Compositions and methods relating to receptor pairing

Abstract

Binds to IL10Rα and IL2Rγ, and anti-IL10Rα V H H antibody and anti-IL2Rγ V H Provided herein are IL10Rα / IL2Rγ binding proteins, including H antibodies. TIFF2024504923000149.tif83128

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED PATENT APPLICATIONS This patent application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 136,098, filed January 11, 2021; U.S. Provisional Patent Application No. 63 / 135,884, filed January 11, 2021, and PCT Patent Application No. PCT / US2021 / 044858, filed August 6, 2021, each of which is incorporated by reference. [Background technology]

[0002] Background of the disclosure Binding of IL10 to the IL10 receptor (IL10R) can induce both immunosuppressive and immunostimulatory effects on various cell types. IL10 can cause a number of harmful and undesirable effects by diverse mechanisms due to, among other factors, the presence of IL10R on various cell types.

[0003] The anti-inflammatory cytokine interleukin-10 (IL-10), also known as human cytokine synthesis inhibitory factor (CSIF), is classified as a type (class)-2 cytokine, a set of cytokines that includes IL-19, IL-20, IL-22, IL-24 (Mda-7), and IL-26, interferons (IFN-α, -β, -γ, -δ, -ε, -κ, -Ω, and -τ), and interferon-like molecules (limitin, IL-28A, IL-28B, and IL-29). Human IL-10 is a homodimer with a molecular weight of 37 kDa, and each 18.5 kDa monomer contains 178 amino acids, the first 18 of which include a signal peptide and two cysteine ​​residues that form two intramolecular disulfide bonds. The IL-10 receptor, a type II cytokine receptor, consists of α (IL10Ra) and β (IL10Rb) subunits, also called R1 and R2, respectively. Binding to both α and β is required for receptor activation. One homodimer of the IL-10 polypeptide binds to α, and the other homodimer of the same IL-10 polypeptide binds to β.

[0004] IL-10 exhibits pleiotropic effects in immune regulation and inflammation through its actions on T cells, B cells, macrophages, and antigen-presenting cells (APCs). IL-10 is produced by mast cells and counteracts the inflammatory effects these cells have at the site of allergic reactions. IL-10 is primarily expressed in macrophages, but expression has also been detected in activated T cells, B cells, mast cells, and monocytes. IL-10 can suppress immune responses by inhibiting the expression of IL-1α, IL-1β, IL-6, IL8, TNFα, GM-CSF, and G-CSF in activated monocytes and activated macrophages, and also suppresses IFN-γ production by NK cells. IL10 can block NF-κB activity and is involved in the control of the JAK-STAT signaling pathway.

[0005] IL2 is a pluripotent cytokine produced by antigen-activated T cells. IL2 exerts a wide range of effects on the immune system and plays a key role in controlling both immune activation, suppression, and homeostasis. IL2 promotes the proliferation and expansion of activated T lymphocytes, induces the proliferation and activation of naive T cells, enhances the proliferation of B cells, and promotes the proliferation and expansion of NK cells. Human interleukin 2 (IL2) is a 133 amino acid, four-alpha helix bundle cytokine. IL2 is a member of the IL2 family of cytokines, which includes IL2, IL-4, IL-7, IL9, IL-15, and IL21.

[0006] IL2 exerts its effects on mammalian immune cells through interactions with three distinct cell surface proteins: (1) CD25 (also called IL2 receptor alpha, IL2Rα, p55), CD122 (also called interleukin-2 receptor beta, IL2Rβ, IL15Rβ, and p70-75), and CD132 (interleukin-2 receptor gamma, IL2Rγ; or common gamma chain, since it is a component of other multimeric receptors in the IL2 receptor family). In addition to the "low affinity" CD25 IL2 receptor, two additional IL2 receptor complexes have been characterized: (a) the "intermediate affinity" dimeric IL2 receptor (also called "IL2Rβγ"), which includes CD122 and CD132, and (b) the "high affinity" trimeric IL2 receptor complex (also called "IL2Rαβγ"), which includes CD25, CD122, and CD132 proteins. hIL2 binds approximately 10 to the intermediate affinity CD122 / CD132 (IL2βγ) receptor complex. -9 hIL2 has a Kd of approximately 10 M for the high IL2 affinity receptor complex. -11 It has a Kd of M.

[0007] In addition to forming a subunit of the high affinity IL2 receptor, CD132 is a type 1 cytokine receptor shared by receptor complexes for IL-4, IL-7, IL-9, IL-15, and IL21, and is therefore referred to in the literature as the "common" gamma chain. Human CD132 (hCD132) is expressed as a 369 amino acid preprotein, including a 22 amino acid N-terminal signal sequence. Amino acids 23-262 (amino acids 1-240 of the mature protein) correspond to the extracellular domain, amino acids 263-283 (amino acids 241-262 of the mature protein) correspond to the 21 amino acid transmembrane domain, and amino acids 284-369 (amino acids 262-347 of the mature protein) correspond to the intracellular domain. hCD132 is referenced in the UniProtKB database as entry P31785. The nucleic acid and protein sequences of human CD132 can be found under Genbank accession numbers: NM_000206 and NP_000197, respectively. Summary of the Invention

[0008] Disclosure Summary In one aspect, anti-IL10Rα V H H antibody and anti-IL2Rγ V H Provided herein are IL10Rα / IL2Rγ binding proteins that specifically bind to IL10Rα and IL2Rγ, including H antibodies.

[0009] In some embodiments, an IL10Rα / IL2Rγ binding protein that specifically binds IL10Rα and IL2Rγ comprises an anti-IL10Rα VHH antibody and an anti-IL2Rγ VHH antibody, wherein: (A) the anti-IL10Rα VHH antibody comprises a complementarity determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:264, a CDR2 comprising the amino acid sequence of SEQ ID NO:2, and a CDR3 comprising the amino acid sequence of SEQ ID NO:3; and The anti-IL2Rγ VHH antibody is i) CDR1 comprising the amino acid sequence of SEQ ID NO:25 or SEQ ID NO:270, CDR2 comprising the amino acid sequence of SEQ ID NO:26, and CDR3 comprising the amino acid sequence of SEQ ID NO:27; ii) a CDR1 comprising the amino acid sequence of SEQ ID NO:29 or SEQ ID NO:271, a CDR2 comprising the amino acid sequence of SEQ ID NO:30, and a CDR3 comprising the amino acid sequence of SEQ ID NO:31; iii) a CDR1 comprising the amino acid sequence of SEQ ID NO:33 or SEQ ID NO:272, a CDR2 comprising the amino acid sequence of SEQ ID NO:34, and a CDR3 comprising the amino acid sequence of SEQ ID NO:35; iv) CDR1 comprising the amino acid sequence of SEQ ID NO:37 or SEQ ID NO:273, CDR2 comprising the amino acid sequence of SEQ ID NO:38, and CDR3 comprising the amino acid sequence of SEQ ID NO:39; v) a CDR1 comprising the amino acid sequence of SEQ ID NO:41 or SEQ ID NO:274, a CDR2 comprising the amino acid sequence of SEQ ID NO:42, and a CDR3 comprising the amino acid sequence of SEQ ID NO:43; or vi) CDR1 comprising the amino acid sequence of SEQ ID NO:45 or SEQ ID NO:275, CDR2 comprising the amino acid sequence of SEQ ID NO:46, and CDR3 comprising the amino acid sequence of SEQ ID NO:47 Contains; or (B) the anti-IL10Rα VHH antibody comprises a complementarity determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:265, a CDR2 comprising the amino acid sequence of SEQ ID NO:6, and a CDR3 comprising the amino acid sequence of SEQ ID NO:7; and The anti-IL2Rγ VHH antibody is i) CDR1 comprising the amino acid sequence of SEQ ID NO:25 or SEQ ID NO:270, CDR2 comprising the amino acid sequence of SEQ ID NO:26, and CDR3 comprising the amino acid sequence of SEQ ID NO:27; ii) a CDR1 comprising the amino acid sequence of SEQ ID NO:29 or SEQ ID NO:271, a CDR2 comprising the amino acid sequence of SEQ ID NO:30, and a CDR3 comprising the amino acid sequence of SEQ ID NO:31; iii) a CDR1 comprising the amino acid sequence of SEQ ID NO:33 or SEQ ID NO:272, a CDR2 comprising the amino acid sequence of SEQ ID NO:34, and a CDR3 comprising the amino acid sequence of SEQ ID NO:35; iv) CDR1 comprising the amino acid sequence of SEQ ID NO:37 or SEQ ID NO:273, CDR2 comprising the amino acid sequence of SEQ ID NO:38, and CDR3 comprising the amino acid sequence of SEQ ID NO:39; v) a CDR1 comprising the amino acid sequence of SEQ ID NO:41 or SEQ ID NO:274, a CDR2 comprising the amino acid sequence of SEQ ID NO:42, and a CDR3 comprising the amino acid sequence of SEQ ID NO:43; or vi) CDR1 comprising the amino acid sequence of SEQ ID NO:45 or SEQ ID NO:275, CDR2 comprising the amino acid sequence of SEQ ID NO:46, and CDR3 comprising the amino acid sequence of SEQ ID NO:47 Contains; or (C) the anti-IL10Rα VHH antibody comprises a complementarity determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:266, a CDR2 comprising the amino acid sequence of SEQ ID NO:10, and a CDR3 comprising the amino acid sequence of SEQ ID NO:11; and The anti-IL2Rγ VHH antibody is i) CDR1 comprising the amino acid sequence of SEQ ID NO:25 or SEQ ID NO:270, CDR2 comprising the amino acid sequence of SEQ ID NO:26, and CDR3 comprising the amino acid sequence of SEQ ID NO:27; ii) a CDR1 comprising the amino acid sequence of SEQ ID NO:29 or SEQ ID NO:271, a CDR2 comprising the amino acid sequence of SEQ ID NO:30, and a CDR3 comprising the amino acid sequence of SEQ ID NO:31; iii) a CDR1 comprising the amino acid sequence of SEQ ID NO:33 or SEQ ID NO:272, a CDR2 comprising the amino acid sequence of SEQ ID NO:34, and a CDR3 comprising the amino acid sequence of SEQ ID NO:35; iv) CDR1 comprising the amino acid sequence of SEQ ID NO:37 or SEQ ID NO:273, CDR2 comprising the amino acid sequence of SEQ ID NO:38, and CDR3 comprising the amino acid sequence of SEQ ID NO:39; v) a CDR1 comprising the amino acid sequence of SEQ ID NO:41 or SEQ ID NO:274, a CDR2 comprising the amino acid sequence of SEQ ID NO:42, and a CDR3 comprising the amino acid sequence of SEQ ID NO:43; or vi) CDR1 comprising the amino acid sequence of SEQ ID NO:45 or SEQ ID NO:275, CDR2 comprising the amino acid sequence of SEQ ID NO:46, and CDR3 comprising the amino acid sequence of SEQ ID NO:47 Contains; or (D) the anti-IL10Rα VHH antibody comprises a complementarity determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO:13 or SEQ ID NO:267, a CDR2 comprising the amino acid sequence of SEQ ID NO:14, and a CDR3 comprising the amino acid sequence of SEQ ID NO:15; and The anti-IL2Rγ VHH antibody is i) CDR1 comprising the amino acid sequence of SEQ ID NO:25 or SEQ ID NO:270, CDR2 comprising the amino acid sequence of SEQ ID NO:26, and CDR3 comprising the amino acid sequence of SEQ ID NO:27; ii) a CDR1 comprising the amino acid sequence of SEQ ID NO:29 or SEQ ID NO:271, a CDR2 comprising the amino acid sequence of SEQ ID NO:30, and a CDR3 comprising the amino acid sequence of SEQ ID NO:31; iii) a CDR1 comprising the amino acid sequence of SEQ ID NO:33 or SEQ ID NO:272, a CDR2 comprising the amino acid sequence of SEQ ID NO:34, and a CDR3 comprising the amino acid sequence of SEQ ID NO:35; iv) CDR1 comprising the amino acid sequence of SEQ ID NO:37 or SEQ ID NO:273, CDR2 comprising the amino acid sequence of SEQ ID NO:38, and CDR3 comprising the amino acid sequence of SEQ ID NO:39; v) a CDR1 comprising the amino acid sequence of SEQ ID NO:41 or SEQ ID NO:274, a CDR2 comprising the amino acid sequence of SEQ ID NO:42, and a CDR3 comprising the amino acid sequence of SEQ ID NO:43; or vi) CDR1 comprising the amino acid sequence of SEQ ID NO:45 or SEQ ID NO:275, CDR2 comprising the amino acid sequence of SEQ ID NO:46, and CDR3 comprising the amino acid sequence of SEQ ID NO:47 Contains; or (E) the anti-IL10Rα VHH antibody comprises a complementarity determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO:17 or SEQ ID NO:268, a CDR2 comprising the amino acid sequence of SEQ ID NO:18, and a CDR3 comprising the amino acid sequence of SEQ ID NO:19; and The anti-IL2Rγ VHH antibody is i) CDR1 comprising the amino acid sequence of SEQ ID NO:25 or SEQ ID NO:270, CDR2 comprising the amino acid sequence of SEQ ID NO:26, and CDR3 comprising the amino acid sequence of SEQ ID NO:27; ii) a CDR1 comprising the amino acid sequence of SEQ ID NO:29 or SEQ ID NO:271, a CDR2 comprising the amino acid sequence of SEQ ID NO:30, and a CDR3 comprising the amino acid sequence of SEQ ID NO:31; iii) a CDR1 comprising the amino acid sequence of SEQ ID NO:33 or SEQ ID NO:272, a CDR2 comprising the amino acid sequence of SEQ ID NO:34, and a CDR3 comprising the amino acid sequence of SEQ ID NO:35; iv) CDR1 comprising the amino acid sequence of SEQ ID NO:37 or SEQ ID NO:273, CDR2 comprising the amino acid sequence of SEQ ID NO:38, and CDR3 comprising the amino acid sequence of SEQ ID NO:39; v) a CDR1 comprising the amino acid sequence of SEQ ID NO:41 or SEQ ID NO:274, a CDR2 comprising the amino acid sequence of SEQ ID NO:42, and a CDR3 comprising the amino acid sequence of SEQ ID NO:43; or vi) CDR1 comprising the amino acid sequence of SEQ ID NO:45 or SEQ ID NO:275, CDR2 comprising the amino acid sequence of SEQ ID NO:46, and CDR3 comprising the amino acid sequence of SEQ ID NO:47 Contains; or (F) the anti-IL10Rα VHH antibody comprises a complementarity determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO:21 or SEQ ID NO:269, a CDR2 comprising the amino acid sequence of SEQ ID NO:22, and a CDR3 comprising the amino acid sequence of SEQ ID NO:23; and The anti-IL2Rγ VHH antibody is i) CDR1 comprising the amino acid sequence of SEQ ID NO:25 or SEQ ID NO:270, CDR2 comprising the amino acid sequence of SEQ ID NO:26, and CDR3 comprising the amino acid sequence of SEQ ID NO:27; ii) a CDR1 comprising the amino acid sequence of SEQ ID NO:29 or SEQ ID NO:271, a CDR2 comprising the amino acid sequence of SEQ ID NO:30, and a CDR3 comprising the amino acid sequence of SEQ ID NO:31; iii) a CDR1 comprising the amino acid sequence of SEQ ID NO:33 or SEQ ID NO:272, a CDR2 comprising the amino acid sequence of SEQ ID NO:34, and a CDR3 comprising the amino acid sequence of SEQ ID NO:35; iv) CDR1 comprising the amino acid sequence of SEQ ID NO:37 or SEQ ID NO:273, CDR2 comprising the amino acid sequence of SEQ ID NO:38, and CDR3 comprising the amino acid sequence of SEQ ID NO:39; v) a CDR1 comprising the amino acid sequence of SEQ ID NO:41 or SEQ ID NO:274, a CDR2 comprising the amino acid sequence of SEQ ID NO:42, and a CDR3 comprising the amino acid sequence of SEQ ID NO:43; or vi) CDR1 comprising the amino acid sequence of SEQ ID NO:45 or SEQ ID NO:275, CDR2 comprising the amino acid sequence of SEQ ID NO:46, and CDR3 comprising the amino acid sequence of SEQ ID NO:47 Includes.

[0010] In some embodiments, the anti-IL10Rα V H The H antibody comprises: (1) a complementarity determining region 1 (CDR1) having the sequence of any one of SEQ ID NOs:1, 5, 9, 13, 1, and 21; (2) a CDR2 having the sequence of any one of SEQ ID NOs:2, 6, 10, 14, 18, and 22; and (3) a CDR3 having the sequence of any one of SEQ ID NOs:3, 7, 11, 15, 19, and 23.

[0011] In some embodiments, the anti-IL10Rα V H The anti-IL10Rα VH antibody is selected from the group consisting of DR235, DR236, DR237, DR239, DR240, and DR241. HIn some embodiments, the anti-IL10Rα V H antibody comprises the CDR1, CDR2, and CDR3 sequences of the anti-IL10Rα V H antibody. H The H antibody comprises a sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to any one of the sequences DR235 (SEQ ID NO:4), DR236 (SEQ ID NO:8), DR237 (SEQ ID NO:12), DR239 (SEQ ID NO:16), DR240 (SEQ ID NO:20), and DR241 (SEQ ID NO:24).

[0012] In some embodiments, the anti-IL10Rα V H The H antibody comprises: (1) a complementarity determining region 1 (CDR1) having the sequence of any one of SEQ ID NOs: 25, 29, 33, 37, 41, and 45; (2) a CDR2 having the sequence of any one of SEQ ID NOs: 26, 30, 34, 38, 42, and 46; and (3) a CDR3 having the sequence of any one of SEQ ID NOs: 27, 31, 35, 39, 43, and 47.

[0013] In some embodiments, anti-IL2Rγ V H The anti-IL2Rγ VH antibody is selected from the group consisting of DR229, DR230, DR231, DR232, DR233, and DR234. H In some embodiments, the anti-IL10Rα V H antibody comprises the CDR1, CDR2, and CDR3 sequences of the anti-IL10Rα V H antibody. H The H antibody comprises a sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to any one of the sequences DR229 (SEQ ID NO:28), DR230 (SEQ ID NO:32), DR231 (SEQ ID NO:36), DR232 (SEQ ID NO:40), DR233 (SEQ ID NO:44), and DR234 (SEQ ID NO:48).

[0014] In some embodiments of this aspect, the anti-IL10Rα V H H antibodies are N-terminal and anti-IL2Rγ V H The H antibody is at the C-terminus. In certain embodiments, the binding protein comprises a sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to any one of SEQ ID NOs:49-59.

[0015] In other embodiments, anti-IL2Rγ V H H antibody is N-terminal and anti-IL10Rα V H The H antibody is at the C-terminus. In some embodiments, the binding protein comprises a sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to any one of SEQ ID NOs:60 and 61.

[0016] In some embodiments, the anti-IL10Rα V H H antibody and anti-IL2Rγ V H The H antibody is linked to the H antibody by a peptide linker. In a particular embodiment, the peptide linker comprises 1 to 50 amino acids.

[0017] In some embodiments, the binding protein comprises a sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to any one of SEQ ID NOs:49-61 or 96-179, and optionally does not include a HHHHHH sequence.

[0018] In some embodiments of this aspect, the binding protein is conjugated to an Fc polypeptide or Fc domain. In some embodiments, the Fc polypeptide or Fc domain is from IgG1, IgG2, IgG3, or IgG4. In some embodiments, the IL10Rα / IL2Rγ binding protein comprises SEQ ID NO:556 or SEQ ID NO:558. In other embodiments, the binding protein is PEGylated.

[0019] Also provided are IL10Rα / IL2Rγ binding proteins that specifically bind to IL10Rα and IL2Rγ, including anti-IL10Rα VHH antibodies and anti-IL2Rγ VHH antibodies, which are linked to an Fc polypeptide or Fc domain from IgG1, IgG2, IgG3, or IgG4.

[0020] Also, the first polypeptide of formula #1: Anti-IL10Rα V H H antibody-L1 a -UH1-Fc1[1] and a second polypeptide of formula #2: Anti-IL2Rγ V H H antibody-L2 b -UH2-Fc2 [2] Also provided is a heterodimeric IL10Rα binding protein / IL2Rγ binding protein pair, comprising: During the ceremony, L1 and L2 are GSA linkers and a and b are independently selected from 0 (absent) or 1 (present); UH1 and UH2 are each an upper hinge domain of a human immunoglobulin independently selected from the group consisting of IgG1, IgG2, IgG3, and IgG4 upper hinges, optionally containing the amino acid substitution C220S (EU numbering); Fc1 is a polypeptide comprising the lower hinge domain, the CH2 domain, and the CH3 domain of a human immunoglobulin selected from the group consisting of IgG1, IgG2, IgG3, and IgG4, and comprising one or more amino acid substitutions that promote heterodimerization with Fc2; and FC2 is a polypeptide comprising the lower hinge domain, the CH2 domain, and the CH3 domain of a human immunoglobulin selected from the group consisting of IgG1, IgG2, IgG3, and IgG4, and comprising one or more amino acid substitutions that promote heterodimerization with Fc1; and The polypeptide of formula 1 and the polypeptide of formula 2 are linked by at least one interchain disulfide bond; and (A) The anti-IL10Rα V H The H antibody comprises a complementarity determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:264, a CDR2 comprising the amino acid sequence of SEQ ID NO:2, and a CDR3 comprising the amino acid sequence of SEQ ID NO:3; and The anti-IL2Rγ V H H antibodies are i) CDR1 comprising the amino acid sequence of SEQ ID NO:25 or SEQ ID NO:270, CDR2 comprising the amino acid sequence of SEQ ID NO:26, and CDR3 comprising the amino acid sequence of SEQ ID NO:27; ii) a CDR1 comprising the amino acid sequence of SEQ ID NO:29 or SEQ ID NO:271, a CDR2 comprising the amino acid sequence of SEQ ID NO:30, and a CDR3 comprising the amino acid sequence of SEQ ID NO:31; iii) a CDR1 comprising the amino acid sequence of SEQ ID NO:33 or SEQ ID NO:272, a CDR2 comprising the amino acid sequence of SEQ ID NO:34, and a CDR3 comprising the amino acid sequence of SEQ ID NO:35; iv) CDR1 comprising the amino acid sequence of SEQ ID NO:37 or SEQ ID NO:273, CDR2 comprising the amino acid sequence of SEQ ID NO:38, and CDR3 comprising the amino acid sequence of SEQ ID NO:39; v) a CDR1 comprising the amino acid sequence of SEQ ID NO:41 or SEQ ID NO:274, a CDR2 comprising the amino acid sequence of SEQ ID NO:42, and a CDR3 comprising the amino acid sequence of SEQ ID NO:43; or vi) CDR1 comprising the amino acid sequence of SEQ ID NO:45 or SEQ ID NO:275, CDR2 comprising the amino acid sequence of SEQ ID NO:46, and CDR3 comprising the amino acid sequence of SEQ ID NO:47 Contains; or (B) The anti-IL10Rα V H The H antibody comprises a complementarity determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:265, a CDR2 comprising the amino acid sequence of SEQ ID NO:6, and a CDR3 comprising the amino acid sequence of SEQ ID NO:7; and The anti-IL2Rγ V H H antibodies are i) CDR1 comprising the amino acid sequence of SEQ ID NO:25 or SEQ ID NO:270, CDR2 comprising the amino acid sequence of SEQ ID NO:26, and CDR3 comprising the amino acid sequence of SEQ ID NO:27; ii) a CDR1 comprising the amino acid sequence of SEQ ID NO:29 or SEQ ID NO:271, a CDR2 comprising the amino acid sequence of SEQ ID NO:30, and a CDR3 comprising the amino acid sequence of SEQ ID NO:31; iii) a CDR1 comprising the amino acid sequence of SEQ ID NO:33 or SEQ ID NO:272, a CDR2 comprising the amino acid sequence of SEQ ID NO:34, and a CDR3 comprising the amino acid sequence of SEQ ID NO:35; iv) CDR1 comprising the amino acid sequence of SEQ ID NO:37 or SEQ ID NO:273, CDR2 comprising the amino acid sequence of SEQ ID NO:38, and CDR3 comprising the amino acid sequence of SEQ ID NO:39; v) a CDR1 comprising the amino acid sequence of SEQ ID NO:41 or SEQ ID NO:274, a CDR2 comprising the amino acid sequence of SEQ ID NO:42, and a CDR3 comprising the amino acid sequence of SEQ ID NO:43; or vi) CDR1 comprising the amino acid sequence of SEQ ID NO:45 or SEQ ID NO:275, CDR2 comprising the amino acid sequence of SEQ ID NO:46, and CDR3 comprising the amino acid sequence of SEQ ID NO:47 Contains; or (C) The anti-IL10Rα V H The H antibody comprises a complementarity determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:266, a CDR2 comprising the amino acid sequence of SEQ ID NO:10, and a CDR3 comprising the amino acid sequence of SEQ ID NO:11; and The anti-IL2Rγ V H H antibodies are i) CDR1 comprising the amino acid sequence of SEQ ID NO:25 or SEQ ID NO:270, CDR2 comprising the amino acid sequence of SEQ ID NO:26, and CDR3 comprising the amino acid sequence of SEQ ID NO:27; ii) a CDR1 comprising the amino acid sequence of SEQ ID NO:29 or SEQ ID NO:271, a CDR2 comprising the amino acid sequence of SEQ ID NO:30, and a CDR3 comprising the amino acid sequence of SEQ ID NO:31; iii) a CDR1 comprising the amino acid sequence of SEQ ID NO:33 or SEQ ID NO:272, a CDR2 comprising the amino acid sequence of SEQ ID NO:34, and a CDR3 comprising the amino acid sequence of SEQ ID NO:35; iv) CDR1 comprising the amino acid sequence of SEQ ID NO:37 or SEQ ID NO:273, CDR2 comprising the amino acid sequence of SEQ ID NO:38, and CDR3 comprising the amino acid sequence of SEQ ID NO:39; v) a CDR1 comprising the amino acid sequence of SEQ ID NO:41 or SEQ ID NO:274, a CDR2 comprising the amino acid sequence of SEQ ID NO:42, and a CDR3 comprising the amino acid sequence of SEQ ID NO:43; or vi) CDR1 comprising the amino acid sequence of SEQ ID NO:45 or SEQ ID NO:275, CDR2 comprising the amino acid sequence of SEQ ID NO:46, and CDR3 comprising the amino acid sequence of SEQ ID NO:47 Contains; or (D) The anti-IL10Rα V H The H antibody comprises a complementarity determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO:13 or SEQ ID NO:267, a CDR2 comprising the amino acid sequence of SEQ ID NO:14, and a CDR3 comprising the amino acid sequence of SEQ ID NO:15; and The anti-IL2Rγ V H H antibodies are i) CDR1 comprising the amino acid sequence of SEQ ID NO:25 or SEQ ID NO:270, CDR2 comprising the amino acid sequence of SEQ ID NO:26, and CDR3 comprising the amino acid sequence of SEQ ID NO:27; ii) a CDR1 comprising the amino acid sequence of SEQ ID NO:29 or SEQ ID NO:271, a CDR2 comprising the amino acid sequence of SEQ ID NO:30, and a CDR3 comprising the amino acid sequence of SEQ ID NO:31; iii) a CDR1 comprising the amino acid sequence of SEQ ID NO:33 or SEQ ID NO:272, a CDR2 comprising the amino acid sequence of SEQ ID NO:34, and a CDR3 comprising the amino acid sequence of SEQ ID NO:35; iv) CDR1 comprising the amino acid sequence of SEQ ID NO:37 or SEQ ID NO:273, CDR2 comprising the amino acid sequence of SEQ ID NO:38, and CDR3 comprising the amino acid sequence of SEQ ID NO:39; v) a CDR1 comprising the amino acid sequence of SEQ ID NO:41 or SEQ ID NO:274, a CDR2 comprising the amino acid sequence of SEQ ID NO:42, and a CDR3 comprising the amino acid sequence of SEQ ID NO:43; or vi) CDR1 comprising the amino acid sequence of SEQ ID NO:45 or SEQ ID NO:275, CDR2 comprising the amino acid sequence of SEQ ID NO:46, and CDR3 comprising the amino acid sequence of SEQ ID NO:47 Contains; or (E) The anti-IL10Rα V H The H antibody comprises a complementarity determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO:17 or SEQ ID NO:268, a CDR2 comprising the amino acid sequence of SEQ ID NO:18, and a CDR3 comprising the amino acid sequence of SEQ ID NO:19; and The anti-IL2Rγ V H H antibodies are i) CDR1 comprising the amino acid sequence of SEQ ID NO:25 or SEQ ID NO:270, CDR2 comprising the amino acid sequence of SEQ ID NO:26, and CDR3 comprising the amino acid sequence of SEQ ID NO:27; ii) a CDR1 comprising the amino acid sequence of SEQ ID NO:29 or SEQ ID NO:271, a CDR2 comprising the amino acid sequence of SEQ ID NO:30, and a CDR3 comprising the amino acid sequence of SEQ ID NO:31; iii) a CDR1 comprising the amino acid sequence of SEQ ID NO:33 or SEQ ID NO:272, a CDR2 comprising the amino acid sequence of SEQ ID NO:34, and a CDR3 comprising the amino acid sequence of SEQ ID NO:35; iv) CDR1 comprising the amino acid sequence of SEQ ID NO:37 or SEQ ID NO:273, CDR2 comprising the amino acid sequence of SEQ ID NO:38, and CDR3 comprising the amino acid sequence of SEQ ID NO:39; v) a CDR1 comprising the amino acid sequence of SEQ ID NO:41 or SEQ ID NO:274, a CDR2 comprising the amino acid sequence of SEQ ID NO:42, and a CDR3 comprising the amino acid sequence of SEQ ID NO:43; or vi) CDR1 comprising the amino acid sequence of SEQ ID NO:45 or SEQ ID NO:275, CDR2 comprising the amino acid sequence of SEQ ID NO:46, and CDR3 comprising the amino acid sequence of SEQ ID NO:47 Contains; or (F) The anti-IL10Rα V H The H antibody comprises a complementarity determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO:21 or SEQ ID NO:269, a CDR2 comprising the amino acid sequence of SEQ ID NO:22, and a CDR3 comprising the amino acid sequence of SEQ ID NO:23; and The anti-IL2Rγ V H H antibodies are i) CDR1 comprising the amino acid sequence of SEQ ID NO:25 or SEQ ID NO:270, CDR2 comprising the amino acid sequence of SEQ ID NO:26, and CDR3 comprising the amino acid sequence of SEQ ID NO:27; ii) a CDR1 comprising the amino acid sequence of SEQ ID NO:29 or SEQ ID NO:271, a CDR2 comprising the amino acid sequence of SEQ ID NO:30, and a CDR3 comprising the amino acid sequence of SEQ ID NO:31; iii) a CDR1 comprising the amino acid sequence of SEQ ID NO:33 or SEQ ID NO:272, a CDR2 comprising the amino acid sequence of SEQ ID NO:34, and a CDR3 comprising the amino acid sequence of SEQ ID NO:35; iv) CDR1 comprising the amino acid sequence of SEQ ID NO:37 or SEQ ID NO:273, CDR2 comprising the amino acid sequence of SEQ ID NO:38, and CDR3 comprising the amino acid sequence of SEQ ID NO:39; v) a CDR1 comprising the amino acid sequence of SEQ ID NO:41 or SEQ ID NO:274, a CDR2 comprising the amino acid sequence of SEQ ID NO:42, and a CDR3 comprising the amino acid sequence of SEQ ID NO:43; or vi) CDR1 comprising the amino acid sequence of SEQ ID NO:45 or SEQ ID NO:275, CDR2 comprising the amino acid sequence of SEQ ID NO:46, and CDR3 comprising the amino acid sequence of SEQ ID NO:47 Contains; or (G) The anti-IL10Rα V H H antibodies are a CDR1 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to any CDR1 sequence in a row of Table 10, or having 0, 1, 2, or 3 amino acid changes, optionally conservative amino acid changes, compared to said sequence; A CDR2 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to any CDR2 sequence in a row of Table 10, or having 0, 1, 2, or 3 amino acid changes, optionally conservative amino acid changes, compared to said sequence; and CDR3 sequences having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to any of the CDR3 sequences listed in Table 10, or having 0, 1, 2, or 3 amino acid changes, optionally conservative amino acid changes, compared to the sequences. and The anti-IL2Rγ V H H antibodies are a CDR1 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to any CDR1 sequence listed in Table 11 or Table 12, or having 0, 1, 2, or 3 amino acid changes, optionally conservative amino acid changes, compared to said sequence; a CDR2 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to any CDR2 sequence listed in Table 11 or Table 12, or having 0, 1, 2, or 3 amino acid changes, optionally conservative amino acid changes, compared to said sequence; and A CDR3 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to any CDR3 sequence listed in Table 11 or Table 12, or having 0, 1, 2, or 3 amino acid changes, optionally conservative amino acid changes, compared to the sequence. Includes.

