacy may have a larger therapeutic index.

[0023] In some embodiments, the fusion protein activates CD8+ T cells 10-fold more potently, or 50-fold more potently, compared to activating monocytes. In some embodiments, the mutant IL-10 polypeptide comprises the sequence of SEQ ID NO: 1 and has one or more amino acid substitutions relative to SEQ ID NO: 1, wherein the substitutions are at positions in SEQ ID NO: 1 selected from the group consisting of P20, L23, R24, R27, D28, K34, T35, Q38, M39, D41, L43, D44, N45, L46, K49, 187, V91, L94, L98, K138, S141, E142, D144, N148, E151, and 1158 (or selected from the group consisting of R24, R27, K34, Q38, D44, 187, K138, E142, D144, N148, and E151). In other embodiments, the mutant IL-10 polypeptide also comprises one or more mutations at one or more positions of SEQ ID NO: 1 selected from the group consisting of N18, N21, M22, R24, D25, D28, S31, R32, D55, M68, 169, L73, E74, M77, P78, Q79, E81, N82, K88, A89, H90, N92, S93, G95, E96, N97, K99, T100, L101, L103, R104, R107, R110 and F111 (or selected from the group consisting of N18, D28, N92, K99, and L103).

[0024] In some embodiments, the IL-10 fusion proteins disclosed herein activate antigen-expressing IL-10R+ cells, such as CD8+ T cells, at least 5-fold, at least 10-fold, at least 50-fold, at least 100-fold, or at least 200-fold more than non-antigen-expressing IL-10R+ cells, such as monocytes. In some embodiments, the fusion proteins activate antigen-expressing IL-10R+ cells at least 50-fold, at least 100-fold, or at least 200-fold more than, for example, a fusion molecule comprising an IL-10 mutant polypeptide and a control antibody that does not bind to any antigen expressed on the cells. In some embodiments, the cell activation by the IL-10 fusion protein is determined by measuring pSTAT3 expression in the cells after treatment with the IL-10 fusion protein.

[0025] In some embodiments, the fusion proteins disclosed herein may also partially reduce the pleiotropic effects of IL-10 on immune cells expressing the IL-10R complex by reducing the effect of IL-10 on a specific immune cell subset of interest, e.g., CD8+ T cells. Such reduction, when administered as a therapeutic, can increase the potency and reduce toxicity of an IL-10 polypeptide by directing the effect on a subset of T cells, including tumor antigen-specific CD8+ T cells or viral antigen-specific CD8+ T cells, thereby sparing 1) T cells that may not contribute to efficacy, or 2) systemically distributed myeloid cells that express IL-10R and may contribute to toxicity or action as a sink, or 3) other immune cells, such as dendritic cells and Tregs, that may negatively contribute to efficacy.

[0026] In some embodiments according to any of the embodiments described herein, the T cell (e.g., a CD8+ T cell) is a human T cell. In some embodiments, the monocyte / other immune cell is a human cell. In some embodiments, the fusion protein comprises a mutated IL-10 polypeptide according to any one of the above embodiments and an antigen binding molecule that binds to an antigen on a T cell, e.g., CD8 (e.g., CD8ab, CD8a, or CD8aa; or CD8b and / or CD8ab), CD4, or PD-1. In some embodiments, the CD8 polypeptide, antigen, or dimer (e.g., CD8a, CD8b, CD8aa, and / or CD8ab) of the present disclosure is a human CD8 polypeptide, antigen, or dimer. In some embodiments, the CD4 polypeptide, antigen, or dimer of the present disclosure is a human CD4 polypeptide. In some embodiments, the PD-1 polypeptide, antigen, or dimer of the present disclosure is a human PD-1 polypeptide.

[0027] In some embodiments, the fusion protein comprises an antigen-binding molecule comprising a heavy chain variable (VH) domain and a light chain variable (VL) domain. In some embodiments, the VH domain comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 110, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 111, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 112; the VL domain comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6. In some embodiments, the VH domain comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 13, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 14, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 15; and the VL domain comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 16, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 17, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 18. In some embodiments, the VH domain comprises 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 the VL domain comprises 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 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 the VL domain comprises 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 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 the VL domain comprises 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 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 the VL domain comprises 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 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 the VL domain comprises 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 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 the VL domain comprises 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 VH domain comprises a CDR-H1 (SEQ ID NO: 259) comprising 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; and a CDR-H1 (SEQ ID NO: 259) comprising the amino acid sequence of X1X2AIS, 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, and X. 10 is A or N and X 11 X1X2X3PX4X5X6X7X8X9YX where is Q or T 10 QKFX 11and CDR-H3 (SEQ ID NO: 261) comprising the amino acid sequence of 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 is X1X2X3GX4X5LFX6X7, 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. It comprises CDR-L1 (SEQ ID NO: 262) comprising the amino acid sequence of 2SX3X4IX5GX6LN; CDR-L2 (SEQ ID NO: 263) comprising the amino acid sequence of GX1X2X3LX4X5, 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 (SEQ ID NO: 264) comprising 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 comprises 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 the VL domain comprises 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 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 the VL domain comprises 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 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 the VL domain comprises 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 a CDR-H1 (SEQ ID NO: 268) comprising the amino acid sequence of X1YX2MS, where X1 is S, D, E, A, or Q, and X2 is A, G, or T; a CDR-H2 (SEQ ID NO: 269) comprising the amino acid sequence of 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 a CDR-H2 (SEQ ID NO: 269) comprising the amino acid sequence of DIX1X2X3GX4X5TX6YADSVKG, where X1 is S or A, and X2 is N, H, A, D, L, Q, Y, or R. and CDR-H3 (SEQ ID NO: 270) comprising the amino acid sequence of X1X2X3YX4WX5X6AX7DX8, where 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; and the VL domain comprises CDR-L1 comprising the amino acid sequence of RASQSVSSNLA (SEQ ID NO: 40), CDR-L2 comprising the amino acid sequence of GASSRAT (SEQ ID NO: 41), and CDR-L3 comprising the amino acid sequence of QQYGSSPPVT (SEQ ID NO: 42). In some embodiments, the VH domain comprises 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 the VL domain comprises CDR-L1 comprising the amino acid sequence of RASQSVSSNLA (SEQ ID NO: 40), CDR-L2 comprising the amino acid sequence of GASSRAT (SEQ ID NO: 41), and CDR-L3 comprising the amino acid sequence of QQYGSSPPVT (SEQ ID NO: 42).In some embodiments, the VH domain comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 229, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 237, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 231, and the VL domain comprises a CDR-L1 comprising the amino acid sequence of RASQSVSSNLA (SEQ ID NO: 40), a CDR-L2 comprising the amino acid sequence of GASSRAT (SEQ ID NO: 41), and a CDR-L3 comprising the amino acid sequence of QQYGSSPPVT (SEQ ID NO: 42). In some embodiments, the VH domain comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 51, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 52, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 15, and the VL domain comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 16, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 17, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 18. In some embodiments, the VH domain comprises 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 the VL domain comprises 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 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 the VL domain comprises 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 a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 54, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 52, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 33, and the VL domain comprises the amino acid sequence of SEQ ID NO: 34. In some embodiments, the VH domain comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 55, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 56, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 39, and the VL domain comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 40, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 41, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 42. In some embodiments, the VH domain comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 55, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 57, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 45, and the VL domain comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 46, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 47, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 48. In some embodiments, the VH domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 49, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 50, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3, and the VL domain comprises 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 VH domain comprises 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 the VL domain comprises 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 VH domain comprises a CDR-H1 (SEQ ID NO: 265) comprising the amino acid sequence of 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; and a CDR-H2 (SEQ ID NO: 265) comprising the amino acid sequence of GX1X2FX3X4X5, where X1 is I, N, or M, X2 is G, N, H, S, R, I, or A, and 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 CDR-H2 (SEQ ID NO: 266) comprising the amino acid sequence of X1PX2X3X4X5, 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 CDR-H3 (SEQ ID NO: 267) comprising the amino acid sequence of X6X7, and the VL domain comprises CDR-L1 (SEQ ID NO: 262) comprising the amino acid sequence of 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, X5 is Y or S, and X6 is A or V. , E, or H, X4 is Q or A, and X5 is S or D; and CDR-L3 (SEQ ID NO: 264) comprising an amino acid sequence comprising 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 comprises 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 the VL domain comprises 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 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 the VL domain comprises 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 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 the VL domain comprises 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 CDR-H1 (SEQ ID NO: 271) comprising the amino acid sequence of GFTFX1X2Y, where X1 is S, D, E, Q, S, or A, and X2 is S, D, E, A, or Q; CDR-H2 (SEQ ID NO: 272) comprising the amino acid sequence of 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; and CDR-H2 (SEQ ID NO: 273) comprising the amino acid sequence of X1X2X3GX4X5, where X1 is S or A, X2 is N, H, A, D, L, Q, Y, or R, and X3 is A, X4 is N, S, or G, X5 is A, V, R, E, or S, X6 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 CDR-H3 (SEQ ID NO: 273) comprising the amino acid sequence of X1X2X3YX4WX5X6AX7DX8, where X1 is 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 comprising the amino acid sequence of RASQSVSSNLA (SEQ ID NO: 40), CDR-L2 comprising the amino acid sequence of GASSRAT (SEQ ID NO: 41), and CDR-L3 comprising the amino acid sequence of QQYGSSPPVT (SEQ ID NO: 42). In some embodiments, the VH domain comprises 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 the VL domain comprises CDR-L1 comprising the amino acid sequence of RASQSVSSNLA (SEQ ID NO: 40), CDR-L2 comprising the amino acid sequence of GASSRAT (SEQ ID NO: 41), and CDR-L3 comprising the amino acid sequence of QQYGSSPPVT (SEQ ID NO: 42). In some embodiments, the VH domain comprises 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 the VL domain comprises CDR-L1 comprising the amino acid sequence of RASQSVSSNLA (SEQ ID NO: 40), CDR-L2 comprising the amino acid sequence of GASSRAT (SEQ ID NO: 41), and CDR-L3 comprising the amino acid sequence of QQYGSSPPVT (SEQ ID NO: 42).In some embodiments, the VH domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 62, and the VL domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 63. In some embodiments, the VH domain comprises the sequence of SEQ ID NO: 62, and the VL domain comprises the sequence of SEQ ID NO: 63. In some embodiments, the VH domain comprises an amino acid sequence 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 at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 65. In some embodiments, the VH domain comprises the sequence of SEQ ID NO: 64, and the VL domain comprises the sequence of SEQ ID NO: 65. In some embodiments, the VH domain comprises an amino acid sequence 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 at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 67. In some embodiments, the VH domain comprises the sequence of SEQ ID NO: 66, and the VL domain comprises the sequence of SEQ ID NO: 67. In some embodiments, the VH domain comprises an amino acid sequence 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 at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 69. In some embodiments, the VH domain comprises the sequence of SEQ ID NO: 68, and the VL domain comprises the sequence of SEQ ID NO: 69. 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 VH domain comprises the sequence of SEQ ID NO:70, and the VL domain comprises the sequence of SEQ ID NO:71. In some embodiments, the VH domain comprises an amino acid sequence 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 at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO:73. In some embodiments, the VH domain comprises the sequence of SEQ ID NO:72, and the VL domain comprises the sequence of SEQ ID NO:73. In some embodiments, the VH domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO:245, and the VL domain comprises an amino acid sequence 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 comprises the sequence of SEQ ID NO:245, and the VL domain comprises the sequence of SEQ ID NO:246. In some embodiments, the VH domain is of SEQ ID NO:251. In some embodiments, the VH domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 251, and the VL domain comprises an amino acid sequence 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 an amino acid sequence 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 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 comprises the sequence of SEQ ID NO: 253 and the VL domain comprises the sequence of SEQ ID NO: 254. In some embodiments, the VH domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 247, and the VL domain comprises an amino acid sequence 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 comprises the sequence of SEQ ID NO: 247, and the VL domain comprises the sequence of SEQ ID NO: 248. In some embodiments, the VH domain comprises an amino acid sequence 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 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 sequence of SEQ ID NO: 249, and the VL domain comprises the sequence of SEQ ID NO: 250. 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 comprises the sequence of SEQ ID NO: 255, and the VL domain comprises the sequence of SEQ ID NO: 256. In some embodiments, the VH domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 257, and the VL domain comprises an amino acid sequence 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 comprises the sequence of SEQ ID NO: 257, and the VL domain comprises the sequence of SEQ ID NO: 258. In some embodiments, the VH domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 58, and the VL domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 59. In some embodiments, the VH domain comprises the sequence of SEQ ID NO: 58, and the VL domain comprises the sequence of SEQ ID NO: 59. 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. In some embodiments, the VH domain comprises the sequence of SEQ ID NO: 185, and the VL domain comprises the sequence of SEQ ID NO: 186.

[0028] In some embodiments, the fusion protein comprises four polypeptide chains, (1) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 113, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 114, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 115, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 113; (2) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 113, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 114, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 116, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 113; (3) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 117, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 118, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 119, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 117; (4) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 117, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 118, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 119, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 117. (5) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 121, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 122, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 123, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 121; (6) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 121, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 122, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 121; (7) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 125, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 126, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 127, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 125;(8) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 125, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 126, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 128, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 125; (9) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 129, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 130, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 131, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 129; (10) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 129, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 130, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 132, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 129; (11) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 133, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 134, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 135, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 133; ( (12) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 133, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 134, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 136, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 133; (13) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 137, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 138, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 139, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 137; (14) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 137, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 138, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 140, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 137; (15) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 141, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 142, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 143, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 141;(16) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 141, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 142, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 144, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 141; (17) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 145, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 146, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 147, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 145; (18) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 145, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 146, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 148, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 145; (19) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 149, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 150, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 151, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 149; (20) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 149, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 150, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 152, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 149; (21) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 153, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 154, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 155, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 153; (22) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 153, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 154, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 156, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 153; (23) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 157, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 158, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 159, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 157;or (24) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 157, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 158, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 160, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 157;

[0029] In some embodiments, the fusion protein comprises a dimer of two mutated IL-10 polypeptides, one of the two mutated IL-10 polypeptides fused to an antigen-binding molecule. In some embodiments, the fusion protein comprises two polypeptides, each comprising an antigen-binding site, one of the mutated IL-10 polypeptides fused to each of the polypeptides. In some embodiments, the fusion protein comprises a mutated IL-10 monomer polypeptide, the mutated IL-10 monomer polypeptide fused to an antigen-binding molecule. In some embodiments, the mutated IL-10 polypeptide is fused to the antigen-binding molecule directly or via a linker.

[0030] In some embodiments, the antigen-binding molecule has formula [I], from the N-terminus to the C-terminus: VH-CH1-hinge-CH2-CH3 [I] and two antibody heavy chain polypeptides comprising a structure according to formula [II], from N-terminus to C-terminus: VL-CL [II] and two antibody light chain polypeptides comprising a structure according to: where VH is an antibody heavy chain variable (VH) domain, CH1 is an antibody CH1 domain, hinge is an antibody hinge domain, CH2 is an antibody CH2 domain, CH3 is an antibody CH3 domain, VL is an antibody light chain variable (VL) domain, and CL is an antibody constant light chain domain, and VH / VL form an antigen-binding site. In some embodiments, the fusion protein comprises two mutated IL-10 polypeptides linked in a dimer, and the N-terminus of one of the two mutated IL-10 polypeptides is fused, either directly or via a linker, to the C-terminus of one of the two CH3 domains. In some embodiments, the fusion protein comprises two mutated IL-10 polypeptides linked in a dimer, wherein the N-terminus of a first of the two mutated IL-10 polypeptides is fused, either directly or via a linker, to the C-terminus of a first of the two CH3 domains, and the N-terminus of a second of the two mutated IL-10 polypeptides is fused, either directly or via a linker, to the C-terminus of a second of the two CH3 domains. In some embodiments, the fusion protein comprises one mutated IL-10 polypeptide, wherein the N-terminus of a mutated IL-10 monomer polypeptide is fused, either directly or via a linker, to the C-terminus of one of the two CH3 domains.

[0031] In some embodiments, the antigen-binding molecule has formula [I], from the N-terminus to the C-terminus: VH-CH1-hinge-CH2-CH3 [I], and a first antibody heavy chain polypeptide comprising a structure according to formula [II], from the N-terminus to the C-terminus: VL-CL [II], and an antibody light chain polypeptide comprising a structure according to formula [III], from the N-terminus to the C-terminus: Hinge-CH2-CH3 [III], and a second antibody heavy chain polypeptide comprising a structure according to the formula: where VH is an antibody heavy chain variable (VH) domain, CH1 is an antibody CH1 domain, hinge is an antibody hinge domain, CH2 is an antibody CH2 domain, CH3 is an antibody CH3 domain, VL is an antibody light chain variable (VL) domain, and CL is an antibody constant light chain domain, and VH / VL form an antigen-binding site. In some embodiments, the fusion protein comprises two mutated IL-10 polypeptides linked in a dimer, and the N-terminus of one of the two mutated IL-10 polypeptides is fused, directly or via a linker, to either the C-terminus of the CH3 domain of the second antibody heavy chain polypeptide or the C-terminus of the CH3 domain of the first antibody heavy chain polypeptide. In some embodiments, the fusion protein comprises two mutated IL-10 polypeptides linked in a dimer, wherein the N-terminus of a first of the two mutated IL-10 polypeptides is fused, either directly or via a linker, to the C-terminus of the CH3 domain of a first antibody heavy chain polypeptide, and the N-terminus of the second of the two mutated IL-10 polypeptides is fused, either directly or via a linker, to the C-terminus of the CH3 domain of a second antibody heavy chain polypeptide. In some embodiments, the fusion protein comprises one mutated IL-10 monomer polypeptide, wherein the N-terminus of the mutated IL-10 monomer polypeptide is fused, either directly or via a linker, to either the C-terminus of the CH3 domain of a second antibody heavy chain polypeptide or the C-terminus of the CH3 domain of the first antibody heavy chain polypeptide.

[0032] In some embodiments, one or both of the antibody heavy chain polypeptides comprise the following amino acid substitutions, as numbered according to the EU index: L234A, L235A, and G237A. In some embodiments, a first of the two Fc domains comprises amino acid substitutions Y349C and T366W, and a second of the two Fc domains comprises amino acid substitutions S354C, T366S, L368A, and Y407V, as numbered according to the EU index. In some embodiments, the linker comprises 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 comprises the sequence GGGGSGGGSGGGGS (SEQ ID NO:79), SGGGGSGGGGSGGGGS (SEQ ID NO:77), or SGGGGSGGGGSGGGG (SEQ ID NO:78). In some embodiments, the antibody heavy chain polypeptide comprises a human IgG1 Fc region.

[0033] Further provided herein are one or more isolated polynucleotides encoding the mutant IL-10 polypeptide or fusion protein of any one of the above embodiments. Further provided herein are one or more vectors comprising one or more polynucleotides of any one of the above embodiments. Further provided herein are host cells (e.g., isolated and / or recombinant host cells) comprising one or more polynucleotides or vectors of any one of the above embodiments. Further provided herein are methods for producing a mutant IL-10 polypeptide or fusion protein, the method comprising culturing a host cell of any one of the above embodiments under conditions suitable for producing the polypeptide or fusion protein. In some embodiments, the method further comprises recovering the polypeptide or fusion protein from the host cell.

[0034] Further provided herein is a pharmaceutical composition comprising a mutant IL-10 polypeptide or fusion protein of any one of the above embodiments and a pharmaceutically acceptable carrier.

[0035] Further provided herein is a method of treating cancer, comprising administering to an individual having cancer an effective amount of a mutant IL-10 polypeptide, fusion protein, or composition thereof according to any one of the above embodiments. Further provided herein is a mutant IL-10 polypeptide, fusion protein, or composition thereof according to any one of the above embodiments for use as a medicament. Further provided herein is a mutant IL-10 polypeptide, fusion protein, or composition thereof according to any one of the above embodiments for use in a method of treating cancer in an individual in need thereof. Further provided herein is a mutant IL-10 polypeptide, fusion protein, or composition thereof according to any one of the above embodiments for use in the manufacture of a medicament for treating cancer in an individual in need thereof. In some embodiments, the method further comprises administering to the individual T cell therapy, a cancer vaccine, a chemotherapeutic agent, an IL-2 polypeptide, or an immune checkpoint inhibitor (ICI). In some embodiments, the ICI is an inhibitor of PD-1, PD-L1, or CTLA-4. In some embodiments, T cell therapy includes chimeric antigen receptor (CAR)-based T cell therapy, tumor infiltrating lymphocyte (TIL)-based therapy, or therapy using T cells with transduced TCRs.

[0036] Further provided herein is a method of treating an infectious disease (e.g., a chronic and / or viral infection), comprising administering to an individual in need thereof an effective amount of a mutated IL-10 polypeptide, fusion protein, or composition thereof according to any one of the above embodiments. Further provided herein is a mutated IL-10 polypeptide, fusion protein, or composition thereof according to any one of the above embodiments for use as a medicament. Further provided herein is a mutated IL-10 polypeptide, fusion protein, or composition thereof according to any one of the above embodiments for use in a method of treating an infectious disease (e.g., a chronic and / or viral infection) in an individual in need thereof. Further provided herein is a mutated IL-10 polypeptide, fusion protein, or composition thereof according to any one of the above embodiments for use in the manufacture of a medicament for treating an infectious disease in an individual in need thereof.

[0037] Further provided herein is a method of expanding T cells (e.g., ex vivo), comprising contacting one or more T cells (e.g., ex vivo) with an effective amount of a mutant IL-10 polypeptide, fusion protein, or composition thereof according to any one of the above embodiments. In some embodiments, the one or more T cells are tumor-infiltrating lymphocytes (TILs).