[0021] In another aspect, the disclosure provides an isolated nucleic acid encoding an IL10Rα / IL2Rγ binding protein or a heterodimeric IL10Rα binding protein / IL2Rγ binding protein pair described herein.

[0022] In another aspect, the disclosure provides an expression vector comprising a nucleic acid described herein.

[0023] In another aspect, the disclosure provides an isolated host cell comprising a vector that includes a nucleic acid described herein.

[0024] In another aspect, the disclosure provides a pharmaceutical composition comprising an IL10Rα / IL2Rγ binding protein or a heterodimeric IL10Rα binding protein / IL2Rγ binding protein pair described herein and a pharma- ceutically acceptable carrier.

[0025] In another aspect, the disclosure provides a method of treating a neoplastic disease in a subject in need thereof comprising administering to the subject a therapeutically effective amount of an IL10Rα / IL2Rγ binding protein or a heterodimeric IL10Rα binding protein / IL2Rγ binding protein pair described herein, or a pharmaceutical composition comprising an IL10Rα / IL2Rγ binding protein or a heterodimeric IL10Rα binding protein / IL2Rγ binding protein pair described herein and a pharma- ceutically acceptable carrier.

[0026] In some embodiments, the method further comprises administering a cofactor to the subject. In some embodiments, the cofactor is selected from the group consisting of a chemotherapeutic agent, an antibody, an immune checkpoint regulator, a TIL, a CAR-T cell, and a physical method.

[0027] In some embodiments of this aspect, the neoplastic disease is selected from the group consisting of adenoma, fibroma, hemangioma, hyperplasia, atypia, metaplasia, dysplasia, carcinoma, leukemia, breast cancer, sarcoma, leukemia, lymphoma, genitourinary cancer, ovarian cancer, urethral cancer, bladder cancer, prostate cancer, gastrointestinal cancer, colon cancer, esophageal cancer, gastric cancer, lung cancer; myeloma; pancreatic cancer; liver cancer; kidney cancer; endocrine cancer; skin cancer; glioma, neuroblastoma, astrocytoma, myelodysplastic disorders; cervical intraepithelial neoplasia; intestinal polyposis; oral leukoplakia; histiocytosis, hyperproliferative scars including keloid scars, respiratory system cancer, gastrointestinal cancer, and the like. Cancers of the gastrointestinal system, genitourinary system, testicular cancer, breast cancer, prostate cancer, endocrine system cancer, melanoma, adenocarcinoma, myeloproliferative neoplasms, myeloid and lymphoid disorders with eosinophilia, myeloproliferative / myelodysplastic neoplasms, myelodysplastic syndromes, acute myeloid leukemia and related precursor neoplasms, and acute leukemia of ambiguous lineage, promyelocytic leukemia (APML), acute myeloid leukemia (AML) and chronic myeloid leukemia (CML), precursor lymphoid neoplasms, mature B-cell neoplasms, mature T-cell neoplasms, Hodgkin's lymphoma, and immunodeficiency-associated lymphoproliferative disorders, The cancer is selected from the group consisting of lymphoblastic leukemia (ALL), including B-lineage lymphoblastic leukemia (ALL) and T-lineage ALL, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), hairy cell leukemia (HLL), and Waldenstrom's macroglobulinemia (WM), erythroblastic leukemia and acute megakaryoblastic leukemia, malignant lymphomas, including but not limited to non-Hodgkin's lymphoma and variants thereof, peripheral T-cell lymphoma, adult T-cell leukemia / lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), large granular lymphocytic leukemia (LGF), Hodgkin's disease, and Reed-Stemberg disease.

[0028] In another aspect, the disclosure provides a method of treating an autoimmune or inflammatory disease, disorder, or condition in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an IL10Rα / IL2Rγ binding protein described herein, or a pharmaceutical composition comprising an IL10Rα / IL2Rγ binding protein described herein and a pharma- ceutically acceptable carrier.

[0029] In some embodiments of this aspect, the method further comprises administering one or more cofactors selected from the group consisting of a corticosteroid, a Janus kinase inhibitor, a calcineurin inhibitor, an mTor inhibitor, an IMDH inhibitor, a biologic, a vaccine, and a therapeutic antibody. In certain embodiments, the therapeutic antibody is an antibody that binds to a protein selected from the group consisting of BLyS, CD11a, CD20, CD25, CD3, CD52, IgEIL12 / IL23, IL17a, IL1β, IL4Rα, IL5, IL6R, integrin-α4β7, RANKL, TNFα, VEGF-A, and VLA-4.

[0030] In some embodiments of this aspect, the disease, disorder, or condition is a viral infection, Helicobacter pylori infection, HTLV, organ rejection, graft versus host disease, autoimmune thyroid disease, multiple sclerosis, allergies, asthma, neurodegenerative diseases including Alzheimer's disease, systemic lupus erythematosus (SLE), autoinflammatory diseases, inflammatory bowel disease (IBD), Crohn's disease, diabetes, cartilage inflammation, arthritis, rheumatoid arthritis, juvenile arthritis, juvenile rheumatoid arthritis, juvenile rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis, systemic juvenile rheumatoid arthritis, juvenile ankylosing spondylitis, juvenile enteropathic arthritis, juvenile reactive arthritis, juvenile Reiter's syndrome, SEA syndrome, juvenile dermatomyositis, juvenile psoriatic arthritis, juvenile scleroderma, juvenile systemic lupus erythematosus, Juvenile vasculitis, small joint rheumatoid arthritis, polyarticular rheumatoid arthritis, systemic rheumatoid arthritis, ankylosing spondylitis, enteropathic arthritis, reactive arthritis, Reiter's syndrome, SEA syndrome, psoriasis, psoriatic arthritis, dermatitis (eczema), exfoliative dermatitis or atopic dermatitis, pityriasis rubra pilaris, pityriasis rosea, parapsoriasis, pityriasis lichenoides, lichen planus, lichen nitidus, ichthyosa-like dermatosis, keratoderma, skin Selected from skin diseases, alopecia areata, pyoderma gangrenosum, vitiligo, pemphigoid, urticaria, porokeratosis, rheumatoid arthritis; seborrheic dermatitis, actinic keratosis, senile keratosis, actinic keratosis, follicular keratosis; acne vulgaris; keloids; birthmarks; warts, including condyloma, or genital warts, and human papillomavirus (HPV) infections.

[0031] In certain embodiments, the methods described herein do not cause anemia.

[0032] In another aspect, the disclosure provides a method of selectively inducing an activity (e.g., phosphorylation) in one or more of a first cell type over one or more of a second cell type, the method comprising contacting a population of cells comprising both the first cell type and the second cell type with an IL10Rα / IL2Rγ binding protein or a heterodimeric IL10Rα binding protein / IL2Rγ binding protein pair described herein, thereby selectively inducing the activity in one or more of the first cell types over one or more of the second cell types.

[0033] In some embodiments, the first cell type is CD4 + In some embodiments, the first cell type is a CD8 T cell. + In some embodiments, the second cell type is a T cell. In some embodiments, the second cell type is a NK cell. In some embodiments, the second cell type is a B cell. In some embodiments, the second cell type is a monocyte. In some embodiments, the first cell type is a CD4 + T cells, CD8 + In certain embodiments, the second cell type is a monocyte.

[0034] In other embodiments of this aspect, the activity of the first cell type is at least 1.2 (e.g., at least 1.4, 1.6, 1.8, 2, 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4, 6, 8, 10, 12, 14, 16, 18, or 20) times greater than the activity of the second cell type. [Brief description of the drawings]

[0035] [Figure 1A] 1A-1D show various configurations of one or two IL10Rα / IL2Rγ binding proteins conjugated to an Fc domain. [Figure 1B]See legend to Figure 1A. [Figure 1C] See legend to Figure 1A. [Figure 1D] See legend to Figure 1A. [Figure 2-1] 2A-2E show dot plots of screening data for induction of pSTAT3 activity by IL10Rα / IL2Rγ binding proteins in various cell types. [Figure 2-2] See description of Figure 2-1. [Figure 2-3] See description of Figure 2-1. [Figure 3-1] 3A-3E show dose-dependent induction of IL10Rα / IL2Rγ binding protein in various cell types. [Figure 3-2] See description of Figure 3-1. [Figure 3-3] See description of Figure 3-1. [Figure 4] FIG. 4 of the accompanying drawings provides a schematic diagram of one embodiment of a bivalent binding molecule of the present disclosure comprising a first single domain antibody (1) and a second single domain antibody (3) and a linker (2), which is depicted as interacting with a cell membrane (10) bound heterodimeric receptor comprising a first receptor subunit comprising an extracellular domain (4), a transmembrane domain (5), and an intracellular domain (6) that interacts with the bivalent binding molecule, and a second first receptor subunit comprising an extracellular domain (7), a transmembrane domain (8), and an intracellular domain (9), where the intracellular domain (6) of the first receptor and the intracellular domain (9) of the second receptor on the bivalent binding molecule are within a proximal distance (11). [Diagram 5]Figure 5 of the accompanying drawings provides schematic diagrams of two illustrative configurations of bivalent binding molecules of the present disclosure. Panel A provides a schematic diagram of an illustrative single polypeptide chain bivalent binding molecule comprising, from amino to carboxy, a first single domain antibody (1) and a second single domain antibody (3) and a linker (2). Panel B provides a schematic diagram of a bivalent binding molecule comprising a first single domain antibody (1) and a second single domain antibody (3) and a linker (2), and a knob-into-hole Fc domain comprising a first subunit which is an Fc knob (13) and a second subunit which is an Fc hole (14), where the single domain antibody is stably associated with the Fc domain via an IgG hinge sequence (12). [Figure 6] Figure 6 of the accompanying drawings provides schematic diagrams of two illustrative configurations of a bivalent binding molecule of the present disclosure. Panel A provides a schematic diagram of an illustrative bivalent binding molecule comprising a first single domain antibody (1) and a second single domain antibody (3) and a linker (2). Panel B provides a schematic diagram of a bivalent binding molecule comprising two polypeptide chains, a first polypeptide chain comprising (amino to carboxy) a first single domain antibody (1), a linker sequence, a second single domain antibody (3), an IgG hinge sequence (12), and an Fc knob domain (13) and a second polypeptide comprising an Fc hole (14), where the first and second polypeptides are stably associated via knob-into-hole Fc domain interactions. [Figure 7]Panel A provides an alternative schematic of the configuration of a bivalent binding molecule of the present disclosure, in which one single domain antibody is attached to each subunit of a knob-into-hole Fc domain comprising two polypeptides, a first polypeptide comprising, amino-to-carboxy, a first single domain antibody (1), an IgG hinge sequence (12), and an Fc knob subunit (13), and a second polypeptide comprising, amino-to-carboxy, a second single domain antibody (3), an IgG hinge sequence (12), and an Fc hole subunit (13), in which the first and second single domain antibodies are stably associated via knob-into-hole Fc domain interactions. Panel B provides a schematic of a bivalent binding molecule in which the binding domains are single domain antibodies associated via a transition metal coordination covalent complex. As shown, the bivalent binding molecule comprises two polypeptide subunits: a first subunit comprising a first single domain antibody (1) attached via a first linker (15) to a first chelating peptide (17) and a second subunit comprising a second single domain antibody (3) attached via a second linker (16) to a second chelating peptide (18), where the first chelating peptide (17) and the second chelating peptide (18) form a coordinate covalent complex with a single transition metal ion ("M"), which may be in a kinetically unstable or kinetically inactive oxidation state. [Figure 8-1] 8A-8E provide STAT3 response dose-response data in various cell types with the indicated IL10Rα / IL2Rγ binding molecule test articles when tested on CD8 T cells (FIG. 8A), monocytes (FIG. 8B), CD4 T cells (FIG. 8C), B cells (FIG. 8D), and NK cells (FIG. 8E), as more fully described in the Examples. [Figure 8-2] See description of Figure 8-1. [Figure 9-1]9A-9E provide STAT3 response dose-response data in various cell types with the A2 and H1 IL10Rα / IL2Rγ binding molecules and their Fc-conjugated counterparts (H1 Fc=DR992, SEQ ID NO:556 and A2 Fc=DR995, SEQ ID NO:558) when tested on CD8 T cells (FIG. 9A), monocytes (FIG. 9B), CD4 T cells (FIG. 9C), B cells (FIG. 9D), and NK cells (FIG. 9E), as more fully described in the Examples. [Figure 9-2] See description of Figure 9-1. [Figure 10-1] 10A-10D provide the results of monocyte function assays showing levels of IL1b expression (FIG. 10A), IL6 (FIG. 10B), IL8 (FIG. 10C), and TNFα (FIG. 10D) in response to various doses of A2 and H1 IL10Rα / IL2Rγ binding molecules and their Fc-conjugated counterparts (H1 Fc=DR992, SEQ ID NO:556 and A2 Fc=DR995, SEQ ID NO:558), as described more fully in the Examples. [Figure 10-2] See description of Figure 10-1. [Figure 11] 11A-11B provide the results of a CD8 T cell blast function assay showing the levels of granzyme A expression (FIG. 11A) and granzyme B expression (FIG. 11B) in response to various doses of A2 and H1 IL10Rα / IL2Rγ binding molecules and their Fc-conjugated counterparts (H1 Fc=DR992, SEQ ID NO:556 and A2 Fc=DR995, SEQ ID NO:558), as described more fully in the Examples. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0036] Detailed Description of the Disclosure I. Introduction The present disclosure provides compositions useful in pairing cell receptors to produce a desired effect useful in the treatment of disease. Generally, binding proteins are provided that include a first domain that binds to IL10Rα and a second domain that binds to IL2Rγ, such that upon contact with a cell expressing IL10Rα (also called IL10R1) and IL2Rγ, the binding protein causes functional association of IL10Rα and IL2Rγ, thereby resulting in functional dimerization of the receptors and downstream signal transduction.

[0037] Several advantages arise from the binding proteins described herein. Unlike IL10, the natural ligand of IL10R, which can induce both immunosuppressive and immunostimulatory effects on various cell types, the binding proteins described herein can separate the immunosuppressive and immunostimulatory effects and selectively provide only the desired effect on the desired cell type. When IL10 is used as a therapeutic agent in mammals, particularly human subjects, it can also induce a number of harmful and undesirable effects by various mechanisms, including the presence of IL10R on various cell types, and binding to IL10R on various cell types can result in undesirable effects and / or undesirable signaling to cells expressing IL10 receptor. The present disclosure is directed to methods and compositions that modulate the multiple effects of IL10R binding so that the desired therapeutic signaling occurs, particularly in the desired cell or tissue subtype, while also minimizing undesirable activity and / or intracellular signaling.

[0038] For example, IL10 stimulates macrophages (e.g., monocytes) as well as T cells (e.g., CD4 + T cells and CD8 +Macrophages are a cell type that express both IL10Rα and IL10Rβ receptors, but when significantly activated, they can result in the phagocytosis of senescent red blood cells, leading to side effects such as anemia in patients undergoing IL10 therapy. In some embodiments, the methods provided herein use binding proteins of the present disclosure that bind IL10Rα and IL2Rγ, resulting in selective activation of T cells relative to activation of macrophages. Selective activation of T cells relative to macrophages is beneficial because IL10-activated macrophages and their associated side effects of anemia can be avoided. Binding proteins as described herein that provide selective substantial activation of T cells while providing minimal activation of macrophages result in a molecule that retains the beneficial properties of the native IL10 ligand, but results in reduced undesirable side effects relative to the native IL10 ligand.

[0039] In some embodiments, the binding molecule that specifically binds to IL10Rα and IL2Rγ is max Reduced E compared to max E max reflects the maximum response level in a cell type that can be obtained with a ligand (e.g., a binding protein described herein or a natural cytokine (e.g., IL10)). In some embodiments, the binding proteins that specifically bind IL10Rα and IL2Rγ described herein enhance the E response produced by IL10. max In some embodiments, the E of the binding protein is at least 1% (e.g., 1% to 100%, 10% to 100%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100%, 90% to 100%, 1% to 90%, 1% to 80%, 1% to 70%, 1% to 60%, 1% to 50%, 1% to 40%, 1% to 30%, 1% to 20%, or 1% to 10%). In some embodiments, the E of the binding protein is at least 1% (e.g., 1% to 100%, 10% to 100%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100%, 90% to 100%, 1% to 90%, 1% to 80%, 1% to 70%, 1% to 60%, 1% to 50%, 1% to 40%, 1% to 30%, 1% to 20%, or 1% to 10%) by varying the linker length of the binding protein that specifically binds to IL10Rα and IL2Rγ. maxThe binding protein can be modified to target the most desired cell type, e.g., CD8 + In T cells, E max In some embodiments, the CD8 binding protein that specifically binds to IL10Rα and IL2Rγ can be provided. + E in T cells max is a signaling pathway that is mediated by E-binding proteins in other T cells. max In another embodiment, the E of a binding protein that specifically binds to IL10Rα and IL2Rγ is 1% to 100% (e.g., 10% to 100%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100%, 90% to 100%, 1% to 90%, 1% to 80%, 1% to 70%, 1% to 60%, 1% to 50%, 1% to 40%, 1% to 30%, 1% to 20%, or 1% to 10%). max is the E of the natural ligand max greater than (e.g., at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% greater).

[0040] The level of downstream signaling caused by the binding protein can also be measured.

[0041] In some embodiments, the IL10Rα / IL2Rγ binding molecules described herein are partial agonists. In some embodiments, the binding molecules described herein are designed such that the binding molecules are full agonists. In some embodiments, the binding molecules described herein are designed such that the binding molecules are superagonists.

[0042] The present disclosure relates to a first single domain antibody (sdAb) that specifically binds to the extracellular domain of IL10Rα of IL10Rα / IL2Rγ ("anti-IL10Rα sdAb"); and A second single domain antibody ("anti-IL2Rγ sdAb") that specifically binds to the extracellular domain IL2Rγ of IL10Rα / IL2Rγ. providing a bivalent IL10Rα / IL2Rγ comprising wherein the anti-IL10Rα sdAb and the anti-IL2Rγ sdAb are stably associated, and contacting a cell expressing IL10Rα and IL2Rγ with an effective amount of the bivalent binding molecule results in dimerization of IL10Rα and IL2Rγ, resulting in intracellular signaling. In some embodiments, one or both of the sdAbs are scFvs. In some embodiments, one or both of the sdAbs are VHHs.

[0043] In some embodiments, one sdAb of the IL10Rα / IL2Rγ bivalent binding molecule is an scFv and the other sdAb is a VHH.

[0044] In some embodiments, the first and second sdAbs are covalently linked via a chemical bond.

[0045] In some embodiments the first and second sdAb are provided as a single contiguous polypeptide.

[0046] In some embodiments the first and second sdAb are provided as a single contiguous polypeptide, optionally including an intervening polypeptide linker between the amino acid sequences of the first and second sdAb.

[0047] In some embodiments, the bivalent binding molecule, optionally including a linker, is expressed as a fusion protein, optionally with an additional amino acid sequence, which in some embodiments is a purification handle, such as a chelating peptide, or an additional protein, such as a subunit of an Fc molecule.

[0048] The disclosure also provides an expression vector comprising a nucleic acid encoding a bispecific binding molecule operably linked to one or more expression control sequences.The disclosure also provides an isolated host cell comprising an expression vector comprising a nucleic acid encoding a bispecific binding molecule operably linked to one or more expression control sequences functional in the host cell.

[0049] In another aspect, the disclosure provides a pharmaceutical composition comprising an IL10Rα / IL2Rγ binding molecule described herein and a pharma- ceutically acceptable carrier.

[0050] In another aspect, the disclosure provides a method of treating an autoimmune or inflammatory disease, disorder, or condition, or a viral infection in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an IL10Rα / IL2Rγ binding molecule described herein, or a pharmaceutical composition described herein.

[0051] In some embodiments, the binding proteins described herein are designed such that the binding proteins provide maximum desirable IL10 intracellular signaling from binding to IL10Rα and IL2Rγ on desirable cell types while providing significantly less IL10 signaling in other undesirable cell types. This can be achieved, for example, by binding to IL10Rα and IL2Rγ with different affinities or by binding to different EGF receptors as compared to the affinity of IL10 for IL10R. maxThis can be achieved by the selection of binding proteins that result in the binding of a ligand to its cognate receptor. Because different cell types respond with different sensitivities to the binding of a ligand to its cognate receptor, modulating the affinity of the dimeric ligand (or its individual binding moieties) for the IL10 receptor compared to wild-type IL10 binding promotes the stimulation of desirable activity while reducing undesirable activity on non-target cells. Numerous methods are available for measuring downstream signaling activity. For example, in some embodiments, JAK / STAT signaling can be measured by the presence of phosphorylated receptor and / or phosphorylated STAT. In other embodiments, the expression of one or more downstream genes, whose expression levels can be affected by the level of downstream signaling caused by the binding protein, can also be measured.

[0052] II. Definition As used herein, the term "antibody" refers collectively to (a) glycosylated and non-glycosylated immunoglobulins (including, but not limited to, mammalian immunoglobulin classes IgG1, IgG2, IgG3, and IgG4) that specifically bind to a target molecule, and (b) IgG(1-4) delta C, which competes with the immunoglobulin from which it was derived for binding to the target molecule. H 2, F(ab')2, Fab, ScFv, V H , V L The term antibody refers to immunoglobulin derivatives, including, but not limited to, F(ab'), ...

[0053] The term antibody also includes so-called "single domain antibodies" or "sdAbs", and "heavy chain antibodies" or "V H Includes "H". V HH may be derived from immunization of camelids (including camels, llamas, and alpacas) (see, e.g., Hamers-Casterman, et al. (1993) Nature 363:446-448), or V H Antibodies with a given specificity can also be obtained by screening libraries (e.g., phage libraries) constructed with the H framework. Antibodies with a given specificity can also be obtained by screening VH framework antibodies obtained from immunization of cartilaginous fish, including but not limited to sharks. H It may be derived from a non-mammalian source, such as H. The term "antibody" encompasses antibodies isolatable from natural sources or from an animal following immunization with an antigen, as well as engineered antibodies, including monoclonal, bispecific, trispecific, chimeric, humanized, human, CDR-grafted, veneered, or deimmunized (e.g., to remove T-cell epitopes) antibodies. The term "human antibody" includes antibodies obtained from humans, as well as antibodies obtained from transgenic mammals that contain human immunoglobulin genes, such that upon stimulation with antigen, the transgenic animal produces antibodies that contain amino acid sequences characteristic of antibodies produced by humans.

[0054] The term antibody refers to a parent antibody and its derivatives, such as affinity matured, veneered, CDR grafted, humanized, camelized (V H H), or binding molecules that comprise the binding domains (e.g., CDRs) of an antibody in a non-immunoglobulin scaffold.

[0055] The term "antibody" should not be construed as limited to any particular synthetic means, but includes naturally occurring antibodies isolatable from natural sources, and engineered antibody molecules prepared by "recombinant" means, including antibodies isolated from transgenic animals transgenic for human immunoglobulin genes or hybridomas prepared therefrom, antibodies isolated from host cells transformed with a nucleic acid construct resulting in the expression of the antibody, and antibodies isolated from combinatorial antibody libraries, including phage display libraries. In one embodiment, the "antibody" is a mammalian immunoglobulin. In some embodiments, the antibody is a "full-length antibody," including variable and constant domains that provide binding and effector functions.

[0056] The term antibody includes antibody conjugates that contain modifications to extend duration of action, such as fusion proteins or conjugation to polymers (eg, PEGylation).

[0057] As used herein, the term "binding protein" refers to a protein that can bind to one or more cell surface receptors or their domains or subunits. In some embodiments, a binding protein specifically binds to two different receptors (or domains or subunits thereof), such that the receptors (or domains or subunits) are maintained in close proximity to each other such that the receptors (or domains or subunits) that contain the domains (e.g., intracellular domains) interact with each other, resulting in downstream signal transduction.

[0058] As used herein, the term "CDR" or "complementarity determining region" is intended to mean the non-contiguous antigen-binding sites found in the variable regions of both heavy and light chain immunoglobulin polypeptides. CDRs are described by Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat et al., US Dept. of Health and Human Services, "Sequences of proteins of immunological interest" (1991) (also referred to herein as Kabat 1991); Chothia et al., J. Mol. Biol. 196:901-917 (1987) (also referred to herein as Chothia 1987); and MacCallum et al., J. Mol. Biol. 262:732-745 (1996), where the definitions include overlapping or subsets of amino acid residues when compared with each other. Nevertheless, the application of either definition to refer to the CDR of an antibody or grafted antibody or variants thereof is intended to be within the scope of the term as defined and used herein. In the context of this disclosure, the numbering of CDR positions is provided according to the Kabat numbering convention. The term "Chothia numbering" as used herein is recognized in the art and refers to a system of numbering amino acid residues based on the location of structural loop regions (Chothia et al. 1986, Science 233:755-758; Chothia & Lesk 1987, JMB 196:901-917; Chothia et al. 1992, JMB 227:799-817). For the purposes of this disclosure, unless otherwise specifically specified, the positioning of CDR2 and CDR3 in the variable region of an antibody is according to Kabat numbering or simply "Kabat".The positioning of CDR1 in the variable region of an antibody can follow Kabat numbering, unless otherwise indicated, as determined by a hybrid of the Kabat and Chothia numbering schemes.

[0059] As used herein, the term "conservative amino acid substitution" refers to the replacement of an amino acid that changes a given amino acid into a different amino acid with similar biochemical properties (e.g., charge, hydrophobicity, and size).For example, the amino acids in each of the following groups can be considered as conservative amino acids of each other: (1) hydrophobic amino acids: alanine, isoleucine, leucine, tryptophan, phenylalanine, valine, proline, and glycine; (2) polar amino acids: glutamine, asparagine, histidine, serine, threonine, tyrosine, methionine, and cysteine; (3) basic amino acids: lysine and arginine; and (4) acidic amino acids: aspartic acid and glutamic acid.

[0060] As used herein, the term "downstream signaling" refers to a cell signaling process that is triggered by the interaction of two or more cell surface receptors that are brought into close proximity to one another.

[0061] As used herein, the term "linker" refers to a link between two elements, e.g., protein domains. The linker can be a covalent bond or a peptide linker. The term "bond" refers to a chemical bond, e.g., an amide bond or a disulfide bond, or any type of bond created from a chemical reaction, e.g., chemical conjugation. The term "peptide linker" refers to an amino acid or polypeptide that can be used to link two protein domains to provide space and / or flexibility between the two protein domains.

[0062] As used herein, the term "multimerization" refers to the bringing into close proximity of two or more cell surface receptors, or domains or subunits thereof, such that the receptors, or domains or subunits thereof, can interact with one another to trigger downstream signaling.

[0063] As used herein, the terms "N-terminal" (or "amino terminal") and "C-terminal" (or "carboxyl terminal") refer to the extreme amino and carboxyl termini of a polypeptide, respectively, while the terms "N-terminal" and "C-terminal" refer to the relative position in the amino acid sequence of a polypeptide toward the N-terminus and C-terminus, respectively, and can include residues at the N-terminus and C-terminus, respectively. "Immediately N-terminal" or "immediately C-terminal" refers to the position of a first amino acid residue relative to a second amino acid residue, when the first and second amino acid residues are covalently linked to provide a contiguous amino acid sequence.

[0064] As used herein, the term "neoplastic disease" refers to a disorder or condition in a subject resulting from excessive cell proliferation or uncontrolled (or dysregulated) cell replication. The term neoplastic disease refers to a disorder resulting from the presence of a neoplasm in a subject. Neoplasms can be classified as (1) benign, (2) pre-malignant (or "pre-cancerous"), and (3) malignant (or "cancerous"). The term "neoplastic disease" includes neoplasia-related diseases, disorders, and conditions that refer to conditions directly or indirectly related to neoplastic disease, including, for example, pre-cancerous conditions such as angiogenesis and dysplasia.

[0065] As used herein, the terms "nucleic acid", "nucleic acid molecule", "polynucleotide", and the like are used interchangeably herein to refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Non-limiting examples of polynucleotides include linear or circular nucleic acids, messenger RNA (mRNA), complementary DNA (cDNA), recombinant polynucleotides, vectors, probes, primers, and the like.

[0066] As used herein, the term "percent sequence identity" used in the context of a nucleic acid or polypeptide refers to a sequence that has at least 50% sequence identity with a reference sequence. Alternatively, the percent sequence identity can be any integer between 50% and 100%. In some embodiments, a sequence has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with a reference sequence as determined by BLAST using standard parameters, as described below.

[0067] For sequence comparison, typically, one sequence serves as a reference sequence, and test sequence is compared to it.When using sequence comparison algorithm, test sequence and reference sequence are input into computer, and coordinates of partial sequence are designated as necessary, and program parameters of sequence algorithm are designated.Default program parameters can be used, or alternative parameters can be designated.Then, sequence comparison algorithm calculates the percent sequence identity of test sequence to reference sequence based on program parameters.

[0068] The comparison window includes reference to any one segment of a number of contiguous positions, e.g., a segment of at least 10 residues. In some embodiments, the comparison window has 10 to 600 residues, e.g., about 10 to about 30 residues, about 10 to about 20 residues, about 50 to about 200 residues, or about 100 to about 150 residues, within which a sequence can be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.

[0069] Algorithms suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and Altschul et al. (1977) Nucleic Acids Res. 25: 3389-3402, respectively. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (NCBI) website. The algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match or meet a certain positive threshold score T when aligned with words of the same length in the database sequence. T is called the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds to initiate searches to find longer HSPs that contain them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of the word hits in each direction is stopped when the cumulative alignment score drops off from its maximum achieved value by an amount X; when the cumulative score falls below zero due to the accumulation of one or more negative scoring residue alignments; or when the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word size (W) of 28, an expectation (E) of 10, M=1, N=-2, and a comparison of both strands.For amino acid sequences, the BLASTP program uses as defaults a word size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

[0070] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the minimum sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, amino acid sequences are selected such that the minimum sum probability in a comparison of a test amino acid sequence to a reference amino acid sequence is less than about 0.01, more preferably less than about 10 -5 and most preferably less than about 10 -20 If the sequence is less than 1, it is considered similar to the reference sequence.

[0071] As used herein, the terms "polypeptide", "peptide" and "protein", which are used interchangeably herein, refer to polymeric forms of amino acids of any length, which can include genetically encoded and non-genetically encoded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides with modified polypeptide backbones. The terms include fusion proteins, including, but not limited to, fusion proteins with heterologous amino acid sequences; fusion proteins with heterologous and homologous leader sequences; fusion proteins with or without N-terminal methionine residues; fusion proteins with immunologically tagged proteins; fusion proteins of immunologically active proteins (e.g., antigenic diphtheria or tetanus toxin fragments), and the like.

[0072] As used herein, the terms "prevent", "preventing", "prevention", and the like generally refer to a course of action initiated with respect to a subject who is predisposed to having a particular disease, disorder, or condition due to genetic, experiential, or environmental factors, prior to the onset of the disease, disorder, condition, or symptoms thereof, such that the subject either temporarily or permanently prevents, inhibits, inhibits, or reduces the risk of developing the disease, disorder, condition, or the like (e.g., as determined by the absence of clinical symptoms), or delays its onset. In certain instances, the terms "prevent", "preventing", and "prevention" are also used to refer to slowing the progression of a disease, disorder, or condition from its current state to a more deleterious state.

[0073] As used herein, the term "single domain antibody" or "sdAb" refers to an antibody having a single monomeric variable antibody domain. An sdAb is capable of selectively binding to a specific antigen. The V domain, as further defined below, H The H antibody is an example of an sdAb.

[0074] As used herein, the term "specifically bind" refers to the degree of selectivity or affinity with which one molecule binds to another molecule. In the context of binding pairs (e.g., binding molecule / receptor, ligand / receptor, antibody / antigen, antibody / ligand, antibody / receptor binding pairs described herein), a first molecule of a binding pair is said to specifically bind to a second molecule of the binding pair when the first molecule of the binding pair does not bind in significant amounts to other components present in the sample. A first molecule of a binding pair is said to specifically bind to a second molecule of the binding pair when the affinity of the first molecule for the second molecule is at least 2 times greater, alternatively at least 5 times greater, alternatively at least 10 times greater, alternatively at least 20 times greater, or alternatively at least 100 times greater than the affinity of the first molecule for other components present in the sample.

[0075] In certain embodiments, the bispecific V H H2 V in binding proteins H H is V H The equilibrium dissociation constant between H and the receptor is approximately 10 as determined, for example, by Scatchard analysis (Munsen, et al. 1980 Analyt. Biochem. 107:220-239). 6 Larger than M, alternatively about 10 8 Larger than M, alternatively about 10 10 Larger than M, alternatively about 10 11 Larger than M, alternatively about 10 10 Larger than M, about 10 12 If M is greater than M, then it binds to a receptor (e.g., a first receptor or a second receptor of a natural or non-natural receptor pair). Specific binding can be assessed using techniques known in the art, including, but not limited to, competitive ELISA, BIACORE® assay, and / or KINEXA® assay.

[0076] As used herein, the terms "subject," "recipient," "individual," or "patient" refer to any mammalian subject, particularly humans, for whom diagnosis, treatment, or therapy is desired. These terms can also be used interchangeably herein. "Mammal" for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, and the like. In some embodiments, the mammal is a human.

[0077] As used herein, the term "T-cell" or "T cell" is used in its conventional sense to refer to lymphocytes that differentiate in the thymus, have specific cell surface antigen receptors, and include those that control the initiation or suppression of cell-mediated and humoral immunity, as well as others that lyse antigen-bearing cells. In some embodiments, T cells include, but are not limited to, naive CD8 + T cells, cytotoxic CD8 + T cells, naive CD4+ T cells, helper T cells, e.g., T H 1. T H 2. T H 9. T H 11. T H 22, T FH ;regulatory T cells, e.g. R 1, Tregs, inducible Tregs; memory T cells, including, for example, central memory T cells, effector memory T cells, NKT cells, tumor infiltrating lymphocytes (TILs), and engineered variants of such T cells, including, but not limited to, CAR-T cells, recombinantly modified TILs, and TCR engineered cells.

[0078] As used herein, the term "therapeutically effective amount" is used herein in reference to the administration of an agent to a subject, either alone or as part of a pharmaceutical composition or treatment regimen, in a single dose or as part of a series of doses, in an amount that can have any detectable positive effect on any symptom, aspect, or characteristic of a disease, disorder, or condition when administered to a subject. The therapeutically effective amount can be confirmed by measuring the relevant physiological effect, which can be adjusted in conjunction with a dosing regimen and depending on diagnostic analysis such as the subject's condition. The parameters for evaluation to determine the therapeutically effective amount of an agent are determined by a physician using diagnostic criteria accepted in the art, including but not limited to age, weight, sex, overall health, ECOG score, observable physiological parameters, blood levels, blood pressure, signs such as electrocardiogram, computed tomography, X-ray, etc. Alternatively, or in addition, other parameters commonly evaluated in a clinical setting may be monitored to determine whether a therapeutically effective amount of an agent has been administered to a subject, such as body temperature, heart rate, normalization of blood chemistry, normalization of blood pressure, normalization of cholesterol levels, or any symptom, aspect, or characteristic of a disease, disorder, or condition, biomarkers (e.g., inflammatory cytokines, IFN-γ, granzymes, etc.), reduction in serum tumor markers, improvement in Response Evaluation Criteria In Solid Tumors (RECIST), improvement in Immune-Related Response Criteria (irRC), increased survival, increased progression-free survival, increased time to progression, increased time to treatment failure, increased event-free survival, increased time to next treatment, improved objective response rate, improved duration of response, reduction in tumor burden, complete response, partial response, stable disease, etc. that are relied upon by clinicians in the art to assess improvement in a subject's condition in response to administration of the agent.As used herein, the terms "complete response (CR)", "partial response (PR)", "stable disease (SD)" and "progressive disease (PD)" for target lesions, and the terms "complete response (CR)", "incomplete response / stable disease (SD)" and "progressive disease (PD)" for non-target lesions are understood to be as defined in RECIST criteria. As used herein, the terms "immune-related complete response (irCR)", "immune-related partial response (irPR)", "immune-related disease progression (irPD)" and "immune-related disease stable (irSD)" are as defined according to immune-related response criteria (irRC). As used herein, the term "immune-related response criteria (irRC)" refers to a system for evaluating response to immunotherapy, as described in Wolchok, et al. (2009) Guidelines for the Evaluation of Immune Therapy Activity in Solid Tumors: Immune-Related Response Criteria, Clinical Cancer Research 15(23): 7412-7420. The therapeutically effective amount can be adjusted over the course of treatment of a subject in conjunction with the dosage regimen and / or assessment of the subject's condition and fluctuations in the aforementioned factors. In one embodiment, a therapeutically effective amount is an amount of an agent that, when used alone or in combination with another agent, does not cause irreversible serious adverse events during the course of administration to a mammalian subject.

[0079] The terms "treat", "treating", "treatment" and the like refer to a course of action (e.g., administering a binding molecule described herein, or a pharmaceutical composition comprising the same) initiated with respect to a subject after a disease, disorder, or condition, or a symptom thereof, has been diagnosed, observed, etc. in a subject, in order to eliminate, reduce, inhibit, alleviate, or ameliorate, either temporarily or permanently, at least one of the underlying causes of, or at least one of the symptoms associated with, the disease, disorder, or condition afflicting the subject. Treatment includes a course of action taken with respect to a subject suffering from a disease, which course of action results in the inhibition of the disease in the subject (e.g., halting the progression of, or ameliorating one or more symptoms associated with, the disease, disorder, or condition).

[0080] As used herein, "V H The term "H" is a type of sdAb that has a single monomeric heavy chain variable antibody domain. Such antibodies can be found in or produced from camelid mammals (e.g., camels, llamas) that are naturally devoid of light chains.

[0081] As used herein, "V H H 2 " refers to two V-linked amines that are joined together by a linker (e.g., a covalent bond or a peptide linker). H H. "Bispecific V H H 2 " refers to a first V that binds to a first receptor, or a domain or subunit thereof. H H, and a second V that binds to a second receptor, or a domain or subunit thereof. H H, V H H 2 Refers to...

[0082] III. Compositions and Methods The present disclosure describes IL10Rα / IL2Rγ binding proteins that bind to IL10Rα and IL2Rγ or domains thereof. Various binding proteins can be screened for binding to IL10Rα and IL2Rγ or domains thereof and for signaling in therapeutically relevant cell types.

[0083] The binding proteins described herein can specifically bind to IL10Rα and IL2Rγ, and are H H antibody and anti-IL2Rγ V H The binding proteins described herein can also include anti-IL10Rα / IL2Rγ V H antibodies. H H 2 The binding protein can trigger multimerization of IL10Rα and IL2Rγ and downstream signaling.

[0084] Anti-IL10Rα V H H antibody In some embodiments, the present disclosure provides an anti-IL10Rα V H A polypeptide comprising any of the anti-IL10Rα VH antibodies, for example, an anti-IL10Rα VH antibody comprising CDR1, CDR2, and CDR3 selected from Table 1 below. H In certain embodiments, the disclosure provides a polypeptide comprising a set of CDR1, CDR2, and CDR3 selected from a row of Table 1 below (e.g., CDR1, CDR2, and CDR3 listed in the same row). In certain embodiments, the disclosure provides a polypeptide comprising an anti-IL10Rα VH selected from Table 1 below. H In some embodiments, the present disclosure provides a polypeptide comprising a sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of an anti-IL10Rα V H antibody. H H antibody, wherein the anti-IL10Rα V H antibody is a dimer or multimer of two or more of the anti-IL10Rα V H antibodies.H The H antibodies can be the same or different.

[0085] In some embodiments, the present disclosure provides a method for the identification of a CDR1, CDR2, CDR3, or VDR4 polypeptide as listed in Table 1 below. H Anti-IL10Rα V H amino acid sequences that may be incorporated into a multivalent binding protein as described herein. H In some embodiments, an anti-IL10Rα V H antibody is provided. H The H antibody has (1) a CDR1 having the sequence of any one of SEQ ID NOs: 1, 5, 9, 13, 17, 21, or 264-269, or a variant thereof having a sequence having one, two, or three amino acid substitutions compared to any one of SEQ ID NOs: 1, 5, 9, 13, 17, 21, or 264-269; (2) a CDR2 having the sequence of any one of SEQ ID NOs: 2, 6, 10, 14, 18, and 22, or a variant thereof having a sequence having one, two, or three amino acid substitutions compared to any one of SEQ ID NOs: 2, 6, 10, 14, 18, and 22; (3) a CDR3 having the sequence of any one of SEQ ID NOs: 3, 7, 11, 15, 19, and 23, or a variant thereof having a sequence having one, two, or three amino acid substitutions compared to any one of SEQ ID NOs: 3, 7, 11, 15, 19, and 23. In some embodiments, the anti-IL10Rα V sequence may include a variant thereof having a sequence having one, two, or three amino acid substitutions compared to any one of the sequences of NOs:3, 7, 11, 15, 19, and 23. H H antibodies can be modified for half-life extension (e.g., Fc conjugation, PEGylation), either alone or in the context of a multivalent binding protein as described herein. In some embodiments, the moiety that provides half-life extension (e.g., PEG, Fc polypeptide, or Fc domain) is conjugated to the N-terminus of the antibody, the C-terminus of the antibody, or to an internal amino acid residue, optionally via a linker (particularly via conjugation to the side chain of a lysine or cysteine ​​residue). In some embodiments, the Fc polypeptide or Fc domain is derived from IgG1, IgG2, IgG3, or IgG4.

[0086] In some embodiments, the anti-IL10Rα V H The H antibody can include a set of CDR1, CDR2, and CDR3 selected from a row of Table 1 below (e.g., CDR1, CDR2, and CDR3 listed in the same row). In each set of CDR1, CDR2, and CDR3, (1) CDR1 can have a sequence shown in the set, or a variant thereof having a sequence with 1, 2, or 3 amino acid substitutions compared to the sequence shown; (2) CDR2 can have a sequence shown in the set, or a variant thereof having a sequence with 1, 2, or 3 amino acid substitutions compared to the sequence shown; (3) CDR3 can have a sequence shown in the set, or a variant thereof having a sequence with 1, 2, or 3 amino acid substitutions compared to the sequence shown.

[0087] In addition, anti-IL10Rα V H The anti-IL10Rα VH antibody can comprise CDR1, CDR2, and CDR3 having the sequences of SEQ ID NOs: 1-3. H The anti-IL10Rα VH antibody can comprise CDR1, CDR2, and CDR3 having the sequences of SEQ ID NOs:5-7. H The anti-IL10Rα VH antibody can comprise CDR1, CDR2, and CDR3 having the sequences of SEQ ID NOs:9 to 11. H The anti-IL10Rα VH antibody can comprise CDR1, CDR2, and CDR3 having the sequences of SEQ ID NOs: 13 to 15. H The anti-IL10Rα VH antibody can comprise CDR1, CDR2, and CDR3 having the sequences of SEQ ID NOs: 17 to 19. H The H antibody can comprise CDR1, CDR2, and CDR3 having the sequences of SEQ ID NOs:21-23.

[0088] In addition, anti-IL10Rα V HThe anti-IL10Rα VH antibody can comprise CDR1, CDR2, and CDR3 having the sequences of SEQ ID NOs:1-3, respectively, and at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:4. H The anti-IL10Rα VH antibody can comprise CDR1, CDR2, and CDR3 having the sequences of SEQ ID NOs:5-7, respectively, and at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:8. H The anti-IL10Rα VH antibody can comprise CDR1, CDR2, and CDR3 having the sequences of SEQ ID NOs:9-11, respectively, and at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:12. H The anti-IL10Rα VH antibody can comprise CDR1, CDR2, and CDR3 having the sequences of SEQ ID NOs:13-15, respectively, and at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:16. H The anti-IL10Rα VH antibody can comprise CDR1, CDR2, and CDR3 having the sequences of SEQ ID NOs:17-19, respectively, and at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:20. HThe H antibody can comprise CDR1, CDR2, and CDR3 having the sequences of SEQ ID NOs:21-23, respectively, and at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:24.

[0089] (Table 1) Anti-IL10Rα V H H antibody sequence TIFF2024504923000002.tif215160

[0090] Anti-IL2Rγ V H H antibody In some embodiments, the present disclosure provides an anti-IL2Rγ V H A polypeptide comprising any of the anti-IL2Rγ VH antibodies, for example, an anti-IL2Rγ VH antibody comprising CDR1, CDR2, and CDR3 selected from Table 2 below. H In certain embodiments, the disclosure provides a polypeptide comprising a set of CDR1, CDR2, and CDR3 selected from a row of Table 2 below (e.g., CDR1, CDR2, and CDR3 listed in the same row). In certain embodiments, the disclosure provides a polypeptide comprising an anti-IL2Rγ VH selected from Table 2 below. H In some embodiments, the polypeptides provided by the present disclosure include polypeptides comprising a sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of an anti-IL2Rγ VH antibody as set forth in Table 2. H H antibodies, wherein the anti-IL2Rγ V H antibodies include dimers or multimers of two or more of the anti-IL2Rγ V H antibodies. H The H antibodies can be the same or different.

[0091] In some embodiments, the present disclosure provides an anti-IL10Rα VHH antibody that may be incorporated into a multivalent binding protein as described herein, comprising one or more of the CDR1, CD2, CDR3, or VHH amino acid sequences as listed in Table 1 below. H In some embodiments, the present disclosure provides anti-IL2Rγ VH antibodies that may be incorporated into a multivalent binding protein as described herein, comprising one or more of the CDR1, CD2, CDR3, or VHH amino acid sequences as listed in Table 2 below. H In some embodiments, an anti-IL2Rγ V H antibody is provided. H The H antibody has (1) a CDR1 having the sequence of any one of SEQ ID NOs: 25, 29, 33, 37, 41, 45, or 270-275, or a variant thereof having a sequence having one, two, or three amino acid substitutions compared to any one of SEQ ID NOs: 25, 29, 33, 37, 41, 45, or 270-275; (2) a CDR2 having the sequence of any one of SEQ ID NOs: 26, 30, 34, 38, 42, and 46, or a variant thereof having a sequence having one, two, or three amino acid substitutions compared to any one of SEQ ID NOs: 26, 30, 34, 38, 42, and 46; (3) a CDR3 having the sequence of any one of SEQ ID NOs: 27, 31, 35, 39, 43, and 47, or a variant thereof having a sequence having one, two, or three amino acid substitutions compared to any one of SEQ ID NOs: 26, 30, 34, 38, 42, and 46; In some embodiments, the anti-IL2Rγ VH1R1 antibody can include an anti-IL2Rγ VH1R2 antibody, an anti-IL2Rγ VH1R3 antibody, or an anti-IL2Rγ VH1R4 antibody, an anti-IL2Rγ VH1R5 antibody, or an anti-IL2Rγ VH1R6 antibody, an anti-IL2Rγ VH1R7 antibody, or an anti-IL2Rγ VH1R8 antibody, an anti-IL2Rγ VH1R9 antibody, or an anti-IL2Rγ VH1R10 antibody, an anti-IL2Rγ VH1R11 antibody, or an anti-IL2Rγ VH1R2 antibody, HH can be modified for half-life extension (e.g., Fc conjugation, PEGylation), either alone or in the context of a multivalent binding protein as described herein. In some embodiments, the moiety that provides half-life extension (e.g., PEG, Fc polypeptide, or Fc domain) is conjugated to the N-terminus of the antibody, the C-terminus of the antibody, or an internal amino acid residue, optionally via a linker (particularly via conjugation to the side chain of a lysine or cysteine ​​residue). In some embodiments, the Fc polypeptide or Fc domain is from IgG1, IgG2, IgG3, or IgG4.

[0092] In some embodiments, anti-IL2Rγ V H The H antibody can include a set of CDR1, CDR2, and CDR3 selected from a row of Table 2 below (e.g., CDR1, CDR2, and CDR3 listed in the same row). In each set of CDR1, CDR2, and CDR3, (1) CDR1 can have a sequence shown in the set, or a variant thereof having a sequence with 1, 2, or 3 amino acid substitutions compared to the sequence shown; (2) CDR2 can have a sequence shown in the set, or a variant thereof having a sequence with 1, 2, or 3 amino acid substitutions compared to the sequence shown; (3) CDR3 can have a sequence shown in the set, or a variant thereof having a sequence with 1, 2, or 3 amino acid substitutions compared to the sequence shown.

[0093] In addition, anti-IL2Rγ V H The anti-IL2Rγ VH antibody can comprise CDR1, CDR2, and CDR3 having the sequences of SEQ ID NOs: 25 to 27. H The anti-IL2Rγ VH antibody can comprise CDR1, CDR2, and CDR3 having the sequences of SEQ ID NOs: 29 to 31. H The anti-IL2Rγ VH antibody can comprise CDR1, CDR2, and CDR3 having the sequences of SEQ ID NOs: 33 to 35. HThe anti-IL2Rγ VH antibody can comprise CDR1, CDR2, and CDR3 having the sequences of SEQ ID NOs: 37 to 39. H The anti-IL2Rγ VH antibody can comprise CDR1, CDR2, and CDR3 having the sequences of SEQ ID NOs: 41 to 43. H The H antibody can include CDR1, CDR2, and CDR3 having the sequences of SEQ ID NOs:45-47.

[0094] In addition, anti-IL2Rγ V H The anti-IL2Rγ VH antibody can comprise CDR1, CDR2, and CDR3 having the sequences of SEQ ID NOs:25-27, respectively, and at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:28. H The anti-IL2Rγ VH antibody can comprise CDR1, CDR2, and CDR3 having the sequences of SEQ ID NOs:29-31, respectively, and at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:32. H The anti-IL2Rγ VH antibody can comprise CDR1, CDR2, and CDR3 having the sequences of SEQ ID NOs:33-35, respectively, and at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:36. H The anti-IL2Rγ VH antibody can comprise CDR1, CDR2, and CDR3 having the sequences of SEQ ID NOs:37-39, respectively, and at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:40. HThe anti-IL2Rγ VH antibody can comprise CDR1, CDR2, and CDR3 having the sequences of SEQ ID NOs:41-43, respectively, and at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:44. H The H antibody can comprise CDR1, CDR2, and CDR3 having the sequences of SEQ ID NOs:45-47, respectively, and at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:48.

[0095] (Table 2) Anti-IL2Rγ V H H antibody sequence TIFF2024504923000003.tif200160

[0096] Anti-IL10Rα / IL2Rγ V H H 2 The IL10Rα / IL2Rγ binding proteins described herein are selected from Table 1. H H antibody and an anti-IL2Rγ V antibody selected from Table 2 H In some embodiments, the N-terminal V of the IL2R binding molecule can include an H antibody. H H is anti-IL10Rα V H H antibody, which binds the C-terminal V H H is anti-IL2Rγ V H H antibody, optionally with two V H A linker can be used between the H antibodies. In some embodiments, the N-terminal V of the IL10Rα / IL2Rγ binding protein H H is anti-IL2Rγ V H H antibody, which binds the C-terminal V H H is anti-IL10Rα V H H antibody, optionally with two V HA linker can be used between the anti-IL10Rα V H antibodies. H H antibody and anti-IL2Rγ V H Examples of linkers that can be used to fuse the IL10Rα / IL2Rγ binding protein to an H antibody (e.g., GGGS (SEQ ID NO:62)) are described in further detail herein. In some embodiments, the IL10Rα / IL2Rγ binding protein may be operably linked to a metal chelating peptide. Chelating peptides include, but are not limited to, Ala-Ser-His-His-His-His-His-His ("ASH6", SEQ ID NO:81) or His-His-His-His-His-His ("H6", SEQ ID NO:82) purification handles to facilitate purification of the binding protein by chelating peptide-immobilized metal affinity chromatography ("CP-IMAC" as described in U.S. Pat. No. 4,569,794).