[0038] It is to be understood that one, some, or all of the features of the various embodiments described herein may be combined to form other embodiments of the present disclosure. These and other aspects of the present disclosure will be apparent to those skilled in the art. These and other embodiments of the present disclosure are further described in the detailed description that follows. [Brief explanation of the drawings]

[0039] [Figure 1-1] 1 shows the amino acid sequence of the polypeptide of mature IL-10 (SEQ ID NO: 1). [Figure 1-2]1 shows the amino acid sequence of the polypeptide of IL-10RA (SEQ ID NO: 2). [Figure 1-3] 1 shows the amino acid sequence of the polypeptide of IL-10RB (SEQ ID NO: 3). [Figure 1-4] 1 shows the amino acid sequence of the polypeptide of mature monomeric IL-10 (SEQ ID NO: 187). [Figure 2-1]

[0023] Figure 1 shows the amino acid sequence of a wild-type mature IL-10 polypeptide (SEQ ID NO: 1). "X" refers to an amino acid that is substituted with another amino acid in the sequence of the wild-type IL-10 polypeptide to generate a mutant IL-10 polypeptide of the present disclosure. [Figure 2-2]

[0033] Figure 1 shows the amino acid sequence of mature monomeric IL-10 (SEQ ID NO: 187). "X" refers to an amino acid that is substituted with another amino acid in the sequence of a wild-type IL-10 polypeptide to generate a mutant IL-10 polypeptide of the present disclosure. [Figure 3-1] 1 shows the amino acid sequence of wild-type mature IL-10 polypeptide (SEQ ID NO: 1). White boxes indicate residues that were substituted to alter the affinity of IL-10 for IL-10RA, and gray shaded boxes indicate residues that were substituted to alter the affinity of IL-10 for IL-10RB. The amino acid substituted for the wild-type residue for each position is indicated. [Figure 3-2]

[0023] Figure 1 shows the amino acid sequence of mature monomeric IL-10 (SEQ ID NO: 187). White boxes indicate residues that were substituted to alter the affinity of IL-10 for IL-10RA, and gray shaded boxes indicate residues that were substituted to alter the affinity of IL-10 for IL-10RB. The amino acid substituted for the wild-type residue for each position is indicated. [Figure 4-1] The general mechanism of how targeted fusions of mutant IL-10 polypeptides with CD8 antigen-binding molecules and non-targeted fusions containing mutant IL-10 polypeptides work to stimulate cells that express the CD8 antigen or cells that do not express the CD8 antigen is shown. [Figure 4-2]

[0023] Figure 1 shows the general mechanism of how targeted fusions of mutant monomeric IL-10 polypeptides with CD8 antigen-binding molecules and non-targeted fusions containing mutant monomeric IL-10 polypeptides work to stimulate cells that express the CD8 antigen or cells that do not express the CD8 antigen. [Figure 5] Figure 5A shows activation of STAT3 by wild-type IL-10 dimer in human PBMCs (Figure 5A) and human whole blood (Figure 5B). STAT3 is shown for CD8+ T cells (filled squares) and monocytes (filled circles). STAT3 activation was measured by flow cytometry. STAT3 activation in both CD8 T cells and monocytes (gated as CD14+CD3-) using human PBMCs was very similar to that using whole blood. Monocytes were found to be more sensitive to IL-10 than CD8+ T cells. [Figure 6] Eight different fusion protein formats (Formats A, B, C, D, E, F, G, and H) according to some embodiments are shown. [Figure 7] Figure 7A shows STAT3 activation in human CD8+ T cells (filled squares) and monocytes (filled circles) by the fusion protein format A, xmCD8a-IL10wt (xmCD8a-IL10wt, Figure 7A), which contains wild-type IL-10 polypeptide and a control antibody targeting mouse CD8, or the fusion protein format A, xhCD8a-IL10wt (Figure 7B), which contains wild-type IL-10 polypeptide and an antibody targeting human CD8. The anti-mouse CD8 antibody, xmCD8a, and the anti-human CD8 antibody, xhCD8a, were previously published (2.43 clone and OKT8 clone, respectively). The anti-mouse CD8 antibody (xmCD8a) does not bind to human CD8 T cells and serves as a non-binding control. STAT3 activation in human PBMCs was measured by flow cytometry. The IL-10 fusion protein format A, xhCD8a-IL10wt, containing an antibody that specifically binds to human CD8, preferentially activated CD8+ T cells over monocytes, whereas the IL-10 fusion protein format A, xmCD8a-IL10wt, containing a control antibody, preferentially activated monocytes. [Figure 8]Figure 8A shows STAT3 activation in human CD8+ T cells (filled squares) and monocytes (filled circles) by fusion proteins of format C, xmCD8a-IL10wt, containing wild-type IL-10 polypeptide and xmCD8a antibody, or format C, xhCD8a-IL10wt, containing wild-type IL-10 polypeptide and xhCD8a antibody, (Figure 8B). Format C was not optimal for IL-10 fusion proteins containing antibodies that bind human CD8, because high concentrations did not fully activate STAT3 in CD8+ T cells, except at low concentrations (up to 0.01 nM) (Figure 8B). [Figure 9] Figure 9A shows STAT3 activation in human CD8+ T cells (filled squares) and monocytes (filled circles) by the fusion protein format D, xmCD8a-IL10wt, which contains wild-type IL-10 polypeptide and xmCD8a antibody, or the fusion protein format D, xhCD8a-IL10wt, which contains wild-type IL-10 polypeptide and xhCD8a antibody, (Figure 9B). The IL-10 fusion protein format D, xhCD8a-IL10wt, which contains an antibody that specifically binds to human CD8, preferentially activated CD8+ T cells over monocytes, whereas the IL-10 fusion protein format D, xmCD8a-IL10wt, which contains a control antibody, preferentially activated monocytes. [Figure 10]Figure 10 shows STAT3 activation in human CD8+ T cells (filled squares), monocytes (filled circles), and CD4+ T cells (filled triangles) by various fusion proteins. Figure 10A shows STAT3 activation by xhCD8b-IL10mono of Format F, which contains a mature monomeric IL-10 polypeptide (SEQ ID NO: 187) and an xhCD8b antibody. Figure 10B shows STAT3 activation by xhCD8b-IL10mono_RBenh of Format F, which contains a monomeric IL-10 polypeptide and an xhCD8b antibody with an amino acid substitution to increase IL-10RB binding affinity. Figure 10C shows STAT3 activation by xhCD8b-IL10mono_RBenh2 of Format F, which contains a monomeric IL-10 polypeptide and an xhCD8b antibody with another amino acid substitution to increase IL-10RB binding affinity. Wild-type monomeric IL-10 fusion proteins containing antibodies against human CD8 preferentially activated CD8 T cells over monocytes and CD4 T cells at concentrations below 1 nM, but the degree of activation was suboptimal. Monomeric IL-10 fusion proteins with enhanced IL-10RB affinity containing antibodies against human CD8 preferentially and efficiently activated CD8 T cells over monocytes and CD4 T cells. [Figure 11-1] Figure 11A shows STAT3 activation by the xhCD8b antibody and a fusion protein (xhCD8b-IL-10mono_RBenh#) of format F of various IL-10 polypeptides with amino acid substitutions to increase IL-10RB binding affinity. Figure 11B shows STAT3 activation of CD8+ T cells and monocytes for a selected set of mutant proteins in human PBMCs. [Figure 11-2] Figure 11B shows STAT3 activation by the xhCD8b antibody and a fusion protein of form F of various IL-10 polypeptides with amino acid substitutions to increase IL-10RB binding affinity (xhCD8b-IL-10mono_RBenh#). Figure 11B shows STAT3 activation of CD8+ T cells and monocytes for a selected set of mutant proteins in human PBMCs. [Figure 11-3]Figure 11C shows STAT3 activation by the xhCD8b antibody and a fusion protein of format F of various IL-10 polypeptides with amino acid substitutions to increase IL-10RB binding affinity (xhCD8b-IL-10mono_RBenh#). Figure 11D shows STAT3 activation of CD8+ T cells and monocytes for an additional set of mutant proteins in human PBMCs. [Figure 11-4] Figure 11D shows STAT3 activation by the xhCD8b antibody and a fusion protein of format F of various IL-10 polypeptides with amino acid substitutions to increase IL-10RB binding affinity (xhCD8b-IL-10mono_RBenh#). Figure 11C shows STAT3 activation of CD8+ T cells and monocytes for an additional set of mutant proteins in human PBMCs. [Figure 11-5] Figure 11E shows STAT3 activation by the xhCD8b antibody and a fusion protein (xhCD8b-IL-10mono_RBenh#) of format F of various IL-10 polypeptides with amino acid substitutions to increase IL-10RB binding affinity. Figure 11F shows STAT3 activation of CD8+ T cells and monocytes in whole blood for three sets of mutant proteins. [Figure 11-6] Figure 11F shows STAT3 activation by the xhCD8b antibody and a fusion protein of form F of various IL-10 polypeptides with amino acid substitutions to increase IL-10RB binding affinity (xhCD8b-IL-10mono_RBenh#). Figure 11F shows STAT3 activation of CD8+ T cells and monocytes in whole blood for three sets of mutant proteins. [Figure 12-1] Figure 12A shows STAT3 activation by xhCD8b antibody and fusion proteins of form F of various IL-10 polypeptides with amino acid substitutions to increase IL-10RB binding affinity and decrease IL-10RA binding affinity. Figure 12B shows STAT3 activation of CD8+ T cells and monocytes for five selected muteins in human PBMCs. [Figure 12-2]Figure 12B shows STAT3 activation by xhCD8b antibody and fusion proteins of form F of various IL-10 polypeptides with amino acid substitutions to increase IL-10RB binding affinity and decrease IL-10RA binding affinity. Figure 12B shows STAT3 activation of CD8+ T cells and monocytes for five selected muteins in human PBMCs. [Figure 12-3] Figure 12C shows STAT3 activation by xhCD8b antibody and fusion proteins of form F of various IL-10 polypeptides with amino acid substitutions to increase IL-10RB binding affinity and decrease IL-10RA binding affinity. Figure 12D shows STAT3 activation of CD8+ T cells and monocytes for five additional selected muteins in human PBMCs. [Figure 12-4] Figure 12D shows STAT3 activation by xhCD8b antibody and fusion proteins of form F of various IL-10 polypeptides with amino acid substitutions to increase IL-10RB binding affinity and decrease IL-10RA binding affinity. Figure 12C shows STAT3 activation of CD8+ T cells and monocytes for five additional selected muteins in human PBMCs. [Figure 12-5] Figure 12B shows STAT3 activation by xhCD8b antibody and fusion proteins of form F of various IL-10 polypeptides with amino acid substitutions to increase IL-10RB binding affinity and decrease IL-10RA binding affinity. Figure 12C shows STAT3 activation of CD8+ T cells and monocytes for five selected muteins in human PBMCs. [Figure 12-6] Figure 12F shows STAT3 activation by xhCD8b antibody and fusion proteins of form F of various IL-10 polypeptides with amino acid substitutions to increase IL-10RB binding affinity and decrease IL-10RA binding affinity. Figure 12F shows STAT3 activation of CD8+ T cells and monocytes for five selected muteins in human PBMCs. [Figure 12-7]Figure 12G shows STAT3 activation by xhCD8b antibody and fusion proteins of form F of various IL-10 polypeptides with amino acid substitutions to increase IL-10RB binding affinity and decrease IL-10RA binding affinity. Figure 12G shows STAT3 activation of CD8+ T cells and monocytes for six selected muteins in whole blood. [Figure 12-8] Figure 12 shows STAT3 activation by xhCD8b antibody and fusion proteins of form F of various IL-10 polypeptides with amino acid substitutions to increase IL-10RB binding affinity and decrease IL-10RA binding affinity. Figure 12H shows STAT3 activation of CD8+ T cells and monocytes for six selected muteins in whole blood. [Figure 13] Figure 1 shows STAT3 activation in CD8 T cells and monocytes by xhCD8b and either IL10mono_RBenh2 or IL10mono_RBenh2_m117 fusion proteins, respectively. These STAT3 activation data were measured in human whole blood. DETAILED DESCRIPTION OF THE INVENTION

[0040] definition As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to a "molecule" optionally includes combinations of two or more such molecules, and the like.

[0041] It is understood that aspects and embodiments of the present disclosure include "comprising," "consisting of," and "consisting essentially of" aspects and embodiments.

[0042] As used herein, the term "about" refers to a normal error range for the respective value, which would be readily understood by one of ordinary skill in the art. Reference herein to "about" a value or parameter includes (and describes) embodiments that are directed to that value or parameter itself.

[0043] As used herein, "immune cells" refer to cells of the immune system that react to microorganisms or other elements considered foreign to the host's immune system. They protect the host from foreign pathogens, microorganisms, and disease. Immune cells, also called white blood cells, are involved in both innate and adaptive immune responses to combat pathogens. The innate immune response occurs immediately upon exposure to a pathogen without further priming or learning processes. The adaptive immune process requires initial priming, then creates memory, which results in enhanced responsiveness upon subsequent encounters with the same pathogen. 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. Adaptive immune cells include B and T lymphocytes / cells. T cell subsets include, but are not limited to, alpha beta CD4+ T (naive CD4+, memory CD4+, effector memory CD4+, effector CD4+, regulatory CD4+) and alpha beta CD8+ T (naive CD8+, memory CD8+, effector memory CD8+, effector CD8+). B cell subsets include, but are not limited to, naive B, memory B, and plasma cells. NK T cells and T gamma delta (Tγδ) cells exhibit characteristics of both innate and adaptive lymphocytes. In some embodiments, any immune cell herein is a human cell.

[0044] "T cells" or "T lymphocytes" are immune cells that play a key role in orchestrating immune responses in health and disease. There are two major T cell subsets with distinct functions and properties: 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 perforin, and T cells that express the CD4 antigen (CD4 + T cells) are CD8 +They are helper T cells that can regulate the function of many other immune cell types, including T cells, B cells, and macrophages. + T cells are further subdivided into several subsets, such as regulatory T (Treg) cells, which can suppress immune responses, and T helper 1 (Th1), T helper 2 (Th2), and T helper 17 (Th17) cells, which control various types of immune responses by secreting immunomodulatory proteins such as cytokines. T cells recognize targets via alpha-beta T cell receptors that bind to unique antigen-specific motifs, and this recognition mechanism is generally required to elicit cytotoxic and cytokine-secreting functions. "Innate lymphocytes" also possess CD8 receptors, which are capable of cytotoxic activity or secretion of Th1, Th2, and Th17 cytokines. + and CD4 + These innate lymphocyte subsets may exhibit characteristics of T cells. Some of these innate lymphocyte subsets include NK cells, as well as ILC1, ILC2, and ILC3 cells; and innate-like T cells, e.g., Tγδ cells; and NK T cells. Generally, these cells can respond rapidly to inflammatory stimuli from infected or damaged tissue, such as immunoregulatory cytokines, but unlike alpha beta T cells, they can respond without the need to recognize antigen-specific patterns.

[0045] A "cytokine" is a type of immunomodulatory polypeptide that mediates crosstalk between progenitor / primary cells and target / effector cells. It functions as a soluble or cell-surface-bound molecule and can bind to a "cytokine receptor" on target immune cells to activate signal transduction. A "cytokine receptor" (i.e., the IL-10 receptor, IL-10R, composed of two subunits, IL-10RA and IL-10RB) as used herein is a polypeptide on the cell surface that activates intracellular signal transduction upon binding to a cytokine on the extracellular cell surface. Cytokines include, but are not limited to, chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors. 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 pleiotropic, and receptors are expressed on multiple immune cell subsets, so a single cytokine can activate signal transduction pathways in multiple cells. However, depending on the cell type, signaling events in response to cytokines can result in different downstream cellular events such as activation, proliferation, survival, apoptosis, effector functions and secretion of other immunomodulatory proteins.

[0046] "Amino acid," as used herein, refers to naturally occurring carboxy α-amino acids, including alanine (three letter code: ala; one 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).

[0047] 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 is included within the definition of "polypeptide," and the term "polypeptide" may be used in place of or interchangeably with any of these terms. The term "polypeptide" is also intended to refer to the product of a polypeptide, whether from a natural biological source or produced by recombinant technology, but not necessarily translated from a designated nucleic acid sequence. It may be produced in any manner, including chemical synthesis. Polypeptides usually have a defined three-dimensional structure, but they do not necessarily have such a structure. Polypeptides of the present disclosure may be about 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 in size. Polypeptides that have a defined three-dimensional structure are referred to as folded, while polypeptides that do not have a defined three-dimensional structure but rather can adopt many different conformations are referred to as unfolded. Polypeptides may also form multimers, i.e., multimers consisting of two or more polypeptide molecules, such as dimers, trimers, and even higher oligomers. The polypeptide molecules forming dimers, trimers, etc. may or may not be identical. Accordingly, the corresponding higher order structures of such multimers are referred to as homo- or heterodimers, homo- or heterotrimers, etc. The terms "polypeptide" and "protein" also refer to modified polypeptides / proteins that are subject to post-expression modifications including, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or modification with non-naturally occurring amino acids.

[0048] "Residue," as used herein, refers to a position in a protein and the identity of its associated amino acid. For example, Leu 234 (also referred to as Leu234 or L234) is the residue at position 234 of the human antibody IgG1.

[0049] "Wild-type" herein means an amino acid or nucleotide sequence found in nature, including allelic variations. A wild-type protein has an amino acid or nucleotide sequence that has not been intentionally modified.

[0050] A "substitution" or "mutation" refers to an alteration of the polypeptide backbone in which an amino acid occurring in the wild-type sequence of the polypeptide is replaced with another amino acid at the same position in the polypeptide. In some embodiments, a mutation(s) is / are introduced to alter the affinity of the polypeptide for a receptor, thereby altering the activity so that it differs from the affinity and activity of the wild-type homologous polypeptide. Mutations may also improve the biophysical properties of the polypeptide. Amino acid mutations can be generated using genetic or chemical methods well known in the art. Genetic methods can include site-directed mutagenesis, PCR, gene synthesis, etc. It is anticipated that methods of modifying amino acid side groups by methods other than genetic engineering, such as chemical modification, may also be useful.

[0051] The terms "interleukin-10" or "IL-10," as used herein, unless otherwise specified, refer to any native IL-10 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). IL-10 typically exists as a homodimer. "IL-10" encompasses unprocessed IL-10 as well as "mature IL-10," the form of IL-10 that results from processing in cells. The sequence of "mature IL-10" is shown in FIG. 1A. An exemplary form of unprocessed human IL-10 contains an additional N-terminal amino acid signal peptide attached to mature IL-10. "IL-10" also includes naturally occurring variants of IL-10, such as alleles or splice variants or variants. The amino acid sequence of an exemplary human IL-10 is shown as UniProt P22301 (IL10_HUMAN).

[0052] "IL-10 homodimer" or "IL-10 dimer" refers to the originally symmetric homodimeric form of wild-type IL-10 polypeptide that binds to the tetrameric IL-10 receptor (IL-10R) complex on cells, consisting of two molecules of the IL-10R α chain (IL-10RA) and two molecules of the IL-10R β chain (IL-10RB). The α-helices of each IL-10 polypeptide chain intertwine such that the first four helices (A–D) of one chain associate with the last two helices (E and F) of the other chain, thereby maintaining the structural integrity of each domain upon dimerization (Walter & Nagabhushan, Biochemistry. 1995 Sep 26;34(38):12118-25). "IL-10 monomer" refers to a monomeric form of IL-10 that can be formed by extending the loops connecting the exchanged secondary structural elements. As described by Josephson et al., Biochemistry 1995 Sep 26;34(38):12118-25, insertion of six amino acids into the loop was sufficient to prevent dimerization and induce IL-10 monomer formation. The resulting IL-10 monomer was biologically active, capable of binding a single IL-10RA molecule and recruiting a single IL-10RB molecule to a signaling complex to elicit IL-10-mediated cellular responses. Thus, insertion of a short amino acid sequence or short linker into the sequence of an IL-10 polypeptide (i.e., wild-type IL-10 or any mutant IL-10 polypeptide of the present disclosure) between loop D (ending at residue C114) and loop E (starting at residue V121) results in a "monomeric isomer" of the IL-10 polypeptide. This additional amino acid sequence or linker can be inserted immediately after C114, E115, N116, K117, S118, K119, or A120. As described herein, the numbering of amino acids for IL-10 monomeric polypeptides is based on the numbering of SEQ ID NO: 1 (i.e., IL-10 dimeric polypeptides), so that the linker sequence / amino acid(s) are not counted. See, e.g., Figures 2B and 3B.

[0053] "Affinity" or "binding affinity" refers to the strength of the total 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 specified, as used herein, "binding affinity" refers to the intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). Affinity is generally determined by the dissociation constant (K), which is the ratio of the dissociation rate constant to the association rate constant (k and k, respectively). D ) Thus, equivalent affinities may involve different rate constants, as long as the ratio of the 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) technology (e.g., BIAcore), biolayer interference (BLI) technology (e.g., Octet), and other conventional binding assays (Heeley, Endocr Res 28, 217-229 (2002)).

[0054] "Binding" or "specific binding," as used herein, refers to the ability of a polypeptide or antigen-binding molecule to selectively interact with a receptor for the polypeptide or a target antigen, respectively, where this specific interaction can be distinguished from non-target or undesired or non-specific interactions.

[0055] A "mutated IL-10 polypeptide" refers to an IL-10 polypeptide having an amino acid sequence that differs from that of wild-type IL-10. For example, a mutant IL-10 polypeptide may have amino acid substitutions, deletions, and insertions. In some embodiments, a mutant IL-10 polypeptide has reduced affinity for the receptor, and such reduced affinity results in reduced biological activity of the mutant. Reduced affinity and therefore reduced activity can be achieved by introducing a small number of amino acid mutations or substitutions. A mutant IL-10 polypeptide may also have other modifications to the peptide backbone, including, but not limited to, amino acid deletions, substitutions, cyclization, disulfide bonds, or post-translational modifications (e.g., glycosylation or modified carbohydrates) of the polypeptide, chemical or enzymatic modifications to the polypeptide (e.g., attachment of PEG to the polypeptide backbone), addition of peptide tags or labels, or fusion with proteins or protein domains, that result in a final construct with desired properties, e.g., reduced affinity for the IL-10R. Desired activity may also include improved biophysical properties compared to wild-type IL-10 polypeptide. Multiple modifications can 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 may be incorporated into a polypeptide to allow glycosylation. Another non-limiting example is that lysine may be incorporated into a polypeptide to allow pegylation. In some embodiments, a mutation(s) is / are introduced into a polypeptide to modify activity by reducing affinity for a receptor.

[0056] "Targeting moiety" and "antigen-binding molecule," as used herein, refer in their broadest sense to a molecule that specifically binds to an antigenic determinant. A targeting moiety or antigen-binding molecule can be a protein, carbohydrate, lipid, or other compound.These include antibodies, antibody fragments (Chames et al., 2009; Chan & Carter, 2010; Leavy, 2010; Holliger & Hudson, 2005), scaffold antigen-binding proteins (Gebauer and Skerra, 2009; Stumpp et al., 2008), single-domain antibodies (sdAbs), minibodies (Tramontano et al., 1994), variable domains of heavy-chain antibodies (nanobodies, VHHs), variable domains of novel antigen receptors (VNARs), carbohydrate-binding domains (CBDs) (Blake et al., 2006), collagen-binding domains (Knight et al., 2000), lectin-binding proteins (tetranectins), collagen-binding proteins, adnectins / fibronectins (Lipovsek, 2011), serum transferrin (transbodies), Evibodies, and protein A-derived molecules, such as the Z-domain of protein A (affibodies) (Nygren et al., 2011). al, 2008), A-domains (avimers / maxibodies), alphabodies (WO2010066740), avimers / maxibodies, designed ankyrin repeat domains (DARPins) (Stumpp et al, 2008), anticalins (Skerra et al, 2008), human gamma-crystallin or ubiquitin (affilin molecules), Kunitz-type domains of human protease inhibitors, knottins (Kolmar et al, 2008), linear or restricted peptides with or without fusions to extend half-life, e.g., (Fc fusions-peptibodies) (Rentero Rebollo & Heinis, 2013; EP1144454B2; Shimamoto et al. al, 2012; US7205275B2), constrained bicyclic peptides (US2018 / 0200378A1), aptamers, engineered CH2 domains (nanobiobodies; Dimitrov, 2009)) and engineered CH3 domains "Fcab" domains (Wozniak-Knopp et al, 2010).

[0057] The terms "antibody" and "immunoglobulin" are used interchangeably and are used herein in the broadest sense to encompass a variety 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 (described in more detail herein), so long as they exhibit the desired antigen-binding activity.

[0058] In some embodiments, an antibody (immunoglobulin) refers to a protein having a structure substantially similar to that of a native antibody. A "native antibody" refers to a naturally occurring immunoglobulin molecule with a variety of structures. For example, the IgG class of native immunoglobulins is a heterotetrameric glycoprotein of approximately 150,000 daltons, composed of two disulfide-bonded 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 domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3), also called a heavy chain constant region. Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called a variable light domain or light chain variable domain, followed by a constant light (CL) domain, also called a light chain constant region. 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., W.H. Freeman and Co., page 91 (2007). Depending on the amino acid sequence of the constant domain of the heavy chain, antibodies (immunoglobulins) are assigned to different classes. There are five major 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 chains of immunoglobulins can be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domains. Immunoglobulins essentially consist of two Fab molecules and one Fc domain linked via an immunoglobulin hinge region.