[0097] Table 3 below shows the N-terminal anti-IL10Rα V H H antibody, and C-terminal anti-IL2Rγ V H Further examples of IL10Rα / IL2Rγ binding proteins described herein include the H antibody.

[0098] In some embodiments, the IL10Rα / IL2Rγ binding protein is H H sequence, V of DR233 H In some embodiments, the IL10Rα / IL2Rγ binding protein comprises the VHHHH sequence of DR235 and has at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:49, optionally excluding the terminal HHHHHH. H H arrangement, DR234 V HIn some embodiments, the IL10Rα / IL2Rγ binding protein comprises the VHHHH sequence of DR236 and has at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:50, optionally excluding the terminal HHHHHH. H H sequence, V of DR231 H In some embodiments, the IL10Rα / IL2Rγ binding protein comprises the VHHHH sequence of DR236 and has at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:51, optionally excluding the terminal HHHHHH. H H arrangement, V of DR232 H In some embodiments, the IL10Rα / IL2Rγ binding protein comprises the VHHHH sequence of DR236 and has at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:52, optionally excluding the terminal HHHHHH. H H arrangement, DR234 V H In some embodiments, the IL10Rα / IL2Rγ binding protein comprises the VHHHH sequence of DR237 and has at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:53, optionally excluding the terminal HHHHHH. H H sequence, V of DR233 H In some embodiments, the IL10Rα / IL2Rγ binding protein comprises the VHHHH sequence of DR240 and has at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:54, optionally excluding the terminal HHHHHH. H H sequence, V of DR231 HIn some embodiments, the IL10Rα / IL2Rγ binding protein comprises the VHHHH sequence of DR240 and has at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:55, optionally excluding the terminal HHHHHH. H H arrangement, V of DR232 H In some embodiments, the IL10Rα / IL2Rγ binding protein comprises the VHHHH sequence of DR240 and has at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:56, optionally excluding the terminal HHHHHH. H H arrangement, DR234 V H In some embodiments, the IL10Rα / IL2Rγ binding protein comprises the VHH sequence of DR241 and has at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:57, optionally excluding the terminal HHHHH. H H sequence, V of DR231 H In some embodiments, the IL10Rα / IL2Rγ binding protein comprises the VHH sequence of DR241 and has at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:58, optionally excluding the terminal HHHHH. H H arrangement, DR234 V H H sequence and has at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:59, and optionally excluding the terminal HHHHHH.

[0099] (Table 3) Anti-IL10Rα / IL2Rγ V H H 2 (Anti-IL10Rα V HH-Linker-Anti-IL2Rγ V H H) TIFF2024504923000004.tif99160TIFF2024504923000005.tif216160TIFF2024504923000006.tif177160

[0100] Table 4 below shows the N-terminal anti-IL2Rγ V H H antibody, and anti-IL10RαV at the C-terminus H Illustrative examples of the IL10Rα / IL2Rγ binding proteins described herein, including the H antibody, are provided.

[0101] In some embodiments, the IL10Rα / IL2Rγ binding protein is the V H H arrangement, V of DR236 H In some embodiments, the IL10Rα / IL2Rγ binding protein comprises a VHHHHH sequence and at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:60, optionally excluding the terminal HHHHHH. H H sequence, V of DR239 H H sequence, and at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the sequence of SEQ ID NO:61, optionally excluding the terminal HHHHHH.

[0102] (Table 4) Anti-IL10Rα / IL2Rγ V H H 2 (Anti-IL2Rγ V H H-Linker-Anti-IL10Rα V H H) TIFF2024504923000007.tif110160

[0103] As shown in the illustrative examples of IL10Rα / IL2Rγ binding proteins in Tables 3 and 4, the IL10Rα / IL2Rγ binding protein sequences contain GGGS (SEQ ID NO:62) as a linker. In some embodiments, GGGS (SEQ ID NO:62) can be replaced by other linkers as further described herein. Additionally, the IL10Rα / IL2Rγ binding protein sequences shown in Tables 3 and 4 can be operably linked to a chelating peptide, such as the "ASH6" (SEQ ID NO:81) metal chelating peptide, which can be used to facilitate purification via metal affinity chromatography. In some embodiments, this purification handle can be removed or replaced by another purification handle (e.g., H6 (SEQ ID NO:82)).

[0104] Additionally, in each of the following SEQ ID NOs: 96-179, the name of each sequence is "anti-IL10Rα / IL2Rγ V H H 2 (N-terminal V H H antibody-C-terminal V H For example, "DR391 (DR229-DR235)" has the DR229 V H DR235 V at H and C termini H Anti-IL10Rα / IL2Rγ V with H antibody H H 2 In each of SEQ ID NOs:96-179 below, the linker is in bold and the N-terminal V H CDR1, CDR2, CDR3 and C-terminal V of H antibody H Each of the CDR1, CDR2, and CDR3 of the H antibody is underlined, respectively. The IL10Rα / IL2Rγ binding proteins described herein have an N-terminal V H H antibody V H H sequence, C-terminal V H H antibody V HThe purification handle "ASH6" (SEQ ID NO:81) at the end of each of SEQ ID NOs:96-179 may be removed or replaced by another purification handle, such as H6 (SEQ ID NO:82). TIFF2024504923000008.tif175159TIFF2024504923000009.tif238160TIFF2024504923000010.tif238160TIFF202 4504923000011.tif238160TIFF2024504923000012.tif238160TIFF2024504923000013.tif238160TIFF2024504923 000014.tif238160TIFF2024504923000015.tif238160TIFF2024504923000016.tif238159TIFF2024504923000017. tif238160TIFF2024504923000018.tif238160TIFF2024504923000019.tif238160TIFF2024504923000020.tif58159

[0105] In some embodiments, the binding proteins described herein comprise one or more anti-IL10Rα V H Two or more anti-IL10Rα V H antibodies may be included. H When an H antibody is present, adjacent antibodies can be conjugated to each other by a linker. In some embodiments, the binding proteins described herein comprise one or more anti-IL2Rγ V H Two or more anti-IL2Rγ V H antibodies may be included. HWhen an H antibody is present, adjacent antibodies can be conjugated to each other by a linker.

[0106] In some embodiments, the binding proteins described herein comprise one or more anti-IL10Rα V H H antibody and one or more anti-IL2Rγ V H In some embodiments, the binding protein can include an anti-IL10Rα V H antibody. Adjacent antibodies can be conjugated to each other by a linker. In some embodiments, the binding protein can include an anti-IL10Rα V H antibody. H Number of H antibodies and anti-IL2Rγ V H In another embodiment, the number of anti-IL10Rα V H antibodies in the binding protein is the same. H Number of H antibodies and anti-IL2Rγ V H The number of H antibodies varies.

[0107] In some embodiments, the binding proteins described herein have the following formula: TIFF2024504923000021.tif6128, where L is a linker, a, b, c are independently selected from 0 or 1, and x is an integer from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). H H#1 and V H H#2 targets the same receptor or a subunit thereof. H H#1 and V H H#2 targets a different receptor or its subunit. H H#1 and V H H#2 can have the same sequence. H H#1 and V H H#2 can have a different sequence.

[0108] In some embodiments, the IL10Rα / IL2Rγ binding protein is linked to an Fc polypeptide or Fc domain. In some embodiments, the Fc polypeptide (e.g., a subunit of an Fc domain) or Fc domain is from IgG1, IgG2, IgG3, or IgG4. In some embodiments, the IL10Rα / IL2Rγ binding protein is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to any one of SEQ ID NOs:49-61 or 96-179, optionally excluding the HHHHHH sequence therein.

[0109] A. "Forward" In some embodiments, the bivalent IL10Rα / IL2Rγ binding molecule has the structure: H2N-[anti-IL10Rα sdAb]-[L] x -[Anti-IL2Rγ sdAb]-[TAG] y -COOH wherein L is a polypeptide linker of 1 to 50 amino acids and x=0 or 1, and TAG is a chelating peptide or a subunit of an Fc domain and y=0 or 1.

[0110] In some embodiments, a bivalent IL10Rα / IL2Rγ binding molecule of the foregoing structure comprises, from amino terminus to carboxy terminus, the following polypeptide: (a) an anti-IL10Rα sdAb, including: a CDR1 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to any CDR1 sequence in a row of Table 10, or having 0, 1, 2, or 3 amino acid changes, optionally conservative amino acid changes, compared to said sequence; a CDR2 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to any CDR2 sequence in a row of Table 10, or having 0, 1, 2, or 3 amino acid changes, optionally conservative amino acid changes, compared to said sequence; and a CDR3 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to any of the CDR3 sequences listed in Table 10, or having 0, 1, 2, or 3 amino acid changes, optionally conservative amino acid changes, compared to said sequence; or (A) a CDR1 comprising the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:264, a CDR2 comprising the amino acid sequence of SEQ ID NO:2, and a CDR3 comprising the amino acid sequence of SEQ ID NO:3; or (B) a CDR1 comprising the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:265, a CDR2 comprising the amino acid sequence of SEQ ID NO:6, and a CDR3 comprising the amino acid sequence of SEQ ID NO:7; or (C) a CDR1 comprising the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:266, a CDR2 comprising the amino acid sequence of SEQ ID NO:10, and a CDR3 comprising the amino acid sequence of SEQ ID NO:11; or (D) a CDR1 comprising the amino acid sequence of SEQ ID NO:13 or SEQ ID NO:267, a CDR2 comprising the amino acid sequence of SEQ ID NO:14, and a CDR3 comprising the amino acid sequence of SEQ ID NO:15; or (E) a CDR1 comprising the amino acid sequence of SEQ ID NO:17 or SEQ ID NO:268, a CDR2 comprising the amino acid sequence of SEQ ID NO:18, and a CDR3 comprising the amino acid sequence of SEQ ID NO:19; or (F) CDR1 comprising the amino acid sequence of SEQ ID NO:21 or SEQ ID NO:269, CDR2 comprising the amino acid sequence of SEQ ID NO:22, and CDR3 comprising the amino acid sequence of SEQ ID NO:23; and (b) optionally a polypeptide linker of 1 to 50 amino acids, alternatively 1 to 40 amino acids, alternatively 1 to 30 amino acids, alternatively 1 to 20 amino acids, alternatively 1 to 15 amino acids, alternatively 1 to 10 amino acids, alternatively 1 to 8 amino acids, alternatively 1 to 6 amino acids, alternatively 1 to 4 amino acids; and (c) an anti-IL2Rγ sdAb, including: a CDR1 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to any CDR1 sequence listed in Table 11 or Table 12, or having 0, 1, 2, or 3 amino acid changes, optionally conservative amino acid changes, compared to said sequence; a CDR2 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to any CDR2 sequence listed in Table 11 or Table 12, or having 0, 1, 2, or 3 amino acid changes, optionally conservative amino acid changes, compared to said sequence; and a CDR3 that has at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to any CDR3 sequence listed in Table 11 or Table 12, or has 0, 1, 2, or 3 amino acid changes, optionally conservative amino acid changes, compared to said sequence; or (A) CDR1 comprising the amino acid sequence of SEQ ID NO:25 or SEQ ID NO:270, CDR2 comprising the amino acid sequence of SEQ ID NO:26, and CDR3 comprising the amino acid sequence of SEQ ID NO:27; (B) CDR1 comprising the amino acid sequence of SEQ ID NO:29 or SEQ ID NO:271, CDR2 comprising the amino acid sequence of SEQ ID NO:30, and CDR3 comprising the amino acid sequence of SEQ ID NO:31; (C) CDR1 comprising the amino acid sequence of SEQ ID NO:33 or SEQ ID NO:272, CDR2 comprising the amino acid sequence of SEQ ID NO:34, and CDR3 comprising the amino acid sequence of SEQ ID NO:35; (D) CDR1 comprising the amino acid sequence of SEQ ID NO:37 or SEQ ID NO:273, CDR2 comprising the amino acid sequence of SEQ ID NO:38, and CDR3 comprising the amino acid sequence of SEQ ID NO:39; (E) a CDR1 comprising the amino acid sequence of SEQ ID NO:41 or SEQ ID NO:274, a CDR2 comprising the amino acid sequence of SEQ ID NO:42, and a CDR3 comprising the amino acid sequence of SEQ ID NO:43; or (F) CDR1 comprising the amino acid sequence of SEQ ID NO:45 or SEQ ID NO:275, CDR2 comprising the amino acid sequence of SEQ ID NO:46, and CDR3 comprising the amino acid sequence of SEQ ID NO:47.

[0111] In some embodiments, the bivalent IL10Rα / IL2Rγ binding molecule comprises an anti-IL10Rα sdAb that comprises CDR1, CDR2, and CDR3 as listed in a row of Table 10, and an anti-IL2Rγ sdAb that comprises CDR1, CDR2, and CDR3 as listed in a row of Table 11 or Table 12.

[0112] In some embodiments, the anti-IL10Rα sdAb of the bivalent IL10Rα / IL2Rγ binding molecule comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the sequence of any one of the anti-IL10Rα sdAbs provided in Table 13. In some embodiments, the anti-IL2Rγ sdAb of the bivalent IL10Rα / IL2Rγ binding molecule comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the sequence of any one of the anti-IL2Rγ sdAbs provided in Table 14 or Table 15.

[0113] In some embodiments, the Fc domain subunit is derived from IgG1, IgG2, IgG3, or IgG4. In some embodiments, the Fc domain subunit comprises one or more amino acid substitutions to reduce effector function, for example, the Fc domain subunit comprises a set of amino acid substitutions selected from the group consisting of: (a) L234A / L235A / P329A ("LALAPA"); L234A / L235A / P329G ("LALAPG"); L234A / L235E / G237A / A330S / P331S ("AEASS"); E233P / L234V / L235A / ΔG237 (PVAdelG); and L234F / L235E / P331S ("FES"). In some embodiments, the Fc domain subunit is modified for multimerization. In some embodiments, the Fc domain subunit comprises an amino acid substitution at position C220 (EU numbering) of the upper hinge domain to delete a sulfhydryl side chain. In some embodiments, the substitution at position C220 is a C220S (EU numbering) substitution. In some embodiments, the Fc domain subunit comprises an amino acid substitution in the Fc domain at positions M428 and / or N434 (EU numbering). In some embodiments, the amino acid substitution at positions M428 and / or N434 is M428L and / or N434S. In some embodiments, the Fc domain subunit comprises an amino acid deletion in the Fc domain at positions G446 and / or K447 (EU numbering).

[0114] B. "Reverse direction" In some embodiments, the bivalent IL10Rα / IL2Rγ binding molecule has the structure: H2N-[anti-IL2Rγ sdAb]-[L] x -[Anti-IL10Rα sdAb]-[TAG] y -COOH wherein L is a polypeptide linker of 1 to 50 amino acids and x=0 or 1, and TAG is a chelating peptide or a subunit of an Fc domain and y=0 or 1.

[0115] In some embodiments, a bivalent IL10Rα / IL2Rγ binding molecule of the foregoing structure comprises, from amino terminus to carboxy terminus, the following polypeptide: (a) an anti-IL2Rγ sdAb, comprising: a CDR1 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to any CDR1 sequence listed in Table 11 or Table 12, or having 0, 1, 2, or 3 amino acid changes, optionally conservative amino acid changes, compared to said sequence; a CDR2 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to any CDR2 sequence listed in Table 11 or Table 12, or having 0, 1, 2, or 3 amino acid changes, optionally conservative amino acid changes, compared to said sequence; and a CDR3 that has at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to any CDR3 sequence listed in Table 11 or Table 12, or has 0, 1, 2, or 3 amino acid changes, optionally conservative amino acid changes, compared to said sequence; or (A) CDR1 comprising the amino acid sequence of SEQ ID NO:25 or SEQ ID NO:270, CDR2 comprising the amino acid sequence of SEQ ID NO:26, and CDR3 comprising the amino acid sequence of SEQ ID NO:27; (B) CDR1 comprising the amino acid sequence of SEQ ID NO:29 or SEQ ID NO:271, CDR2 comprising the amino acid sequence of SEQ ID NO:30, and CDR3 comprising the amino acid sequence of SEQ ID NO:31; (C) CDR1 comprising the amino acid sequence of SEQ ID NO:33 or SEQ ID NO:272, CDR2 comprising the amino acid sequence of SEQ ID NO:34, and CDR3 comprising the amino acid sequence of SEQ ID NO:35; (D) CDR1 comprising the amino acid sequence of SEQ ID NO:37 or SEQ ID NO:273, CDR2 comprising the amino acid sequence of SEQ ID NO:38, and CDR3 comprising the amino acid sequence of SEQ ID NO:39; (E) a CDR1 comprising the amino acid sequence of SEQ ID NO:41 or SEQ ID NO:274, a CDR2 comprising the amino acid sequence of SEQ ID NO:42, and a CDR3 comprising the amino acid sequence of SEQ ID NO:43; or (F) CDR1 comprising the amino acid sequence of SEQ ID NO: 45 or SEQ ID NO: 275, CDR2 comprising the amino acid sequence of SEQ ID NO: 46, and CDR3 comprising the amino acid sequence of SEQ ID NO: 47; and (b) optionally a polypeptide linker of 1 to 50 amino acids, alternatively 1 to 40 amino acids, alternatively 1 to 30 amino acids, alternatively 1 to 20 amino acids, alternatively 1 to 15 amino acids, alternatively 1 to 10 amino acids, alternatively 1 to 8 amino acids, alternatively 1 to 6 amino acids, alternatively 1 to 4 amino acids; and (c) an anti-IL10Rα sdAb, including: a. a CDR1 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to any CDR1 sequence in a row of Table 10, or having 0, 1, 2, or 3 amino acid changes, optionally conservative amino acid changes, compared to said sequence; b. a CDR2 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to any CDR2 sequence in a row of Table 10, or having 0, 1, 2, or 3 amino acid changes, optionally conservative amino acid changes, compared to said sequence; and c. a CDR3 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to any of the CDR3 sequences listed in Table 10, or having 0, 1, 2, or 3 amino acid changes, optionally conservative amino acid changes, compared to said sequences; or d. (A) a CDR1 comprising the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:264, a CDR2 comprising the amino acid sequence of SEQ ID NO:2, and a CDR3 comprising the amino acid sequence of SEQ ID NO:3; or e. (B) a CDR1 comprising the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:265, a CDR2 comprising the amino acid sequence of SEQ ID NO:6, and a CDR3 comprising the amino acid sequence of SEQ ID NO:7; or f. (C) a CDR1 comprising the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:266, a CDR2 comprising the amino acid sequence of SEQ ID NO:10, and a CDR3 comprising the amino acid sequence of SEQ ID NO:11; or g. (D) a CDR1 comprising the amino acid sequence of SEQ ID NO:13 or SEQ ID NO:267, a CDR2 comprising the amino acid sequence of SEQ ID NO:14, and a CDR3 comprising the amino acid sequence of SEQ ID NO:15; or h. (E) a CDR1 comprising the amino acid sequence of SEQ ID NO:17 or SEQ ID NO:268, a CDR2 comprising the amino acid sequence of SEQ ID NO:18, and a CDR3 comprising the amino acid sequence of SEQ ID NO:19; or i. (F) a CDR1 comprising the amino acid sequence of SEQ ID NO:21 or SEQ ID NO:269, a CDR2 comprising the amino acid sequence of SEQ ID NO:22, and a CDR3 comprising the amino acid sequence of SEQ ID NO:23.

[0116] In some embodiments, the anti-IL2Rγ sdAb comprises a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a sequence listed in a row of Table 14 or Table 15. In certain embodiments, the anti-IL10Rα sdAb comprises a sequence having at least 90% sequence identity to any one of the sequences listed in a row of Table 13.

[0117] In some embodiments, the Fc domain subunit is derived from IgG1, IgG2, IgG3, or IgG4. In some embodiments, the Fc domain subunit comprises one or more amino acid substitutions to reduce effector function, for example, the Fc domain subunit comprises a set of amino acid substitutions selected from the group consisting of: (a) L234A / L235A / P329A ("LALAPA"); L234A / L235A / P329G ("LALAPG"); L234A / L235E / G237A / A330S / P331S ("AEASS"); E233P / L234V / L235A / ΔG237 (PVAdelG); and L234F / L235E / P331S ("FES"). In some embodiments, the Fc domain subunit is modified for multimerization. In some embodiments, the Fc domain subunit comprises an amino acid substitution at position C220 (EU numbering) of the upper hinge domain to delete a sulfhydryl side chain. In some embodiments, the substitution at position C220 is a C220S (EU numbering) substitution. In some embodiments, the Fc domain subunit comprises an amino acid substitution in the Fc domain at positions M428 and / or N434 (EU numbering). In some embodiments, the amino acid substitution at positions M428 and / or N434 is M428L and / or N434S. In some embodiments, the Fc domain subunit comprises an amino acid deletion in the Fc domain at positions G446 and / or K447 (EU numbering).

[0118] IV. Single Domain Antibodies and VH H A single domain antibody (sdAb) is an antibody that contains a single monomeric variable antibody domain. Like a full-length antibody, it can selectively bind to a specific antigen. The complementarity determining regions (CDRs) of an sdAb are located within a single domain polypeptide. Single domain antibodies are characterized by the V H H can be engineered from the heavy chain antibodies found in camelids. Cartilaginous fish also have heavy chain antibodies (IgNAR, "immunoglobulin new antigen receptor"), which can then be engineered into V. NAR Single domain antibodies, called sdAbs, can be obtained. Dimeric variable domains from common immunoglobulin G (IgG) of humans or mice can also be split into monomers to make sdAbs. Most research on sdAbs is currently based on heavy chain variable domains, but sdAbs derived from light chains have also been shown to specifically bind targets, see, for example, Moller et al., J Biol Chem. 285(49):38348-38361, 2010. In some embodiments, sdAbs are composed of a single monomeric light chain variable antibody domain.

[0119] sdAb is a heavy chain antibody (V H H). H H is a type of sdAb that has a single monomeric heavy chain variable antibody domain. Like traditional antibodies, V H The H can selectively bind to a specific antigen. The binding proteins described herein are comprised of two V linked together by a linker (e.g., a peptide linker). H H (e.g., V H H 2 The binding protein can include a first V that binds to a first receptor or a domain or subunit thereof. H H, and a second V that binds to a second receptor or a domain or subunit thereof. H Bispecific V, including H H H 2 where the two V HThe H's are joined together by a linker.

[0120] Exemplary V H H has a molecular weight of approximately 12-15 kDa, much smaller than traditional mammalian antibodies (150-160 kDa), which are composed of two heavy chains and two light chains. H H can be found in or produced from mammals of the Camillidae family (e.g., camels, llamas, dromedaries, alpacas, and guanacos) that are naturally devoid of light chains. H Descriptions of HS can be found, for example, in De Greve et al., Curr Opin Biotechnol. 61:96-101, 2019; Ciccarese, et al., Front Genet. 10:997, 2019; Chanier and Chames, Antibodies (Basel) 8(1), 2019; and De Vlieger et al., Antibodies (Basel) 8(1), 2018.

[0121] Bispecific V H H 2 In some embodiments, to prepare a binding protein, H The bispecific VH can be synthesized separately and then joined together by a linker. H H 2 V with different binding activities and receptor targets can be synthesized as fusion proteins. H H is paired with bispecific V H H 2 The binding proteins can be screened for signaling on cells bearing one or both of the relevant receptors.

[0122] V. Linker As previously described, the binding domains of the dimeric binding proteins of the present disclosure may be contiguously linked (e.g., the first V HThe C-terminal amino acid of H binds the second V in the protein. H The binding domains of the binding protein may be optionally joined via a linker (eg, to the N-terminal amino acid of H). A linker is a link between two elements, e.g., protein domains. The bispecific V H H 2 In the binding protein, the linker is a linker between two V H H. The linker can be a covalent bond or a peptide linker. In some embodiments, the linker between two V H H is directly (i.e., via a covalent bond) linked to two V in the binding protein. H The length of the linker between the two V H The linker can be used to adjust the proximity of H. By varying the length of the linker, the overall size and length of the binding protein can be tailored to bind to a specific cell receptor or its domain or subunit. For example, a short linker can be used when the binding protein is designed to bind to two receptors or their domains or subunits located near each other on the same cell. In another example, a long linker can be used when the binding protein is designed to bind to two receptors or their domains or subunits located on two different cells.

[0123] In some embodiments, the linker is a peptide linker. The peptide linker can include 1 to 50 amino acids (e.g., 2 to 50, 5 to 50, 10 to 50, 15 to 50, 20 to 50, 25 to 50, 30 to 50, 35 to 50, 40 to 50, 45 to 50, 2 to 45, 2 to 40, 2 to 35, 2 to 30, 2 to 25, 2 to 20, 2 to 15, 2 to 10, 2 to 5 amino acids). The linker can also be a chemical linker, such as a synthetic polymer, for example, a polyethylene glycol (PEG) polymer.

[0124] In some embodiments, the linker is a first V H The C-terminus of H is linked to a second V in the binding protein. H In another embodiment, the linker is joined to the N-terminus of the second V H The C-terminus of H is linked to the first V H Link to the N-terminus of H.

[0125] Suitable peptide linkers are known in the art and include, for example, peptide linkers that contain flexible amino acid residues, such as glycine and serine. In certain embodiments, the peptide linker is In certain embodiments, the peptide linker can contain 2 to 12 amino acids including a GS motif, e.g., In certain other embodiments, the peptide linker can contain 3 to 12 amino acids including a GGS motif, e.g., In yet another embodiment, the peptide linker can contain 4 to 20 amino acids including the motif GGSG (SEQ ID NO:65), e.g., In other embodiments, the peptide linker can contain the motif GGGGS (SEQ ID NO:63), e.g., TIFF2024504923000026.tif12128 may be included.

[0126] Modulation of the activity of receptor binding molecules In some embodiments, the design of the IL10Rα / IL2Rγ binding molecules of the present disclosure can be modulated by structural variations in the design of the receptor binding molecules, for example, to achieve partial agonism or selective activation of specific cell types. This variation in activity can be used to modulate the binding and activity of the IL10Rα / IL2Rγ binding molecules to optimize the activity of the IL10Rα / IL2Rγ binding molecules to achieve partial agonism, selective cell type activation, or to provide molecules with increased or decreased binding compared to the cognate ligand for each of the IL10Rα and IL2Rγ sdAbs to their respective receptor subunits. The ability to modulate the activity of the IL10Rα / IL2Rγ binding molecules of the present disclosure provides substantial benefits in multiple therapeutic applications. The IL10Rα / IL2Rγ binding molecules of the present disclosure can induce different levels of downstream signaling in different cell types. For example, by varying the length of the linker between the IL10Ra sdAb antibody and the IL2Rg sdAb antibody in the IL10Rα / IL2Rγ binding molecule, the IL10Rα / IL2Rγ binding molecule provides a higher level of downstream signaling in desirable cell types compared to undesirable cell types. In other embodiments, different IL10Rα sdAb antibodies with different binding affinities and different IL IL2Rγ sdAb antibodies with different binding affinities can be used to modulate the activity of the IL10R binding molecule. Furthermore, when the IL10Rα / IL2Rγ binding molecule is provided as a single polypeptide, the orientation of the two antibodies in the polypeptide can also be changed to alter the properties of the molecule.

[0127] In some embodiments, the IL10Rα / IL2Rγ binding molecules of the present disclosure have an Emax for T cells that is at least 5 times greater than the Emax of signaling in monocytes, alternatively 10 times greater, alternatively 100 times greater, alternatively at least 1000 times greater. max T cells with + This results in increased levels of downstream signaling in T cells.

[0128] In one embodiment, the disclosure provides IL10Rα / IL2Rγ binding molecules that preferentially activate T cells, particularly CD8+ T cells, relative to monocytes. In some embodiments, the IL10Rα / IL2Rγ binding molecules of the disclosure have an Emax for T cells that is at least 5 times greater, alternatively 10 times greater, alternatively 100 times greater, alternatively at least 1000 times greater than the Emax of signaling in monocytes. max T cells with + This results in increased levels of downstream signaling in T cells.