[0059] "Fc," "Fc region," or "Fc domain," as used herein, refers to the C-terminal region of an antibody heavy chain containing at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions. Fc can refer to all or part of the last two constant region immunoglobulin domains (e.g., CH2 and CH3) of IgA, IgD, and IgG, the last three constant region immunoglobulin domains of IgE and IgM, and optionally the flexible hinge N-terminal to these domains. For IgA and IgM, Fc may also include the J chain. An IgG Fc region includes the IgG CH2 and IgG CH3 domains, and optionally the hinge. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, as described in Kabat et 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 the amino acid residue at about position 216 to the amino acid residue at about position 230. The hinge region may be a native hinge domain or a mutant hinge domain herein. The "CH2 domain" of a human IgG Fc region typically extends from the amino acid residue at about position 231 to the amino acid residue at about position 340. The CH2 domain may be a native-sequence CH2 domain or a mutant CH2 domain herein. The "CH3 domain" comprises the stretch of residues from the C-terminus of the Fc region to the CH2 domain, from the amino acid residue at about position 341 to the amino acid residue at about position 447 of IgG. The CH3 region, as used herein, may be a native sequence CH3 domain or a variant CH3 domain (e.g., a CH3 domain having a "protrusion" ("knob") incorporated into one of its chains and a corresponding "void" ("hole") incorporated into the other chain; see U.S. Pat. No. 5,821,333, expressly 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. An "Fc fragment," in this context, may include a small number of amino acids at either the N-terminus or C-terminus, or both, but still retains the ability to form dimers with another Fc domain or Fc fragment, which is also generally detectable using standard size-based methods (e.g., non-denaturing chromatography, size-exclusion chromatography, etc.). Human IgG Fc domains are particularly useful in the present disclosure and may be Fc domains derived from human IgG1, IgG2, or IgG4.

[0060] A "mutated Fc domain" or "Fc variant" or "mutated Fc" comprises amino acid modifications (e.g., substitutions, additions, and deletions) compared to a parent Fc domain. The term also includes naturally occurring allelic variants of the Fc region of an immunoglobulin. Generally, a mutant Fc domain has at least about 80, 85, 90, 95, 97, 98, or 99 percent identity (using the identity algorithms described below; one embodiment utilizes the BLAST algorithm known in the art using default parameters) with a corresponding parent human IgG Fc domain. Alternatively, a mutant Fc domain can 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 substantial loss of biological function. Moreover, as described herein, the variant Fc domain still retains the ability to form a dimer with another Fc domain, as determined using known techniques described herein, such as non-denaturing gel electrophoresis.

[0061] "Fc gamma receptor," "FcγR," or "Fc gamma R," as used herein, refers to any member of a family of proteins that binds the Fc region of an IgG antibody and are encoded by the FcγR gene. In humans, this family includes FcγRI (CD64), which includes the isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32), which includes the isoforms FcγRIIa (including allotypes H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII (CD16), which includes the isoforms FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIb-NA1 and FcγRIIb-NA2) (Jefferis et al., 2002, Immunol Lett 82:57-65, incorporated by reference in its entirety), and any undiscovered human FcγR or FcγR isoform or allotype. FcγRs may be derived from any organism, including, but not limited to, humans, mice, rats, rabbits, and monkeys. Mouse FcγRs include, but are not limited to, FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), and FcγRIII-2 (CD16-2), as well as any unidentified mouse FcγR or FcγR isoform or allotype.

[0062] As used herein, "effector function" refers to a biochemical event that occurs as a result of the interaction of an antibody Fc region with an Fc receptor or ligand, and varies depending on the antibody isotype. Effector functions include, but are not limited to, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), cytokine secretion, immune complex-associated 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 (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize antibody-bound on target cells and subsequently cause lysis of the target cells. ADCC correlates with FcγRIIIa binding, and increased FcγRIIIa binding leads to increased ADCC activity. To assess ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in U.S. Patent No. 5,500,362 or U.S. Patent No. 5,821,337, may be performed. "ADCP" or antibody-dependent cellular phagocytosis, as used herein, refers to a cell-mediated reaction in which nonspecific cytotoxic cells expressing FcγR recognize antibody bound on target cells, resulting in the subsequent phagocytosis of the target cells.

[0063] "Fc null" and "Fc null variant" are used interchangeably and are used herein to refer to a modified Fc with reduced or abolished effector function. Such Fc null or Fc null variants have reduced or abolished FcγR and / or complement receptor activity. In some embodiments, such Fc null or Fc null variants have abolished effector function. Exemplary methods for modification include, but are not limited to, chemical modification, amino acid residue substitution, insertion, and deletion. Exemplary amino acid positions in an Fc molecule (numbering according to the EU numbering system) into which one or more modifications have been incorporated to reduce the effector function of the resulting variant are 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.Exemplary Fc molecules with reduced effector function include those with 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 / L235A, L234F / L235A, L234F / L235B, L234F / L235C, L234F / L235D, L234F / L235E, L234F / L235F, L234F / L235F, L234F / L235C, L234F / L235F ... 35E / P331S, L234A / L235A, L234A / L235A / G237A, L234A / L235A / G237A / K322A, L234A / L235A / G237A / A 330S / A331S, L234A / L235A / P329G, E233P / L234V / L235A / G236del / S239K, E233P / L234V / L235A / G236 del / S267K, E233P / L234V / L235A / G236del / S239K / A327G, E233P / L234V / L235A / G236del / S267K / A32 7G and E233P / L234V / L235A / G236del, L234A / L235A / G237 deletion; ii) IgG2:A330S / A331S, V234A / G237A, V2 34A / 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.

[0064] As used herein, "epitope" refers to a determinant capable of specific binding to the variable region of an antibody molecule, known as the paratope. An epitope is a collection of molecules, such as amino acids or sugar side chains, that typically possess specific structural and charge characteristics. A single antigen may have more than one epitope. An epitope may include both amino acid residues directly involved in binding and other amino acid residues not directly involved in binding, such as those effectively blocked by the antigen-binding peptide (i.e., within the footprint of the antigen-binding peptide). Epitopes can be either conformational or linear. Epitopes generally contain at least three, more usually at least five or eight to ten amino acids. Antibodies that recognize the same epitope can be identified by simple immunoassays, such as "binning," that demonstrate the ability of an antibody to block the binding of another antibody to its target antigen.

[0065] 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, those disclosed in Protein Engineering, 9(3), 299-305, 1996). The length and sequence of the polypeptide linker are not particularly limited and can be selected by those skilled in the art according to the purpose. The polypeptide linker comprises one or more amino acids. In some embodiments, the polypeptide linker is a peptide at least 5 amino acids long, in some embodiments, 5 to 100 or 10 to 50 amino acids long. In one embodiment, the peptide linker is G, S, GS, SG, SGG, GGS, or 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), 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 is (GGGGS)xGn, where x=2, 3, or 4, and n=0 (SEQ ID NO:85), and in some embodiments, the linker is (GGGGS)xGn, where x=3, and n=0 (SEQ ID NO:86). In some embodiments, the linker comprises the sequence GGGGSGGGSGGGGS (SEQ ID NO: 79), SGGGGSGGGGSGGGGS (SEQ ID NO: 77), or SGGGGSGGGGSGGGG (SEQ ID NO: 78).Synthetic chemical linkers include cross-linking agents commonly used to cross-link 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-(succinimidooxycarbonyloxy)ethyl]sulfone (BSOCOES), and bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone (sulfo-BSOCOES).

[0066] "Percent (%) amino acid sequence identity" with respect to protein sequences is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in a particular (parent) sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, not counting any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be accomplished in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared. One particular program is the ALIGN-2 program outlined in paragraphs

[0279] to

[0280] of U.S. Patent Application Publication No. 20160244525, incorporated herein by reference.

[0067] The term "polynucleotide" refers to an isolated nucleic acid molecule or construct, e.g., messenger RNA (mRNA), viral-derived RNA, or plasmid DNA (pDNA), encoding a polypeptide of the present disclosure. A polynucleotide 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, e.g., DNA or RNA fragments, present in a polynucleotide. In some embodiments, one or more vectors (particularly expression vectors) containing such nucleic acids are provided. In one embodiment, a method for producing a polypeptide of the present disclosure is provided, comprising culturing a host cell containing a nucleic acid encoding the polypeptide under conditions suitable for expression of the polypeptide and recovering the polypeptide from the host cell. "Recombinant" means that the protein is produced in a foreign host cell using recombinant nucleic acid technology. A recombinantly produced protein expressed in a host cell is considered isolated for purposes of the present invention, as is a naturally occurring or recombinant protein that has been separated, fractionated, or partially or substantially purified by any appropriate technique.

[0068] The term "isolated," when used to describe various polypeptides disclosed herein, refers to a polypeptide that has been identified, separated, and / or recovered from the cell or cell culture in which it is expressed. Generally, an isolated polypeptide will have been purified by at least one purification step. No level of purity is required; "purified" or "purified" refers to increasing the concentration of the target protein relative to the concentration of contaminants in the composition compared to the starting material. As used herein, an "isolated protein" refers to a target protein that is substantially free of other proteins with different binding specificities.

[0069] The term "cancer" generally refers to a physiological condition in mammals characterized by uncontrolled and abnormal cell growth that may invade or spread to other parts of the body. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specific examples of such cancers include intractable types of any of the following cancers, or a combination of one or more of the following cancers: lung cancer, small cell lung cancer, non-small cell lung (NSCL) cancer, bronchioloalveolar carcinoma, squamous cell carcinoma, adenocarcinoma of the lung, squamous cell carcinoma of the lung, cancer of the peritoneum, head and neck cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, thyroid cancer, uterine cancer, gastrointestinal cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, gastric cancer, and the like, including refractory forms of any of the following cancers: cancer), colon cancer, breast cancer, endometrial cancer, uterine cancer, fallopian tube cancer, cervical carcinoma, vaginal carcinoma, vulvar cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, cancer of the endocrine system, thyroid cancer, parathyroid cancer, adrenal gland cancer, sarcoma of soft tissue, urethral cancer, penile cancer, prostate cancer, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvis carcinoma, mesothelioma, bladder cancer, liver cancer, hepatoma, hepatocellular carcinoma, cervical cancer, salivary gland cancer, bile duct cancer, neoplasms of the central nervous system (CNS), spinal axis tumors, brain stem glioma, glioblastoma multiforme, astrocytoma, schwannoma, ependymoma, medulloblastoma, meningioma, squamous cell carcinoma, pituitary adenoma, and Ewing's sarcoma.

[0070] Mutant IL-10 polypeptides In some embodiments, the present disclosure relates to mutated IL-10 polypeptides and fusion proteins thereof. In some embodiments, the mutated IL-10 polypeptides comprise one or more mutations (e.g., relative to SEQ ID NO: 1) that increase binding affinity to an IL-10RB polypeptide (e.g., comprising the sequence of SEQ ID NO: 3). In some embodiments, the mutated IL-10 polypeptides comprise one or more mutations (e.g., relative to SEQ ID NO: 1) that decrease binding affinity to an IL-10RA polypeptide (e.g., comprising the sequence of SEQ ID NO: 2). In some embodiments, the mutated IL-10 polypeptides comprise one or more mutations (e.g., relative to SEQ ID NO: 1) that increase binding affinity to an IL-10RB polypeptide (e.g., comprising the sequence of SEQ ID NO: 3) and one or more mutations (e.g., relative to SEQ ID NO: 1) that decrease binding affinity to an IL-10RA polypeptide (e.g., comprising the sequence of SEQ ID NO: 2). In some embodiments, the mutant IL-10 polypeptide comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence of either the wild-type mature IL-10 shown in FIG. 1A or the mature monomeric IL-10 shown in FIG. 1D.

[0071] In some embodiments, the mutant IL-10 polypeptide comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence of either the wild-type mature IL-10 shown in FIG. 1A or the mature monomeric IL-10 shown in FIG. 1D. In some embodiments, the mutant IL-10 polypeptide i) exhibits reduced binding affinity for an IL-10RA polypeptide having the amino acid sequence depicted in Figure 1B, and ii) has one or more amino acid substitutions selected from the group consisting of P20, L23, R24, R27, D28, K34, T35, Q38, M39, D41, L43, D44, N45, L46, K49, 187, V91, L94, L98, K138, S141, E142, D144, N148, E151, and 1158 relative to the amino acid sequence of the wild-type IL-10 polypeptide depicted in Figure 1A or the mature monomeric IL-10 depicted in Figure 1D, as depicted in Figures 2A-3B. In some embodiments, the mutant IL-10 polypeptides of the present disclosure exhibit binding affinity that is reduced by 50% or more for an IL-10RA polypeptide having the amino acid sequence depicted in Figure 1B. The difference in binding affinity of wild-type and mutant IL-10 polypeptides for IL-10RA is measured using standard SPR assays that measure the affinity of protein-protein interactions, as is well known to those skilled in the art.

[0072] In some embodiments, the mutant IL-10 polypeptide comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence of either the wild-type mature IL-10 shown in FIG. 1A or the mature monomeric IL-10 shown in FIG. 1D. In some embodiments, the mutant IL-10 polypeptide i) exhibits increased binding affinity to an IL-10RB polypeptide having the amino acid sequence depicted in FIG. 1C; and ii) may have one or more amino acid substitutions selected from the group consisting of N18, N21, M22, R24, D25, D28, S31, R32, D55, M68, I69, L73, E74, M77, P78, Q79, E81, N82, K88, A89, H90, N92, S93, G95, E96, N97, K99, T100, L101, L103, R104, R107, R110, and F111 relative to the amino acid sequence of the wild-type mature IL-10 polypeptide depicted in FIG. 1A (FIGS. 2A-3B). In yet another embodiment, the mutant IL-10 polypeptide exhibits a binding affinity that is increased by 150% or more for an IL-10RB polypeptide having the amino acid sequence shown in Figure 1C.

[0073] The locations of possible amino acid substitutions in the sequence of a wild-type mature IL-10 polypeptide are shown in Figure 2. In some embodiments, the amino acid shown in the sequence of a wild-type mature IL-10 polypeptide has been substituted with alanine or another amino acid as shown in Figure 3.

[0074] In some embodiments, the mutant IL-10 polypeptides also contain other modifications, including, but not limited to, mutations and deletions, that provide additional advantages, such as improved biophysical properties, including, but not limited to, improved thermal stability, aggregation tendency, acid reversibility, viscosity, and production in mammalian or bacterial or yeast cells.

[0075] In some embodiments, the mutated IL-10 polypeptide is a monomer, e.g., as described herein. See, e.g., SEQ ID NO: 187 shown in Figures 1D, 2B, and 3B. In some embodiments, the mutated IL-10 monomer polypeptide comprises the amino acid substitutions N18I, N92I, K99N, and F111L, e.g., as shown in Table 4A below as SEQ ID NO: 188. In some embodiments, the mutated IL-10 monomer polypeptide comprises the amino acid sequence listed in Table 4A. In some embodiments, the mutated IL-10 monomer polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 188-201. In some embodiments, the mutated IL-10 monomer polypeptide comprises the amino acid substitutions N18I, N92I, K99N, and F111L and one or more additional amino acid substitutions. In some embodiments, the one or more additional amino acid substitutions are at position(s) R24, R27, Q38, I87, K138, E142, D144, and / or E151. In some embodiments, the mutated IL-10 monomer polypeptide comprises the amino acid substitutions N18I, Q38A, N92I, K99N, and F111L. In some embodiments, the mutated IL-10 monomer polypeptide comprises the sequence of SEQ ID NO: 189. In some embodiments, the mutated IL-10 monomer polypeptide comprises the amino acid substitutions N18I, R24A, N92I, K99N, and F111L. In some embodiments, the mutated IL-10 monomer polypeptide comprises the sequence of SEQ ID NO: 190. In some embodiments, the mutated IL-10 monomer polypeptide comprises the amino acid substitutions N18I, R24A, Q38A, N92I, K99N, and F111L. In some embodiments, the mutated IL-10 monomer polypeptide comprises the sequence of SEQ ID NO: 191. In some embodiments, the mutated IL-10 monomer polypeptide comprises the amino acid substitutions N18I, N92I, K99N, F111L, and E151A. In some embodiments, the mutated IL-10 monomer polypeptide comprises the sequence of SEQ ID NO: 192. In some embodiments, the mutated IL-10 monomer polypeptide comprises the amino acid substitutions N18I, R24A, N92I, K99N, F111L, and E151A.In some embodiments, the mutated IL-10 monomer polypeptide comprises the sequence of SEQ ID NO: 193. In some embodiments, the mutated IL-10 monomer polypeptide comprises the amino acid substitutions N18I, I87A, N92I, K99N, and F111L. In some embodiments, the mutated IL-10 monomer polypeptide comprises the sequence of SEQ ID NO: 194. In some embodiments, the mutated IL-10 monomer polypeptide comprises the amino acid substitutions N18I, N92I, K99N, F111L, and K138A. In some embodiments, the mutated IL-10 monomer polypeptide comprises the sequence of SEQ ID NO: 195. In some embodiments, the mutated IL-10 monomer polypeptide comprises the amino acid substitutions N18I, R27A, N92I, K99N, and F111L. In some embodiments, the mutated IL-10 monomer polypeptide comprises the sequence of SEQ ID NO: 196. In some embodiments, the mutated IL-10 monomer polypeptide comprises the amino acid substitutions N18I, N92I, K99N, F111L, and E142A. In some embodiments, the mutated IL-10 monomer polypeptide comprises the sequence of SEQ ID NO: 197. In some embodiments, the mutated IL-10 monomer polypeptide comprises the amino acid substitutions N18I, N92I, K99N, F111L, and D144A. In some embodiments, the mutated IL-10 monomer polypeptide comprises the sequence of SEQ ID NO: 198. In some embodiments, the mutated IL-10 monomer polypeptide comprises the amino acid substitutions N18I, Q38A, N92I, K99N, F111L, and E142A. In some embodiments, the mutated IL-10 monomer polypeptide comprises the sequence of SEQ ID NO: 199. In some embodiments, the mutated IL-10 monomer polypeptide comprises the amino acid substitutions N18I, N92I, K99N, F111L, E142A, and K138A. In some embodiments, the mutated IL-10 monomer polypeptide comprises the sequence of SEQ ID NO: 200. In some embodiments, the mutated IL-10 monomer polypeptide comprises the amino acid substitution N92I. In some embodiments, the mutated IL-10 monomer polypeptide comprises the sequence of SEQ ID NO: 201.In some embodiments, the mutated IL-10 monomer polypeptide comprises a sequence selected from the group consisting of SEQ ID NOs: 87-89 and 188-201. In some embodiments, the mutated IL-10 monomer polypeptide comprises the sequence of a mutated monomeric IL-10 polypeptide shown in Table 4A. In some embodiments, the mutated IL-10 monomer polypeptide comprises an amino acid substitution and / or amino acid insertion sequence of a mutated monomeric IL-10 polypeptide shown in Table 4A. [Table 1-1] [Table 1-2] [Table 2-1] [Table 2-2]

[0076] In some embodiments, the mutated IL-10 monomer polypeptide comprises a sequence selected from the group consisting of SEQ ID NOs: 310-318. In some embodiments, the mutated IL-10 monomer polypeptide comprises the sequence of a mutated monomeric IL-10 polypeptide shown in Table 8. In some embodiments, the mutated IL-10 monomer polypeptide comprises an amino acid substitution and / or amino acid insertion sequence of a mutated monomeric IL-10 polypeptide shown in Table 8. [Table 3]

[0077] In some embodiments, the mutated IL-10 monomer polypeptide comprises a sequence selected from the group consisting of SEQ ID NOs: 422-428. In some embodiments, the mutated IL-10 monomer polypeptide comprises the sequence of a mutated monomeric IL-10 polypeptide shown in Table 11. In some embodiments, the mutated IL-10 monomer polypeptide comprises an amino acid substitution and / or amino acid insertion sequence of a mutated monomeric IL-10 polypeptide shown in Table 11.

[0078] Table 4B shows exemplary amino acid insertions and positions of insertions for IL-10 monomer polypeptides of the present disclosure (insertions are underlined). In some embodiments, a mutant IL-10 monomer polypeptide comprises an amino acid sequence listed in Table 4B. In some embodiments, a mutant IL-10 monomer polypeptide comprises a sequence selected from the group consisting of SEQ ID NOs: 91-101. In some embodiments, a mutant IL-10 monomer polypeptide comprises an amino acid insertion listed in Table 4B and / or at a position listed in Table 4B. In some embodiments, the insertion is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in length. In some embodiments, the mutated IL-10 monomer polypeptide comprises the amino acid sequence of a mutated IL-10 monomer polypeptide of the present disclosure with a peptide insertion of one amino acid, or between 1 and 15 amino acids, immediately following residues C114, E115, N116, K117, S118, K119, or A120, numbered based on SEQ ID NO: 1. Examples of insertions can include, but are not limited to, G, GG, GGG, GGGG (SEQ ID NO: 80), GGGSG (SEQ ID NO: 81), GGGGG (SEQ ID NO: 82), GGGGGG (SEQ ID NO: 83), and GGGSGG (SEQ ID NO: 84). [Table 4-1] [Table 4-2]

[0079] fusion proteins Further provided herein is a fusion protein comprising any one of the mutated IL-10 polypeptides of the present disclosure and an antigen binding molecule that binds to an antigen on T cells. In some embodiments, the fusion protein preferentially stimulates T cells over monocytes. In some embodiments, a fusion protein of the present disclosure comprises a mutated IL-10 polypeptide and an antigen binding molecule that binds to CD8+ T cells, and the fusion protein preferentially stimulates CD8+ T cells over monocytes. In some embodiments, the antigen binding molecule binds to CD8 (e.g., CD8ab, CD8a, or CD8aa), CD4, or PD-1, e.g., human CD8 (e.g., human CD8ab, human CD8a, or human CD8aa), human CD4, or human PD-1. Human CD8, CD4, and PD-1 sequences are known in the art, see e.g., NP_001139345 for human CD8a, NP_001171571 for human CD8b, NP_000607 for human CD4, and NP_005009 for human PD-1.

[0080] In another embodiment, a fusion protein comprises a mutant IL-10 polypeptide and an antigen binding molecule that binds to CD8ab and / or CD8a antigens, wherein the fusion protein preferentially stimulates CD8+ T cells over monocytes.

[0081] The preferential activity of the targeted IL-10 fusion proteins of the present disclosure against antigen-expressing cells is demonstrated in assays including antigen-expressing cells and non-antigen-expressing cells that also express IL-10R. One such assay is an in vitro assay that measures STAT3 phosphorylation (pSTAT3) in human immune cells, such as human peripheral blood cells and / or tumor-infiltrating immune cells, upon exposure to an IL-10 polypeptide. In one assay format, the activity of the targeted IL-10 fusion protein against antigen-expressing and non-antigen-expressing cells is measured to demonstrate selectivity for antigen-expressing cells. In another assay format, the activity of the targeted IL-10 fusion protein comprising a mutant IL-10 polypeptide against antigen-expressing cells is compared to the activity of a non-targeted IL-10 fusion protein comprising the same mutant IL-10 polypeptide and a control antibody that does not recognize any antigen on antigen-expressing cells to demonstrate the magnitude of signaling rescue of the mutant IL-10 polypeptide when fused to an antigen-binding molecule.

[0082] In some embodiments, a fusion protein of the present disclosure comprising a CD8ab antigen binding molecule activates CD8ab+ IL-10R+ cells at least 5-fold, at least 10-fold, at least 50-fold, or at least 100-fold more than CD8ab- IL-10R+ cells. In some embodiments, the fusion protein activates CD8ab+ IL-10R+ cells more than 50-fold, or more desirably at least 100-fold, or even more desirably at least 200-fold, compared to a fusion molecule comprising the IL-10 mutant polypeptide and a control antibody that does not bind to any antigen expressed on the cells. The cell activation by the IL-10 fusion protein is determined by measuring pSTAT3 expression in the cells after treatment with the IL-10 fusion protein.

[0083] In some embodiments, a fusion protein of the present disclosure comprising a CD8a antigen binding molecule activates CD8a+ IL-10R+ cells at least 5-fold, at least 10-fold, at least 50-fold, or at least 100-fold more than CD8a- IL-10R+ cells. In some embodiments, the fusion protein activates CD8a+ IL-10R+ cells more than 50-fold, or more desirably at least 100-fold, or even more desirably at least 200-fold, compared to a fusion molecule comprising the IL-10 mutant polypeptide and a control antibody that does not bind to any antigen expressed on the cells. The cell activation by the IL-10 fusion protein is determined by measuring pSTAT3 expression in the cells after treatment with the IL-10 fusion protein.

[0084] Fusion protein format The present disclosure relates, inter alia, to fusion proteins comprising an antigen-binding molecule (e.g., an antibody or other antigen-binding protein) and a mutated IL-10 polypeptide of the present disclosure. In some embodiments, the IL-10 fusion protein has various formats, for example, as shown in FIG. 6. In some embodiments, the fusion protein comprises a dimer of two mutated IL-10 polypeptides, one of the two mutated IL-10 polypeptides being fused to the antigen-binding molecule (e.g., an IL-10 dimer is fused to the antigen-binding molecule by a single bond). In some embodiments, the fusion protein comprises a single mutated monomeric IL-10 polypeptide fused to an antigen-binding molecule. In some embodiments, the fusion protein comprises two antigen-binding molecules, one mutated IL-10 polypeptide being fused to each of the two antigen-binding molecules (e.g., an antigen-binding molecule comprises two polypeptide chains, each fused to a single mutated IL-10 polypeptide, and the two mutated IL-10 polypeptides are associated as a dimer upon assembly of the fusion protein). In some embodiments, the mutant IL-10 polypeptide and the antigen binding molecule are fused (eg, covalently) via a linker.

[0085] In some embodiments, the fusion protein has formula [I], from N-terminus to C-terminus: VH-CH1-hinge-CH2-CH3 [I] and two antibody heavy chain polypeptides comprising a structure according to formula [II], from N-terminus to C-terminus: VL-CL [II] and two antibody light chain polypeptides comprising a structure according to the formula: where VH is an antibody heavy chain variable (VH) domain, CH1 is an antibody CH1 domain, hinge is an antibody hinge domain, CH2 is an antibody CH2 domain, CH3 is an antibody CH3 domain, VL is an antibody light chain variable (VL) domain, and CL is an antibody constant light chain domain. See, e.g., Figure 6A, D, and F. In some embodiments, VH / VL form an antigen-binding site.

[0086] In some embodiments, the fusion protein comprises two mutant IL-10 polypeptides linked in a dimer, wherein the N-terminus of one of the two mutant IL-10 polypeptides is fused, directly or via a linker, to the C-terminus of one of the two CH3 domains, for example, as shown in Figure 6A. In some embodiments, the fusion protein comprises two mutant IL-10 polypeptides linked in a dimer, wherein the N-terminus of a first of the two mutant IL-10 polypeptides is fused, directly or via a linker, to the C-terminus of a first of the two CH3 domains, and the N-terminus of a second of the two mutant IL-10 polypeptides is fused, directly or via a linker, to the C-terminus of a second of the two CH3 domains, for example, as shown in Figure 6D. In some embodiments, the fusion protein comprises one mutant IL-10 monomer polypeptide, wherein the N-terminus of the mutant IL-10 monomer polypeptide is fused, directly or via a linker, to the C-terminus of one of the two CH3 domains, for example, as shown in Figure 6F.

[0087] In some embodiments, the fusion protein has formula [I], from N-terminus to C-terminus: VH-CH1-hinge-CH2-CH3 [I], and a first antibody heavy chain polypeptide comprising a structure according to formula [II], from the N-terminus to the C-terminus: VL-CL [II], and an antibody light chain polypeptide comprising a structure according to formula [III], from the N-terminus to the C-terminus: Hinge-CH2-CH3 [III], and a second antibody heavy chain polypeptide comprising a structure according to: where VH is an antibody heavy chain variable (VH) domain, CH1 is an antibody CH1 domain, hinge is an antibody hinge domain, CH2 is an antibody CH2 domain, CH3 is an antibody CH3 domain, VL is an antibody light chain variable (VL) domain, and CL is an antibody constant light chain domain. See, e.g., Figure 6, B, C, E, G, and H. In some embodiments, VH / VL form an antigen-binding site.

[0088] In some embodiments, the fusion protein comprises two mutated IL-10 polypeptides linked in a dimer, wherein the N-terminus of one of the two mutated IL-10 polypeptides is fused, directly or via a linker, to either the C-terminus of the CH3 domain of a second antibody heavy chain polypeptide or the C-terminus of the CH3 domain of a first antibody heavy chain polypeptide, for example, as shown in Figure 6B. In some embodiments, the fusion protein comprises two mutated IL-10 polypeptides linked in a dimer, wherein the N-terminus of one of the two mutated IL-10 polypeptides is fused, directly or via a linker, to the N-terminus of the hinge region of a second antibody heavy chain polypeptide, for example, as shown in Figure 6C. In some embodiments, the fusion protein comprises two mutated IL-10 polypeptides linked in a dimer, wherein the N-terminus of a first of the two mutated IL-10 polypeptides is fused, directly or via a linker, to the C-terminus of the CH3 domain of a first antibody heavy chain polypeptide, and the N-terminus of the second of the two mutated IL-10 polypeptides is fused, directly or via a linker, to the C-terminus of the CH3 domain of a second antibody heavy chain polypeptide, e.g., as shown in Figure 6E. In some embodiments, the fusion protein comprises one mutated IL-10 monomer polypeptide, wherein the N-terminus of the mutated IL-10 monomer polypeptide is fused, directly or via a linker, to either the C-terminus of the CH3 domain of a second antibody heavy chain polypeptide or the C-terminus of the CH3 domain of a first antibody heavy chain polypeptide, e.g., as shown in Figure 6G and H.

[0089] In some embodiments, the fusion protein is as shown in Figure 6A. For example, in some embodiments, the fusion protein comprises the following structure: VH-CH1-hinge-CH2-CH3 [I] and two antibody heavy chain polypeptides comprising a structure according to formula [II], from N-terminus to C-terminus: VL-CL [II] and two antibody light chain polypeptides comprising a structure according to the formula: where VH is an antibody heavy chain variable (VH) domain, CH1 is an antibody CH1 domain, hinge is an antibody hinge domain, CH2 is an antibody CH2 domain, CH3 is an antibody CH3 domain, VL is an antibody light chain variable (VL) domain, and CL is an antibody constant light chain domain; the fusion protein comprises two mutant IL-10 polypeptides linked in a dimer, and the N-terminus of one of the two mutant IL-10 polypeptides is fused to the C-terminus of one of the two CH3 domains (e.g., covalently fused via a linker of the present disclosure). In some embodiments, each heavy chain is paired with a light chain. In some embodiments, the VH domain of each heavy chain forms an antigen-binding site with the VL domain of the respective paired light chain.

[0090] In some embodiments, the fusion protein is as shown in Figure 6B. For example, in some embodiments, the fusion protein comprises the formula [I], from N-terminus to C-terminus: VH-CH1-hinge-CH2-CH3 [I] and a first antibody heavy chain polypeptide comprising a structure according to formula [II], from the N-terminus to the C-terminus: VL-CL [II] and an antibody light chain polypeptide comprising a structure according to formula [III], from the N-terminus to the C-terminus: Hinge-CH2-CH3 [III], and a second antibody heavy chain polypeptide comprising a structure according to the formula: wherein VH is an antibody heavy chain variable (VH) domain, CH1 is an antibody CH1 domain, hinge is an antibody hinge domain, CH2 is an antibody CH2 domain, CH3 is an antibody CH3 domain, VL is an antibody light chain variable (VL) domain, and CL is an antibody constant light chain domain, and the fusion protein comprises two mutated IL-10 polypeptides linked in a dimer. In some embodiments, the N-terminus of one of the two mutated IL-10 polypeptides is fused to the C-terminus of the CH3 domain of the second antibody heavy chain polypeptide (e.g., covalently fused via a linker of the present disclosure). In some embodiments, the N-terminus of one of the two mutated IL-10 polypeptides is fused to the C-terminus of the CH3 domain of the first antibody heavy chain polypeptide (e.g., covalently fused via a linker of the present disclosure). In some embodiments, the first heavy chain is paired with a light chain. In some embodiments, the VH domain of the first heavy chain forms an antigen-binding site with the VL domain of the light chain.

[0091] In some embodiments, the fusion protein is as shown in Figure 6D. For example, in some embodiments, the fusion protein comprises the formula [I], from N-terminus to C-terminus: VH-CH1-hinge-CH2-CH3 [I] and two antibody heavy chain polypeptides comprising a structure according to formula [II], from N-terminus to C-terminus: VL-CL [II] and two antibody light chain polypeptides having a structure according to the formula: where VH is an antibody heavy chain variable (VH) domain, CH1 is an antibody CH1 domain, hinge is an antibody hinge domain, CH2-CH3 are antibody Fc domains, VL is an antibody light chain variable (VL) domain, and CL is an antibody constant light chain domain; the fusion protein comprises two mutant IL-10 polypeptides linked in a dimer, wherein the N-terminus of a first of the two mutant IL-10 polypeptides is fused to the C-terminus of a first of the two CH3 domains, and the N-terminus of a second of the two mutant IL-10 polypeptides is fused to the C-terminus of a second of the two CH3 domains (e.g., covalently fused via a linker of the present disclosure). In some embodiments, each heavy chain is paired with a light chain. In some embodiments, the VH domain of each heavy chain forms an antigen-binding site with the VL domain of the respective paired light chain.

[0092] In some embodiments, the fusion protein is as shown in Figure 6E. For example, in some embodiments, the fusion protein comprises the following structure: VH-CH1-hinge-CH2-CH3 [I] and a first antibody heavy chain polypeptide comprising a structure according to formula [II], from the N-terminus to the C-terminus: VL-CL [II] and an antibody light chain polypeptide comprising a structure according to formula [III], from the N-terminus to the C-terminus: Hinge-CH2-CH3 [III], and a second antibody heavy chain polypeptide comprising a structure according to the formula: wherein VH is an antibody heavy chain variable (VH) domain, CH1 is an antibody CH1 domain, hinge is an antibody hinge domain, CH2 is an antibody CH2 domain, CH3 is an antibody CH3 domain, VL is an antibody light chain variable (VL) domain, and CL is an antibody constant light chain domain; the fusion protein comprises two mutated IL-10 polypeptides linked in a dimer, wherein the N-terminus of a first of the two mutated IL-10 polypeptides is fused to the C-terminus of the CH3 domain of the first antibody heavy chain polypeptide, and the N-terminus of a second of the two mutated IL-10 polypeptides is fused to the C-terminus of the CH3 domain of the second antibody heavy chain polypeptide (e.g., covalently fused via a linker of the present disclosure). In some embodiments, the first heavy chain is paired with a light chain. In some embodiments, the VH domain of the first heavy chain forms an antigen-binding site with the VL domain of the light chain.

[0093] In some embodiments, the fusion protein is as shown in Figure 6F. For example, in some embodiments, the fusion protein comprises the formula [I], from N-terminus to C-terminus: VH-CH1-hinge-CH2-CH3 [I] and two antibody heavy chain polypeptides comprising a structure according to formula [II], from N-terminus to C-terminus: VL-CL [II] and two antibody light chain polypeptides having a structure according to the formula: where VH is an antibody heavy chain variable (VH) domain, CH1 is an antibody CH1 domain, hinge is an antibody hinge domain, CH2 is an antibody CH2 domain, CH3 is an antibody CH3 domain, VL is an antibody light chain variable (VL) domain, and CL is an antibody constant light chain domain; the fusion protein comprises one mutant monomeric IL-10 polypeptide, the N-terminus of which is fused to the C-terminus of one of the two CH3 domains (e.g., covalently fused via a linker of the present disclosure). In some embodiments, each heavy chain is paired with a light chain. In some embodiments, the VH domain of each heavy chain forms an antigen-binding site with the VL domain of the respective paired light chain.

[0094] In some embodiments, the first and second Fc domains of the fusion protein comprise one or more of the following Fc mutations according to EU numbering: L234A, L235A, G237A, and K322A to reduce effector function. In some embodiments, the first and second Fc domains of the fusion protein comprise the following Fc mutations according to EU numbering: L234A, L235A, and G237A to reduce effector function. In some embodiments, the first and second Fc domains of the fusion protein comprise the following Fc mutations according to EU numbering: L234A, L235A, G237A, and K322A to reduce effector function. In some embodiments, the first and second Fc domains of the fusion protein comprise the following amino acid substitutions: Y349C / T366W (knob) and S354C, T366S, L368A, and Y407V (hole) to promote heterodimer formation. In some embodiments, one or both of the antibody Fc domains does not have a C-terminal lysine. In some embodiments, the first and second Fc domains are human IgG1 Fc domains.

[0095] In some embodiments, bispecific antibodies can be formed by post-production assembly of half antibodies, thereby resolving the problem of mispairing of heavy and light chains. These antibodies often contain modifications to promote heterodimerization of the half 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 duobodies (F405L-K409R). In such cases, the half antibodies are individually produced and purified in separate cell lines. The purified antibodies are then subjected to mild reduction to obtain the half antibodies that are subsequently assembled into the bispecific antibody. The heterodimeric bispecific antibody is then purified from the mixture using conventional purification methods.

[0096] In some embodiments, strategies for generating bispecific antibodies that do not rely on preferential chain pairing can also be used. These strategies generally involve introducing genetic modifications into antibodies in such a way that the heterodimer has different biochemical or biophysical properties than the homodimer, allowing the constructed or expressed heterodimer to be selectively purified from the homodimer. One example is introducing H435R / Y436F into the IgG1 CH3 domain to abolish Fc binding to Protein A resin, followed by co-expression of the H435R / Y436F variant with wild-type Fc. The resulting homodimeric antibody containing two copies of H435R / Y436F will be unable to bind to a Protein A column, while a heterodimeric antibody containing one copy of the H435R / Y436F mutation will have reduced affinity for Protein A compared to the strong interaction of the homodimeric wild-type antibody (Tustian et al., 2016). Other examples include kappa / lambda antibodies (Fischer et al., Nature Communication 2015) and the introduction of differential charges into each chain (E357Q, S267K or N208D / Q295E / N384D / Q418E / N421D) (US2018 / 0142040A1; (Strop et al. J Mol Biol (2012)).

[0097] In some embodiments, bispecific antibodies can be generated by fusing additional binding sites to either the immunoglobulin heavy or light chain, including, but not limited to, variable regions, scFv, Fab, VHH, and peptides.

[0098] In some embodiments, heterodimerization mutations and / or mutations altering Fc gamma receptor binding resulted in decreased Fc stability. Therefore, additional mutation(s) that enhance stability were added to the Fc region. For example, one or more pairs of disulfide bonds, such as A287C and L306C, V259C and L306C, R292C and V302C, and V323C and I332C, are introduced into the Fc region. Another example is the introduction of S228P into an IgG4-based bispecific antibody to stabilize the hinge disulfide. Further examples include the introduction of K338I, A339K, and K340S mutations to improve Fc stability and aggregation resistance (Gao et al., 2019 Mol Pharm. 2019;16:3647).

[0099] In some embodiments, the fusion proteins of the present disclosure include a linker. In some embodiments, the linker can be a chemical linker (see, for example, those disclosed in Protein Engineering, 9(3), 299-305, 1996). Synthetic chemical linkers include cross-linking agents commonly used to cross-link 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-(succinimidooxycarbonyloxy)ethyl]sulfone (BSOCOES), and bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone (sulfo-BSOCOES).

[0100] In some embodiments, the linker is an amino acid or peptide-based linker. In some embodiments, the polypeptide linker is a peptide at least 5 amino acids in length, or 5 to 100, or 10 to 50 amino acids in length. 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 comprises 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 comprises the sequence GGGGSGGGSGGGGS (SEQ ID NO: 79), SGGGGSGGGGSGGGGS (SEQ ID NO: 77), or SGGGGSGGGGSGGGG (SEQ ID NO: 78).

[0101] antigen binding molecule In some embodiments, an antigen binding molecule of the present disclosure binds to an epitope on CD8a, and binding of the antigen binding molecule to CD8a does not interfere with interaction of CD8aa or CD8ab with an MHC class I molecule on a target cell or antigen-presenting cell. In some embodiments, an antigen binding molecule of the present disclosure binds to an epitope on CD8b, and binding of the antigen binding molecule to CD8b does not interfere with interaction of CD8ab with an MHC class I molecule on a target cell or antigen-presenting cell.

[0102] In some embodiments, the fusion protein binds to human CD8, and the binding of the fusion protein to CD8 does not interfere with the interaction of CD8 with MHC class I. In some embodiments, the antigen binding molecule of the present disclosure binds to an epitope on CD8ab, and the binding of the antigen binding molecule to CD8ab does not interfere with the interaction of CD8aa or CD8ab with an MHC class I molecule on a target cell or antigen-presenting cell. In some embodiments, the antigen binding molecule of the present disclosure binds to an epitope on CD8a, and the binding of the antigen binding molecule to CD8a does not interfere with the interaction of CD8ab with an MHC class I molecule on a target cell or antigen-presenting cell.

[0103] In some embodiments, the fusion protein binds to human CD8, and the binding of the fusion protein to CD8 does not interfere with the interaction of CD8 with MHC class I. In some embodiments, an antigen binding molecule of the present disclosure binds to an epitope on CD8α, and the binding of the antigen binding molecule to CD8α does not interfere with the interaction of CD8αα or CD8αβ with an MHC class I molecule on a target cell or antigen-presenting cell. In some embodiments, an antigen binding molecule of the present disclosure binds to an epitope on CD8β, and the binding of the antigen binding molecule to CD8β does not interfere with the interaction of CD8αβ with an MHC class I molecule on a target cell or antigen-presenting cell. In some embodiments, whether an anti-CD8 antibody or fusion protein of the present disclosure interferes with the interaction of CD8 with MHC class I can be assayed, for example, by assaying the activation state of CD8+ T cells (e.g., upon antigen stimulation) in the presence or absence of the anti-CD8 antibody or fusion protein.

[0104] In some embodiments, an anti-CD8 antibody or fusion protein of the disclosure comprises a VH domain comprising the sequence of EVQLVESGGGLVQPGRSLKLSCAASGFTFSNYYMAWVRQAPTKGLEWVAYINTGGGTTYYRDSVKGRFTISRDDAKSTLYLQMDSLRSEDTATYYCTTAIGYYFDYWGQGVMVTVSS (SEQ ID NO: 102) and a VL domain comprising the sequence of DIQLTQSPASLSASLGETVSIECLASEDIYSYLAWYQQKPGKSPQVLIYAANRLQDGVPSRFSGSGSGTQYSLKISGMQPEDEGDYFCLQGSKFPYTFGAGTKLELK (SEQ ID NO: 103). In some embodiments, an anti-CD8 antibody or fusion protein of the disclosure comprises a VH domain comprising the sequence of EVKLQESGPSLVQPSQTLSLTCSVSGFSLISDSVHWVRQPPGKGLEWMGGIWADGSTDYNSALKSRLSISRDTSKSQGFLKMNSLQTDDTAIYFCTSNRESYYFDYWGQGTMVTVSS (SEQ ID NO: 104) and a VL domain comprising the sequence of DIQMTQSPASLSASLGDKVTITCQASQNIDKYIAWYQQKPGKAPRQLIHYTSTLVSGTPSRFSGSGSGRDYSFSISSVESEDIASYYCLQYDTLYTFGAGTKLELK (SEQ ID NO: 105). In some embodiments, an anti-CD8 antibody or fusion protein of the disclosure comprises a VH domain comprising the sequence of EVKLQESGPSLVQPSQTLSLTCSVSGFSLISDSVHWVRQPPGKGLEWMGGIWADGSTDYNSALKSRLSISRDTSKSQGFLKMNSLQTDDTAIYFCTSARESYYFDYWGQGTMVTVSS (SEQ ID NO: 106) and a VL domain comprising the sequence of DIQMTQSPASLSASLGDKVTITCQASQNIDKYIAWYQQKPGKAPRQLIHYTSTLVSGTPSRFSGSGSGRDYSFSISSVESEDIASYYCLQYATLYTFGAGTKLELK (SEQ ID NO: 107).In some embodiments, an anti-CD8 antibody or fusion protein of the disclosure comprises a VH domain comprising the sequence of EVQLVESGGALVQPGRSLKLSCAASGLTFSDCYMAWVRQTPTKGLEWVSYISSDGGSTYYGDSVKGRFTISRDNAKSTLYLQMNSLRSEDMATYYCACATDLSSYWSFDFWGPGTMVTVSS (SEQ ID NO: 108) and a VL domain comprising the sequence of DIQMTQSPSSLPVSLGERVTISCRASQGISNNLNWYQQKPDGTIKPLIYHTSNLQSGVPSRFSGSGSGTDYSLTISSLEPEDFAMYYCQQDATFPLTFGSGTKLEIK (SEQ ID NO: 109).

[0105] In some embodiments, an anti-CD8 antibody or fusion protein of this disclosure comprises a VH domain comprising the sequence of SEQ ID NO: 58 and a VL domain comprising the sequence of SEQ ID NO: 59. In some embodiments, an anti-CD8 antibody or fusion protein of this disclosure comprises a VH domain comprising the sequence of SEQ ID NO: 62 and a VL domain comprising the sequence of SEQ ID NO: 63. In some embodiments, an anti-CD8 antibody or fusion protein of this disclosure comprises a VH domain comprising the sequence of SEQ ID NO: 64 and a VL domain comprising the sequence of SEQ ID NO: 65. In some embodiments, an anti-CD8 antibody or fusion protein of this disclosure comprises a VH domain comprising the sequence of SEQ ID NO: 66 and a VL domain comprising the sequence of SEQ ID NO: 67. In some embodiments, an anti-CD8 antibody or fusion protein of this disclosure comprises a VH domain comprising the sequence of SEQ ID NO: 68 and a VL domain comprising the sequence of SEQ ID NO: 69. In some embodiments, an anti-CD8 antibody or fusion protein of this disclosure comprises a VH domain comprising the sequence of SEQ ID NO: 70 and a VL domain comprising the sequence of SEQ ID NO: 71. In some embodiments, an anti-CD8 antibody or fusion protein of this disclosure comprises a VH domain comprising the sequence of SEQ ID NO: 72 and a VL domain comprising the sequence of SEQ ID NO: 73. In some embodiments, an anti-CD8 antibody or fusion protein of this disclosure comprises a VH domain comprising the sequence of SEQ ID NO: 185 and a VL domain comprising the sequence of SEQ ID NO: 186. In some embodiments, an anti-CD8 antibody or fusion protein of this disclosure comprises a VH domain comprising the sequence of SEQ ID NO: 245 and a VL domain comprising the sequence of SEQ ID NO: 246. In some embodiments, an anti-CD8 antibody or fusion protein of this disclosure comprises a VH domain comprising the sequence of SEQ ID NO: 247 and a VL domain comprising the sequence of SEQ ID NO: 248. In some embodiments, an anti-CD8 antibody or fusion protein of this disclosure comprises a VH domain comprising the sequence of SEQ ID NO: 249 and a VL domain comprising the sequence of SEQ ID NO: 250. In some embodiments, an anti-CD8 antibody or fusion protein of this disclosure comprises a VH domain comprising the sequence of SEQ ID NO: 251 and a VL domain comprising the sequence of SEQ ID NO: 252.In some embodiments, an anti-CD8 antibody or fusion protein of this disclosure comprises a VH domain comprising the sequence of SEQ ID NO: 253 and a VL domain comprising the sequence of SEQ ID NO: 254. In some embodiments, an anti-CD8 antibody or fusion protein of this disclosure comprises a VH domain comprising the sequence of SEQ ID NO: 255 and a VL domain comprising the sequence of SEQ ID NO: 256. In some embodiments, an anti-CD8 antibody or fusion protein of this disclosure comprises a VH domain comprising the sequence of SEQ ID NO: 257 and a VL domain comprising the sequence of SEQ ID NO: 258.

[0106] In some embodiments, the antigen binding molecules (and fusion proteins) of the present disclosure specifically bind to human CD8b and / or human CD8ab.

[0107] In some embodiments, the anti-CD8 antibodies of the present disclosure are human antibodies or antibody fragments. In some embodiments, the anti-CD8 antibodies of the present disclosure are humanized antibodies or antibody fragments.

[0108] In some embodiments, an anti-CD8 antibody of the present disclosure specifically binds to human CD8b and / or human CD8ab with an affinity that is at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold, or at least 200-fold higher than the binding to human CD8a and / or human CD8aa, e.g., expressed on natural killer (NK) cells (e.g., human NK cells). In some embodiments, an anti-CD8 antibody of the present disclosure specifically binds to human CD8b and / or human CD8ab with an affinity that is at least 10-fold higher than the binding to human CD8a and / or human CD8aa, e.g., expressed on natural killer (NK) cells. In some embodiments, human CD8b and / or human CD8ab are expressed on the surface of human cells, e.g., human T cells.