[0129] In some embodiments, for a given cell type, the E produced by IL10, the cognate ligand of the IL10 receptor (i.e., an IL10R binding molecule that is an IL10 partial agonist) is max Reduced E compared to max In some embodiments, the IL10Ra / IL2Rg binding proteins described herein have an E associated with wild-type hIL10. max of at least 1% (e.g., 1% to 100%, 10% to 100%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100%, 90% to 100%, 1% to 90%, 1% to 80%, 1% to 70%, 1% to 60%, 1% to 50%, 1% to 40%, 1% to 30%, 1% to 20%, or 1% to 10%).

[0130] Modulation of activity by adjusting linker length In some embodiments, for example, varying the linker length between the binding domains of the binding molecule can be used to modulate the activity of the dimeric binding protein, both in terms of specific activity in a given cell type and between cell types. In some embodiments, for example, by varying the linker length, the IL10Rα / IL2Rγ binding molecule can be used to modulate the level of downstream signaling in T cells (e.g., CD8 +The ability to modulate the activity of IL10Rα / IL2Rγ binding molecules can result in higher levels of downstream signaling in T cells (e.g., CD8 T cells) compared to the levels of downstream signaling in monocytes. + The present invention provides molecules that have higher levels of downstream signaling in T cells.

[0131] The ability to modulate the activity of a dimeric binding molecule may be achieved by increasing the level of a downstream molecule in one particular cell type (e.g., CD8 + The present invention provides molecules that have higher levels of downstream signaling in T cells (T cells). A series of representative IL10Ra / IL2Rg dimeric binding molecules were constructed to evaluate and demonstrate the effect of linker length on various biological activities regulated in T cells and monocytes. The results of these studies are presented in Tables 20-29 below, providing details regarding the specific binding protein test article components, linker amino acid sequences and lengths, concentrations of test articles evaluated, cell types used, and the resulting biological responses measured. Each of these molecules was recombinantly produced and purified substantially in accordance with the examples provided herein. Parameters evaluated included pSTAT3 induction in CD8+ T cells (Table 20), pSTAT3 induction on CD4 T cells (Table 21), pSTAT3 induction in monocytes (Table 22), IFNγ secretion in CD8+ T cells (Table 23), Granzyme A secretion in CD8+ T cells (Table 24), Granzyme B secretion in CD8+ T cells (Table 25), IL9 secretion in CD8+ T cells (Table 26), IL-1β secretion in LPS-treated monocytes (Table 27), IL6 secretion in LPS-treated monocytes (Table 28), and TNF-α secretion in LPS-treated monocytes (Table 29). These data demonstrate the ability to modulate the function of IL10Ra / IL2Rg dimeric binding molecules within a given cell type or bias function towards one cell type or the other by variation of the linker between the binding domains.

[0132] Modulation of activity by adjusting the binding affinity of sdAbs: In some embodiments, the activity and / or specificity of the bivalent IL10Rα / IL2Rγ binding molecules of the present disclosure may be modulated by the binding affinity of each of the sdAbs to their respective receptor subunits. It will be understood by those skilled in the art that the binding of the first sdAb of the bivalent IL10Rα / IL2Rγ binding molecule to the ECD of the first receptor subunit on the cell surface increases the probability of a binding interaction between the second sdAb of the bivalent IL10Rα / IL2Rγ binding molecule and the ECD of the second receptor subunit. This cooperative binding effect may result in a bivalent IL10Rα / IL2Rγ binding molecule with very high affinity for the receptor and a very slow "off-rate" from the receptor. A typical VHH single domain antibody has a binding affinity of about 10 -5 M~about 10 -10 M. In these instances, such slow off-rate kinetics are desirable in a bivalent IL10Rα / IL2Rγ binding molecule, and high affinity (approximately 10 -7 M~about 10 -10 Selection of sdAbs having the following structure is preferred:

[0133] Naturally occurring cytokine ligands typically do not exhibit similar affinities for each subunit of the heterodimeric receptor. Thus, in designing a bivalent IL10Rα / IL2Rγ binding molecule, selection of sdAbs against the first and second IL10Rα / IL2Rγ receptor subunits that have similar affinities to the cognate ligand for each receptor subunit (e.g., about 10-fold, alternatively about 20-fold, or alternatively about 50-fold higher or lower affinity) can be used.

[0134] In some embodiments, the bivalent IL10Rα / IL2Rγ binding molecules of the present disclosure are partial agonists of the IL10Rα / IL2Rγ receptor. Thus, the activity of the bivalent binding molecules can be modulated by selecting sdAbs with greater or lesser affinity for either or both of the IL10Rα / IL2Rγ receptor subunits. Because some heterodimeric cytokine receptors are composed of a "unique subunit" (i.e., a subunit that is not naturally a subunit of another multimeric receptor) and a second "common" subunit (such as CD132) that is a shared component of multiple cytokine receptors, the selectivity for the formation of such receptors can be enhanced by using a first sdAb with a higher affinity for the unique receptor subunit and a second sdAB that exhibits a lower affinity for the common receptor subunit. Additionally, the common receptor subunit can be expressed on a wider variety of cell types than the unique receptor subunits. In some embodiments where the receptor is a heterodimeric receptor comprising a unique subunit and a common subunit, a first sdAb of the bivalent IL10Rα / IL2Rγ binding molecule exhibits significantly greater affinity (more than 10-fold greater, alternatively more than 100-fold greater, alternatively more than 1000-fold greater) for the unique receptor than a second sdAb of the bivalent IL10Rα / IL2Rγ binding molecule for the common receptor subunit. In one embodiment, the disclosure provides a bivalent IL10Rα / IL2Rγ binding molecule, wherein the affinity of the anti-IL10Rα sdAb is more than 10-fold greater, alternatively more than 100-fold greater, alternatively more than 1000-fold greater than the affinity of the anti-IL2Rγ sdAb for the common receptor subunit.

[0135] In one embodiment, the disclosure provides an IL10Rα / IL2Rγ binding molecule, in which the affinity of the IL10Rα sdAb has a higher affinity for the extracellular domain of IL10Rα than the affinity of the IL2Rg sdAb for the extracellular domain of IL2Rγ. In some embodiments, the disclosure provides an IL10Rα sdAb with an affinity of about 10 -8 ~about 10 -10M, alternatively about 10 -9 ~about 10 -10 M, or alternatively about 10 -10 M, and the IL2Rγ sdAb has an affinity for the extracellular domain of IL10Rα of approximately 10 -6 ~about 10 -9 M, alternatively about 10 -7 ~about 10 -9 M, alternatively about 10 -7 ~about 10 -8 M, alternatively about 10 -9 M, alternatively about 10 -8 M. In some embodiments, the present disclosure provides an IL10Rα sdAb having an affinity for the extracellular domain of IL2Rγ of about 10 -8 ~about 10 -10 M, alternatively about 10 -9 ~about 10 -10 M, or alternatively about 10 -10 M, and the IL2Rg sdAb has an affinity for the extracellular domain of IL10Rα of about 10 -6 ~about 10 -9 M, alternatively about 10 -7 ~about 10 -9 M, alternatively about 10 -7 ~about 10 -8 M, alternatively about 10 -9 M, alternatively about 10 -8 M for the extracellular domain of IL2Rγ, and wherein the affinity of the IL10Ra sdAb for the ECD of IL10Ra is more than 2 fold higher, alternatively more than 5 fold higher, alternatively more than 10 fold higher, alternatively more than 20 fold higher, alternatively more than 40 fold higher, alternatively more than 50 fold higher, alternatively more than 60 fold higher, alternatively more than 70 fold higher, alternatively more than 80 fold higher, alternatively more than 90 fold higher, alternatively more than 100 fold higher, alternatively more than 150 fold higher, alternatively more than 200 fold higher, or alternatively more than 500 fold higher than the affinity of the IL2Rγ sdAb for the ECD of IL2Rγ.

[0136] VI. Modifications to Extend Duration of Action In Vivo The binding protein described herein can be modified to provide extended in vivo life span and / or extended duration of action in subjects.In some embodiments, the binding protein can be conjugated to a carrier molecule to provide desirable pharmacological properties such as extended half-life.In some embodiments, the binding protein can be covalently bound to the Fc domain of IgG, albumin, or other molecules, for example, by pegylation, glycosylation, etc., as known in the art, to extend its half-life.

[0137] In some embodiments, the binding protein, optionally including an intervening linker, is conjugated to an Fc polypeptide or Fc domain (a dimer of two Fc polypeptides). Fc fusion conjugates have been shown to increase the systemic half-life of biologics, and thus biologic products may require less frequent administration. Fc binds to the neonatal Fc receptor (FcRn) in endothelial cells lining blood vessels, and upon binding, the Fc fusion molecule is protected from degradation and re-released into circulation, keeping the molecule in circulation longer. This Fc binding is thought to be the mechanism by which endogenous IgG retains its long plasma half-life. Recent Fc-fusion technology links a single copy biologic to the Fc region of an antibody, optimizing the pharmacokinetic and pharmacodynamic properties of the biologic compared to traditional Fc-fusion conjugates. Fc polypeptides or Fc domains useful in the preparation of Fc fusions can be naturally occurring or synthetic polypeptides that are homologous to the IgG C-terminal domain produced by digestion of IgG with papain. IgG Fc has a molecular weight of approximately 50 kDa. The binding proteins described herein can be conjugated to the entire Fc polypeptide or Fc domain, or to smaller portions that retain the ability to extend the circulating half-life of the chimeric polypeptide of which it is a part. In addition, the full-length or fragmented Fc polypeptides can be variants of the wild-type molecule. In a typical presentation, each Fc polypeptide in an Fc domain can carry a heterologous polypeptide; the two heterologous polypeptides in an Fc domain can be the same or different (e.g., one is anti-IL10Rα V H H antibody and the other anti-IL2Rγ V H H antibody or one or both of the heterologous polypeptides are H H antibody / anti-IL2Rγ V HH antibody dimeric polypeptide). As shown, linking of the IL10Rα / IL2Rγ bivalent binding molecule to the Fc subunit may incorporate a linker molecule as described below between the bivalent sdAb and the Fc subunit. In some embodiments, the IL10Rα / IL2Rγ bivalent binding molecule is expressed as a fusion protein with an Fc domain incorporating the amino acid sequence of the hinge region of an IgG antibody. The Fc domain engineered according to the foregoing may be derived from IgG1, IgG2, IgG3, and IgG4 mammalian IgG species. In some embodiments, the Fc domain may be derived from human IgG1, IgG2, IgG3, and IgG4 IgG species. In some embodiments, the hinge region is an IgG1 hinge region. In one particular embodiment, the IL10Rα / IL2Rγ bivalent binding molecule is linked to the Fc domain using a human IgG1 hinge domain.

[0138] In some embodiments, the bivalent binding molecules of the present disclosure may be conjugated to one (as shown in Figures 1C and 1D) or both domains of Fc (as shown in Figures 1C and 1D). In one embodiment, the Fc domain is an Fc domain derived from the IgG4 heavy chain constant region of human IgG4 (UniProt Reference P01861). The use of hIgG4 as a source of Fc offers advantages such as very low FcγR binding, thereby reducing the need for mutations, immunogenicity, or effector function. In some embodiments, when hIgG4 is used as a source of Fc domain, hIgG4 Fc may contain the amino acid substitution S228P (EU numbering), which is useful for stabilizing Fc dimers. Additionally or alternatively, hIgG4 Fc may contain the amino acid substitution N297G (EU numbering), which reduces FcγR binding. Additionally, or alternatively, the hIgG4 Fc may contain a deletion of a C-terminal lysine residue (K447del) (EU numbering) that reduces FcγR binding.

[0139] In one embodiment, the disclosure is comprised of two of the same IL10Ra / IL2Rg dimeric binding molecules (H1, DR240-G3S-DR231), each attached via an AS linker to a domain of hIgG4 Fc (including the hIgG4 hinge, CH2, and CH3 domains) containing the amino acid substitutions S228P and N297G and a deletion of K447, and having the following amino acid sequence: The present invention provides a homodimeric binding protein having the TIFF2024504923000027.tif43159, herein referred to as DR992. The nucleic acid sequence encoding DR992 is represented by the following DNA sequence: I have TIFF2024504923000028.tif131160.

[0140] In another embodiment, the disclosure is comprised of two of the same IL10Ra / IL2Rg dimeric binding molecules (A2, DR229-G3S-DR239), each appended to a domain of IgG4 Fc containing the S228P amino acid substitution, and the following amino acid sequence: The present invention provides a homodimeric binding protein having the TIFF2024504923000029.tif43160, herein referred to as DR995. The nucleic acid sequence encoding DR995 is represented by the following DNA sequence: I have TIFF2024504923000030.tif131159. Alternatively, the following amino acid sequence: Wild type human IgG4 Fc (hIgG4 hinge-CH2-CH3) having TIFF2024504923000031.tif24147 may be used.

[0141] In some embodiments, the present disclosure provides at least one anti-IL10Rα V H H antibody and at least one anti-IL2Rγ V H A heterodimeric Fc comprising an anti-IL10Rα V H antibody, wherein the heterodimeric Fc H H antibody / Fc fusion polypeptide and anti-IL2Rγ V HThe anti-IL10Rα V H antibody / Fc fusion polypeptide is covalently linked via one disulfide bond, optionally two disulfide bonds, optionally three disulfide bonds, or optionally four disulfide bonds. H H antibody / FC fusion polypeptide and anti-IL2Rγ V H The H antibody / Fc fusion polypeptide is an anti-IL10Rα V H The sulfhydryl group at amino acid C226 in the lower hinge domain of the H antibody / Fc fusion and the anti-IL2Rγ V H In some embodiments, the two fusions are covalently linked via a disulfide bond between the sulfhydryl group at amino acid C226 of the lower hinge domain of the anti-IL10Rα V H antibody / Fc fusion. H The sulfhydryl group at amino acid C229 in the lower hinge domain of the H antibody / Fc fusion and the anti-IL2Rγ V H The H antibody / Fc fusion is covalently linked via a disulfide bond between the sulfhydryl group of amino acid C229 of the lower hinge domain. In some embodiments, the first Fc domain comprises the amino acid substitution S354C and the second Fc domain comprises the amino acid substitution Y349C. In some embodiments, the heterodimeric Fc comprises a first Fc domain comprising the amino acid substitution S354C and a second Fc domain comprising the amino acid substitution Y349C, and the fusion is linked via a disulfide bond between S354C of the first Fc domain and Y349C of the second Fc domain. In some embodiments, the two polypeptides of the heterodimeric Fc are covalently linked via one or more, optionally two or more, optionally three or more, optionally four or more disulfide bonds between the side chains of the following groups of cystine pairs: (a) between C96 of the first Fc fusion and C199 of the second Fc fusion; (b) between C226 of the first Fc fusion and C226 of the second Fc fusion; (c) between C229 of the first Fc fusion and C229 of the second Fc fusion; and (d) between S354C of the first Fc fusion comprising the S354C amino acid substitution and Y349C of the second Fc fusion comprising the Y349C amino acid substitution.

[0142] In some embodiments, the present disclosure provides a heterodimeric Fc, wherein either or both of the fusion subunits of the heterodimeric Fc comprise one or more amino acid substitutions to reduce effector function. In some embodiments, the fusion polypeptide comprises a set of amino acid substitutions selected from the group consisting of: (a) L234A / L235A / P329A ("LALAPA"); L234A / L235A / P329G ("LALAPG"); L234A / L235E / G237A / A330S / P331S ("AEASS"); E233P / L234V / L235A / ΔG237 (PVAdelG); and L234F / L235E / P331S ("FES").

[0143] In some embodiments, the disclosure provides a heterodimeric Fc, wherein either or both of the fusion subunits of the heterodimeric Fc comprise an amino acid substitution at position C220 (EU numbering) of the upper hinge domain to eliminate a sulfhydryl side chain. In some embodiments, the substitution at position C220 is a C220S (EU numbering) substitution.

[0144] In some embodiments, the disclosure provides a heterodimeric Fc, wherein either or both of the fusion subunits of the heterodimeric Fc comprise an amino acid substitution in the Fc domain at positions M428 and / or N434 (EU numbering). In some embodiments, the amino acid substitution at positions M428 and / or N434 is M428L and / or N434S.

[0145] In some embodiments, the disclosure provides a heterodimeric Fc, wherein either or both of the fusion subunits of the heterodimeric Fc comprise an amino acid deletion in the Fc domain at positions G446 and / or K447 (EU numbering).

[0146] Illustrative examples of Fc formats useful in binding molecules of the present disclosure are provided generally in Figures 4-7 of the accompanying drawings.

[0147] In some embodiments, the present disclosure provides a heterodimeric Fc, in which either or both of the fusion subunits of the heterodimeric Fc are PEGylated, in some embodiments, either or both of the fusion subunits are PEGylated via the sulfhydryl side chain of amino acid C220 of the upper hinge.

[0148] In some embodiments, the present disclosure provides an anti-IL10Rα V polypeptide operably linked to one or more heterologous nucleic acid sequences. H H antibody / Fc fusion polypeptide and anti-IL2Rγ V H An expression cassette encoding a heterodimeric Fc is provided, comprising a nucleic acid sequence encoding an anti-IL10Rα V H antibody / Fc fusion polypeptide, H H antibody / Fc fusion polypeptide and anti-IL2Rγ V H The nucleic acid sequences encoding the H antibody / Fc fusion polypeptides are (a) under the control of a single promoter and (b) linked via intervening sequences that facilitate co-expression. H H antibody / Fc fusion polypeptide and anti-IL2Rγ V H In some embodiments, the nucleic acid sequences encoding the anti-IL10Rα V H antibody / Fc fusion polypeptides are linked via intervening sequences that facilitate coexpression. H The nucleic acid sequence encoding the H antibody / Fc fusion polypeptide is H 5' to the nucleic acid sequence encoding the H antibody / Fc fusion polypeptide. H H antibody / Fc fusion polypeptide and anti-IL2Rγ V H In some embodiments, the nucleic acid sequences encoding the anti-IL2Rγ V H antibody / Fc fusion polypeptides are linked via intervening sequences that facilitate co-expression. H The nucleic acid sequence encoding the H antibody / Fc fusion polypeptide is H It is 5' to the nucleic acid sequence encoding the H antibody / Fc fusion polypeptide. In some embodiments, the intervening sequence to facilitate co-expression is an IRES element or a T2A sequence.

[0149] In some embodiments, the present disclosure provides an anti-IL10Rα V polypeptide operably linked to one or more heterologous nucleic acid sequences. H H antibody / Fc fusion polypeptide and anti-IL2Rγ V H An expression cassette encoding a heterodimeric Fc is provided, comprising a nucleic acid sequence encoding an anti-IL10Rα V H antibody / Fc fusion polypeptide, H H antibody / Fc fusion polypeptide and anti-IL2Rγ V H The nucleic acid sequences encoding the H antibody / Fc fusion polypeptides are (a) under the control of a single promoter and (b) linked via intervening sequences that facilitate co-expression in mammalian cells.

[0150] The present disclosure further provides a recombinant vector encoding a heterodimeric Fc, the vector comprising an anti-IL10Rα V H A first expression cassette encoding an anti-IL2Rγ V antibody / Fc fusion polypeptide, and H A second expression cassette comprising a nucleic acid sequence encoding an H antibody / Fc fusion polypeptide. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a non-viral vector.

[0151] Further provided is a recombinant modified cell comprising the nucleic acid molecule or vector of the present disclosure.In some embodiments, the cell is a prokaryotic cell, such as a bacterial cell.In some embodiments, the cell is a eukaryotic cell, such as a mammalian cell.Also provided is a cell culture, comprising at least one recombinant modified cell of the present disclosure and a culture medium.

[0152] In some embodiments, the recombinant engineered cells are transformed with a recombinant vector encoding a heterodimeric Fc, the vector comprising an anti-IL2Rγ V H A first expression cassette encoding an anti-IL10Rα V antibody / Fc fusion polypeptide, and HIn some embodiments, the recombinant engineered cell is transformed with a recombinant vector encoding an anti-IL10Rα V H antibody / Fc fusion polypeptide, the vector comprising a second expression cassette comprising a nucleic acid sequence encoding an anti-IL10Rα V H antibody / Fc fusion polypeptide. H A first expression cassette encoding an anti-IL2Rγ V antibody / Fc fusion polypeptide, and H A second expression cassette comprising a nucleic acid sequence encoding an H antibody / Fc fusion polypeptide.

[0153] In some embodiments, the recombinant modified cells comprise an anti-IL2Rγ V operably linked to one or more expression control sequences. H A first vector comprising a nucleic acid sequence encoding an anti-IL10Rα V H antibody / Fc fusion polypeptide operably linked to one or more expression control sequences. H In some embodiments, the recombinant modified cells are transformed with a second vector comprising an expression cassette comprising a nucleic acid sequence encoding an anti-IL10Rα V H antibody / Fc fusion polypeptide. In some embodiments, the recombinant modified cells are transformed with a second vector comprising an expression cassette comprising an anti-IL10Rα V H antibody / Fc fusion polypeptide operably linked to one or more expression control sequences. H A first vector comprising a nucleic acid sequence encoding an anti-IL2Rγ V H antibody / Fc fusion polypeptide, and an anti-IL2Rγ V H antibody / Fc fusion polypeptide operably linked to one or more expression control sequences. H The second vector is transformed with an expression cassette comprising a nucleic acid sequence encoding an H antibody / Fc fusion polypeptide. In some embodiments, the cell is a prokaryotic cell, such as a bacterial cell. In some embodiments, the cell is a eukaryotic cell, such as a mammalian cell. Also provided is a cell culture, comprising at least one recombinant modified cell of the present disclosure and a culture medium.

[0154] The present disclosure further provides the method for recombinant production, isolation, purification and characterization of heterodimeric Fc.Therefore, the method for producing the heterodimeric Fc of the present disclosure is provided herein.In some embodiments, the method includes: a) providing one or more recombinant modified cells, comprising the nucleic acid molecule or vector comprising the nucleic acid sequence encoding heterodimeric Fc as disclosed herein; and b) culturing one or more cells in culture medium, so that the cell produces the heterodimeric Fc encoded by the nucleic acid sequence.

[0155] Also provided is a pharmaceutical composition comprising the heterodimeric Fc of the present disclosure. In some embodiments, the pharmaceutical composition comprises the heterodimeric Fc of the present disclosure and a pharma- ceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises the nucleic acid molecule or vector of the present disclosure. In some embodiments, the pharmaceutical composition comprises the recombinant modified cell of the present disclosure. In some embodiments, the recombinant modified cell is a mammalian cell.

[0156] The present disclosure provides a first polypeptide of formula #1: Anti-IL10Rα V H H antibody-L1 a -UH1-Fc1[1] and a second polypeptide of formula #2: Anti-IL2Rγ V H H antibody-L2 b -UH2-Fc2 [2] and providing a heterodimeric Fc comprising: During the ceremony, L1 and L2 are GSA linkers and a and b are independently selected from 0 (absent) or 1 (present); UH1 and UH2 are each an upper hinge domain of a human immunoglobulin independently selected from the group consisting of IgG1, IgG2, IgG3, and IgG4 upper hinges, optionally containing the amino acid substitution C220S (EU numbering); Fc1 is a polypeptide comprising the lower hinge domain, the CH2 domain, and the CH3 domain of a human immunoglobulin selected from the group consisting of IgG1, IgG2, IgG3, and IgG4, and comprising one or more amino acid substitutions that promote heterodimerization with Fc2; and FC2 is a polypeptide comprising the lower hinge domain, the CH2 domain, and the CH3 domain of a human immunoglobulin selected from the group consisting of IgG1, IgG2, IgG3, and IgG4, and comprising one or more amino acid substitutions that promote heterodimerization with Fc1; and The polypeptide of formula 1 and the polypeptide of formula 2 are linked by at least one interchain disulfide bond.

[0157] Top Hinge: The heterodimeric Fc of the present disclosure is a heterodimer comprising the polypeptides of formulas [1] and [2], each incorporating an upper hinge region of a human immunoglobulin molecule. The term "upper hinge" or "UH" refers to an amino acid sequence corresponding to amino acid residues 216-220 (EU numbering) of a human immunoglobulin molecule. In some embodiments, the upper hinge region is a naturally occurring upper hinge region of a human immunoglobulin selected from the LH region of a human IgG1, human IgG2, human IgG3, and human IgG4 upper hinge domain. In some embodiments, the upper hinge region is an upper hinge region of a human IgG1 immunoglobulin. In some embodiments, the upper hinge region is an upper hinge region of a human IgG1 immunoglobulin comprising the pentameric amino acid sequence: EPKSC (SEQ ID NO:11).

[0158] In some embodiments, the upper hinge region contains an unpaired cysteine ​​residue at position 220 (EU numbering), which typically binds to a cysteine ​​on the light chain in a complete immunoglobulin molecule. If only the Fc domain containing the hinge domain is used, the unpaired cysteine ​​in the hinge domain creates the possibility of improper disulfide bond formation. Thus, in some embodiments, the cysteine ​​at position 220 (C220, numbered according to EU numbering) is substituted with an amino acid that does not promote disulfide bonds. In some embodiments, the Fc domain comprises a C220S mutation with the amino acid sequence EPKSS.

[0159] Fc1 and Fc2: The heterodimeric Fc of the present disclosure is a heterodimer comprising the polypeptides of formulas [1] and [2], each of which comprises the Fc region (Fc1 and Fc2) of a human immunoglobulin molecule, modified to promote heterodimerization.

[0160] As used herein, the terms "Fc" and "Fc monomer" are used interchangeably herein to designate a monomeric polypeptide subunit of an Fc dimer. Fc comprises an amino acid sequence that includes (from amino terminus to carboxy terminus) the lower hinge domain and the CH2 and CH3 domains of a human immunoglobulin molecule. In some embodiments, an Fc monomer is a polypeptide that includes the lower hinge domain and the CH2 and CH3 domains of a human immunoglobulin molecule domain of human IgG1, human IgG2, human IgG3, and human IgG4 hinge domain. The CH2 domain of hIgG1 corresponds to amino acid residues 231-340 (EU numbering) and is provided as SEQ ID NO:14. The CH3 domain of hIgG1 corresponds to amino acid residues 341-447 (EU numbering). The polypeptides of formulas [1] and [2] each incorporate a lower hinge region of a human immunoglobulin. As used herein, the term "lower hinge" or "LH" refers to an amino acid sequence corresponding to amino acid residues 221-229 (EU numbering) of a human immunoglobulin molecule. In some embodiments, the lower hinge region is a naturally occurring lower hinge region of a human immunoglobulin selected from the LH region of an IgG1, IgG2, IgG3, and IgG4 lower hinge domain. In some embodiments, the lower hinge region is a lower hinge region of a human IgG1 immunoglobulin. In some embodiments, the lower hinge region is a lower hinge region of a human IgG1 immunoglobulin comprising the decameric amino acid sequence: DKTHTCPPCP.

[0161] In some embodiments, Fc1 and Fc2 have the following amino acid sequence (EU numbering shown): It is derived from a polypeptide corresponding to amino acids 221 to 447 (EU numbering) of human IgG1 immunoglobulin, having TIFF2024504923000032.tif62138.

[0162] As shown in the sequence above, the C-terminal residue of the wild-type form of the IgG1 Fc domain is a lysine, designated K447 according to EU numbering. K447 is inconsistently removed by the producing cells during recombinant production. As a result, the population of recombinant Fc monomers may be heterogeneous in that some fraction of recombinantly produced Fc monomers contain K447 and others do not. Such inconsistent proteolytic processing by the producing cells may therefore result in a heterogeneous population of Fc. Typically, such heterogeneity of pharmacoactive ingredients should be avoided, especially in the context of human medicine. Thus, in addition to modifications to Fc monomer sequences that promote heterodimerization, the disclosure provides nucleic acid sequences encoding Fc monomers that further comprise a deletion of the C-terminal K447 or a deletion of G446 and K447, as well as Fc monomers that comprise (a) a deletion of the lysine residue at position 447 (K447, EU numbering, abbreviated as ΔK447 or des-K447), or (b) a deletion of both the glycine at position 456 (G446, EU numbering, abbreviated as des-G446) and K447 (this double deletion of G446 and K447 is referred to herein as des-G446 / des-K447 or ΔG446 / ΔK447).