[0109] In some embodiments, an anti-CD8 antibody of the present disclosure specifically binds to cells expressing human CD8ab heterodimers on their surface (e.g., human T cells) with an EC50 of less than 1000 nM. In some embodiments, an anti-CD8 antibody of the present disclosure specifically binds to human CD8+ T cells.

[0110] In some embodiments, an anti-CD8 antibody of the disclosure comprises a VH domain comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 110, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 111, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 112, 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, an anti-CD8 antibody of the disclosure comprises one, two, or three heavy chain CDRs of antibody xhCD8v1 (e.g., as shown in Tables 1-3) and / or one, two, or three light chain CDRs of antibody xhCD8v1 (e.g., as shown in Tables 1-3). In some embodiments, the antibody is humanized.

[0111] In some embodiments, an anti-CD8 antibody of the 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, an anti-CD8 antibody of the disclosure comprises one, two, or three heavy chain CDRs of antibody xhCD8v8 (e.g., as shown in Tables 1-3) and / or one, two, or three light chain CDRs of antibody xhCD8v8 (e.g., as shown in Tables 1-3). In some embodiments, the antibody is humanized.

[0112] In some embodiments, an anti-CD8 antibody of the 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 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 at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 63. In some embodiments, an anti-CD8 antibody of the disclosure comprises one, two, or three heavy chain CDRs of antibody xhCD8v2 (e.g., as shown in Tables 1-3) and / or one, two, or three light chain CDRs of antibody xhCD8v2 (e.g., as shown in Tables 1-3). In some embodiments, the antibody is humanized.

[0113] In some embodiments, an anti-CD8 antibody of the 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 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 at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 65. In some embodiments, an anti-CD8 antibody of the disclosure comprises one, two, or three heavy chain CDRs of antibody xhCD8v3 (e.g., as shown in Tables 1-3) and / or one, two, or three light chain CDRs of antibody xhCD8v3 (e.g., as shown in Tables 1-3). In some embodiments, the antibody is humanized.

[0114] In some embodiments, an anti-CD8 antibody of the 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 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 at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 67. In some embodiments, an anti-CD8 antibody of the disclosure comprises one, two, or three heavy chain CDRs of antibody xhCD8v4 (e.g., as shown in Tables 1-3) and / or one, two, or three light chain CDRs of antibody xhCD8v4 (e.g., as shown in Tables 1-3). In some embodiments, the antibody is humanized.

[0115] In some embodiments, an anti-CD8 antibody of the 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 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 at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 69. In some embodiments, an anti-CD8 antibody of the disclosure comprises one, two, or three heavy chain CDRs of antibody xhCD8v5 (e.g., as shown in Tables 1-3) and / or one, two, or three light chain CDRs of antibody xhCD8v5 (e.g., as shown in Tables 1-3). In some embodiments, the antibody is humanized.

[0116] In some embodiments, an anti-CD8 antibody of the 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 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 at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 71. In some embodiments, an anti-CD8 antibody of the disclosure comprises one, two, or three heavy chain CDRs of antibody xhCD8v6 (e.g., as shown in Tables 1-3) and / or one, two, or three light chain CDRs of antibody xhCD8v6 (e.g., as shown in Tables 1-3). In some embodiments, the antibody is human.

[0117] In some embodiments, an anti-CD8 antibody of the 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 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 at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 73. In some embodiments, an anti-CD8 antibody of the disclosure comprises one, two, or three heavy chain CDRs of antibody xhCD8v7 (e.g., as shown in Tables 1-3) and / or one, two, or three light chain CDRs of antibody xhCD8v7 (e.g., as shown in Tables 1-3). In some embodiments, the antibody is human.

[0118] In some embodiments, an anti-CD8 antibody of the disclosure comprises a CDR-H1 (SEQ ID NO: 259) comprising 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; and a CDR-H2 (SEQ ID NO: 259) comprising the amino acid sequence of X1X2AIS, 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, and X 10 is A or N and X 11 X1X2X3PX4X5X6X7X8X9YX where is Q or T 10 QKFX 11and CDR-H3 (SEQ ID NO: 261) comprising the amino acid sequence of 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 a VH domain comprising a CDR-H2 (SEQ ID NO: 260) comprising the amino acid sequence of 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 a VH domain comprising a CDR-H3 (SEQ ID NO: 261) comprising the amino acid sequence of X1X2X3GX4X5LFX6X7, 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. X4IX5GX6LN (SEQ ID NO: 262); CDR-L2 (SEQ ID NO: 263) comprising the amino acid sequence of GX1X2X3LX4X5, 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 (SEQ ID NO: 264) comprising 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 further comprises an FW-1 comprising the sequence QVQLVQSGAEVKKPGSSVKVSCKASGGTFS (SEQ ID NO: 274), an FW-2 comprising the sequence WVRQAPGQGLEWMG (SEQ ID NO: 275), an FW-3 comprising the sequence RVTITADESTSTAYMELSSLRSEDTAVYYCAR (SEQ ID NO: 276), and / or an FW-4 comprising the sequence WGQGTLVTVSS (SEQ ID NO: 277). In some embodiments, the VL domain further comprises an FW-1 comprising the sequence DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 289), an FW-2 comprising the sequence WYQQKPGKAPKLLIY (SEQ ID NO: 290), an FW-3 comprising the sequence GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 291), and / or an FW-4 comprising the sequence FGGGTKVEIK (SEQ ID NO: 292).

[0119] In some embodiments, an anti-CD8 antibody of the 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 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 at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 246. In some embodiments, an anti-CD8 antibody of the disclosure comprises one, two, or three heavy chain CDRs of antibody xhCD8v9 (e.g., as shown in Tables 1-3) and / or one, two, or three light chain CDRs of antibody xhCD8v9 (e.g., as shown in Tables 1-3). In some embodiments, the antibody is humanized. In some embodiments, an anti-CD8 antibody of the disclosure comprises a VH domain comprising CDR-H1, CDR-H2, and CDR-H3 of the sequence of SEQ ID NO: 245 and a VL domain comprising CDR-L1, CDR-L2, and CDR-L3 of the sequence of SEQ ID NO: 246. In some embodiments, the VH domain further comprises an FW-1 comprising the sequence QVQLVQSGAEVKKPGSSVKVSCKASGGTFS (SEQ ID NO: 274), an FW-2 comprising the sequence WVRQAPGQGLEWMG (SEQ ID NO: 275), an FW-3 comprising the sequence RVTITADESTSTAYMELSSLRSEDTAVYYCAR (SEQ ID NO: 276), and / or an FW-4 comprising the sequence WGQGTLVTVSS (SEQ ID NO: 277). In some embodiments, the VL domain further comprises an FW-1 comprising the sequence DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 289), an FW-2 comprising the sequence WYQQKPGKAPKLLIY (SEQ ID NO: 290), an FW-3 comprising the sequence GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 291), and / or an FW-4 comprising the sequence FGGGTKVEIK (SEQ ID NO: 292).

[0120] In some embodiments, an anti-CD8 antibody of the disclosure comprises a VH domain comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 225, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 232, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 233, and a VL domain comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 234, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 235, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 236. In some embodiments, the VH domain comprises an amino acid sequence 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 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, an anti-CD8 antibody of the disclosure comprises one, two, or three heavy chain CDRs of antibody xhCD8v12 (e.g., as shown in Tables 1-3) and / or one, two, or three light chain CDRs of antibody xhCD8v12 (e.g., as shown in Tables 1-3). In some embodiments, the antibody is humanized. In some embodiments, an anti-CD8 antibody of the disclosure comprises a VH domain comprising CDR-H1, CDR-H2, and CDR-H3 from the sequence of SEQ ID NO: 251 and a VL domain comprising CDR-L1, CDR-L2, and CDR-L3 from the sequence of SEQ ID NO: 252. In some embodiments, the VH domain further comprises an FW-1 comprising the sequence QVQLVQSGAEVKKPGSSVKVSCKASGGTFS (SEQ ID NO: 274), an FW-2 comprising the sequence WVRQAPGQGLEWMG (SEQ ID NO: 275), an FW-3 comprising the sequence RVTITADESTSTAYMELSSLRSEDTAVYYCAR (SEQ ID NO: 276), and / or an FW-4 comprising the sequence WGQGTLVTVSS (SEQ ID NO: 277).In some embodiments, the VL domain further comprises an FW-1 comprising the sequence DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 289), an FW-2 comprising the sequence WYQQKPGKAPKLLIY (SEQ ID NO: 290), an FW-3 comprising the sequence GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 291), and / or an FW-4 comprising the sequence FGGGTKVEIK (SEQ ID NO: 292).

[0121] In some embodiments, an anti-CD8 antibody of the 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 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 at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 254. In some embodiments, an anti-CD8 antibody of the disclosure comprises one, two, or three heavy chain CDRs of antibody xhCD8v13 (e.g., as shown in Tables 1-3) and / or one, two, or three light chain CDRs of antibody xhCD8v13 (e.g., as shown in Tables 1-3). In some embodiments, the antibody is humanized. In some embodiments, an anti-CD8 antibody of the disclosure comprises a VH domain comprising CDR-H1, CDR-H2, and CDR-H3 from the sequence of SEQ ID NO: 253 and a VL domain comprising CDR-L1, CDR-L2, and CDR-L3 from the sequence of SEQ ID NO: 254. In some embodiments, the VH domain further comprises an FW-1 comprising the sequence QVQLVQSGAEVKKPGSSVKVSCKASGGTFS (SEQ ID NO: 274), an FW-2 comprising the sequence WVRQAPGQGLEWMG (SEQ ID NO: 275), an FW-3 comprising the sequence RVTITADESTSTAYMELSSLRSEDTAVYYCAR (SEQ ID NO: 276), and / or an FW-4 comprising the sequence WGQGTLVTVSS (SEQ ID NO: 277). In some embodiments, the VL domain further comprises an FW-1 comprising the sequence DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 289), an FW-2 comprising the sequence WYQQKPGKAPKLLIY (SEQ ID NO: 290), an FW-3 comprising the sequence GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 291), and / or an FW-4 comprising the sequence FGGGTKVEIK (SEQ ID NO: 292).

[0122] In some embodiments, an anti-CD8 antibody of the disclosure comprises a CDR-H1 (SEQ ID NO: 268) comprising the amino acid sequence of X1YX2MS, where X1 is S, D, E, A, or Q, and X2 is A, G, or T; and a CDR-H2 (SEQ ID NO: 269) comprising the amino acid sequence of 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 CDR-H3 (SEQ ID NO: 270) having the amino acid sequence of X1X2X3YX4WX5X6AX7DX8, where 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; and a VL domain having CDR-L1 having the amino acid sequence of RASQSVSSNLA (SEQ ID NO: 40), CDR-L2 having the amino acid sequence of GASSRAT (SEQ ID NO: 41), and CDR-L3 having the amino acid sequence of QQYGSSPPVT (SEQ ID NO: 42). In some embodiments, the VH domain further comprises an FW-1 comprising the sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFS (SEQ ID NO:281), an FW-2 comprising the sequence WVRQAPGKGLEWVS (SEQ ID NO:282), an FW-3 comprising the sequence RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR (SEQ ID NO:283), and / or an FW-4 comprising the sequence WGQGTMVTVSS (SEQ ID NO:284) or WGQGTLVTVSS (SEQ ID NO:285). In some embodiments, the VL domain further comprises an FW-1 comprising the sequence EIVLTQSPGTLSLSPGERATLSC (SEQ ID NO:293), an FW-2 comprising the sequence WYQQKPGQAPRLLIY (SEQ ID NO:294), an FW-3 comprising the sequence GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC (SEQ ID NO:295), and / or an FW-4 comprising the sequence FGQGTKVEIK (SEQ ID NO:296).

[0123] In some embodiments, an anti-CD8 antibody of the 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 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 at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 248. In some embodiments, an anti-CD8 antibody of the disclosure comprises one, two, or three heavy chain CDRs of antibody xhCD8v10 (e.g., as shown in Tables 1-3) and / or one, two, or three light chain CDRs of antibody xhCD8v10 (e.g., as shown in Tables 1-3). In some embodiments, the antibody is humanized. In some embodiments, an anti-CD8 antibody of the disclosure comprises a VH domain comprising CDR-H1, CDR-H2, and CDR-H3 from the sequence of SEQ ID NO:247 and a VL domain comprising CDR-L1, CDR-L2, and CDR-L3 from the sequence of SEQ ID NO:248. In some embodiments, the VH domain further comprises an FW-1 comprising the sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFS (SEQ ID NO: 281), an FW-2 comprising the sequence WVRQAPGKGLEWVS (SEQ ID NO: 282), an FW-3 comprising the sequence RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 283), and / or an FW-4 comprising the sequence WGQGTMVTVSS (SEQ ID NO: 284) or WGQGTLVTVSS (SEQ ID NO: 285).In some embodiments, the VL domain further comprises an FW-1 comprising the sequence EIVLTQSPGTLSLSPGERATLSC (SEQ ID NO: 293), an FW-2 comprising the sequence WYQQKPGQAPRLLIY (SEQ ID NO: 294), an FW-3 comprising the sequence GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC (SEQ ID NO: 295), and / or an FW-4 comprising the sequence FGQGTKVEIK (SEQ ID NO: 296).

[0124] In some embodiments, an anti-CD8 antibody of the 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 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 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, an anti-CD8 antibody of the disclosure comprises one, two, or three heavy chain CDRs of antibody xhCD8v11 (e.g., as shown in Tables 1-3) and / or one, two, or three light chain CDRs of antibody xhCD8v11 (e.g., as shown in Tables 1-3). In some embodiments, the antibody is humanized. In some embodiments, an anti-CD8 antibody of the disclosure comprises a VH domain comprising CDR-H1, CDR-H2, and CDR-H3 from the sequence of SEQ ID NO: 249 and a VL domain comprising CDR-L1, CDR-L2, and CDR-L3 from the sequence of SEQ ID NO: 250. In some embodiments, the VH domain further comprises an FW-1 comprising the sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFS (SEQ ID NO: 281), an FW-2 comprising the sequence WVRQAPGKGLEWVS (SEQ ID NO: 282), an FW-3 comprising the sequence RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 283), and / or an FW-4 comprising the sequence WGQGTMVTVSS (SEQ ID NO: 284) or WGQGTLVTVSS (SEQ ID NO: 285).In some embodiments, the VL domain further comprises an FW-1 comprising the sequence EIVLTQSPGTLSLSPGERATLSC (SEQ ID NO: 293), an FW-2 comprising the sequence WYQQKPGQAPRLLIY (SEQ ID NO: 294), an FW-3 comprising the sequence GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC (SEQ ID NO: 295), and / or an FW-4 comprising the sequence FGQGTKVEIK (SEQ ID NO: 296).

[0125] In some embodiments, an anti-CD8 antibody of the 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 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 at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 256. In some embodiments, an anti-CD8 antibody of the disclosure comprises one, two, or three heavy chain CDRs of antibody xhCD8v14 (e.g., as shown in Tables 1-3) and / or one, two, or three light chain CDRs of antibody xhCD8v14 (e.g., as shown in Tables 1-3). In some embodiments, the antibody is humanized. In some embodiments, an anti-CD8 antibody of the disclosure comprises a VH domain comprising CDR-H1, CDR-H2, and CDR-H3 from the sequence of SEQ ID NO: 255 and a VL domain comprising CDR-L1, CDR-L2, and CDR-L3 from the sequence of SEQ ID NO: 256. In some embodiments, the VH domain further comprises an FW-1 comprising the sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFS (SEQ ID NO: 281), an FW-2 comprising the sequence WVRQAPGKGLEWVS (SEQ ID NO: 282), an FW-3 comprising the sequence RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 283), and / or an FW-4 comprising the sequence WGQGTMVTVSS (SEQ ID NO: 284) or WGQGTLVTVSS (SEQ ID NO: 285).In some embodiments, the VL domain further comprises an FW-1 comprising the sequence EIVLTQSPGTLSLSPGERATLSC (SEQ ID NO: 293), an FW-2 comprising the sequence WYQQKPGQAPRLLIY (SEQ ID NO: 294), an FW-3 comprising the sequence GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC (SEQ ID NO: 295), and / or an FW-4 comprising the sequence FGQGTKVEIK (SEQ ID NO: 296).

[0126] In some embodiments, an anti-CD8 antibody of the 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 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 at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 258. In some embodiments, an anti-CD8 antibody of the disclosure comprises one, two, or three heavy chain CDRs of antibody xhCD8v15 (e.g., as shown in Tables 1-3) and / or one, two, or three light chain CDRs of antibody xhCD8v15 (e.g., as shown in Tables 1-3). In some embodiments, the antibody is humanized. In some embodiments, an anti-CD8 antibody of the disclosure comprises a VH domain comprising CDR-H1, CDR-H2, and CDR-H3 from the sequence of SEQ ID NO:257 and a VL domain comprising CDR-L1, CDR-L2, and CDR-L3 from the sequence of SEQ ID NO:258. In some embodiments, the VH domain further comprises an FW-1 comprising the sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFS (SEQ ID NO: 281), an FW-2 comprising the sequence WVRQAPGKGLEWVS (SEQ ID NO: 282), an FW-3 comprising the sequence RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 283), and / or an FW-4 comprising the sequence WGQGTMVTVSS (SEQ ID NO: 284) or WGQGTLVTVSS (SEQ ID NO: 285).In some embodiments, the VL domain further comprises an FW-1 comprising the sequence EIVLTQSPGTLSLSPGERATLSC (SEQ ID NO: 293), an FW-2 comprising the sequence WYQQKPGQAPRLLIY (SEQ ID NO: 294), an FW-3 comprising the sequence GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC (SEQ ID NO: 295), and / or an FW-4 comprising the sequence FGQGTKVEIK (SEQ ID NO: 296).

[0127] Multiple definitions for CDR sequences of antibody variable domains are known in the art. Unless otherwise specified, CDR sequences are described herein according to the definitions of Kabat (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 contemplated for use. For example, in some embodiments, CDR sequences may be described according to the definitions of Chothia (see, e.g., Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)).

[0128] In some embodiments, an anti-CD8 antibody of the 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: 50, 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, an anti-CD8 antibody of the disclosure comprises a VH domain comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 51, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 52, 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, an anti-CD8 antibody of the 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, an anti-CD8 antibody of the 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, an anti-CD8 antibody of the 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, an anti-CD8 antibody of the 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, an anti-CD8 antibody of the 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, an anti-CD8 antibody of the 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.

[0129] In some embodiments, an anti-CD8 antibody of the disclosure comprises a CDR-H1 (SEQ ID NO: 265) comprising the amino acid sequence of 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. , F, or Y, X4 is A, D, or G, and X5 is T, E, K, V, Q, or A; and CDR-H2 (SEQ ID NO: 266) comprising the amino acid sequence of X1PX2X3X4X5, 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 CDR-L1 (SEQ ID NO: 262) comprising the amino acid sequence of 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, X5 is Y or S, and X6 is A or V. and CDR-L3 (SEQ ID NO: 264) comprising an amino acid sequence comprising 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 an FW-1 comprising the sequence QVQLVQSGAEVKKPGSSVKVSCKAS (SEQ ID NO:278), an FW-2 comprising the sequence AISWVRQAPGQGLEWMGGI (SEQ ID NO:279), an FW-3 comprising the sequence ANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAR (SEQ ID NO:280), and / or an FW-4 comprising the sequence WGQGTLVTVSS (SEQ ID NO:277). In some embodiments, the VL domain further comprises an FW-1 comprising the sequence DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO:289), an FW-2 comprising the sequence WYQQKPGKAPKLLIY (SEQ ID NO:290), an FW-3 comprising the sequence GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO:291), and / or an FW-4 comprising the sequence FGGGTKVEIK (SEQ ID NO:292).

[0130] In some embodiments, an anti-CD8 antibody of the disclosure comprises a VH domain comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 238, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 239, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 233, and a VL domain comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 16, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 17, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 228. In some embodiments, the VH domain further comprises an FW-1 comprising the sequence QVQLVQSGAEVKKPGSSVKVSCKAS (SEQ ID NO: 278), an FW-2 comprising the sequence AISWVRQAPGQGLEWMGGI (SEQ ID NO: 279), an FW-3 comprising the sequence ANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAR (SEQ ID NO: 280), and / or an FW-4 comprising the sequence WGQGTLVTVSS (SEQ ID NO: 277). In some embodiments, the VL domain further comprises an FW-1 comprising the sequence DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 289), an FW-2 comprising the sequence WYQQKPGKAPKLLIY (SEQ ID NO: 290), an FW-3 comprising the sequence GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 291), and / or an FW-4 comprising the sequence FGGGTKVEIK (SEQ ID NO: 292).

[0131] In some embodiments, an anti-CD8 antibody of the disclosure comprises a VH domain comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 238, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 243, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 233, and a VL domain comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 234, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 235, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 236. In some embodiments, the VH domain further comprises an FW-1 comprising the sequence QVQLVQSGAEVKKPGSSVKVSCKAS (SEQ ID NO: 278), an FW-2 comprising the sequence AISWVRQAPGQGLEWMGGI (SEQ ID NO: 279), an FW-3 comprising the sequence ANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAR (SEQ ID NO: 280), and / or an FW-4 comprising the sequence WGQGTLVTVSS (SEQ ID NO: 277). In some embodiments, the VL domain further comprises an FW-1 comprising the sequence DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 289), an FW-2 comprising the sequence WYQQKPGKAPKLLIY (SEQ ID NO: 290), an FW-3 comprising the sequence GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 291), and / or an FW-4 comprising the sequence FGGGTKVEIK (SEQ ID NO: 292).

[0132] In some embodiments, an anti-CD8 antibody of the disclosure comprises a VH domain comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 238, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 243, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 233, and a VL domain comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 16, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 17, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 228. In some embodiments, the VH domain further comprises an FW-1 comprising the sequence QVQLVQSGAEVKKPGSSVKVSCKAS (SEQ ID NO: 278), an FW-2 comprising the sequence AISWVRQAPGQGLEWMGGI (SEQ ID NO: 279), an FW-3 comprising the sequence ANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAR (SEQ ID NO: 280), and / or an FW-4 comprising the sequence WGQGTLVTVSS (SEQ ID NO: 277). In some embodiments, the VL domain further comprises an FW-1 comprising the sequence DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 289), an FW-2 comprising the sequence WYQQKPGKAPKLLIY (SEQ ID NO: 290), an FW-3 comprising the sequence GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 291), and / or an FW-4 comprising the sequence FGGGTKVEIK (SEQ ID NO: 292).

[0133] In some embodiments, an anti-CD8 antibody of the disclosure comprises a CDR-H1 (SEQ ID NO: 271) comprising the amino acid sequence of GFTFX1X2Y, where X1 is S, D, E, Q, S, or A, and X2 is S, D, E, A, or Q; a CDR-H2 (SEQ ID NO: 272) comprising the amino acid sequence of 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; and a CDR-H3 (SEQ ID NO: 273) comprising the amino acid sequence of X1X2X3GX4X5, where X1 is S or A, X2 is N, H, A, D, L, Q, Y, or R, and 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; and a VL domain comprising a CDR-L1 having the amino acid sequence of RASQSVSSNLA (SEQ ID NO: 40), a CDR-L2 having the amino acid sequence of GASSRAT (SEQ ID NO: 41), and a CDR-L3 having the amino acid sequence of QQYGSSPPVT (SEQ ID NO: 42). In some embodiments, the VH domain further comprises an FW-1 comprising the sequence EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO:286), an FW-2 comprising the sequence AMSWVRQAPGKGLEWVSDI (SEQ ID NO:287), an FW-3 comprising the sequence TAYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR (SEQ ID NO:288), and / or an FW-4 comprising the sequence WGQGTMVTVSS (SEQ ID NO:284) or WGQGTLVTVSS (SEQ ID NO:285). In some embodiments, the VL domain further comprises an FW-1 comprising the sequence EIVLTQSPGTLSLSPGERATLSC (SEQ ID NO:293), an FW-2 comprising the sequence WYQQKPGQAPRLLIY (SEQ ID NO:294), an FW-3 comprising the sequence GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC (SEQ ID NO:295), and / or an FW-4 comprising the sequence FGQGTKVEIK (SEQ ID NO:296).

[0134] In some embodiments, an anti-CD8 antibody of the disclosure comprises a VH domain comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 240, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 241, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 242, and a VL domain comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 40, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 41, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 42. In some embodiments, the VH domain further comprises an FW-1 comprising the sequence EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 286), an FW-2 comprising the sequence AMSWVRQAPGKGLEWVSDI (SEQ ID NO: 287), an FW-3 comprising the sequence TAYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 288), and / or an FW-4 comprising the sequence WGQGTMVTVSS (SEQ ID NO: 284) or WGQGTLVTVSS (SEQ ID NO: 285). In some embodiments, the VL domain further comprises an FW-1 comprising the sequence EIVLTQSPGTLSLSPGERATLSC (SEQ ID NO: 293), an FW-2 comprising the sequence WYQQKPGQAPRLLIY (SEQ ID NO: 294), an FW-3 comprising the sequence GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC (SEQ ID NO: 295), and / or an FW-4 comprising the sequence FGQGTKVEIK (SEQ ID NO: 296).

[0135] In some embodiments, an anti-CD8 antibody of the disclosure comprises a VH domain comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 240, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 244, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 242, and a VL domain comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 40, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 41, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 42. In some embodiments, the VH domain further comprises an FW-1 comprising the sequence EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 286), an FW-2 comprising the sequence AMSWVRQAPGKGLEWVSDI (SEQ ID NO: 287), an FW-3 comprising the sequence TAYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 288), and / or an FW-4 comprising the sequence WGQGTMVTVSS (SEQ ID NO: 284) or WGQGTLVTVSS (SEQ ID NO: 285). In some embodiments, the VL domain further comprises an FW-1 comprising the sequence EIVLTQSPGTLSLSPGERATLSC (SEQ ID NO: 293), an FW-2 comprising the sequence WYQQKPGQAPRLLIY (SEQ ID NO: 294), an FW-3 comprising the sequence GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC (SEQ ID NO: 295), and / or an FW-4 comprising the sequence FGQGTKVEIK (SEQ ID NO: 296). In some embodiments, the disclosure provides an anti-CD8 antibody comprising a VH domain comprising the CDR-H1, CDR-H2, and CDR-H3 sequences of a single antibody listed in Table 1, and a VL domain comprising the CDR-L1, CDR-L2, and CDR-L3 sequences of a single antibody listed in Table 1. For example, the anti-CD8 antibody comprises the six CDRs of antibody 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 present disclosure provides an anti-CD8 antibody comprising a VH domain comprising the CDR-H1, CDR-H2, and CDR-H3 sequences of a single antibody listed in Table 2, and a VL domain comprising the CDR-L1, CDR-L2, and CDR-L3 sequences of a single antibody listed in Table 2. For example, the anti-CD8 antibody comprises the six CDRs of antibody xhCD8v1, xhCD8v1.1, xhCD8v2, xhCD8v3, xhCD8v4, xhCD8v5, xhCD8v6, xhCD8v7, xhCD8v8, xhCD8v9, xhCD8v10, xhCD8v11, xhCD8v12, xhCD8v13, xhCD8v14, ​​xhCD8v15, V9 family, or V11 family, as shown in Table 2. In some embodiments, the present disclosure provides fusion proteins comprising an anti-CD8 antibody comprising a VH domain comprising the CDR-H1, CDR-H2, and CDR-H3 sequence of a single antibody listed in Table 1, and a VL domain comprising the CDR-L1, CDR-L2, and CDR-L3 sequence of a single antibody listed in Table 1. For example, the anti-CD8 antibody of the fusion protein comprises six CDRs of antibody xhCD8v1, xhCD8v1.1, xhCD8v2, xhCD8v3, xhCD8v4, xhCD8v5, xhCD8v6, xhCD8v7, xhCD8v8, xhCD8v9, xhCD8v10, xhCD8v11, xhCD8v12, xhCD8v13, xhCD8v14, ​​xhCD8v15, V9 family, or V11 family, as shown in Table 1. In some embodiments, the disclosure provides fusion proteins comprising an anti-CD8 antibody comprising a VH domain comprising the CDR-H1, CDR-H2, and CDR-H3 sequence of a single antibody listed in Table 2, and a VL domain comprising the CDR-L1, CDR-L2, and CDR-L3 sequence of a single antibody listed in Table 2.For example, the anti-CD8 antibody of the fusion protein comprises the six CDRs of antibody 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 an anti-CD8 antibody comprising a VH domain comprising the CDR-H1, CDR-H2, and CDR-H3 sequences of the VH domain listed in Table 3, and a VL domain comprising 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 from the same single antibody listed in Table 3). For example, the anti-CD8 antibody comprises the VH and VL of antibody 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 fusion proteins comprising an anti-CD8 antibody comprising a VH domain comprising the CDR-H1, CDR-H2, and CDR-H3 sequences of the VH domain listed in Table 3, and a VL domain comprising 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 from the same single antibody listed in Table 3). In some embodiments, the disclosure provides anti-CD8 antibodies comprising the VH domain sequence and VL domain sequence for a single antibody listed in Table 3. In some embodiments, the disclosure provides fusion proteins comprising an anti-CD8 antibody comprising the VH domain sequence and VL domain sequence for a single antibody listed in Table 3.For example, the anti-CD8 antibody of the fusion protein comprises the VH and VL of antibody xhCD8v1, xhCD8v1.1, xhCD8v2, xhCD8v3, xhCD8v4, xhCD8v5, xhCD8v6, xhCD8v7, xhCD8v8, xhCD8v9, xhCD8v10, xhCD8v11, xhCD8v12, xhCD8v13, xhCD8v14, ​​or xhCD8v15 shown in Table 3. [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4] [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4] [Table 7-1] [Table 7-2] [Table 7-3]

[0136] Further provided herein is a fusion protein comprising any one of the anti-CD8 antibodies, or antigen-binding domains, or antibody fragments disclosed herein. In some embodiments, the fusion protein of the present disclosure comprises:

[0137] In some embodiments, the disclosure provides a fusion protein comprising two heavy chain sequences and two light chain sequences of a single fusion protein listed in Table 13, where one of the heavy chain sequences has an IL-10 fusion and the other heavy chain sequence does not have an IL-10 fusion, and the two light chain sequences are identical. In some embodiments, the heavy chain sequence that does not have the IL-10 fusion comprises a lysine at its C-terminus. In some embodiments, the fusion protein is of Format F, as shown in Figure 6. For example, in some embodiments, the fusion protein comprises four polypeptide chains, (1) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 113, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 114, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 115, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 113; (2) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 113, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 114, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 116, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 113; (3) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 117, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 118, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 119, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 119. (4) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 117, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 118, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 120, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 117; (5) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 121, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 122, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 123, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 121; (6) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 121, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 122, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 121;(7) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 125, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 126, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 127, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 125; (8) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 125, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 126, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 128, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 125; (9) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 129, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 130, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 131, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 129; (10) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 129, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 130, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 132, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 129; ( (11) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 133, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 134, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 135, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 133; (12) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 133, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 134, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 136, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 133; (13) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 137, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 138, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 139, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 137; (14) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 137, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 138, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 140, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 137;(15) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 141, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 142, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 143, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 141; (16) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 141, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 142, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 144, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 141; (17) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 145, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 146, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 147, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 145; (18) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 145, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 146, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 148, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 145; (19) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 149, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 150, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 151, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 149; (20) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 149, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 150, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 152, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 149; (21) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 153, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 154, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 155, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 153; (22) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 153, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 154, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 156, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 153;(23) The first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 157, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 158, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 159, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 157; or (24) The first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 157, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 158, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 160, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 157. [Table 8-1] [Table 8-2] [Table 8-3] [Table 8-4] [Table 8-5] [Table 8-6] [Table 8-7] [Table 8-8] [Table 8-9] [Table 8-10] [Table 8-11] [Table 8-12]

[0138] Further provided herein are polynucleotides (e.g., isolated polynucleotides) encoding any of the antibodies, antibody fragments, and fusion proteins described herein. Further provided herein are vectors (e.g., expression vectors) encoding any of the antibodies, antibody fragments, and fusion proteins described herein.

[0139] Further provided herein are host cells (eg, isolated host cells or host cell lines) comprising any of the polynucleotides or vectors described herein.

[0140] Further provided herein are methods for producing any of the antibodies, antibody fragments, and fusion proteins described herein. In some embodiments, the methods comprise culturing a host cell of the present disclosure under conditions suitable for producing the antibody, antibody fragment, or fusion protein. In some embodiments, the methods further comprise recovering the antibody, antibody fragment, or fusion protein.

[0141] Antibodies, antibody fragments, and fusion proteins can be produced using recombinant methods, for example, as exemplified below. In some embodiments, nucleic acids encoding the antibody / fusion protein can be isolated and inserted into a replicable vector for further cloning or expression. DNA encoding the antibody / fusion protein can be readily isolated and sequenced using conventional procedures (e.g., by oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the antibody / fragment). Many vectors are known in the art. Vector components generally include, but are not limited to, one or more of a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. Suitable host cells for cloning or expressing the DNA in the vectors herein are prokaryote, yeast, or higher eukaryote cells. When using recombinant techniques, the antibody / fusion protein can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the antibody / fragment is produced intracellularly, particulate debris, either host cells or lysed fragments, is removed, for example, by centrifugation or ultrafiltration. Where the antibody / fusion protein is secreted into the medium, supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter.

[0142] In some embodiments, the fusion proteins of the present disclosure are part of a pharmaceutical composition, for example, comprising the fusion protein and one or more pharmaceutically acceptable carriers. The pharmaceutical compositions and formulations described herein can be prepared in the form of a lyophilized formulation or an aqueous solution by mixing an active ingredient (e.g., a fusion protein) having a desired purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations used and include, but are not limited to, buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives; low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes), and / or nonionic surfactants such as polyethylene glycol (PEG). In some embodiments, the fusion proteins of the present disclosure are lyophilized.

[0143] Certain aspects of the present disclosure relate to methods for treating cancer or chronic infections. In some embodiments, the methods comprise administering to a patient an effective amount of a fusion protein or antibody, or a pharmaceutical composition comprising the fusion protein or antibody and a pharmaceutical-acceptable carrier. In some embodiments, the patient in need of treatment has been diagnosed with cancer.

[0144] In some embodiments, the fusion protein or composition is administered in combination with a T cell therapy, a cancer vaccine, a chemotherapeutic agent, or an immune checkpoint inhibitor (ICI). In some embodiments, the chemotherapeutic agent is a kinase inhibitor, an antimetabolite, a cytotoxic or cytostatic agent, an antihormonal agent, a platinum-based chemotherapeutic agent, a methyltransferase inhibitor, an antibody, or an anti-cancer peptide. In some embodiments, the immune checkpoint inhibitor targets PD-L1, PD-1, CTLA-4, CEACAM, LAIR1, CD160, 2B4, CD80, CD86, CD276, VTCN1, HVEM, KIR, A2AR, MHC class I, MHC class II, GALS, adenosine, TGFR, OX40, CD137, CD40, CD47, TREM1, TREM2, HLA-G, CCR4, CCR8, CD39, CD73, IDO, CSF1R, TIM-3, BTLA, VISTA, LAG-3, TIGIT, IDO, MICA / B, LILRB4, SIGLEC-15, or arginase, and includes, but is not limited to, inhibitors of PD-1 (e.g., an anti-PD-1 antibody), PD-L1 (e.g., an anti-PD-L1 antibody), or CTLA-4 (e.g., an anti-CTLA-4 antibody). In some embodiments, the fusion protein or composition is administered in combination with an IL-2 polypeptide (including muteins or variants thereof), or a fusion protein comprising an IL-2 polypeptide (including muteins or variants thereof), such as, but not limited to, an antibody:IL-2 fusion protein (e.g., an anti-CD8:IL-2 fusion protein).

[0145] Examples of anti-PD-1 antibodies include, but are not limited to, pembrolizumab, nivolumab, cemiplimab, zimberelimab (Arcus), sasanlimab (Pfizer), JTX-4014, spartalizumab (PDR001; Novartis), camrelizumab (SHR1210; Jiangsu HengRui Medicine), sintilimab (IBI308; Innovent and Eli Lilly), tislelizumab (BGB-A317), toripalimab (JS 001), dostallimab (TSR-042, WBP-285), INCMGA00012 (MGA012), AMP-224, and AMP-514 (MEDI0680). Examples of anti-PD-L1 antibodies include, but are not limited to, atezolizumab, avelumab, durvalumab, KN035, and CK-301 (checkpoint therapy). Examples of PD-L1 inhibitors (non-antibody-based) include, but are not limited to, AUNP12, CA-170, and BMS-986189. Examples of anti-CTLA-4 antibodies include, but are not limited to, ipilimumab, tremelimumab, BMS-986218, BMS-986249, BMS-986288, HBM4003, ONC-392, KN044, ADG116, ADU-1604, AGEN1181, AGEN1884, MK-1308, and REGN4659.

[0146] Examples of T cell therapies include, but are not limited to, CD4+ or CD8+ T cell-based therapies, adoptive T cell therapies, chimeric antigen receptor (CAR)-based T cell therapies, tumor-infiltrating lymphocyte (TIL)-based therapies, autologous T cell therapies, allogeneic T cell therapies, and therapies using T cells with transduced TCRs. Exemplary cancer vaccines include, but are not limited to, dendritic cell vaccines, vaccines comprising one or more polynucleotides encoding one or more cancer antigens, and vaccines comprising one or more cancer antigen peptides.

[0147] Certain aspects of the present disclosure relate to methods of expanding T cells, e.g., ex vivo. In some embodiments, the method comprises contacting one or more T cells, e.g., ex vivo, with an effective amount of an antibody or fusion protein of the present disclosure. In some embodiments, the one or more T cells are tumor-infiltrating lymphocytes (TILs). In some embodiments, the method further comprises isolating the tumor-infiltrating lymphocytes (TILs) from a tumor or tumor sample. In some embodiments, the method comprises contacting one or more T cells, e.g., ex vivo, with an effective amount of an antibody or fusion protein of the present disclosure in combination with an IL-2 polypeptide (including a mutein or variant thereof) or a fusion protein comprising an IL-2 polypeptide (including a mutein or variant thereof), such as, but not limited to, an antibody:IL-2 fusion protein (e.g., an anti-CD8:IL-2 fusion protein). [Example]

[0148] Example 1: Preparation of IL-10 fusion proteins and the ability of IL-10 to activate STAT3 by phosphorylating STAT3 Materials and Methods recombinant DNA technology Techniques involving recombinant DNA manipulation have been previously described in Sambrook et al., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989. All reagents were used according to the manufacturer's instructions. DNA sequences were determined by double-strand sequencing.

[0149] Gene synthesis Desired gene segments were generated by PCR using appropriate templates or synthesized from synthetic oligonucleotides by Genewiz (South Plainfield, NJ), Integrated DNA Technologies (Coralville, IA), or GeneScript (Piscataway, NJ). Gene segments were cloned into expression vectors using the Gibson assembly® method or by restriction digestion followed by ligation. DNA was purified from transformed bacteria, and concentrations were determined by UV-visible spectroscopy. DNA sequencing was used to confirm the DNA sequences of subcloned gene fragments.

[0150] Isolation of antibody genes Antibodies that bind to the CD8 antigen were generated using either humanization of mouse antibodies or an in vitro phage display system.

[0151] For humanization, the complementarity determining regions (CDRs) of murine residues were grafted onto selected human framework(s) that showed close sequence similarity and good stability to the parent murine framework. The resulting CDR-grafted antibodies were further humanized to remove any unwanted non-human mutations.

[0152] For the in vitro display method, a non-immune human single-chain Fv phage library generated from naive B cells was panned five to six times to isolate antibodies against the CD8 antigen. After panning, individual phage clones that showed specific binding to the target antigen over nonspecific antigens were identified by ELISA. DNA fragments of the heavy and light chain V domains of specific binders were then cloned and sequenced.

[0153] Cloning of antibody constructs General information regarding the nucleotide sequences of human immunoglobulin light and heavy chains is given in IMGT® (the international ImMunoGeneTics information system®) in Lefranc et al. IMGT®, the international ImMunoGeneTics information system® 25 years on. Nucleic Acids Res. 2015 Jan;43. DNA fragments of the heavy and light chain V domains were inserted in frame into mammalian expression vectors containing human IgG1 and CK.

[0154] Cloning of fusion constructs The IL-10 portion of the construct was cloned in-frame with the heavy chain using a glycine-serine-based linker between the C-terminus of the IgG heavy chain and the N-terminus of IL-10. The C-terminal lysine residue of the IgG heavy chain was removed after fusion of the IL-10 portion. To generate constructs with asymmetric structures, knob-into-hole modifications were introduced into the CH3 domain of the Fc region to promote heterodimerization. Specifically, the "hole" domain contained Y349C, T366S, L368A, and Y407V mutations in the CH3 domain, while the "knob" domain contained S354C and T366W mutations in the CH3 domain (EU numbering). To abolish FcγR binding / effector function and prevent FcR coactivation, the mutations L234A / L235A / G237A (EU numbering) were introduced into the CH2 domain of each IgG heavy chain or Fc region. Expression of the antibody·IL-10 fusion construct was driven by a CMV promoter, and transcription was terminated by a synthetic poly(A) signal sequence located downstream of the coding sequence.

[0155] Purification of fusion proteins with IL-10 polypeptides Constructs encoding fusion proteins with IL-10 polypeptides used in the examples were generated by cotransfecting exponentially growing Expi293 cells with mammalian expression vectors using polyethyleneimine (PEI). Supernatants were harvested after 4–5 days of culture. IL-10 fusion constructs were first purified by affinity chromatography using a protein A matrix. The protein A column was equilibrated and washed with phosphate-buffered saline (PBS). The fusion constructs were eluted with 20 mM sodium citrate, 50 mM sodium chloride, pH 3.6. The eluted fractions were pooled and dialyzed against 10 mM MES, 25 mM sodium chloride, pH 6. The proteins were further purified using an ion-exchange chromatograph (Mono-S, GE Healthcare) to purify heterodimers over homodimers. After loading the protein, the column was washed with 10 mM MES, 25 mM sodium chloride, pH 6. The protein was then eluted with an increasing sodium chloride gradient from 25 to 500 mM in 10 mM MES, pH 6 buffer. The major eluate peak corresponding to the heterodimer was collected and concentrated. The purified protein was then purified by size exclusion chromatography (Superdex200, GE Healthcare) in PBS.

[0156] Protein concentrations of purified IL-10 fusion constructs were determined by measuring absorbance (OD) at 280 nm using the molar extinction coefficient calculated based on the amino acid sequence. The purity, integrity, and monomeric state of the fusion constructs were analyzed by SDS-PAGE in the presence and absence of a reducing agent (5 mM 1,4-dithiothreitol) and stained with Coomassie Blue (SimpleBlue™ SafeStain, Invitrogen). The NuPAGE® Pre-Cast Gel System (Invitrogen) was used according to the manufacturer's instructions (4-20% Tris-glycine gels or 3-12% Bis-Tris). The aggregate content of immunoconjugate samples was analyzed using a Superdex200 10 / 300GL analytical size-exclusion column (GE Healthcare).

[0157] Binding affinity measurements for CD8, IL-10RA, and IL-10RB by surface plasmon resonance (SPR) The kinetic rate constants (k and k) and affinity (K) of IL-10 fusion proteins for human and cynomolgus monkey CD8, IL-10RA, and IL-10RB. D ) was measured by surface plasmon resonance (SPR) using a BIAcore® T200 (Cytiva) at 37°C. Briefly, to measure affinity for human and cynomolgus monkey CD8, antibodies or fusion proteins were captured via Fc onto a CM4 sensor chip using a covalently immobilized anti-human Fc capture antibody generated using the Human Antibody Capture Kit (Cytiva). No protein was captured on flow cell 1, which served as the reference surface. Soluble antigen diluted in HBS-EP+ buffer at four or more concentrations ranging from 0.1x to 10x the KD was flowed over the antibody / fusion protein-captured surface for 1–2 min. Dissociation was monitored for 5–10 min, and the anti-hIgG-Fc surface was regenerated with 3 M MgCl2 before recapturing the antibody / fusion protein in each subsequent cycle. Binding data were analyzed using Biacore® Evaluation Software version 3.2 using a 1:1 Langmuir with mass transport model.

[0158] To measure affinity for IL-10RA, antibodies or fusion proteins were captured via Fc by a covalently immobilized anti-human Fc antibody onto a CM4 sensor chip (Southern Biotech, catalog no. 2081-01). No protein was captured on flow cell 1, which served as the reference surface. In-house produced human IL-10RA ECD diluted at concentrations of 8, 40, 200, 1000, and 5000 nM in HBS-P+ buffer supplemented with 1 g / L BSA, or buffer, was flowed over the fusion protein-captured surface at 30 μL / min for 2 min. Dissociation was monitored for 4–5 min, and the anti-hIgG-Fc surface was regenerated between analysis cycles with three 30-s injections of 75 mM phosphoric acid. Binding data were analyzed using Biacore® Evaluation Software version 3.2 or steady-state affinity analysis using a 1:1 Langmuir with mass transport model.

[0159] To measure binding to IL-10RB, biotinylated human IL-10RA produced in-house was captured on a CAP sensor chip (Cytiva) with biotin CAPture reagent (Cytiva) first immobilized on the chip according to the manufacturer's instructions. After blocking the surface with 20 μM amine-PEG2-biotin (ThermoFisher Scientific) for 60 seconds, IL-10 fusion protein was injected at 10 μL / min for 3 minutes. No IL-10 fusion protein was captured on flow cell 1, which served as the reference surface. In-house produced human IL-10RB ECD diluted at concentrations of 0.2, 1, 5, 10, and 20 μM in HBS-EP+ buffer supplemented with 1 g / L BSA, or buffer, was flowed as the analyte at 30 μL / min for 2 minutes, followed by a 4-minute dissociation period. Between analysis cycles, the CAP sensor chip surface was regenerated by a 2-minute injection of a mixture of 3 parts 8 M guanidine-HCl and 1 part 1 M NaOH. Sensorgrams were double-referenced. To rank the binding, after normalization to the capture response units of the IL-10 fusion protein, the binding response units of IL-10RB were recorded using Biacore Evaluation Software version 3.2 5 seconds before the end of the binding step at the highest concentration.

[0160] STAT3 phosphorylation assay in primary human cells The ability of IL-10 to activate various immune cell subsets was measured in an assay measuring STAT3 phosphorylation by flow cytometry in human PBMCs or human whole blood.

[0161] PBMCs were isolated from healthy donor blood using Ficoll-Paque Plus (GE Healthcare) and red blood cells were lysed using ACK lysis buffer (Gibco) according to the manufacturer's instructions. Generally, PBMCs were cultured in serum-free RPMI 1640 medium at a concentration of 20 × 10 6The cells were resuspended at 1000 cells / ml and aliquoted into 96-well U-bottom plates (50 μl per well). IL-10 fusion proteins and control proteins, e.g., wild-type IL-10 dimer and control fusion proteins, were diluted to the desired concentrations and added to the wells (50 μl added as 2x stimulation). Incubation was generally performed at 37°C for 30 minutes. For staining with CD8 antibodies, e.g., CD8a (SK1, Biolegend; RPA-T8, Biolegend), the antibody was added directly to the wells and incubated on ice for 10 minutes. Staining was stopped with 100 μl of ice-cold 8% PFA (4% final) for 10 minutes on ice. Cells were washed three times with wash buffer (2% FBS in PBS). Cells were permeabilized in 100 ml of pre-chilled Phosflow Perm Buffer III (BD Biosciences) according to the manufacturer's protocol and stored at -20°C overnight. The next day, cells were washed twice with wash buffer and stained with antibodies against CD3 (UCHT1, BD Biosciences), CD4 (RPA-T4, Biolegend), CD14 (M5E2, Biolegend), CD25 (M-A251, Biolegend), CD56 (HCD56, Biolegend), and / or perforin (clone δG9, BD Biosciences), Foxp3 (259D, Biolegend), and pSTAT3 [pY705] (clone 4, BD Biosciences) for 30–45 min at 4°C. Cells were then analyzed by flow cytometer. Data are presented as percent pSTAT3 positivity and, in some cases, as pSTAT3 mean fluorescence intensity (MFI) and imported into GraphPad Prism.