[0163] Engineering the Fc subunit to promote heterodimerization As provided in the above formulas [1] and [2], the Fc1 and Fc2 monomers of the dimeric Fc contain amino acid substitutions that promote heterodimerization between Fc1 and Fc2. A variety of techniques have been established for promoting heterodimerization of Fc domains. See, for example, US Patent to Gillies et al., US Patent No. 1,108,7249 to Kim et al., issued on August 3, 2021. In some embodiments, the modification to promote heterodimerization between Fc1 and Fc2 monomers is HF-TA mutation and HA-TF mutation as described in Moore, et al. (2011) mAbs 3(6):546-557. The HF-TA method uses S364H / T394F substitutions on one Fc monomer and Y349T / F405A substitutions on the complementary Fc monomer. The (HA-TF) method uses S364H / F405A substitutions on one Fc monomer and Y349T / T394F substitutions on the complementary Fc monomer. Alternatively, Fc1 and Fc2 monomers are modified to promote heterodimerization by the ZW1 heterodimerization method, using T350V / L351Y / F405A / Y407V substitutions on one Fc monomer and T350V / T366L / K392L / T394W substitutions on the complementary Fc monomer. Von Kreudenstein, et al (2013) mAbs, 5(5):646-654. Alternatively, the Fc1 and Fc2 monomers are engineered to promote heterodimerization by the EW-RVT heterodimerization method, using K360E / K409W substitutions on one Fc monomer and Q347R / D399V / F405T substitutions on the complementary Fc monomer. Choi, et al (2015) Molecular Immunology 65(2):377-83.

[0164] In one embodiment, Fc1 and Fc2 are modified to promote heterodimerization by the use of "knob-into-hole" (abbreviated KiH) modifications as exemplified herein. KiH modifications include one or more amino acid substitutions in a first Fc monomer (e.g., Fc1) that create a bulky "knob" domain on the first Fc, and one or more amino acid substitutions to a second Fc monomer (e.g., Fc2) that create a complementary pocket or "hole" for receiving the "knob" of the first Fc monomer.

[0165] The knobs-into-holes format is used to facilitate expression of a first polypeptide on a first Fc monomer having a "knob" modification and a second polypeptide on a second Fc monomer having a "hole" modification to facilitate expression of a heterodimeric polypeptide conjugate. In some embodiments, an IL10Rα / IL2Rγ bivalent binding molecule covalently linked to a single subunit of Fc, as shown in Figure 6, is provided for each of the subunits of Fc, as shown in Figure 7A.

[0166] Various amino acid substitutions have been established to generate complementary knob and hole Fc monomers. See, e.g., Ridgway, et al (1996) Protein Engineering 9(7):617-921; Atwell, et al (1997) J. Mol. Biol. 270:26-35; Carter et al., U.S. Pat. No. 5,807,706, issued Sep. 15, 1998; Carter et al., U.S. Pat. No. 7,695,936, issued Apr. 13, 2010; Zhao et al. "A new approach to produce IgG4-like bispecific antibodies," Scientific Reports 11: 18630 ​​(2021); Cao et al. "Characterization and Monitoring of a Novel Light-heavy-light Chain Mispair in a Therapeutic Bispecific Antibody," and Liu et al. "Fc Engineering for Developing Therapeutic Bispecific Antibodies and Novel Scaffolds." Frontiers in Immunology. 8: 38. See doi:10.3389 / fimmu.2017.00038 (2017).

[0167] In some embodiments, the Fc domain comprises two Fc monomers, where the CH3 domain of a first Fc monomer has the threonine at position 366 (EU numbering) altered with a bulky residue (e.g., T366W) creating a "knob" and substitution, and a second Fc monomer includes one or more substitutions at complementary residues in the CH3 domain of the second Fc monomer, e.g., by amino acid substitutions such as T366S, L368A, and / or Y407V, creating a pocket or "hole" to accommodate the bulky residue.

[0168] In one embodiment, the Fc1 monomer of formula 1 is a "knob" modified Fc monomer comprising the amino acid substitution T366W, and the Fc2 monomer of formula 2 is a "hole" modified Fc comprising the set of amino acid substitutions T366S / L368A / Y407V.

[0169] Alternatively, the Fc1 monomer of formula 1 is a "hole" modified Fc monomer comprising the set of amino acid substitutions T366S / L368A / Y407V, and the Fc2 monomer of formula 2 is a "knob" modified Fc monomer comprising the amino acid substitution T366W.

[0170] Examples of engineered Fc heterodimer pairs containing complementary KiH modifications are provided in the table below.

[0171] (Table) Set of amino acid substitutions for complementary IgG1 KiH heterodimer pairs TIFF2024504923000033.tif24169

[0172] As stated, the heterodimeric Fc of the present disclosure is provided as a complementary heterodimeric pair of polypeptides of formula [1] and [2], where the first and second polypeptides are linked by at least one disulfide bond. In some embodiments, the incorporation of a disulfide bond between the polypeptides of formula [1] and [2] can be achieved by cysteine ​​substitution at specific points in the Fc1 and Fc2 domains. In one embodiment, the Fc1 domain of the polypeptide of formula [1] is derived from the Fc domain of hIgG1 containing the amino acid substitution S354C (EU numbering), and the Fc2 domain of the polypeptide of formula [2] is derived from the Fc domain of hIgG1 containing the amino acid substitution Y349C (EU numbering), providing a disulfide bond between S354C of Fc1 and Y349C of Fc2. Alternatively, the Fc1 domain of the polypeptide of formula [1] is derived from the Fc domain of hIgG1 containing the amino acid substitution Y349C (EU numbering), and the Fc2 domain of the polypeptide of formula [2] is derived from the Fc domain of hIgG1 containing the amino acid substitution S354C (EU numbering), providing a disulfide bond between S354C of Fc1 and Y349C of Fc2.

[0173] Further examples of complementary KiH engineered heterodimeric Fc pairs that can be used in the practice of the present disclosure are provided in the table below.

[0174] (Table) Knob-into-hole Fc dimer pair TIFF2024504923000034.tif75166

[0175] Additional Fc modifications In addition to modifications of the Fc1 and Fc2 domains to promote heterodimerization, Fc1 and Fc2 can optionally be provided with additional amino acid modifications to attenuate effector function or to eliminate the glycosylation site at N297, e.g., N297Q.

[0176] Modifications to reduce effector function In some embodiments, the amino acid sequence of the Fc1 monomer and / or Fc2 monomer modified to promote heterodimerization may be further modified to reduce effector function. In some embodiments, the Fc domain may be modified to substantially reduce binding to Fc receptors (FcyR and FcR), which reduces or abolishes antibody-directed cellular cytotoxicity (ADCC) effector function. Modification of the Fc domain to reduce effector function is well known in the art. See, for example, Wang, et al. (2018) IgG Fc engineering to modulate antibody effector functions, Protein Cell 9(1):63-73. For example, mutation of the lysine residue at position 235 (EU numbering) from leucine (L) to glutamic acid (E) is known to reduce effector function by reducing binding of FcgR and C1q. Alegre, et al. (1992) J. Immunology 148:3461-3468.

[0177] Additionally, substitution of two leucine (L) residues at positions 234 and 235 (EU numbering) in the IgG1 hinge region with alanine (A), i.e., L234A and L235A, results in reduced complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC). Hezereh et al., (2001) J. Virol 75(24):12161-68. Furthermore, mutation of the proline at position 329 (EU numbering) to alanine (P329A) or glycine (P329G) attenuates effector function and may be combined with the L234A and L235A substitutions. In some embodiments, the Fc domains (Fc1 and Fc2) of the compositions of the present disclosure may contain amino acid substitutions L234A / L235A / P329A (EU numbering), referred to as "LALAPA" substitutions, or L234A / L235A / P329G (EU numbering), referred to as "LALAPG" substitutions. In some embodiments, the Fc domains (Fc1 and Fc2) of the compositions of the present disclosure may contain amino acid substitutions E233P / L234V / L235A / ΔG237, referred to in the scientific literature as PVAdelG mutations.

[0178] In some embodiments, the Fc domains (Fc1 and Fc2) of the compositions of the present disclosure are derived from hIgG4. In such instances where the Fc domain of the heterodimeric Fc is derived from hIgG4, attenuation of effector function can be achieved by introduction of S228P and / or L235E mutations (EU numbering).

[0179] Examples of paired KiH Fc dimer constructs that can be incorporated into the Fc of this disclosure are provided in the table below.

[0180] (Table) Set of amino acid substitutions of complementary IgG1 KiH UH / Fc heterodimer pairs containing mutations to reduce effector function. TIFF2024504923000035.tif181167

[0181] Sequence modifications to increase duration of action: In some embodiments, the amino acid sequence of the Fc1 monomer and / or Fc2 monomer modified to promote heterodimerization can be further modified to incorporate amino acid substitutions that extend the duration of action of the molecule and prevent clearance. In some embodiments, such modifications to the Fc monomer include amino acid substitutions M428L and N434S (EU numbering), referred to as "LS" modifications. The LS modifications can optionally be combined with amino acid substitutions to reduce effector function and provide a disulfide bond between Fc1 and Fc2. The following table provides exemplary Fc1 and Fc1 heterodimer pairs with complementary sequence modifications to promote heterodimerization that can be used in designing the Fc1 and Fc2 polypeptides of formulas [1] and [2].

[0182] The following table provides exemplary Fc heterodimer pairs that may be used in preparing the Fc1 and Fc2 polypeptides of the heterodimeric Fc of the disclosure.

[0183] (Table) Set of amino acid substitutions for complementary IgG1 KiH UH / Fc heterodimer pairs containing mutations for reduced effector function and extended LS half-life. TIFF2024504923000036.tif185170

[0184] In some embodiments, the Fc domains (Fc1 and Fc2) of the compositions of the present disclosure are derived from hIgG4. In such examples where the Fc domains of the heterodimeric Fc are derived from hIgG4, the heterodimerization of the Fc1 and Fc2 domains is by introduction of the mutations K370E, K409W, and E357N, D399V, F405T (EU numbering) in the complementary Fc sequence comprising the heterodimeric Fc domain.

[0185] Modification to remove glycosylation sites In some embodiments, the amino acid sequence of the Fc1 monomer and / or Fc2 monomer modified to promote heterodimerization may be further modified to eliminate N-linked or O-linked glycosylation sites. Aglycosylation variants of the Fc domain, particularly of the IgG1 subclass, are known to be poor mediators of effector function. Jefferies et al. 1998, Immol. Rev., vol. 163, 50-76. Glycosylation at position 297 (EU numbering) has been shown to contribute to effector function. Edelman, et al (1969) PNAS (USA) 63:78-85. In some embodiments, the Fc domain of the composition of the present disclosure comprises one or more modifications to eliminate N-linked or O-linked glycosylation sites. Examples of modifications at N297 to eliminate glycosylation sites in the Fc domain include the amino acid substitutions N297Q and N297G.

[0186] In some embodiments, when the binding proteins described herein are to be administered in the form of an Fc domain fusion, particularly in situations where the polypeptides conjugated to each Fc polypeptide of the Fc domain dimer are different, the Fc domain may be engineered to have a "knob-into-holes modification." Knob-into-hole modifications are more fully described in Ridgway, et al. (1996) Protein Engineering 9(7):617-621 and U.S. Patent No. 5,731,168, issued March 24, 1998. Knob-into-hole modification refers to a modification at the interface between two immunoglobulin heavy chains in the CH3 domain, where: i) in the CH3 domain of the first heavy chain, an amino acid residue is replaced with an amino acid residue having a larger side chain (e.g., tyrosine or tryptophan) to create a protrusion ("knob") from the surface, and ii) in the CH3 domain of the second heavy chain, an amino acid residue is replaced with an amino acid residue having a smaller side chain (e.g., alanine or threonine), thereby generating a cavity ("hole") at the interface in the second CH3 domain, in which the protruding side chain ("knob") of the first CH3 domain is received by the cavity in the second CH3 domain. In one embodiment, the "knob-into-hole modification" comprises the amino acid substitution T366W and optionally the amino acid substitution S354C in one of the antibody heavy chains, and the amino acid substitutions T366S, L368A, Y407V, and optionally Y349C in the other antibody heavy chain. Additionally, the Fc domain may be modified by the introduction of cysteine ​​residues at positions S354 and Y349, which results in stabilizing disulfide bridges between the two antibody heavy chains in the Fc region (Carter, et al. (2001) Immunol Methods 248, 7-15). The knob-into-hole format involves the insertion of a first polypeptide (e.g., a first V domain in a binding protein described herein) on a first Fc polypeptide having a "knob" modification. H H), and a second polypeptide on a second Fc polypeptide having a "hole" modification (e.g., a second V in a binding protein described herein HH) to facilitate expression of the heterodimeric polypeptide conjugate.

[0187] In some embodiments, the binding proteins described herein can have a format as shown in Figures 1A-1D. In one example, the binding proteins include an anti-IL10Rα V H H antibody, and C-terminal anti-IL2Rγ V H A first binding protein comprising an H antibody (e.g., N-terminal-anti-IL10Rα V H H antibody-linker-anti-IL2Rγ V H H antibody-C-terminus) can be fused to a first Fc polypeptide, and an anti-IL2Rγ V H H antibody, and anti-IL10Rα V H A second binding protein comprising an H antibody (e.g., N-terminal-anti-IL2Rγ V H H antibody-linker-anti-IL10Rα V H A first Fc polypeptide and a second Fc polypeptide can be fused to the second Fc polypeptide (FIG. 1A), where the first Fc polypeptide and the second Fc polypeptide form an Fc domain. In this example, one Fc polypeptide can contain T366W as a knob mutation and the other Fc polypeptide can contain T366S, L368A, Y407V as hole mutations to promote Fc heterodimer formation.

[0188] In another example, two identical binding proteins can each be conjugated to an Fc polypeptide. The two identical binding protein-Fc polypeptide conjugates can then dimerize to form a homodimer (FIG. 1B). In some embodiments, both binding proteins contain an anti-IL10Rα V H H antibody, and C-terminal anti-IL2Rγ V H H antibody (e.g., N-terminal-anti-IL10Rα V H H antibody-linker-anti-IL2Rγ V HIn another embodiment, both binding proteins have an anti-IL2Rγ VH antibody at the N-terminus. H H antibody, and anti-IL10Rα V H H antibodies (e.g., N-terminal-anti-IL2Rγ V H H antibody-linker-anti-IL10Rα V H H antibody-C terminus).

[0189] In yet another example, the binding protein can be conjugated to one of two Fc polypeptides at the Fc domain (FIGS. 1C and 1D). In this case, one Fc polypeptide can contain T366W as a knob mutation and the other Fc polypeptide can contain T366S, L368A, Y407V as hole mutations to promote heterodimer formation. The binding protein can contain anti-IL10Rα V at the N-terminus. H H antibody, and C-terminal anti-IL2Rγ V H H antibody (e.g., N-terminal-anti-IL10Rα V H H antibody-linker-anti-IL2Rγ V H In other embodiments, the binding protein comprises an anti-IL2Rγ VH antibody at the N-terminus. H H antibody, and anti-IL10Rα V H H antibodies (e.g., N-terminal-anti-IL2Rγ V H H antibody-linker-anti-IL10Rα V H H antibody-C terminus).

[0190] In some embodiments, the binding protein can be conjugated to one or more water-soluble polymers, optionally including an intervening linker. Examples of water-soluble polymers useful in the practice of the present disclosure include polyethylene glycol (PEG), poly-propylene glycol (PPG), polysaccharides (polyvinylpyrrolidone, copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyols), polyolefin alcohols), polysaccharides), poly-alpha-hydroxy acids), polyvinyl alcohols (PVA), polyphosphazenes, polyoxazolines (POZ), poly(N-acryloylmorpholines), or combinations thereof.

[0191] In some embodiments, the binding protein can be conjugated or "PEGylated" to one or more polyethylene glycol molecules. The method or site of PEG attachment to the binding protein can vary, but in certain embodiments, PEGylation does not alter or only minimally alters the activity of the binding protein. As reviewed in Dozier, JK and Distefano, MD (2015) "Site Specific Pegylation of Therapeutic Proteins" International Journal of Molecular Science 16(10):25832-25864, a variety of techniques are available for site-specific incorporation of PEG moieties.

[0192] In some embodiments, selective PEGylation of a binding protein may be used, for example, by incorporation of unnatural amino acids with side chains that facilitate selective PEG conjugation. A specific PEGylation site can be selected such that PEGylation of the binding protein does not affect its binding to the target receptor.

[0193] In certain embodiments, the increase in half-life is greater than any decrease in biological activity. PEG suitable for conjugation to polypeptide sequences is generally water soluble at room temperature and has the general formula R(O-CH2-CH2) n The PEG has the formula OR, where R is hydrogen or a protecting group such as an alkyl or alkanol group, and n is an integer from 1 to 1000. When R is a protecting group, it generally has 1 to 8 carbons. The PEG conjugated to the polypeptide sequence can be linear or branched. Branched PEG derivatives, "star PEGs," and multi-armed PEGs are contemplated by the present disclosure.

[0194] The molecular weight of PEG used in the present disclosure is not limited to any particular range. The PEG component of the binding protein can have a molecular mass of greater than about 5 kDa, greater than about 10 kDa, greater than about 15 kDa, greater than about 20 kDa, greater than about 30 kDa, greater than about 40 kDa, or greater than about 50 kDa. In some embodiments, the molecular mass is about 5 kDa to about 10 kDa, about 5 kDa to about 15 kDa, about 5 kDa to about 20 kDa, about 10 kDa to about 15 kDa, about 10 kDa to about 20 kDa, about 10 kDa to about 25 kDa, or about 10 kDa to about 30 kDa. The linear or branched PEG molecule has a molecular weight of about 2,000 to about 80,000 daltons, alternatively about 2,000 to about 70,000 daltons, alternatively about 5,000 to about 50,000 daltons, alternatively about 10,000 to about 50,000 daltons, alternatively about 20,000 to about 50,000 daltons, alternatively about 30,000 to about 50,000 daltons, alternatively about 20,000 to about 40,000 daltons, or alternatively about 30,000 to about 40,000 daltons. In one embodiment of the disclosure, the PEG is a 40 kD branched PEG comprising two 20 kD arms.

[0195] The present disclosure also contemplates compositions of conjugates in which PEG has different n values, and thus various different PEGs are present in specific ratios. For example, some compositions contain a mixture of conjugates with n=1, 2, 3, and 4. In some compositions, the percentage of conjugates with n=1 is 18-25%, the percentage of conjugates with n=2 is 50-66%, the percentage of conjugates with n=3 is 12-16%, and the percentage of conjugates with n=4 is up to 5%. Such compositions can be made by reaction conditions and purification methods known in the art. Chromatography can be used to separate fractions of conjugates, and then, for example, fractions containing conjugates with the desired number of PEGs attached are identified and purified to be free of unmodified protein sequences and conjugates with other numbers of PEGs attached.

[0196] PEG suitable for conjugation to polypeptide sequences is generally water soluble at room temperature and has the general formula R(O-CH2-CH2) n It has the formula OR, where R is hydrogen or a protecting group such as an alkyl or alkanol group, and n is an integer from 1 to 1000. When R is a protecting group, it generally has 1 to 8 carbons.

[0197] Two widely used first generation activated monomethoxy PEGs (mPEGs) are succinimidyl carbonate PEG (SC-PEG; see, e.g., Zalipsky, et al. (1992) Biotehnol. Appl. Biochem 15:100-114) and benzotriazole carbonate PEG (BTC-PEG; see, e.g., U.S. Pat. No. 5,650,234 to Dolence et al.), which react preferentially with lysine residues to form carbamate linkages, but are also known to react with histidine and tyrosine residues. The use of PEG-aldehyde linkers targets a single site at the N-terminus of a polypeptide through reductive amination.

[0198] PEGylation most frequently occurs at the α-amino group at the N-terminus of a polypeptide, the ε-amino group on the side chain of a lysine residue, and the imidazole group on the side chain of a histidine residue.Since most recombinant polypeptides possess a single α-amino group and multiple ε-amino groups and imidazole groups, numerous positional isomers can be generated depending on the chemical nature of the linker.General PEGylation strategies known in the art can be applied herein.

[0199] PEG can be attached to the binding proteins of the present disclosure via a terminal reactive group ("spacer") that mediates a bond between one or more free amino or carboxyl groups of the polypeptide sequence and polyethylene glycol. PEGs having a spacer that can be attached to a free amino group include N-hydroxysuccinylimide polyethylene glycol, which can be prepared by activating the succinic acid ester of polyethylene glycol with N-hydroxysuccinylimide.

[0200] In some embodiments, the PEGylation of binding protein is facilitated by the incorporation of unnatural amino acids that carry unique side chains to facilitate site-specific PEGylation.The incorporation of unnatural amino acids into polypeptides to provide functional moieties to achieve site-specific PEGylation of such polypeptides is known in the art.See, for example, PCT International Application No. PCT / US2018 / 045257 to Ptacin et al., filed August 3, 2018, and published February 7, 2019 under International Publication No. WO 2019 / 028419Al.

[0201] The PEG conjugated to the polypeptide sequence can be linear or branched. Branched PEG derivatives, "star PEGs," and multi-armed PEGs are contemplated by the present disclosure.Specific embodiment PEGs useful in the practice of the present disclosure include 10 kDa linear PEG-aldehyde (e.g., Sunbright® ME-100AL, NOF America Corporation, One North Broadway, White Plains, NY 10601 USA), 10 kDa linear PEG-NHS ester (e.g., Sunbright® ME-100CS, Sunbright® ME-100AS, Sunbright® ME-100GS, Sunbright® ME-100HS, NOF), 20 kDa linear PEG-aldehyde (e.g., Sunbright® ME-200AL, NOF), 20 kDa linear PEG-NHS ester (e.g., Sunbright® ME-200CS, Sunbright® ME-200AS, Sunbright® ME-200GS, Sunbright® ME-200HS, NOF), two 10 20 kDa 2-arm branched PEG-aldehyde, which is a 20 kDA PEG-aldehyde containing two 10 kDA linear PEG molecules (e.g., Sunbright® GL2-200AL3, NOF); 20 kDa 2-arm branched PEG-NHS ester, which is a 20 kDA PEG-NHS ester containing two 10 kDA linear PEG molecules (e.g., Sunbright® GL2-200TS, Sunbright® GL200GS2, NOF); 40 kDa 2-arm branched PEG-aldehyde, which is a 40 kDA PEG-aldehyde containing two 20 kDA linear PEG molecules (e.g., Sunbright® GL2-400AL3); 40 kDa PEG-NHS ester, which is a 40 kDA PEG-NHS ester containing two 20 kDA linear PEG molecules (e.g., Sunbright® GL2-400AL3). These include 2-arm branched PEG-NHS ester (e.g., Sunbright® GL2-400AL3, Sunbright® GL2-400GS2, NOF), linear 30 kDa PEG-aldehyde (e.g., Sunbright® ME-300AL), and linear 30 kDa PEG-NHS ester.

[0202] In some embodiments, a linker can be used to link the binding protein and the PEG molecule. Suitable linkers include "flexible linkers," which are generally long enough to allow some movement between the modified polypeptide sequence and the components and molecules to be linked. The linker molecule is generally about 6-50 atoms long. The linker molecule can also be, for example, an aryl acetylene, an ethylene glycol oligomer containing 2-10 monomer units, a diamine, a diacid, an amino acid, or a combination thereof. A suitable linker can be readily selected and can be of any suitable length, for example, 1 amino acid (e.g., Gly), 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-20, 20-30, 30-50, or more than 50 amino acids.

[0203] Examples of flexible linkers include glycine polymers (G)n, glycine-alanine polymers, alanine-serine polymers, glycine-serine polymers (e.g., (GmSo)n, (GSGGS)n, (GmSoGm)n, (GmSoGmSoGm)n, (GSGGSm)n, (GSGSmG)n, and (GGGSm)n, and combinations thereof, where m, n, and o are each independently selected from integers of at least 1-20, e.g., 1-18, 216, 3-14, 4-12, 5-10, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10), and other flexible linkers. Glycine and glycine-serine polymers are relatively unstructured and therefore can act as neutral tethers between components. Examples of flexible linkers are provided in Section V.

[0204] Additional examples of flexible linkers include glycine polymers (G)n, or glycine-serine polymers (e.g., (GS)n, (GSGGS)n, (GGGS)n, and (GGGGS)n, where n=1-50, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-20, 20-30, 30-50). Multimers (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-20, 20-30, or 30-50) of these linker sequences may be linked together to provide a flexible linker that can be used to conjugate two molecules. Alternatively to a polypeptide linker, the linker can be a chemical linker, e.g., a PEG-aldehyde linker. In some embodiments, the binding protein is acetylated at the N-terminus by enzymatic reaction with an N-terminal acetyltransferase and, for example, acetyl-CoA. Alternatively, or in addition to N-terminal acetylation, the binding protein can be acetylated at one or more lysine residues, for example, by enzymatic reaction with a lysine acetyltransferase. See, e.g., Choudhary et al. (2009) Science 325 (5942):834-840.

[0205] In other embodiments, the binding protein can be modified to include an additional polypeptide sequence that functions as an antigenic tag, such as a FLAG sequence. The FLAG sequence is recognized by a biotinylated high-specificity anti-FLAG antibody, as described herein (see, for example, Blanar et al. (1992) Science 256:1014 and LeClair, et al. (1992) PNAS-USA 89:8145). In some embodiments, the binding protein further includes a C-terminal c-myc epitope tag.

[0206] In some embodiments, the binding proteins are expressed as fusion proteins with albumin molecules (eg, human serum albumin) as known in the art to facilitate extended exposure in vivo.

[0207] In some embodiments, the binding proteins (including fusion proteins of the binding proteins) of the present disclosure are expressed as fusion proteins with one or more transition metal chelating polypeptide sequences. The incorporation of such transition metal chelating domains facilitates purification immobilized metal affinity chromatography (IMAC), as described in U.S. Patent No. 4,569,794 to Smith et al., issued February 11, 1986. Examples of transition metal chelating polypeptides useful in the practice of the present disclosure are described in Smith et al., supra, and in U.S. Patent No. 5,320,663 to Dobeli et al., issued May 10, 1995, the entire teachings of which are incorporated herein by reference. A particular transition metal chelating polypeptide useful in the practice of the present disclosure is a peptide containing 3-6 consecutive histidine residues, such as the 6-histidine peptide (His)6, frequently referred to in the art as a "His tag."

[0208] The above fusion proteins can be readily produced by recombinant DNA methods according to techniques known in the art by constructing a recombinant vector containing a nucleic acid sequence encoding the binding protein in frame with a nucleic acid sequence encoding a fusion partner at either the N-terminus or C-terminus of the binding protein, optionally further comprising a nucleic acid sequence encoding a linker or spacer polypeptide in frame.

[0209] VII. Pharmaceutical Compositions The binding protein of the present disclosure can be administered to a subject in a pharma- ceutically acceptable dosage form. The preferred formulation depends on the intended mode of administration and therapeutic application. The pharmaceutical dosage form of the binding protein described herein comprises a physiologically acceptable carrier that is essentially non-toxic and non-therapeutic. Examples of such carriers include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts, or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, and PEG. Carriers for topical or gel-based forms of polypeptides include polysaccharides, such as sodium carboxymethylcellulose or methylcellulose, polyvinylpyrrolidone, polyacrylates, polyoxyethylene-polyoxypropylene-block polymers, PEG, polymeric amino acids, amino acid copolymers, and lipid aggregates (such as oil droplets or liposomes).

[0210] The pharmaceutical composition may also include a pharma- ceutically acceptable, non-toxic carrier, excipient, stabilizer, or diluent, which are defined as vehicles commonly used to formulate pharmaceutical compositions for administration to animals or humans. The diluent is selected so as not to affect the biological activity of the combination. Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations used, and include buffers, such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives, such as octadecyidimethylbenzyl ammonium chloride; chloride); hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; 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 non-ionic surfactants, such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG).