[0162] For assays in human whole blood, 90 μL of human blood was used per well in a 1 mL 96-well deep plate well and pre-warmed to 37°C for 10 minutes. IL-10 fusion proteins and control proteins, e.g., wild-type IL-10 dimer and control fusion proteins, were prepared and pre-warmed to 37°C at 10x strength. 10 μL of pre-warmed 10x stimulus was added to each well for a total volume of 100 μL at 1x stimulus concentration. Incubation was typically performed for 25 minutes at 37°C. Stimulation was quenched by adding prefix antibody staining cocktail, vortexing briefly, and incubating on ice in the dark for 10 minutes. The prefix staining cocktail contained TruStain FcX (Biolegend) and antibodies against CD4 (RPA-T4, Biolegend), CD19 (HIB19, BD), CD56 (NCAM16.2, BD), CD16 (3G8, Biolegend), and CD8 (SK1, Biolegend). 900 μL of prewarmed Lyse Fix (BD) was added to the sample wells and incubated at 37°C for 10 minutes. Cells were washed in pre-chilled wash buffer containing PBS + 0.5% bovine serum albumin and 2 mM EDTA. Pre-chilled Perm Buffer III (BD) was added and incubated at -20°C for 60 minutes, followed by two washes in wash buffer and one wash in TFP Perm / Wash (BD). Cells were resuspended in 25 μL of a "post-methanol" staining cocktail prepared in TFP Perm / Wash containing antibodies against CD3 (UCHT, BD), CD14 (MΦP9, BD), CD11c (B-ly6, BD), HLADR (L243, Biolegend), and pSTAT3 pY705 (4 / P-STAT3, BD). Cells were incubated in the dark at 4°C for 30 minutes, washed in TFP Perm / Wash buffer, and subsequently fixed in 100 μL of 4% PFA for 10 minutes at room temperature. Cells were washed twice in wash buffer and analyzed on a flow cytometer. Data were expressed as percent pSTAT3 positivity and imported into GraphPad Prism.

[0163] Polyreactivity assessment by ELISA To assess the polyreactivity of candidate fusion proteins, ELISA assays were used to test for binding to a panel of unrelated antigens, including dsDNA salmon sperm, human serum albumin, keyhole limpet hemocyanin, lipopolysaccharide, insulin, and heparin biotin sodium salt, all purchased from Sigma.

[0164] Antigens were diluted in PBS to concentrations ranging from 0.3 to 10 μg / mL and coated onto 384-well Nunc MaxiSorp plates (Thermo Fisher Scientific) at a volume of 25 μL per well. 25 μL of PBS alone was used as a no-antigen control. The plates were incubated overnight at 4°C. The antigens were removed, and the plates were washed with milli-Q water (Millipore). The wells were filled with PBS supplemented with 0.05% Tween® and 1 mM EDTA (assay buffer) and then incubated at room temperature for 1 hour. The assay buffer was removed, and the wells were washed with milli-Q water. 25 μL of 10 μg / mL of the fusion protein or bococizumab, a positive control for polyreactivity, diluted in assay buffer was added and incubated at room temperature for 1 hour. The samples were removed, and the plates were washed with milli-Q water. 25 μl of detection antibody, a 1:25,000 diluted horseradish peroxidase-conjugated goat anti-human IgG (Jackson ImmunoResearch), was added and incubated for 1 hour at room temperature. The reagent was removed, and the wells were washed with milli-Q water. Wells were developed with 25 μL of KPL SureBlue TMB Microwell Substrate (SeraCare) for 5–7 minutes and quenched with 25 μL of 0.1 M HCl. Absorbance at 450 nm was recorded on a SpectraMax iD5 plate reader (Molecular Devices) and normalized to no-antigen control wells.

[0165] result The ability of wild-type IL-10 dimers to activate STAT3 in monocytes and CD8+ T cells is shown in Figures 5A and 5B. PBMCs from two healthy donors were used, and representative data are shown in Figure 5A. Whole blood from two different healthy donors was used, and representative data are shown in Figure 5B. The extent of activation and EC50 of activation in each cell type are comparable across both assays. In both assays, monocytes (gated as CD14+CD3-) were found to be more sensitive to IL-10 than CD8+ T cells.

[0166] Example 2: Preferential activation of STAT3 in CD8+ T cells by fusion proteins containing IL-10 dimers Fusion proteins containing CD8 antibody and IL-10 dimeric polypeptides were produced in one of five dimeric formats (A, B, C, D, and E shown in Figure 6).

[0167] The selectivity and potency of STAT3 activation in hPBMCs by IL-10 fusion proteins of format A are shown in Figure 7. Fusion proteins tested included xmCD8a-IL10wt of format A, which contains wild-type IL-10 polypeptide and a control antibody targeting mouse CD8 (Figure 7A), and xhCD8a-IL10wt of format A, which contains wild-type IL-10 polypeptide and an antibody targeting human CD8 (Figure 7B). The antibody xmCD8a was a previously published anti-mouse CD8 antibody (2.43 clone), and xhCD8a was a previously published anti-human CD8 antibody (OKT8). STAT3 activation in human PBMCs was measured as described in Example 1. The IL-10 fusion protein format A, xhCD8a-IL10wt, containing an antibody that specifically binds to human CD8, preferentially activated CD8+ T cells over monocytes, whereas the IL-10 fusion protein format A, xmCD8a-IL10wt, containing a control antibody, preferentially activated monocytes.

[0168] We generated the IL-10 fusion proteins xmCD8a-IL10wt and xhCD8a-IL10wt in format C and evaluated their ability to activate STAT3 in human PBMCs. The results for format C xmCD8a-IL10wt are shown in Figure 8A, and the results for format C xhCD8a-IL10wt are shown in Figure 8B. The potency of the format C fusion protein xmCD8a-IL10wt was approximately 10-fold lower than that of format A xmCD8a-IL10wt (compare Figures 7A and 8A), suggesting that fusion of IL-10 at the N-terminus of human Fc reduced activity compared with that when IL-10 was fused at the C-terminus of human Fc. Furthermore, except at low concentrations (up to 0.01 nM), format C was not optimal for IL-10 fusion proteins containing antibodies that bind to human CD8. High concentrations of Form C xhCD8a-IL10wt did not fully activate STAT3 in CD8+ T cells and did not preferentially activate CD8+ T cells over monocytes (Fig. 8B).

[0169] The selectivity and potency of STAT3 activation in hPBMCs by IL-10 fusion proteins of format D are shown in Figure 9. The results for format D xmCD8a-IL10wt are shown in Figure 9A, and the results for format D xhCD8a-IL10wt are shown in Figure 9B. The IL-10 fusion protein of format D, xhCD8a-IL10wt, containing an antibody that specifically binds human CD8, preferentially activated CD8+ T cells over monocytes, whereas the IL-10 fusion protein of format D, xmCD8a-IL10wt, containing a control antibody, preferentially activated monocytes.

[0170] Example 3: Preferential activation of STAT3 in CD8+ T cells by fusion proteins containing IL-10 monomers Fusion proteins containing CD8 antibodies and IL-10 monomer polypeptides were generated according to Format F shown in Figure 6. The amino acid sequences of the IL-10 monomer polypeptides constructed as part of the fusion proteins are shown in Figure 1D for the unmodified monomeric IL-10 polypeptide, referred to as IL10mono, or in Table 4A above for the mutant monomeric IL-10 polypeptides. IL10mono_RBenh is a previously described mutant monomeric IL-10 polypeptide (Gorby et al. Sci Signal. 2020 Sep 15;13(649):eabc0653). It contains the amino acid substitutions N18I, N92I, K99N, and F111L in the IL10mono background, increasing binding affinity for IL-10RB. IL10mono_RBenh2 is a mutant monomeric IL-10 polypeptide containing a single amino acid substitution, N92I, in the IL10mono background.

[0171] The selectivity and potency of STAT3 activation in hPBMCs by IL-10 monomer fusion proteins are shown in Figures 10A-10C. The fusion proteins tested included format F, xhCD8b-IL10mono, which contains the IL10mono polypeptide described above and an antibody targeting human CD8 (Figure 10A); format F, xhCD8b-IL10mono_RBenh, which contains the IL10mono_RBenh mutant monomeric IL-10 polypeptide described above and an antibody targeting human CD8 (Figure 10B); and format F, xhCD8b-IL10mono_RBenh2, which contains the IL10mono_RBenh2 mutant monomeric IL-10 polypeptide described above and an antibody targeting human CD8 (Figure 10C). The antibody xhCD8b was an antibody with specificity for human CD8b. STAT3 activation in human PBMCs was measured as described in Example 2.

[0172] The fusion protein xhCD8b-IL10mono preferentially activated CD8+ T cells over monocytes and CD4+ T cells at concentrations of 1 nM or less, but the degree of activation was relatively low (Fig. 10A). The mutant monomeric fusion protein xhCD8b-IL10mono_RBenh, which contains an antibody specifically binding to human CD8 and has enhanced IL-10RB affinity, also preferentially activated CD8+ T cells over monocytes and CD4+ T cells (Fig. 10B). The difference in the potency of CD8+ T cell activation by xhCD8b-IL10mono_RBenh versus monocytes / CD4+ T cells was significantly greater than the difference in the potency of CD8+ T cell activation by xhCD8b-IL10mono versus monocytes / CD4+ T cells. Furthermore, the potency of CD8+ T cell activation by xhCD8b-IL10mono_RBenh was much higher than that by xhCD8b-IL10mono and comparable to that of the wild-type dimeric IL-10 fusion protein. This indicates that the increased binding affinity for IL-10RB favors both the potency of CD8+ T cell activation and the selectivity of CD8+ T cell activation over monocytes and CD4+ T cells compared to unmodified IL-10RB. The fusion protein xhCD8b-IL10mono_RBenh2, which contains only a single N92I substitution in the IL10mono background, also showed increased potency and selective activation of CD8+ T cells over monocytes and CD4+ T cells (Figure 10C).

[0173] Example 4: Attenuated binding affinity to IL-10RA As shown in Example 3 and Figure 10, enhanced binding to IL-10RB could enhance STAT3 activation by monomeric IL-10. To this end, mutations on the IL-10mono background that would enhance IL-10RB binding were identified using a combination of existing structures of IL-10 and its receptor (e.g., PDB IDs: 1J7V, 6X93, 3LQM) and homology modeling against similar cytokine / receptor complexes (e.g., PDB IDs: 5T5W, 4DOH, 1Y6K). The amino acid sequences of representative mutant monomeric IL-10 polypeptides evaluated are shown in Table 5.

[0174] STAT3 activation in hPBMCs by fusion proteins of a panel of IL-10RB-enhanced IL-10 monomers and the xhCD8b antibody, IL10mono_RBenh3 to IL10mono_RBenh20, is shown in Figures 11A to 11D. All fusion proteins were constructed in Format F, and STAT3 activation in human PBMCs was measured as described in Example 1. STAT3 activation by fusion proteins IL10mono_RBenh3 to IL10mono_RBenh13 is shown in Figure 11A for CD8 T cells and in Figure 11B for monocytes. STAT3 activation by fusion proteins IL10mono_RBenh14 to IL10mono_RBenh20 is shown in Figure 11C for CD8 T cells and in Figure 11D for monocytes. In all figures, STAT3 activation is compared to that of xhCDb-IL10mono_RBenh2. From this panel, all fusions, CD8b-IL10mono_RBenh3 to CD8b-IL10mono_RBenh13 and CD8b-IL10mono_RBenh17 to CD8b-IL10mono_RBenh20, showed enhanced STAT3 activation in CD8 T cells and CD8 T cell selectivity over monocytes compared to xCD8b-IL10mono. Furthermore, the fusion proteins IL10mono_RBenh3, IL10mono_RBenh4, IL10mono_RBenh6, IL10mono_RBenh7, IL10mono_RBenh8, IL10mono_RBenh18, and IL10mono_RBenh19 showed comparable potency of STAT3 activation and CD8 selectivity to the IL10mono_RBenh2 fusion protein.

[0175] Additional mutations predicted for IL-10RB enhancement were constructed as described in Example 1 and screened using a BIAcore-based assay. Due to the low binding affinity to IL-10RB, precise kinetics could not be determined. Instead, to rank the relative binding affinity of the IL10mono muteins to IL-10RB, the binding responses to 20 μM IL-10RB were measured and normalized to the capture level of the IL10mono fusion protein. The normalized binding responses to IL-10RB for the fusion proteins IL10mono_RBenh21 through IL10mono_RBenh60, along with several controls, are shown in Table 6. Notably, IL10mono_RBenh38, IL10mono_RBenh40, and IL10mono_RBenh60 show enhanced binding compared to IL10mono.

[0176] STAT3 activation in human whole blood was assessed for selected fusion proteins identified by BIAcore-based screening. STAT3 activation is shown in Figure 11E for CD8 T cells and in Figure 11F for monocytes. Here, the IL10mono_RBenh38 fusion protein showed similar, though slightly lower, STAT3 activation in CD8 T cells than that of IL10mono_RBenh2. [Table 9-1] [Table 9-2] [Table 9-3] [Table 9-4] [Table 9-5] [Table 9-6] [Table 9-7] [Table 10-1] [Table 10-2]

[0177] Example 5: Attenuated binding affinity to IL-10RA As shown in Example 3, fusion proteins comprising a CD8 antibody and an IL-10 monomer polypeptide selectively activated CD8+ T cells over monocytes and CD4+ T cells. To further reduce activity against nonspecific cells, a panel of amino acid substitutions was designed to reduce binding affinity for IL-10RA against the background of the IL-10RB-enhating polypeptide, IL10mono_RBenh, or IL10mono_RBenh2. The amino acid substitutions and amino acid sequences of representative mutant monomeric IL-10 polypeptides evaluated are shown in Table 4A. The binding affinity of these constructs for IL-10RA was measured by BIAcore as described in Example 1, and a summary of these data is shown in Table 7. In all cases, the IL-10 polypeptide is expressed as a fusion protein with the format indicated in parentheses, based on the antibody binding domain specified in column 2 and the schematic diagram shown in Figure 6. The Kd of binding is listed in column 3, where "ND" indicates that binding was detected but the affinity was too low to allow reliable calculation of the Kd. "NT" indicates that the mutein was not evaluated by BIAcore.

[0178] In all cases, the mutations reduced the binding affinity of the IL-10 polypeptide, indicating that these amino acid substitutions are useful in reducing activity against monocytes and other non-specific cell types.

[0179] The selectivity and potency of STAT3 activation in hPBMCs by fusion proteins of the xhCD8b antibody against a panel of IL-10RA-attenuated constructs in either the IL10mono_RBenh or IL10mono_RBenh2 background are shown in Figures 12A to 12F. All fusion proteins were constructed in Format F, and STAT3 activation in human PBMCs was measured as described in Example 1. Figures 12A and 12B show STAT3 activation in CD8 T cells and monocytes, respectively, for fusion proteins with insufficiently attenuated IL-10RA binding. As shown in Figure 12B, activity on monocytes remains largely unchanged. Figures 12C, 12D, 12E, and 12F show STAT3 activation in CD8 T cells and monocytes, respectively, for fusion proteins with varying degrees of attenuation of IL-10RA binding. The results in Figures 12D and 12F show that the reduction in potency in monocytes ranged from approximately 2-fold reduction for IL10mono_RBenh_m10 to over 100-fold reduction for IL10mono_RBenh_m11. Similar levels of reduced potency were observed in both monocytes and CD8 T cells for each of these IL-10RA muteins compared to IL-10mono_RBenh, which does not contain any IL-10RA mutations. The affinity of selected fusions for IL-10RA is also reported in Table 7.

[0180] Based on these results, selected IL-10RA-attenuating mutations were combined with various IL-10RB-enhancing mutations and evaluated in a STAT3 assay in human whole blood. The amino acid sequences of representative mutant monomeric IL-10 polypeptides evaluated are shown in Table 8. Figures 12G and 12H show STAT3 activation in CD8 T cells and monocytes, respectively, for these representative fusion proteins. For all constructs tested, there was selectivity for activation of CD8 T cells over monocytes, with greater potency and selectivity observed for the IL10mono_RBenh2_m10 or IL10mono_RBenh7_m10 fusion proteins. [Table 11] [Table 12]

[0181] Example 6: Further screening of constructs with reduced binding affinity to IL-10RA Mutations predicted to attenuate IL-10RA binding were designed and engineered into the IL10mono_RBenh6 background. The amino acid sequences of representative mutant monomeric IL-10 polypeptides evaluated are shown in Table 9.

[0182] Screening for mutant proteins with reduced binding affinity to IL-10RA was performed by BIAcore as described in Example 1. Affinity data are shown in Table 10. Constructs with weak or undetectable binding to IL-10RA were assessed for binding to protein G and for integrity by SDS-PAGE to rule out the possibility of degraded proteins. "ND" indicates that binding was detected but the affinity was too low to allow reliable calculation of Kd. "NB" indicates that no binding was detected above the buffer-only control. [Table 13-1] [Table 13-2] [Table 13-3] [Table 13-4] [Table 13-5] [Table 13-6] [Table 13-7] [Table 13-8] [Table 13-9] [Table 13-10] [Table 14-1] [Table 14-2] [Table 14-3]

[0183] Example 7: Removal of glycosaminoglycan binding sites on IL-10 IL-10 contains a positively charged patch that has been shown to bind glycosaminoglycans, with heparin being the most potent binder for IL-10 (Kunze et al. J Biol Chem, 2016). This property of IL-10 is speculated to help regulate IL-10 function, but reduced exposure may also limit its therapeutic efficacy. R107 has been identified as the most important residue for interacting with glycosaminoglycans, and based on molecular modeling, other residues cannot compensate for the loss of R107 (Gehrcke et al. J Mol Graph Model, 2015).

[0184] The R107A mutation, designated m117, was engineered into the IL10mono_RBenh2 background as a fusion with the format F xhCD8b antibody and tested in a STAT3 assay in human whole blood. The sequence of this construct is shown in Table 11. STAT3 activation data for CD8 T cells and monocytes are shown in Figures 13A and 13B, respectively. STAT3 activation for both CD8 T cells and monocytes was comparable between IL10mono_RBenh2 and IL10mono_RBenh2_m117, indicating that the addition of the m117 mutation does not significantly affect activity.

[0185] The results of a polyreactive ELISA assay measuring binding to a panel of unrelated proteins are shown in Table 12. Bococizumab, which has been shown to be polyreactive, is included as a positive control. Comparing IL10mono_RBenh2 to IL10mono_RBenh2_m117, there is a decrease in nonspecific binding to many unrelated targets. This is also true for heparin. Altogether, this indicates that the introduction of m117 reduces reactivity with nonspecific, unrelated proteins, potentially leading to improved exposure. [Table 15] [Table 16]

Claims

1. 1. A mutant IL-10 polypeptide comprising an amino acid sequence having at least 80% amino acid sequence identity with the amino acid sequence of SEQ ID NO:1 and having one or more amino acid substitutions relative to the amino acid sequence of SEQ ID NO:1, (a) the one or more amino acid substitutions are at a position(s) selected from the group consisting of N18, N21, M22, R24, D25, D28, S31, R32, D55, M68, I69, L73, E74, M77, P78, Q79, E81, N82, K88, A89, H90, N92, S93, G95, E96, N97, K99, T100, L101, L103, R104, R107, R110 and F111, numbered according to SEQ ID NO:1; or (b) the one or more amino acid substitutions are at a position(s) selected from the group consisting of P20, L23, R24, R27, D28, K34, T35, Q38, M39, D41, L43, D44, N45, L46, K49, I87, V91, L94, L98, K138, S141, E142, D144, N148, E151, and I158, numbered according to SEQ ID NO:1; The mutant IL-10 polypeptide.

2. said one or more amino acid substitutions are at a position(s) selected from the group consisting of N18, D28, N92, K99, and L103, numbered according to SEQ ID NO:1; Optionally, the one or more amino acid substitutions are selected from the group consisting of N18F, N18L, N18Y, D28Q, D28R, N92F, N92H, N92I, N92K, N92L, N92R, N92S, N92T, N92V, N92Y, K99N, L103N, and L103Q, numbered according to SEQ ID NO:

1. The mutant IL-10 polypeptide of claim 1.

3. the mutant IL-10 polypeptide exhibits increased binding affinity to the IL-10RB polypeptide comprising the amino acid sequence of SEQ ID NO:3 compared to the binding affinity of a wild-type mature IL-10 polypeptide comprising the amino acid sequence of SEQ ID NO:1 to an IL-10RB polypeptide comprising the amino acid sequence of SEQ ID NO:3, and optionally the polypeptide exhibits a binding affinity for the IL-10RB polypeptide comprising the amino acid sequence of SEQ ID NO:3 that is increased by 50% or more compared to the binding affinity of the wild-type mature IL-10 polypeptide comprising the amino acid sequence of SEQ ID NO:1 for the IL-10RB polypeptide comprising the amino acid sequence of SEQ ID NO:3; or the polypeptide exhibits a binding affinity for the IL-10RB polypeptide comprising the amino acid sequence of SEQ ID NO:3 that is increased by 150% or more compared to the binding affinity of the wild-type mature IL-10 polypeptide comprising the amino acid sequence of SEQ ID NO:1 for the IL-10RB polypeptide comprising the amino acid sequence of SEQ ID NO:3; A mutant IL-10 polypeptide according to claim 1 or claim 2.

4. the mutant IL-10 polypeptide comprises one or more additional amino acid substitutions relative to the amino acid sequence of SEQ ID NO:1, wherein the one or more additional amino acid substitutions are at a position(s) selected from the group consisting of P20, L23, R24, R27, D28, K34, T35, Q38, M39, D41, L43, D44, N45, L46, K49, I87, V91, L94, L98, K138, S141, E142, D144, N148, E151, and I158; Optionally, the mutant IL-10 polypeptide comprises one or more additional amino acid substitutions relative to the amino acid sequence of SEQ ID NO:1, wherein the one or more additional amino acid substitutions are at a position(s) selected from the group consisting of R24, R27, K34, Q38, D44, I87, K138, E142, D144, N148, and E151; Optionally, the one or more further amino acid substitutions are R24A, R27A, K34A, K34D, K34E, K34S, K34P, K34G, K34T, K34H, K34L, K34N, K34F, K34R, K34Q, K34V, K34Y, Q38A, Q38D, Q38P, Q38G, Q38H, Q38I, Q38L, Q38R, Q38K, Q38N, Q38F, Q38T, Q38E, Q38S, Q38V, Q38Y, D44A, D44E, D44S, D44V, D44G, D44H, D44I, D44K, D 44P, D44L, D44N, D44F, D44T, D44R, D44Q, I87A, K138A, E142A, E142G, E142N, E142L, E142F, E1 42I, E142V, E142K, E142R, E142P, E142Q, E142T, E142S, E142Y, D144A, D144E, D144G, D144H, D144R, D144I, D144K, D144N, D144Q, D144P, D144S, D144L, D144T, D144V, D144Y, N148G, N148P , N148S, N148D, N148T, N148K, N148V, N148I, N148E, N148F, E151A, E151G, E151H, E151I, E151N, E151F, E151L, E151V, E151R, E151K, E151P, E151Q, E151S, E151T, and E151Y; and Optionally, the one or more further amino acid substitutions are selected from the group consisting of R24A, R27A, K34A, K34D, K34E, K34S, K34P, K34G, K34T, K34H, K34L, K34N, K34F, K34V, K34Y, Q38A, Q38D, Q38P, Q38G, Q38I, Q38L, Q38R, Q38K, Q38F, Q38T, Q38E, Q38S, Q38V, Q38Y, I87A, K138A, E142A, E142G, E142N, E142L, E142F, E142I, E142V, E142K, E142R, E142 P, E142Q, E142T, E142S, E142Y, D144A, D144E, D144G, D144H, D144R, D144I, D144K, D144N, D144Q, D144P, D144S, D144L, D144T, D144V, D144Y, N148P, N148D, N148I, E151A, E151G, E151H, E151I, E151N, E151F, E151L, E151V, E151R, E151K, E151P, E151Q, E151S, E151T, and E151Y; A mutant IL-10 polypeptide according to any one of claims 1 to 3.

5. The mutated IL-10 polypeptide of any one of claims 1 to 4, wherein the mutated IL-10 polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 87-89, 188-201, and 310-318.

6. the one or more amino acid substitutions are at a position(s) selected from the group consisting of R24, R27, K34, Q38, D44, I87, K138, E142, D144, N148, and E151; Optionally, the one or more amino acid substitutions are selected from the group consisting of R24A, R27A, K34A, K34D, K34E, K34S, K34P, K34G, K34T, K34H, K34L, K34N, K34F, K34R, K34Q, K34V, K34Y, Q38A, Q38D, Q38P, Q38G, Q38H, Q38I, Q38L, Q38R, Q38K, Q38 N, Q38F, Q38T, Q38E, Q38S, Q38V, Q38Y, D44A, D44E, D44S, D44V, D44G, D44H, D44I, D44K, D44P , D44L, D44N, D44F, D44T, D44R, D44Q, I87A, K138A, E142A, E142G, E142N, E142L, E142F, E142 I, E142V, E142K, E142R, E142P, E142Q, E142T, E142S, E142Y, D144A, D144E, D144G, D144H, D1 44R, D144I, D144K, D144N, D144Q, D144P, D144S, D144L, D144T, D144V, D144Y, N148G, N148P, N148S, N148D, N148T, N148K, N148V, N148I, N148E, N148F, E151A, E151G, E151H, E151I, E151N, E151F, E151L, E151V, E151R, E151K, E151P, E151Q, E151S, E151T, and E151Y; and Optionally, the one or more amino acid substitutions are selected from the group consisting of R24A, R27A, K34A, K34D, K34E, K34S, K34P, K34G, K34T, K34H, K34L, K34N, K34F, K34V, K34Y, Q38A, Q38D, Q38P, Q38G, Q38I, Q38L, Q38R, Q38K, Q38F, Q38T, Q38E, Q38S, Q38V, Q38Y, I87A, K138A, E142A, E142G, E142N, E142L, E142F, E142I, E142V, E142K, E142R, E142P, selected from the group consisting of E142Q, E142T, E142S, E142Y, D144A, D144E, D144G, D144H, D144R, D144I, D144K, D144N, D144Q, D144P, D144S, D144L, D144T, D144V, D144Y, N148P, N148D, N148I, E151A, E151G, E151H, E151I, E151N, E151F, E151L, E151V, E151R, E151K, E151P, E151Q, E151S, E151T, and E151Y; The mutant IL-10 polypeptide of claim 1.

7. the mutant IL-10 polypeptide exhibits a reduced binding affinity to the IL-10RA polypeptide comprising the amino acid sequence of SEQ ID NO:2 compared to the binding affinity of the wild-type mature IL-10 polypeptide comprising the amino acid sequence of SEQ ID NO:1 to the IL-10RA polypeptide comprising the amino acid sequence of SEQ ID NO:2; Optionally, the polypeptide exhibits a binding affinity for the IL-10RA polypeptide comprising the amino acid sequence of SEQ ID NO:2 that is reduced by 100 / 150 or less, 100 / 250 or less, 2-fold or more, or 10-fold or more, compared to the binding affinity of the wild-type mature IL-10 polypeptide comprising the amino acid sequence of SEQ ID NO:1 for the IL-10RA polypeptide comprising the amino acid sequence of SEQ ID NO:

2. A mutant IL-10 polypeptide according to any one of claims 4 to 6. (a) the mutant IL-10 polypeptide further comprises an amino acid substitution at position R107 relative to the amino acid sequence of SEQ ID NO:1, optionally an R107A mutation; or (b) the mutant IL-10 polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 422-428; A mutant IL-10 polypeptide according to any one of claims 1 to 7. (a) the mutant IL-10 polypeptide is a dimer, optionally a homodimer or a heterodimer; or (b) the mutated IL-10 polypeptide is monomeric, and optionally the mutated IL-10 monomer polypeptide comprises the amino acid sequence of SEQ ID NO: 1 with a peptide insertion of between 1 and 15 amino acids immediately following residues C114, E115, N116, K117, S118, K119, or A120, numbered according to SEQ ID NO: 1, and / or the mutated IL-10 monomer polypeptide comprises an amino acid substitution at position N92, numbered according to SEQ ID NO: 1, and optionally the mutated IL-10 monomer polypeptide comprises an amino acid substitution N92I, N92F, N92H, N92F, N92G, N92H, N92I, N92F ... 2K, N92L, N92R, N92S, N92T, N92V, or N92Y, and optionally said mutant IL-10 monomer polypeptide further comprises one or more of the amino acid substitutions N18I, K99N, and F111L, numbering based on SEQ ID NO:1, and / or one or more amino acid substitutions at position(s) R24, R27, Q38, I87, K138, E142, D144, and / or E151, numbering based on SEQ ID NO:1, and optionally comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:87-89, 188-201, 310-318, and 422-428. A mutant IL-10 polypeptide according to any one of claims 1 to 8.

10. 10. A fusion protein comprising a mutant IL-10 polypeptide according to any one of claims 1 to 9 and an antigen binding molecule that binds to an antigen on a T cell, optionally wherein the fusion protein selectively stimulates T cells over monocytes, and optionally wherein the T cells are human T cells.

11. The antigen-binding molecule binds to CD8, CD4 or PD-1, and optionally (1) The antigen-binding molecule binds to CD8ab, CD8a, or CD8aa; (2) The antigen-binding molecule binds to CD8b and / or CD8ab. (3) The antigen-binding molecule comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain; (a) the VH domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 110, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 111, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 112, and the VL domain comprises 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; (b) the VH domain comprises 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 the VL domain comprises 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; (c) the VH domain comprises 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 the VL domain comprises 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; (d) the VH domain comprises 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 the VL domain comprises 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; (e) the VH domain comprises 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 the VL domain comprises 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; (f) the VH domain comprises 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 the VL domain comprises 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; (g) the VH domain comprises 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 the VL domain comprises 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; (h) the VH domain comprises 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 the VL domain comprises 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; (i) the VH domain comprises a CDR-H1 (SEQ ID NO:259) comprising the amino acid sequence of X 1 X 2 AIS, where X 1 is S, K, G, N, R, D, T, or G, and X 2 is Y, L, H, or F; and X 1 is G or H, X 2 is I or F, X 3 is I, N, or M, X 4 is G, N, H, S, R, I, or A, X 5 is A, N, H, S, T, F, or Y, X 6 is A, D, or G, X 7 is T, E, K, V, Q, or A, X 8 is A or T, X 9 is N or K, X 10 is A or N, and X 11 is Q or T. and a CDR-H3 (SEQ ID NO:261) comprising the amino acid sequence of X 1 X 2 X 3 GX 4 X 5 LFX 6 X 7 , in which X 1 is D or A, X 2 is A, G, E, R, Y, K, N, Q, L, or F, X 3 is A, L, P, or Y, X 4 is I or L, X 5 is R, A, Q, or S, X 6 is A or D, and X 7 is D, E, A, or S. CDR-L1 (SEQ ID NO:262) comprising the amino acid sequence of X 1 X 2 SX 3 X 4 IX 5 GX 6 LN, where X 1 is A or S, X 2 is T, S, E, Q, or D, X 3 is N, R, A, E, or H, X 4 is Q or A, and X 5 is S or D; and CDR-L2 (SEQ ID NO:263) comprising the amino acid sequence of GX 1 X 2 X 3 LX 4 X 5, where X 1 is S, N, D, Q, A, or E, X 2 is T, I, or S, X 3 is Y, L, or F, and X 4 is QX 1 X 2 X 3 X 4 X 5 is D, G, T, E, Q, A, or Y, and X 5 is A, T, R, S, K, or Y.and CDR-L3 comprising the amino acid sequence of PWT (SEQ ID NO:264); (j) the VH domain comprises 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 the VL domain comprises 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; (k) the VH domain comprises 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 the VL domain comprises 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; (l) the VH domain comprises 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 the VL domain comprises 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; (m) the VH domain comprises a CDR-H1 (SEQ ID NO: 268) comprising an amino acid sequence of X 1 YX 2 MS, where X 1 is S, D, E, A, or Q, and X 2 is A, G, or T; a CDR-H2 (SEQ ID NO: 269) comprising an amino acid sequence of DIX 1 X 2 X 3 GX 4 X 5 TX 6 YADSVKG, where X 1 is S or A, X 2 is N, H, A, D, L, Q, Y, or R, and X 3 is A, S, Q, E, or T, X 4 is G or E, X 5 is S or I, and X 6 is A or G; and CDR-H3 (SEQ ID NO:270) comprising the amino acid sequence of X 1 X 2 X 3 YX 4 WX 5 X 6 AX 7 DX 8 , where X 4 is A, N, S, or G, X 5 is A, V, R, E, or S, X 5 is D or S, X 6 is D, N, Q, E, S, T, or L, X 7 is L, F, or M, and X 8 is I, Y, or V; said VL domain comprises CDR-L1 comprising the amino acid sequence of RASQSVSSNLA (SEQ ID NO:40), CDR-L2 comprising the amino acid sequence of GASSRAT (SEQ ID NO:41), and CDR-L3 comprising the amino acid sequence of QQYGSSPPVT (SEQ ID NO:42); (n) the VH domain comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 229, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 230, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 231, and the VL domain comprises a CDR-L1 comprising the amino acid sequence of RASQSVSSNLA (SEQ ID NO: 40), a CDR-L2 comprising the amino acid sequence of GASSRAT (SEQ ID NO: 41), and a CDR-L3 comprising the amino acid sequence of QQYGSSPPVT (SEQ ID NO: 42); or (o) the VH domain comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 229, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 237, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 231; and the VL domain comprises a CDR-L1 comprising the amino acid sequence of RASQSVSSNLA (SEQ ID NO: 40), a CDR-L2 comprising the amino acid sequence of GASSRAT (SEQ ID NO: 41), and a CDR-L3 comprising the amino acid sequence of QQYGSSPPVT (SEQ ID NO: 42); (4) The antigen-binding molecule comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain; (a) the VH domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO:51, CDR-H2 comprising the amino acid sequence of SEQ ID NO:52, and CDR-H3 comprising the amino acid sequence of SEQ ID NO:15, and the VL domain comprises 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; (b) the VH domain comprises 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 the VL domain comprises 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; (c) the VH domain comprises 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 the VL domain comprises 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; (d) the VH domain comprises 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 the VL domain comprises 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; (e) the VH domain comprises 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 the VL domain comprises 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; (f) the VH domain comprises 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 the VL domain comprises 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; (g) the VH domain comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 49, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 50, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3, and the VL domain comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6; (h) the VH domain comprises 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 the VL domain comprises 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; (i) the VH domain comprises a CDR-H1 (SEQ ID NO:265) comprising an amino acid sequence of GX 1 X 2 FX 3 X 4 X 5 , where X 1 is G, Y, S, or A, X 2 is T, S, G, R, N, or H, X 3 is S, T, R, H, Y, G, or P, X 4 is S, K, G, N, R, D, T, or G, and X 5 is Y, L, H, or F; and a CDR-H2 (SEQ ID NO:265) comprising an amino acid sequence of X 1 PX 2 X 3 X 4 X 5 , where X 1 is I, N, or M, X 2 is G, N, H, S, R, I, or A, X 3 is A, N, H, S, T, F, or Y, X 4 is A, D, or G, and X 5 is T, E, K, V, Q, or A. and a CDR-H3 (SEQ ID NO:267) comprising an amino acid sequence of X 1 X 2 X 3 GX 4 X 5 LFX 6 X 7 , in which X 1 is D or A, X 2 is A, G, E, R, Y, K, N, Q, L, or F, X 3 is A, L, P, or Y, X 4 is I or L, X 5 is R, A, Q, or S, X 6 is A or D, and X 7 is D, E, A, or S. The VL domain comprises a CDR-H2 (SEQ ID NO:266) comprising an amino acid sequence of X 5 , and a CDR-H3 (SEQ ID NO:267) comprising an amino acid sequence of X 1 X 2 X 3 GX 4 X 5 LFX 6 X 7 , in which X 1 is D or A, X 2 is A or T, X 3 is Q or E, X 4 is E, N, T, S, A, K, D, G, R, or Q, and X 5 CDR-L1 (SEQ ID NO:262) comprising the amino acid sequence of X 1 X 2 SX 3 X 4 IX 5 GX 6 LN, where X 1 is A or S, X 2 is T, S, E, Q, or D, X 3 is N, R, A, E, or H, X 4 is Q or A, and X 5 is S or D; and CDR-L2 (SEQ ID NO:263) comprising the amino acid sequence of GX 1 X 2 X 3 LX 4 X 5, where X 1 is S, N, D, Q, A, or E, X 2 is T, I, or S, X 3 is Y, L, or F, X 4 is D, G, T, E, Q, A, or Y, and X 5 is and a CDR-L3 (SEQ ID NO:264) comprising an amino acid sequence comprising QX 1 X 2 X 3 X 4 X 5 PWT, wherein QX 1 X 2 X 3 X 4 X 5 is A, T, R, S, K, or Y; (j) the VH domain comprises 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 the VL domain comprises 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; (k) the VH domain comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 238, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 243, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 233, and the VL domain comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 234, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 235, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 236; (l) the VH domain comprises 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 the VL domain comprises 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; (m) the VH domain comprises a CDR-H1 (SEQ ID NO: 271) comprising the amino acid sequence of GFTFX 1 X 2 Y, where X 1 is S, D, E, Q, S, or A, and X 2 is S, D, E, A, or Q; a CDR-H2 (SEQ ID NO: 272) comprising the amino acid sequence of X 1 X 2 X 3 GX 4 X 5, where X 1 is T, N, S, Q, E, H, R, or A, X 2 is Y, W, F, or H, X 3 is A, S, Q, E, or T, X 4 is G or E, and X 5 is S or I; and CDR-H3 (SEQ ID NO:273) comprising the amino acid sequence of X 1 X 2 X 3 YX 4 WX 5 X 6 AX 7 DX 8 , where X 5 is A, V, R, E, or S, X 5 is D or S, X 6 is D, N, Q, E, S, T, or L, X 7 is L, F, or M, and X 8 is I, Y, or V; said VL domain comprises CDR-L1 comprising the amino acid sequence of RASQSVSSNLA (SEQ ID NO:40), CDR-L2 comprising the amino acid sequence of GASSRAT (SEQ ID NO:41), and CDR-L3 comprising the amino acid sequence of QQYGSSPVT (SEQ ID NO:42); (n) the VH domain comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 240, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 241, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 242, and the VL domain comprises a CDR-L1 comprising the amino acid sequence of RASQSVSSNLA (SEQ ID NO: 40), a CDR-L2 comprising the amino acid sequence of GASSRAT (SEQ ID NO: 41), and a CDR-L3 comprising the amino acid sequence of QQYGSSPPVT (SEQ ID NO: 42); or (o) the VH domain comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 240, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 244, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 242; and the VL domain comprises a CDR-L1 comprising the amino acid sequence of RASQSVSSNLA (SEQ ID NO: 40), a CDR-L2 comprising the amino acid sequence of GASSRAT (SEQ ID NO: 41), and a CDR-L3 comprising the amino acid sequence of QQYGSSPVT (SEQ ID NO: 42); or (5) The antigen-binding molecule comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain; (a) the VH domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO:62, and the VL domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO:63; (b) the VH domain comprises an amino acid sequence 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 at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO:65; (c) the VH domain comprises an amino acid sequence 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 at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO:67; (d) the VH domain comprises an amino acid sequence 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 at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO:69; (e) the VH domain comprises an amino acid sequence 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 at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 71; (f) the VH domain comprises an amino acid sequence 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 at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 73; (g) the VH domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO:245, and the VL domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO:246; (h) the VH domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO:251, and the VL domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO:252; (i) the VH domain comprises an amino acid sequence 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 at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO:254; (j) the VH domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO:247, and the VL domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO:248; (k) the VH domain comprises an amino acid sequence 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 at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO:250; (l) the VH domain comprises an amino acid sequence 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 at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO:256; (m) the VH domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO:257, and the VL domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO:258; (n) the VH domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO:58, and the VL domain comprises an amino acid sequence at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO:59; or (o) the VH domain comprises an amino acid sequence 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 at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 186; The fusion protein of claim 10. (a) the fusion protein comprises a dimer of two mutant IL-10 polypeptides, one of which is fused to the antigen-binding molecule directly or via a linker; or (b) the fusion protein comprises two polypeptides, each comprising an antigen-binding site, and one mutant IL-10 polypeptide is fused, either directly or via a linker, to each of the polypeptides; or (c) the fusion protein comprises a mutated IL-10 monomer polypeptide, the mutated IL-10 monomer polypeptide being fused directly or via a linker to the antigen-binding molecule; Optionally, (i) The antigen-binding molecule has formula [I], VH-CH1-hinge-CH2-CH3 [I] and two antibody heavy chain polypeptides comprising a structure according to formula [II], from N-terminus to C-terminus: VL-CL [II] and two antibody light chain polypeptides comprising a structure according to: wherein VH is an antibody heavy chain variable (VH) domain, CH1 is an antibody CH1 domain, hinge is an antibody hinge domain, CH2 is an antibody CH2 domain, CH3 is an antibody CH3 domain, VL is an antibody light chain variable (VL) domain, and CL is an antibody constant light chain domain, and VH / VL form an antigen binding site, and optionally said fusion protein comprises two mutated IL-10 polypeptides linked in a dimer, wherein the N-terminus of one of the two mutated IL-10 polypeptides is fused, either directly or via a linker, to the C-terminus of one of the two CH3 domains, or said fusion protein comprises two mutated IL-10 polypeptides linked in a dimer, wherein the first N-terminus of the two mutated IL-10 polypeptides is fused, either directly or via a linker, to the C-terminus of one of the two CH3 domains. or the fusion protein comprises one mutated IL-10 monomer polypeptide and the N-terminus of the mutated IL-10 monomer polypeptide is fused directly or via a linker to the C-terminus of one of the two CH3 domains, and optionally one or both of the antibody heavy chain polypeptides comprise the following amino acid substitutions, as numbered according to the EU index: L234A, L235A, and G237A; and / or the first of the two Fc domains comprises the amino acid substitutions Y349C and T366W and the second of the two Fc domains comprises the amino acid substitutions S354C, T366S, L368A, and Y407V, as numbered according to the EU index; (ii) The antigen-binding molecule has formula [I], VH-CH1-hinge-CH2-CH3 [I], and a first antibody heavy chain polypeptide comprising a structure according to formula [II], from the N-terminus to the C-terminus: VL-CL [II], and an antibody light chain polypeptide comprising a structure according to formula [III], from the N-terminus to the C-terminus: Hinge-CH2-CH3 [III], and a second antibody heavy chain polypeptide comprising a structure according to: wherein VH is an antibody heavy chain variable (VH) domain, CH1 is an antibody CH1 domain, hinge is an antibody hinge domain, CH2 is an antibody CH2 domain, CH3 is an antibody CH3 domain, VL is an antibody light chain variable (VL) domain, CL is an antibody constant light chain domain, and VH / VL form an antigen binding site, and optionally said fusion protein comprises two mutated IL-10 polypeptides linked in a dimer, wherein the N-terminus of one of the two mutated IL-10 polypeptides is fused, directly or via a linker, to one of the C-terminus of the CH3 domain of the second antibody heavy chain polypeptide or the C-terminus of the CH3 domain of the first antibody heavy chain polypeptide, or said fusion protein comprises two mutated IL-10 polypeptides linked in a dimer, wherein the N-terminus of a first of the two mutated IL-10 polypeptides is fused, directly or via a linker, to the C-terminus of the CH3 domain of the first antibody heavy chain polypeptide. the N-terminus of a second of the two mutated IL-10 polypeptides is fused, directly or via a linker, to the C-terminus of the CH3 domain of the second antibody heavy chain polypeptide, or the fusion protein comprises one mutated IL-10 monomer polypeptide and the N-terminus of the mutated IL-10 monomer polypeptide is fused, directly or via a linker, to the C-terminus of the CH3 domain of the second antibody heavy chain polypeptide or the C-terminus of the first antibody heavy chain polypeptide. and optionally one or both of the antibody heavy chain polypeptides comprises the following amino acid substitutions, as numbered according to the EU index: L234A, L235A, and G237A; and / or a first of the two Fc domains comprises the amino acid substitutions Y349C and T366W and a second of the two Fc domains comprises the amino acid substitutions S354C, T366S, L368A, and Y407V, as numbered according to the EU index. A fusion protein according to claim 10 or claim 11.

13. The fusion protein comprises four polypeptide chains, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:113, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:114, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:115, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:113; the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:113, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:114, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:116, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:113; the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:117, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:118, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:119, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:117; the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:117, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:118, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:120, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:117; the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:121, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:122, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:123, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:121; the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:121, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:122, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:124, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:121; the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:125, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:126, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:127, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:125; the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:125, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:126, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:128, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:125; the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:129, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:130, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:131, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:129; the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:129, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:130, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:132, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:129; the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:133, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:134, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:135, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:133; the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:133, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:134, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:136, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:133; the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:137, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:138, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:139, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:137; the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:137, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:138, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:140, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:137; the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:141, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:142, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:143, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:141; the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:141, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:142, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:144, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:141; the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:145, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:146, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:147, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:145; the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:145, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:146, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:148, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:145; the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:149, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:150, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:151, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:149; the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:149, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:150, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:152, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:149; the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:153, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:154, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:155, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:153; the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:153, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:154, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:156, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:153; the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:157, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:158, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:159, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:157; or The fusion protein of any one of claims 10 to 12, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 157, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 158, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 160, and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO:

157.

14. One or more isolated polynucleotides encoding a mutant IL-10 polypeptide according to any one of claims 1 to 9 or a fusion protein according to any one of claims 10 to 13.

15. 15. One or more vectors comprising one or more polynucleotides of claim 14, optionally wherein the vector(s) is / are expression vector(s).

16. 16. A host cell comprising one or more polynucleotides according to claim 14 or one or more vectors according to claim 15.

17. 17. A method for producing a mutant IL-10 polypeptide or fusion protein, comprising culturing a host cell according to claim 16 under conditions suitable for production of said polypeptide or said fusion protein, and optionally said method further comprising recovering said polypeptide or said fusion protein from said host cell.

18. A pharmaceutical composition comprising a mutated IL-10 polypeptide according to any one of claims 1 to 9 or a fusion protein according to any one of claims 10 to 13 and a pharma- ceutically acceptable carrier.

19. A composition comprising a mutated IL-10 polypeptide according to any one of claims 1 to 9 or a fusion protein according to any one of claims 10 to 13, or a composition according to claim 18, for use as a medicament.

20. A composition comprising a mutant IL-10 polypeptide according to any one of claims 1 to 9 or a fusion protein according to any one of claims 10 to 13 for use in (a) a method for treating cancer, or (b) a method for treating an infectious disease, optionally a viral infection, or the composition according to claim 18, characterized in that the composition is administered to an individual having cancer or an individual in need of treatment for an infectious disease.

21. 19. A method of expanding T cells ex vivo comprising contacting one or more T cells ex vivo with an effective amount of a mutated IL-10 polypeptide of any one of claims 1 to 9, or a fusion protein of any one of claims 10 to 13, or a composition of claim 18, optionally wherein the one or more T cells are tumor infiltrating lymphocytes (TIL).

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