[0211] Formulations to be used for in vivo administration are typically sterile. Sterilization of the compositions of the present disclosure can be readily accomplished by filtration through sterile filtration membranes.

[0212] Typically, the composition is prepared as an injectable, either as a liquid solution or suspension; solid forms suitable for solution or suspension in liquid vehicles before injection can also be prepared.The preparation can also be emulsified or encapsulated in liposomes or microparticles, such as polylactide, polyglycolide, or copolymers, for enhanced adjuvant effect, as discussed above (Langer, Science 249: 1527, 1990 and Hanes, Advanced Drug Delivery Reviews 28: 97-119, 1997).The agent of the present disclosure can be administered in the form of a depot injection or implant preparation, which can be formulated in such a manner that it allows sustained or pulsed release of active ingredient.The pharmaceutical composition is generally formulated as sterile, substantially isotonic, and in full compliance with all Good Manufacturing Practice (GMP) regulations of the United States Food and Drug Administration.

[0213] Administration of the binding proteins described herein may be accomplished through any of a variety of art-recognized methods, including, but not limited to, topical, intravascular injection (including intravenous or intraarterial infusion), intradermal injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, intracranial injection, intratumoral injection, intranodal injection, transdermal, transmucosal, iontophoretic delivery, intralymphatic injection (Senti and Kundig (2009) Current Opinions in Allergy and Clinical Immunology 9(6):537-543), intragastric injection, intraprostatic injection, intracapsular injection (e.g., bladder), respiratory system inhalers including nebulizers, intraocular injection, intraperitoneal injection, intralesional injection, intraovarian injection, intracerebral infusion or injection, intraventricular injection (ICVI), and the like. In some embodiments, administration includes administration of the binding protein itself (e.g., parenterally), and administration of a recombinant vector (e.g., viral or non-viral vector) to cause in situ expression of the binding protein in a subject. Alternatively, cells, such as cells isolated from a subject, can also be recombinantly modified to express a binding protein of the present disclosure.

[0214] The dosage of the pharmaceutical composition depends on factors including the route of administration, the disease to be treated, and the physical characteristics of the subject, such as age, weight, and general health. Typically, the amount of binding protein contained in a single dose can be an amount that effectively prevents, delays, or treats the disease without inducing significant toxicity. The pharmaceutical composition of the present disclosure may be administered in a concentration of 0.01 to 500 mg / kg (e.g., 0.01 to 450 mg, 0.01 to 400 mg, 0.01 to 350 mg, 0.01 to 300 mg, 0.01 to 250 mg, 0.01 to 200 mg, 0.01 to 150 mg, 0.01 to 100 mg, 0.01 to 50 mg, 0.01 to 10 mg, 0.01 to 1 mg, 0.1 to 500 mg / kg, 1 to 500 mg / kg, 5 to 500 mg / kg, 10 to 500 mg / kg, 50 to 500 mg / kg, 100 to 500 mg / kg, 150 to 500 mg / kg, 200 to 500 mg / kg, 250 to 500 mg / kg, 300 to 500 mg / kg, 350 to 500 mg / kg, 400 to 500 mg / kg, 500 to 500 mg / kg, 600 to 650 mg / kg, 700 to 750 mg / kg, 800 to 850 mg / kg, 900 to 950 mg / kg, 1000 to 1000 mg / kg, 1500 to 1000 mg / kg, 2000 to 500 mg / kg, 2500 to 500 mg / kg, 3000 to 500 mg / kg, 3500 to 500 mg / kg, 4000 to 500 mg / kg, 5000 to 500 mg / kg, 6000 to 750 mg / kg, 7000 to mg / kg, 400 to 500 mg / kg, or 450 to 500 mg / kg), and in a more specific embodiment, about 1 to about 100 mg / kg (e.g., about 1 to about 90 mg / kg, about 1 to about 80 mg / kg, about 1 to about 70 mg / kg, about 1 to about 60 mg / kg, about 1 to about 50 mg / kg, about 1 to about 40 mg / kg, about 1 to about 30 mg / kg, about 1 to about 20 mg / kg, about 1 to about 10 mg / kg, about 10 to about 100 mg / kg, about 20 to about 100 mg / kg, about 30 to about 100 mg / kg, about 40 to about 100 mg / kg, about 50 to about 100 mg / kg, about 60 to about 100 mg / kg, about 70 to about 100 mg / kg, about 80 to about 100 mg / kg, or about 90 to about 100 mg / kg). The dosages may include doses of the binding proteins described herein in the range of 1 mg / kg.In some embodiments, the pharmaceutical composition of the present disclosure may comprise a dosage of the binding protein described herein in the range of 0.01-20 mg / kg (e.g., 0.01-15 mg / kg, 0.01-10 mg / kg, 0.01-8 mg / kg, 0.01-6 mg / kg, 0.01-4 mg / kg, 0.01-2 mg / kg, 0.01-1 mg / kg, 0.01-0.1 mg / kg, 0.01-0.05 mg / kg, 0.05-20 mg / kg, 0.1-20 mg / kg, 1-20 mg / kg, 2-20 mg / kg, 4-20 mg / kg, 6-20 mg / kg, 8-20 mg / kg, 10-20 mg / kg, 15-20 mg / kg). The dosage may be adapted by the physician according to conventional factors such as the extent of the disease and various parameters of the subject.

[0215] A pharmaceutical composition containing a binding protein described herein can be administered to a subject in need thereof, for example, one or more times (e.g., 1-10 or more times) daily, weekly, monthly, twice yearly, annually, or as medically necessary. Dosages can be provided in either single or multiple dosing regimens. The timing between doses may decrease as the medical condition improves, or increase as the patient's health declines. The course of treatment may be a single dose, or multiple doses over a period of time. In some embodiments, a single dose is used. In some embodiments, two or more split doses are used, administered over a period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 21, 28, 30, 60, 90, 120, or 180 days. Each dose administered in such split dosing protocols may be the same or different in each administration. A multi-day administration protocol over a period of time can be provided by a person skilled in the art (e.g., a physician) who monitors administration, taking into account the subject's response to treatment, including adverse effects of treatment and its adjustment, as discussed above.In some embodiments, the serum trough concentration of the binding molecule is maintained above a threshold level corresponding to about 0.1 pg / ml, alternatively 0.1 ng / ml, alternatively about 0.5 ng / ml, alternatively 1 ng / ml, alternatively 2 ng / ml, for at least 80%, alternatively at least 85%, alternatively at least 90%, alternatively at least 95% of a period of at least 24 hours, alternatively 48 hours, alternatively 72 hours, alternatively 1 week, alternatively 1 month.See, for example, US Patent Application Publication US2016 / 0193300A1, published July 7, 2016, by Mumm et al.

[0216] VIII. Method of Use Neoplastic disease The present disclosure provides a method of use of a binding protein that binds IL10Rα and IL2Rγ in treating a subject suffering from a neoplastic disease, disorder, or condition by administering a therapeutically effective amount of the binding protein (or a nucleic acid encoding the binding protein, including a recombinant vector encoding the binding protein) as described herein.IL10 agonists have been identified as useful in the treatment of neoplastic diseases, as described in Oft, M. (2014) Cancer Immunology Research 2(3):194-199;Naing, et al. (2108) Cancer Cell 34(5):775-791;and Mumm, J. and Oft, M (2013) Bioessays 35(7):623-631.

[0217] The compositions and methods of the present disclosure are useful in treating subjects suffering from neoplastic diseases characterized by the presence of a neoplasm, including benign and malignant neoplasms, as well as neoplastic diseases. In certain embodiments, the methods do not cause anemia.

[0218] Examples of benign neoplasms suitable for treatment using the compositions and methods of the present disclosure include, but are not limited to, adenoma, fibroma, hemangioma, and lipoma. Examples of premalignant neoplasms suitable for treatment using the compositions and methods of the present disclosure include, but are not limited to, hyperplasia, atypia, metaplasia, and dysplasia. Examples of malignant neoplasms suitable for treatment using the compositions and methods of the present disclosure include, but are not limited to, carcinomas (cancers arising from epithelial tissues such as skin or tissues lining internal organs), leukemia, lymphoma, and sarcomas that typically originate from bone, fat, muscle, blood vessels, or connective tissue. The term neoplasm also includes virus-induced neoplasms such as warts and EBV-induced diseases (i.e., infectious mononucleosis), scar formation, hyperproliferative vascular diseases (including intimal smooth muscle cell hyperplasia, restenosis, and vascular occlusion), and the like.

[0219] The term "neoplastic disease" includes cancers characterized by solid and non-solid tumors, including, but not limited to, breast cancer, sarcoma (including, but not limited to, osteosarcoma and angiosarcoma and fibrosarcoma), leukemia, lymphoma, genitourinary cancer (including, but not limited to, ovarian, urethral, ​​bladder, and prostate cancer), gastrointestinal cancer (including, but not limited to, colon, esophageal, and gastric cancer), lung cancer, myeloma, pancreatic cancer, liver cancer, kidney cancer, endocrine cancer, skin cancer, and malignant or benign brain or central nervous system (CNS) and peripheral nervous system tumors, including gliomas and neuroblastomas, astrocytomas, myelodysplastic disorders, cervical intraepithelial neoplasia, intestinal polyposis, oral leukoplakia, histiocytosis, hyperproliferative scars including keloid scars, hemangiomas, hyperproliferative arterial stenosis, psoriasis, inflammatory arthritis, hyperkeratosis, and papular-squamous rashes including arthritis.

[0220] The term "neoplastic disease" includes carcinomas. The term "carcinoma" refers to malignant tumors of epithelial or endocrine tissues, including cancers of the respiratory system, digestive system, genitourinary system, testicular cancer, breast cancer, prostate cancer, endocrine system cancer, and melanoma. The term neoplastic disease includes adenocarcinomas. "Adenocarcinoma" refers to carcinomas that originate from glandular tissue or in which the tumor cells form recognizable glandular structures.

[0221] As used herein, the term "hematopoietic neoplastic disorder" refers to a neoplastic disease involving hyperplastic / neoplastic cells of hematopoietic origin, e.g., arising from the myeloid, lymphoid, or erythroid lineages, or precursor cells thereof.

[0222] Myeloid neoplasms include, but are not limited to, myeloproliferative neoplasms, myeloid and lymphoid disorders with eosinophilia, myeloproliferative / myelodysplastic neoplasms, myelodysplastic syndromes, acute myeloid leukemia and related precursor neoplasms, and acute leukemia of ill-defined lineage.Exemplary myeloid disorders suitable for treatment according to the present disclosure include, but are not limited to, acute promyelocytic leukemia (APML), acute myeloid leukemia (AML), and chronic myeloid leukemia (CML).

[0223] Lymphoid neoplasms include, but are not limited to, precursor lymphoid neoplasms, mature B-cell neoplasms, mature T-cell neoplasms, Hodgkin's lymphoma, and immunodeficiency-associated lymphoproliferative disorders.Exemplary lymphoid disorders suitable for treatment according to the present disclosure include, but are not limited to, acute lymphoblastic leukemia (ALL), including B-lineage acute lymphoblastic leukemia (ALL) and T-lineage ALL, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), hairy cell leukemia (HLL), and Waldenstrom's microglobulinemia (WM).

[0224] In some cases, hematopoietic neoplastic disorders arise from poorly differentiated acute leukemias (e.g., erythroblastic leukemia and acute megakaryoblastic leukemia). As used herein, the term "hematopoietic neoplastic disorder" refers to malignant lymphomas, including, but not limited to, non-Hodgkin's lymphoma and its variants, peripheral T-cell lymphoma, adult T-cell leukemia / lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), large granular lymphocytic leukemia (LGF), Hodgkin's disease, and Reed-Stenberg disease.

[0225] A determination of whether a subject is "suffering from a neoplastic disease" refers to a determination made by a physician regarding a subject based on available information accepted in the art for identifying a disease, disorder, or condition, including, but not limited to, x-rays, CT scans, conventional laboratory diagnostic tests (e.g., blood counts, etc.), genomic data, protein expression data, immunohistochemistry, that the subject requires or would benefit from treatment.

[0226] Assessment of Anti-Neoplastic Efficacy The determination of the effectiveness of the disclosed methods in the treatment of cancer generally relates to the achievement of one or more art-recognized parameters, such as reduction in lesions, particularly metastatic lesions, reduction in metastasis, reduction in tumor volume, improvement in ECOG score, etc. The determination of response to treatment can be evaluated through the measurement of biomarkers that can provide reproducible information useful in any aspect of binding protein therapy, including the presence and degree of subject response to such therapy, and the presence and degree of inappropriate effects caused by such therapy. By way of example and not by way of limitation, biomarkers include enhancement of IFNγ, and upregulation of granzyme A, granzyme B, and perforin; CD8 + Increased T cell numbers and function; enhanced IFNγ, CD8 + Increased ICOS expression on T cells, IL-10-expressing T Reg The response to treatment may be characterized by an improvement in conventional measures of clinical efficacy, such as complete response (CR), partial response (PR), stable disease (SD), etc., and with respect to target lesions, complete response (CR), incomplete response / stable disease (SD) as defined by RECIST, and immune-related complete response (irCR), immune-related partial response (irPR), and immune-related stable disease (irSD) as defined by immune-related response criteria (irRC) are considered by those skilled in the art to demonstrate efficacy in treating neoplastic disease in mammalian (e.g., human) subjects.

[0227] Further embodiments include methods or models for determining the optimal amount of agents in combination. The optimal amount can be, for example, an amount that achieves optimal effect in a subject or subject population, or an amount that achieves a therapeutic effect while minimizing or eliminating adverse effects associated with one or more of the agents. In some embodiments, the method includes a combination of a binding protein as described herein with a cofactor that is known or determined to be effective in treating or preventing a disease, disorder, or condition (e.g., a cancerous condition) as described herein in a subject (e.g., a human) or subject population, and the amount of one agent is titrated while the amount of the other agent is held constant. By manipulating the amount of agents in this way, a clinician can determine the most effective ratio of agents, for example, to treat a particular disease, disorder, or condition, or to eliminate adverse effects, or to reduce adverse effects as tolerated under the circumstances.

[0228] Combination of Binding Proteins with Adjunctive Therapeutic Agents The present disclosure provides for the use of the binding proteins of the present disclosure in combination with one or more additional active agents ("cofactors"). Such further combinations are interchangeably referred to as "co-combinations" or "co-therapy", and those therapeutic agents used in combination with the binding proteins of the present disclosure are referred to as "cofactors". As used herein, the term "cofactors" includes agents that can be administered or introduced separately, for example, formulated separately for separate administration (e.g., can be provided in a kit), and / or therapeutic agents that can be administered or introduced in combination with the binding proteins.

[0229] As used herein, the term "in combination with" when used in reference to administering multiple agents to a subject refers to administering a first agent and at least one additional (i.e., second, third, fourth, fifth, etc.) agent to a subject. For purposes of the present invention, an agent (e.g., a binding protein described herein) is considered to be administered in combination with a second agent (e.g., a regulator of an immune checkpoint pathway) if the biological effect resulting from administration of the first agent persists in the subject upon administration of the second agent, such that the therapeutic effects of the first and second agents overlap. For example, PD1 immune checkpoint inhibitors (e.g., nivolumab or pembrolizumab) are typically administered by IV infusion every 2 or 3 weeks, while the binding proteins of the present disclosure are typically administered more frequently, e.g., daily, BID, or weekly. However, administration of a first agent (e.g., pembrolizumab) provides a therapeutic effect over an extended period of time, and administration of a second agent (e.g., a binding protein described herein) provides its therapeutic effect while the therapeutic effect of the first agent continues, and thus, even if the first agent is administered at a time significantly separated (e.g., days or weeks) from the time of administration of the second agent, the second agent is considered to be administered in combination with the first agent. In one embodiment, an agent is considered to be administered in combination with a second agent if the first and second agents are administered in parallel (within 30 minutes of each other), simultaneously, or sequentially. In some embodiments, a first agent is considered to be administered "concurrently" with a second agent if the first and second agents are administered within about 24 hours of each other, preferably within about 12 hours of each other, preferably within about 6 hours of each other, preferably within about 2 hours of each other, or preferably within about 30 minutes of each other. The term "in combination with" is also understood to apply to the situation where the first agent and the second agent are co-formulated in a single pharma- ceutically acceptable formulation, and the co-formulation is administered to a subject. In certain embodiments, the binding protein and the cofactor are administered or applied sequentially, e.g., one agent is administered before one or more other agents.In other embodiments, the binding protein and the cofactor are administered in parallel, e.g., two or more agents are administered at or about the same time; the two or more agents may be in two or more separate formulations or may be combined in a single formulation (i.e., a co-formulation). Regardless of whether the agents are administered sequentially or in parallel, they are considered to be administered in combination for the purposes of this disclosure.

[0230] Chemotherapeutic agents In some embodiments, the cofactor is a chemotherapeutic agent. In some embodiments, the cofactor is a "cocktail" of multiple chemotherapeutic agents. The use of IL-10 agents in combination with chemotherapeutic agents is described in U.S. Patent No. 9,833,514 B2, issued December 5, 2017 to Oft et al., the teachings of which are incorporated herein by reference. In some embodiments, the chemotherapeutic agent or cocktail is administered in combination with one or more physical methods (e.g., radiation therapy). The term "chemotherapeutic agent" includes alkylating agents such as thiotepa and cyclophosphamide, alkylsulfonates such as busulfan, improsulfan, and piposulfan, aziridines such as benzodopa, carboquone, meturedopa, and uredopa, altretamine, ethylenimines and methylamelamines including triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine, nitrogen mustards such as chiorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembich in), phenesterine, prednimustine, trofosfamide, uracil mustard, nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine, antibiotics such as aclacinomycin, actinomycin, autramycin, azaserine, bleomycins such as bleomycin A2, cactinomycin, caliche mycin, carabicin, caminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin and derivatives such as demethoxy-daunomycin, 11-deoxydaunorubicin, 13-deoxydaunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin,Mitomycins such as mitomycin C, N-methylmitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin, antimetabolites such as methotrexate and 5-fluorouracil (5-FU), folic acid analogues such as denopterin, methotrexate, Pteropterin, trimetrexate, dideazatetrahydrofolic acid, and folinic acid, purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine, pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU, androgens such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone, anti-adrenal drugs such as For example, aminoglutethimide, mitotane, trilostane, folic acid supplements such as floric acid, aceglatone, aldophosphamide glycosides, aminolevulinic acid, amsacrine, bestravcil, bisantrene, edatraxate, defofamine, demecolcine, diaziquone, elformithine, elliptinium acetate, etoglucide, gallium nitrate, hydroxyurea, lentinan, lonidamine, mitoguazone, mitoxantrone, mopidamol, nitracrine, pentostatin phenameth, pirarubicin, podophyllic acid, 2-ethylhydrazide, procarbazine, razoxane, schizophyllan, spirogermanium, tenuazonic acid, triaziquone, 2,2',2''-trichlorotriethylamine, urethane, vindesine, dacarbazine, mannomustine, mitobronitol, mitolactol, pipobroman, gacytosine, arabinoside (Ara-C), cyclophosphamide, thiotepa, taxoids such as paclitaxel, nab-paclitaxel, and doxetaxel, chlorambucil,Gemcitabine, 6-thioguanine, mercaptopurine, methotrexate, platinum and platinum coordination compounds such as cisplatin, oxaplatin, and carboplatin, vinblastine, etoposide (VP-16), ifosfamide, mitomycin C, mitoxantrone, vincristine, vinorelbine, navelbine, novantrone, teniposide, daunomycin, aminopterin, xeloda, ibandronate, CPT11, topoisomerase inhibitors, dif Examples of suitable anti-inflammatory agents include, but are not limited to, difluoromethylornithine (DMFO), retinoic acid, esperamicin, capecitabine, taxanes such as paclitaxel, docetaxel, cabazitaxel, carminomycin, adriamycins such as 4'-epidadriamycin, 4-adriamycin-14-benzoate, adriamycin-14-octanoate, adriamycin-14-naphthalene acetate, colchicine, as well as pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0231] The term "chemotherapeutic agent" also includes anti-hormonal agents that act to control or inhibit hormone action on tumors, such as anti-estrogens, including, for example, tamoxifen, raloxifene, aromatase-inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxyphene, ketoxifene, onapristone, and toremifene; and anti-androgens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0232] In some embodiments, the cofactor is a cytokine or cytokine antagonist, such as IL12, INFα, or anti-epidermal growth factor receptor, irinotecan; a tetrahydrofolate antimetabolite, such as pemetrexed; an antibody against a tumor antigen, a monoclonal antibody-toxin conjugate, a T cell adjuvant, bone marrow transplant, or an antigen presenting cell (e.g., dendritic cell therapy), as practiced in known chemotherapy treatment regimens, including, but not limited to, TAC, FOLFOX, TPC, FEC, ADE, FOLFOX-6, EPOCH, CHOP, CMF, CVP, BEP, OFF, FLOX, CVD, TC, FOLFIRI, PCV, FOLFOXIRI, ICE-V, XELOX, and others readily recognized by a skilled clinician in the art. ), anti-tumor vaccines, replication competent viruses, signal transduction inhibitors (e.g., Gleevec® or Herceptin®) or immunomodulators to achieve additive or synergistic suppression of tumor growth, nonsteroidal anti-inflammatory drugs (NSAIDs), cyclooxygenase-2 (COX-2) inhibitors, steroids, TNF antagonists (e.g., Remicade® and Enbrel®), interferon-β1a (Avonex®), and interferon-β1b (Betaseron®), as well as combinations of one or more of the foregoing.

[0233] In some embodiments, the binding protein is administered in combination with a BRAF / MEK inhibitor, a kinase inhibitor such as sunitinib, a PARP inhibitor such as olaparib, an EGFR inhibitor such as osimertinib (Ahn, et al. (2016) J Thorac Oncol 11:S115), an IDO inhibitor such as epacadostat, and an oncolytic virus such as talimogene laherparepvec (T-VEC).

[0234] Combination with therapeutic antibodies In some embodiments, the "cofactor" is a therapeutic antibody (including bispecific and trispecific antibodies that bind to one or more tumor-associated antigens, including but not limited to bispecific T cell engagers (BITEs), dual affinity retargeting (DART) constructs, and trispecific killer engager (TriKE) constructs). The use of IL10 agents in combination with therapeutic antibodies in the treatment of neoplastic diseases is described in U.S. Patent No. 10,618,970 B2, issued April 14, 2020 to Mumm et al.

[0235] In some embodiments, the therapeutic antibody is selected from the group consisting of HER2 (e.g., trastuzumab, pertuzumab, ado-trastuzumab emtansine), nectin-4 (e.g., enfortumab), CD79 (e.g., polatuzumab vedotin), CTLA4 (e.g., ipilumumab), CD22 (e.g., moxetumomab pasudotox), CCR4 (e.g., magamuizumab), IL23p19 (e.g., tildrakizumab), PDL1 (e.g., durvalumab, avelumab, atezolizumab), IL17a (e.g., ixekizumab) , CD38 (e.g., daratumumab), SLAMF7 (e.g., elotuzumab), CD20 (e.g., rituximab, tositumomab, ibritumomab, and ofatumumab), CD30 (e.g., brentuximab vedotin), CD33 (e.g., gemtuzumab ozogamicin), CD52 (e.g., alemtuzumab), EpCam, CEA, fpA33, TAG-72, CAIX, PSMA, PSA, folate binding protein, GD2 (e.g., dinuntuximab), GD3, IL6 (e.g., silutxumab), GM2, Le y, VEGF (e.g., bevacizumab), VEGFR, VEGFR2 (e.g., ramucirumab), PDGFRα (e.g., olartumumab), EGFR (e.g., cetuximab, panitumumab, and necitumumab), ERBB2 (e.g., trastuzumab), ERBB3, MET, IGF1R, EPHA3, TRAIL R1, TRAIL R2, RANKL RAP, tenascin, integrin αVβ3, and integrin α4β1.

[0236] Examples of antibody therapeutics that have been approved by the FDA and can be used as cofactors for use in the treatment of neoplastic disease indications include those provided in Table 5 below.

[0237] Table 5. FDA Anti-Neoplastic Disease Antibodies and Indications TIFF2024504923000037.tif234163TIFF2024504923000038.tif219163

[0238] In some embodiments, the antibody is a bispecific antibody targeting a first tumor antigen and a second tumor antigen such as HER2 and HER3 (abbreviated as HER2×HER3), FAP×DR-5 bispecific antibody, CEA×CD3 bispecific antibody, CD20×CD3 bispecific antibody, EGFR-EDV-miR16 trispecific antibody, gp100×CD3 bispecific antibody, Ny-eso×CD3 bispecific antibody, EGFR×cMet bispecific antibody, BCMA×CD3 bispecific antibody, EGFR-EDV bispecific antibody, CLEC12A×CD3 bispecific antibody, HER2×HER3 bispecific antibody, Lgr5×EGFR bispecific antibody, PD1×CTLA-4 bispecific antibody, CD123×CD3 bispecific antibody, gpA33×CD3 bispecific antibody, B7-H3×CD3 bispecific antibody, LAG-3×PD1 bispecific antibody, DLL4×VEGF bispecific antibody, Cadherin-P x CD3 bispecific antibody, BCMA x CD3 bispecific antibody, DLL4 x VEGF bispecific antibody, CD20 x CD3 bispecific antibody, Ang-2 x VEGF-A bispecific antibody, CD20 x CD3 bispecific antibody, CD123 x CD3 bispecific antibody, SSTR2 x CD3 bispecific antibody, PD1 x CTLA-4 bispecific antibody, HER2 x HER2 bispecific antibody, GPC3 x CD3 bispecific antibody, PSMA x CD3 bispecific antibody, LAG-3 x PD-L1 bispecific antibody, CD38 x CD3 bispecific antibody, HER2 x CD3 bispecific antibody, GD2 x CD3 bispecific antibody, and CD33 x CD3 bispecific antibody.

[0239] Such therapeutic antibodies may be further conjugated to one or more chemotherapeutic agents (e.g., antibody drug conjugates or ADCs), either directly or through a linker, particularly an acid-, base-, or enzymatically labile linker.

[0240] Combination with physical methods In some embodiments, the cofactor is one or more non-pharmacological modalities (e.g., local or total body radiation therapy or surgery). By way of example, the present disclosure contemplates a treatment regimen in which a radiation step precedes or follows treatment with a treatment regimen that includes a binding protein and one or more cofactors. In some embodiments, the present disclosure further contemplates the use of the binding protein in combination with surgery (e.g., tumor resection). In some embodiments, the present disclosure further contemplates the use of the binding protein in combination with bone marrow transplantation, peripheral blood stem cell transplantation, or other types of transplantation therapy.

[0241] Combination with immune checkpoint modulators: In some embodiments, the "cofactor" is an immune checkpoint regulator for the treatment and / or prevention of neoplastic disease in a subject, as well as diseases, disorders, or conditions associated with neoplastic disease. The use of IL10 agents in combination with immune checkpoint regulators in the treatment of neoplastic disease is described in U.S. Patent Publication US2020 / 0353050 to Oft, published November 12, 2020. The term "immune checkpoint pathway" refers to a biological response elicited by the binding of a first molecule (e.g., a protein such as PD1) expressed on an antigen-presenting cell (APC) to a second molecule (e.g., a protein such as PDL1) expressed on an immune cell (e.g., T cell) that regulates the immune response through either stimulation (e.g., upregulation of T cell activity) or inhibition (e.g., downregulation of T cell activity). Molecules involved in the formation of a binding pair that regulates the immune response are commonly referred to as "immune checkpoints." The biological responses regulated by such immune checkpoint pathways are mediated by intracellular signaling pathways that lead to downstream immune effector pathways, such as cell activation, cytokine production, cell migration, secretion of cytotoxic factors, and antibody production. Immune checkpoint pathways are generally triggered by the binding of a first cell surface expression molecule to a second cell surface molecule associated with an immune checkpoint pathway (e.g., PD1 binding to PDL1, CTLA4 binding to CD28, etc.). Activation of immune checkpoint pathways can result in the stimulation or inhibition of immune responses.

[0242] The immune checkpoint whose activation results in the inhibition or downregulation of immune response is referred to herein as "negative immune checkpoint pathway regulator". The inhibition of immune response caused by the activation of negative immune checkpoint regulator reduces the host immune system's ability to recognize foreign antigens, such as tumor-associated antigens. The term negative immune checkpoint pathway includes, but is not limited to, the biological pathway regulated by the binding of PD1 to PDL1, PD1 to PDL2, and CTLA4 to CDCD80 / 86. Examples of such negative immune checkpoint antagonists include, but are not limited to, antagonists (e.g., antagonist antibodies) that bind to T cell inhibitory receptors, including, but not limited to, PD1 (also known as CD279), TIM3 (T cell membrane protein 3; also known as HAVcr2), BTLA (B and T lymphocyte attenuator; also known as CD272), VISTA (B7-H5) receptor, LAG3 (lymphocyte activation gene 3; also known as CD233), and CTLA4 (cytotoxic T lymphocyte-associated antigen 4; also known as CD152).

[0243] In one embodiment, the immune checkpoint pathway whose activation results in the stimulation of immune response is referred to herein as "positive immune checkpoint pathway regulator".The term positive immune checkpoint pathway regulator includes, but is not limited to, the biological pathway regulated by the binding of ICOSL to ICOS (CD278), B7-H6 to NKp30, CD155 to CD96, OX40L to OX40, CD70 to CD27, CD40 to CD40L, and GITRL to GITR.Molecules that agonize positive immune checkpoints (such as the natural or synthetic ligands of the components of the binding pair that stimulate immune response) are useful for upregulating immune response. Examples of such positive immune checkpoint agonists include, but are not limited to, agonist antibodies that bind to T cell activation receptors such as ICOS (such as JTX-2011, Jounce Therapeutics), OX40 (such as MEDI6383, Medinum), CD27 (such as varlilumab, Celldex Therapeutics), CD40 (such as dacetuzmumab CP-870,893, Roche, Chi Lob 7 / 4), HVEM, CD28, CD137 4-1BB, CD226, and GITR (such as MEDI1873, Medinum; INCAGN1876, Agenus).

[0244] As used herein, the term "immune checkpoint pathway regulator" refers to a molecule that inhibits or stimulates the activity of immune checkpoint pathway in a biological system, including an immune-competent mammal. An immune checkpoint pathway regulator may exert its effect by binding to immune checkpoint proteins (such as immune checkpoint proteins expressed on the surface of antigen-presenting cells (APCs) and / or immune T effector cells, such as cancer cells), or may exert its effect on upstream and / or downstream responses in immune checkpoint pathways. For example, an immune checkpoint pathway regulator may regulate the activity of SHP2, a tyrosine phosphatase involved in PD-1 and CTLA-4 signaling. The term "immune checkpoint pathway regulator" encompasses both immune checkpoint pathway regulators that can at least partially downregulate the function of an inhibitory immune checkpoint (herein referred to as "immune checkpoint pathway inhibitors" or "immune checkpoint pathway antagonists"), and immune checkpoint pathway regulators that can at least partially upregulate the function of a stimulatory immune checkpoint (herein referred to as "immune checkpoint pathway effectors" or "immune checkpoint pathway agonists").

[0245] The immune response mediated by immune checkpoint pathways is not limited to T cell-mediated immune response. For example, KIR receptors on NK cells regulate the immune response against tumor cells mediated by NK cells. Tumor cells express a molecule called HLA-C, which inhibits KIR receptors on NK cells, resulting in the attenuation of anti-tumor immune response. The administration of an agent that antagonizes the binding of HLA-C to KIR receptors, such as anti-KIR3 mab (e.g., lirilumab, BMS), inhibits the ability of HLA-C to bind to NK cell inhibitory receptors (KIR), thereby restoring the ability of NK cells to detect and attack cancer cells. Thus, the immune response mediated by the binding of HLA-C to KIR receptors is an example of a negative immune checkpoint pathway, whose inhibition results in the activation of non-T cell-mediated immune response.

[0246] In one embodiment, the immune checkpoint pathway regulator is a negative immune checkpoint pathway inhibitor / antagonist.In another embodiment, the immune checkpoint pathway regulator used in combination with the binding protein is a positive immune checkpoint pathway agonist.In another embodiment, the immune checkpoint pathway regulator used in combination with the binding protein is an immune checkpoint pathway antagonist.

[0247] The term "negative immune checkpoint pathway inhibitor" refers to an immune checkpoint pathway regulator that interferes with the activation of a negative immune checkpoint pathway, resulting in upregulation or enhancement of an immune response. Exemplary negative immune checkpoint pathway inhibitors include, but are not limited to, programmed death-1 (PD1) pathway inhibitors, programmed death-ligand-1 (PDL1) pathway inhibitors, TIM3 pathway inhibitors, and anti-cytotoxic T-lymphocyte antigen 4 (CTLA4) pathway inhibitors.

[0248] In one embodiment, the immune checkpoint pathway regulator is a negative immune checkpoint pathway antagonist ("PD1 pathway inhibitor") that inhibits the binding of PD1 to PDL1 and / or PDL2. PD1 pathway inhibitors result in the stimulation of various favorable immune responses, such as reversing T cell exhaustion, restoring cytokine production, and antigen-dependent T cell expansion and proliferation. PD1 pathway inhibitors have been recognized as effective against a variety of cancers, and have been approved by the USFDA for the treatment of various cancers, including melanoma, lung cancer, kidney cancer, Hodgkin's lymphoma, head and neck cancer, bladder cancer, and urothelial cancer.

[0249] The term PD1 pathway inhibitor includes monoclonal antibodies that interfere with the binding of PD1 to PDL1 and / or PDL2. Antibody PD1 pathway inhibitors are well known in the art. Examples of commercially available PD1 pathway inhibitors that are monoclonal antibodies that interfere with the binding of PD1 to PDL1 and / or PDL2 include nivolumab (Opdivo®, BMS-936558, MDX1106, available from BristolMyers Squibb, Princeton NJ), pembrolizumab (Keytruda® MK-3475, lambrolizumab, available from Merck and Company, Kenilworth NJ), and atezolizumab (Tecentriq®, Genentech / Roche, South San Francisco CA). Additional PD1 pathway inhibitor antibodies are in clinical development, including, but not limited to, durvalumab (MEDI4736, Mediimmune / AstraZeneca), pidilizumab (CT-011, CureTech), PDR001 (Novartis), BMS-936559 (MDX1105, BristolMyers Squibb), as well as avelumab (MSB0010718C, Merck Serono / Pfizer) and SHR-1210 (Incyte). Additional antibody PD1 pathway inhibitors are described in U.S. Patent No. 8,217,149, issued July 10, 2012 (Genentech, Inc.); U.S. Patent No. 8,168,757, issued May 1, 2012 (Merck Sharp and Dohme Corp.), U.S. Patent No. 8,008,449, issued August 30, 2011 (Medarex), and U.S. Patent No. 7,943,743, issued May 17, 2011 (Medarex, Inc.).

[0250] The term PD1 pathway inhibitor is not limited to antagonistic antibodies. Non-antibody biological PD1 pathway inhibitors are also in clinical development, including PD-L2 IgG2a fusion protein AMP-224 and PDL2 fusion protein AMP-514, which are being clinically developed by Amplimmune and Glaxo SmithKline. Aptamer compounds useful as PD1 pathway inhibitors are also described in the literature (Wang, et al. (2018) 145:125-130).

[0251] The term PD1 pathway inhibitors includes peptidyl PD1 pathway inhibitors, such as those described in U.S. Patent No. 9,422,339 to Sasikumar et al., issued August 23, 2016, and U.S. Patent No. 8,907,053 to Sasilkumar et al., issued December 9, 2014. CA-170 (AUPM-170, Aurigene / Curis) is an orally bioavailable small molecule that reportedly targets the immune checkpoints PDL1 and VISTA. Pottayil Sasikumar, et al. Oral immune checkpoint antagonists targeting PD-L1 / VISTA or PD-L1 / Tim3 for cancer therapy. Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): [Abstract] at AACR;Cancer Res 2016;76(14 Suppl): Abstract #4861. CA-327 (AUPM-327, Aurigene / Curis) is an orally available small molecule that reportedly inhibits the immune checkpoints programmed death ligand-1 (PDL1) and T-cell immunoglobulin and mucin domain-containing protein-3 (TIM3).

[0252] The term PD1 pathway inhibitors includes small molecule PD1 pathway inhibitors. Examples of small molecule PD1 pathway inhibitors useful in the practice of the present invention include Sasikumar, et al., 1,2,4-oxadiazole and thiadiazole compounds as immunomodulators (PCT / IB2016 / 051266, filed March 7, 2016, published September 15, 2016 as WO2016142833A1) and Sasikumar, et al. 3-substituted-1,2,4-oxadiazole and thiadiazole (PCT / IB2016 / 051343, filed March 9, 2016, published as WO2016142886A2), BMS-1166, and Chupak LS and Zheng X. Compounds useful as immunomodulators. Bristol-Myers Squibb Co. (2015) WO 2015 / 034820 A1, EP3041822 B1 granted Aug. 9, 2017; WO2015034820 A1; and Chupak, et al. Compounds useful as immunomodulators. Bristol-Myers Squibb Co. (2015) WO 2015 / 160641 A2. WO 2015 / 160641 A2, Chupak, et al. Compounds useful as immunomodulators. Bristol-Myers Squibb Co. Sharpe, et al. Modulators of immunoinhibitory receptor PD-1, and methods of use thereof, WO 2011082400 A2 published Jul. 7, 2011; U.S. Patent No. 7,488,802 (Wyeth), issued Feb. 10, 2009, have been described in the art.

[0253] In some embodiments, the combination of the binding proteins described herein with one or more PD1 immune checkpoint regulators is useful in the treatment of neoplastic conditions in which PD1 pathway inhibitors have demonstrated clinical efficacy in humans, either through FDA approval for the treatment of the disease or through demonstration of clinical efficacy in clinical trials, including, but not limited to, melanoma, non-small cell lung cancer, small cell lung cancer, head and neck cancer, renal cell carcinoma, bladder cancer, ovarian cancer, endometrial cancer, cervical cancer, uterine sarcoma, gastric cancer, esophageal cancer, DNA mismatch repair deficient colon cancer, DNA mismatch repair deficient endometrial cancer, hepatocellular carcinoma, breast cancer, Merkel cell carcinoma, thyroid cancer, Hodgkin's lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, mycosis fungoides, peripheral T-cell lymphoma. In some embodiments, the combination of the binding proteins and PD1 immune checkpoint regulators is useful in the treatment of tumors characterized by high levels of PDL1 expression, where the tumor has a tumor mutation burden and has high levels of CD8 + There is the presence of T cells, an immune activation signature associated with IFNγ, and a lack of metastatic disease, particularly liver metastases.

[0254] In some embodiments, the binding protein is administered in combination with an antagonist of a negative immune checkpoint pathway that inhibits the binding of CTLA4 to CD28 (a "CTLA4 pathway inhibitor"). Examples of CTLA4 pathway inhibitors are well known in the art (see, for example, U.S. Patent No. 6,682,736 (Abgenix), issued January 27, 2004; U.S. Patent No. 6,984,720 (Medarex, Inc.), issued May 29, 2007; U.S. Patent No. 7,605,238 (Medarex, Inc.), issued October 20, 2009).

[0255] In some embodiments, the binding protein is administered in combination with an antagonist of a negative immune checkpoint pathway that inhibits the binding of BTLA to HVEM ("BTLA pathway inhibitor"). A number of approaches to target the BTLA / HVEM pathway using anti-BTLA antibodies and antagonistic HVEM-Ig have been evaluated, and such approaches have shown promising utility in a number of diseases, disorders, and conditions, including transplantation, infectious diseases, tumors, and autoimmune diseases (see, for example, Wu, et al., (2012) Int. J. Biol. Sci. 8:1420-30).

[0256] In some embodiments, the binding protein is administered in combination with an antagonist of a negative immune checkpoint pathway that inhibits the ability of TIM3 to bind a TIM3-activating ligand (a "TIM3 pathway inhibitor"). Examples of TIM3 pathway inhibitors are known in the art, and representative non-limiting examples are described in U.S. Patent Application Publication No. PCT / US2016 / 021005, published September 15, 2016; U.S. Patent Application Publication No. US 20160257749 A1, published September 8, 2016, to Lifke et al. (F. Hoffman-LaRoche); U.S. Patent No. 9,631,026, issued April 27, 2017, to Karunsky; U.S. Patent No. 8,841,418, issued September 23, 2014, to Karunsky, Sabatos-Peyton et al.; U.S. Patent No. 9,605,070; and U.S. Patent No. 8,552,156, issued October 8, 2013, to Takayanagi et al.

[0257] In some embodiments, blockade of LAG3 and PD1 inhibits tumor-specific CD8 + T cells and virus-specific CD8 in the context of chronic infection +The binding protein is administered in combination with both LAG3 and PD1 inhibitors, as it is suggested to synergistically reverse the anergy between T cells.IMP321 (ImmuFact) has been evaluated in melanoma, breast cancer, and renal cell carcinoma.See generally Woo et al., (2012) Cancer Res 72:917-27;Goldberg et al., (2011) Curr. Top. Microbiol. Immunol. 344:269-78;Pardoll (2012) Nature Rev. Cancer 12:252-64;Grosso et al., (2007) J. Clin. Invest. 117:3383-392.

[0258] In some embodiments, the binding protein is administered in combination with an A2aR inhibitor. A2aR is an A2aR inhibitor that inhibits CD4 + T cells Reg A2aR inhibits T cell responses by stimulating cells to develop. A2aR is particularly important in tumor immunity because in tumors there is a high rate of cell death from cell turnover, and dying cells release adenosine, which is a ligand for A2aR. In addition, loss of A2aR is associated with enhanced and sometimes pathological inflammatory responses to infection. Inhibition of A2aR can be achieved by administration of molecules such as antibodies that block adenosine binding, or by adenosine analogues. Such agents can be used in combination with binding proteins for use in treating disorders such as cancer and Parkinson's disease.

[0259] In some embodiments, the binding protein is administered in combination with an inhibitor of IDO (indoleamine 2,3-dioxygenase).IDO downregulates immune response mediated through the oxidation of tryptophan, resulting in the inhibition of T cell activation and the induction of T cell apoptosis, creating an environment in which tumor-specific cytotoxic T lymphocytes are functionally inactivated or can no longer attack the cancer cells of the subject.Indoximod (NewLink Genetics) is an IDO inhibitor that is being evaluated in metastatic breast cancer.

[0260] As previously described, the present invention provides methods of treating neoplastic disease (e.g., cancer) in a mammalian subject by administration of a binding protein in combination with an agent that modulates at least one immune checkpoint pathway, including immune checkpoint pathway regulators that modulate two, three, or more immune checkpoint pathways.

[0261] In some embodiments, the binding protein is administered in combination with an immune checkpoint regulator that can regulate multiple immune checkpoint pathways. Multiple immune checkpoint pathways can be regulated by administering a multifunctional molecule that can act as a regulator of multiple immune checkpoint pathways. Examples of such multiple immune checkpoint pathway regulators include, but are not limited to, bispecific or multispecific antibodies. Examples of multispecific antibodies that can act as regulators of multiple immune checkpoint pathways are known in the art. For example, US Patent Application Publication No. 2013 / 0156774 describes bispecific and multispecific agents (e.g., antibodies) and methods of their use for targeting cells that co-express PD1 and TIM3. Furthermore, dual blockade of BTLA and PD1 has been shown to enhance anti-tumor immunity (Pardoll, (April 2012) Nature Rev. Cancer 12:252-64). The present disclosure contemplates the use of binding proteins in combination with immune checkpoint pathway regulators that target multiple immune checkpoint pathways, including but not limited to bispecific antibodies that bind both PD1 and LAG3. Thus, anti-tumor immunity can be enhanced at multiple levels and combination strategies can be generated taking into account various mechanistic considerations.

[0262] In some embodiments, the binding proteins may be administered in combination with two, three, four, or more checkpoint pathway regulators. Such combinations may be advantageous in that immune checkpoint pathways may have distinct mechanisms of action, providing an opportunity to attack the underlying disease, disorder, or condition from multiple distinct therapeutic angles.

[0263] It should be noted that therapeutic response to immune checkpoint pathway inhibitors often appears much later than response to traditional chemotherapy such as tyrosine kinase inhibitors. In some cases, after the initiation of treatment with immune checkpoint pathway inhibitors, it may take more than six months before objective signs of therapeutic response are observed. Therefore, the decision on treatment with immune checkpoint pathway inhibitors in combination with the binding proteins of the present disclosure must be made over a progression-free period that is often longer than that of traditional chemotherapy. The desired response can be any outcome that is considered favorable under the circumstances. In some embodiments, the desired response is prevention of progression of a disease, disorder, or condition, while in other embodiments, the desired response is regression or stabilization of one or more characteristics of a disease, disorder, or condition (e.g., reduction in tumor size). In still other embodiments, the desired response is reduction or elimination of one or more adverse effects associated with one or more agents of the combination.

[0264] Cofactor Cellular Therapeutic Agents and Methods In some embodiments, the method of the present disclosure may include the combination of binding protein and cofactor administration in the form of cell therapy for the treatment of neoplastic disease, autoimmune disease, or inflammatory disease.Examples of cell therapy suitable for use in combination with the method of the present disclosure include, but are not limited to, engineered T cell products, including one or more activated CAR-T cells, engineered TCR cells, tumor infiltrating lymphocytes (TIL), engineered Treg cells.Since engineered T cell products are generally activated ex vivo before their administration to subjects, thus providing upregulated levels of CD25, cell products including such activated engineered T cell types are suitable for further support via administration of CD25-biased binding proteins as described herein.

[0265] CAR-T cells In some embodiments of the methods of the present disclosure, the cofactors are "chimeric antigen receptor T cells" and "CAR-T cells," which are used interchangeably to refer to T cells that have been recombinantly modified to express a chimeric antigen receptor. The use of IL10 agents in combination with CAR-T cells for the treatment of neoplastic disease is described in U.S. Patent No. 10,195,274, issued February 5, 2019 to Mumm et al. As used herein, the terms "chimeric antigen receptor" and "CAR" are used interchangeably to refer to a chimeric polypeptide that includes multiple functional domains arranged in sequence from amino-terminus to carboxy-terminus: (a) an antigen binding domain (ABD), (b) a transmembrane domain (TD); and (c) one or more cytoplasmic signaling domains (CSDs), where the aforementioned domains may optionally be linked by one or more spacer domains. CARs may also further include a signal peptide sequence, which is routinely removed during post-translational processing and presentation of the CAR on the cell surface of cells transformed with an expression vector containing a nucleic acid sequence encoding the CAR. CARs useful in the practice of the present invention are prepared according to principles well known in the art.See, for example, U.S. Patent No. 7,741,465 B1 to Eshhaar et al., issued June 22, 2010; Sadelain, et al. (2013) Cancer Discovery 3(4):388-398; Jensen and Riddell (2015) Current Opinions in Immunology 33:9-15; Gross, et al. (1989) PNAS(USA) 86(24):10024-10028; Curran, et al. (2012) J Gene Med 14(6):405-15. Examples of commercially available CAR-T cell products that can be modified to incorporate the orthogonal receptors of the present invention include axicabtagene ciloreucel (commercially available as Yescarta® from Gilead Pharmaceuticals) and tisagenlecleucel (commercially available as Kymriah® from Novartis).

[0266] As used herein, the term antigen binding domain (ABD) refers to a polypeptide that specifically binds to an antigen expressed on the surface of a target cell. ABD can be any polypeptide that specifically binds to one or more cell surface molecules (e.g., tumor antigens) that are expressed on the surface of a target cell. In some embodiments, ABD is a polypeptide that specifically binds to a cell surface molecule associated with tumor cells, selected from the group consisting of GD2, BCMA, CD19, CD33, CD38, CD70, GD2, IL3Rα2, CD19, mesothelin, Her2, EpCam, Muc1, ROR1, CD133, CEA, EGRFRVIII, PSCA, GPC3, Pan-ErbB, and FAP. In some embodiments, the ABD is an antibody (as defined herein above to include molecules such as one or more VHHs, scFvs, etc.) that specifically binds to at least one cell surface molecule associated with a tumor cell (i.e., at least one tumor antigen), where the cell surface molecule associated with a tumor cell is selected from the group consisting of GD2, BCMA, CD19, CD33, CD38, CD70, GD2, IL3Rα2, CD19, mesothelin, Her2, EpCam, Muc1, ROR1, CD133, CEA, EGRFRVIII, PSCA, GPC3, Pan-ErbB, and FAP. Examples of CAR-T cells useful as cofactors in the practice of the methods of the present disclosure include, but are not limited to, CAR-T cells expressing a CAR comprising an ABD further comprising at least one of the following: anti-GD2 antibody, anti-BCMA antibody, anti-CD19 antibody, anti-CD33 antibody, anti-CD38 antibody, anti-CD70 antibody, anti-GD2 antibody and IL3Rα2 antibody, anti-CD19 antibody, anti-mesothelin antibody, anti-Her2 antibody, anti-EpCam antibody, anti-Muc1 antibody, anti-ROR1 antibody, anti-CD133 antibody, anti-CEA antibody, anti-PSMA antibody, anti-EGRFRVIII antibody, anti-PSCA antibody, anti-GPC3 antibody, anti-Pan-ErbB antibody, anti-FAP antibody.

[0267] The CAR of the CAR-T cell useful in the practice of the disclosed method further comprises a transmembrane domain that connects the ABD (or linker, if used, see the linker discussion below) to the intracellular cytoplasmic domain of the CAR. The transmembrane domain is composed of any polypeptide sequence that is thermodynamically stable in the cell membrane of eukaryotic organisms. The transmembrane domain may be derived from the transmembrane domain of naturally occurring transmembrane proteins or may be synthetic. In designing synthetic transmembrane domains, amino acids that favor α-helical structure are preferred. The transmembrane domains useful in constructing CARs are composed of approximately 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 22, 23, or 24 amino acids that favor the formation of α-helical secondary structure. Amino acids that favor α-helical conformation are well known in the art. See, for example, Pace, et al. (1998) Biophysical Journal 75: 422-427. The amino acids that are particularly favorable in α-helical conformation include methionine, alanine, leucine, glutamic acid, and lysine. In some embodiments, the CAR transmembrane domain can be derived from the transmembrane domain from type I transmembrane proteins, such as CD3ζ, CD4, CD8, CD28, etc.

[0268] The cytoplasmic domain of the CAR polypeptide comprises one or more intracellular signal domains. In one embodiment, the intracellular signal domain comprises the cytoplasmic sequence of the T cell receptor (TCR) and co-receptor, which initiates signal transduction after antigen receptor binding, and its functional derivatives and subfragments. Cytoplasmic signal transduction domains such as those derived from the T cell receptor zeta chain are used as part of the CAR to generate stimulatory signals for T lymphocyte proliferation and effector function after the chimeric receptor binds to the target antigen. Examples of cytoplasmic signaling domains include, but are not limited to, the cytoplasmic domain of CD27, the cytoplasmic domain S of CD28, the cytoplasmic domain of CD137 (also known as 4-1BB and TNFRSF9), the cytoplasmic domain of CD278 (also known as ICOS), the p110α, β, or δ catalytic subunit of PI3 kinase, human CD3 ζ chain, the cytoplasmic domain of CD134 (also known as OX40 and TNFRSF4), the CD3 polypeptides (δ, Δ, and ε), such as the FcεR1 γ and β chains, MB1 (Igα) chain, B29 (Igβ) chain, syk family tyrosine kinases (Syk, ZAP 70, etc.), src family tyrosine kinases (Lck, Fyn, Lyn, etc.), and other molecules involved in T cell signaling, such as CD2, CD5, and CD28.

[0269] In some embodiments, the CAR may also provide a costimulatory domain. The term "costimulatory domain" ("CSD") refers to the stimulatory domain, typically an endodomain, of the CAR, which provides a secondary non-specific activation mechanism through which the primary specific stimulation is propagated. The costimulatory domain refers to a portion of the CAR that enhances the proliferation, survival, or development of memory cells. Examples of costimulation include antigen non-specific T cell costimulation after antigen-specific signaling through the T cell receptor, and antigen non-specific B cell costimulation after signaling through the B cell receptor. Costimulation, e.g., T cell costimulation, and factors involved, are described in Chen & Flies (2013) Nat Rev Immunol 13(4):227-42. In some embodiments of the disclosure, the CSD comprises one or more of the following members of the TNFR superfamily: CD28, CD137 (4-1BB), CD134 (OX40), Dap10, CD27, CD2, CD5, ICAM-1, LFA-1 (CD11a / CD18), Lck, TNFR-I, TNFR-II, Fas, CD30, CD40, or a combination thereof.

[0270] CARs useful in the practice of the disclosed methods may optionally include one or more polypeptide spacers linking the domains of the CAR, particularly the link between the ABD and the transmembrane domain of the CAR. Although not a required element of the CAR structure, the inclusion of a spacer domain is generally considered desirable to facilitate antigen recognition by the ARD. When used with the CAR-T cell technology described herein, the terms "linker," "linker domain," and "linker region" refer to a polypeptide of about 1-100 amino acids in length. Linkers are typically composed of amino acid residues (e.g., glycine and serine) that allow flexibility of the polypeptide so that adjacent domains of the CAR are provided with greater freedom of movement relative to one another. There is no particular defined length or sequence of amino acids that is required for the spacer to achieve its function, but a typical property of the spacer is flexibility that allows freedom of movement of the ABD to facilitate target antigen recognition. Similarly, it has been found that there is substantial latitude in the length of the spacer while retaining the function of the CAR. Jensen and Riddell (2014) Immunol. Review 257(1) 127-144. Sequences useful as spacers in the construction of CARs useful in the practice of the present invention include, but are not limited to, the hinge region of IgG1, immunoglobulin 1 CH2-CH3 region, IgG4 hinge-CH2-CH3, IgG4 hinge-CH3, and IgG4 hinge. The hinge and transmembrane domains can be derived from the same molecule, such as the hinge and transmembrane domain of CD8-α. Imai, et al. (2004) Leukemia 18(4):676-684.

[0271] CARs are often referred to as first, second, third, or fourth generation. The term first generation CAR refers to CARs whose cytoplasmic domain transmits signals from antigen binding only through a single signaling domain, e.g., a signaling domain derived from the high affinity receptor FcεR1γ or CD3ζ chain of IgE. The domain contains one or three immunoreceptor tyrosine-based activation motifs [ITAMs] for antigen-dependent T cell activation. ITAM-based activation signals confer the T cells the ability to lyse target tumor cells and secrete cytokines in response to antigen binding. Second generation CARs contain costimulatory signals in addition to the CD3ζ signal. Simultaneous delivery of costimulatory signals enhances cytokine secretion and antitumor activity induced by CAR-transduced T cells. The costimulatory domain is usually membrane proximal to the CD3ζ domain. Third generation CARs contain tripartite signaling domains, e.g., including CD28, CD3ζ, OX40, or 4-1BB signaling regions. Fourth generation, or "armored car" CAR T cells are further engineered to express or block molecules and / or receptors that enhance immune activity, such as expression of IL12, IL18, IL7, and / or IL10; 4-1BB ligand, CD-40 ligand. Examples of intracellular signaling domains that can be incorporated into the CARs of the invention include (amino to carboxy): CD3ζ; CD28-41BB-CD3ζ; CD28-OX40-CD3ζ; CD28-41BB-CD3ζ; 41BB-CD-28--CD3ζ and 41BB-CD3ζ.

[0272] The term CAR includes CAR variants, including but not limited to split CAR, ON-switch CAR, bispecific or tandem CAR, inhibitory CAR (iCAR), and induced pluripotent stem (iPS) CAR-T cells. The term "split CAR" refers to a CAR in which the extracellular portion, ABD, and cytoplasmic signaling domain of the CAR are present on two separate molecules. CAR variants also include ON-switch CAR, which is a conditionally activatable CAR, including, for example, a split CAR in which the conditional heterodimerization of the two parts of the split CAR is pharmacologically controlled. CAR molecules and their derivatives (i.e., CAR variants) are described, for example, in PCT Application Nos. US2014 / 016527, US1996 / 017060, US2013 / 063083;Fedorov et al. Sci Transl Med (2013) ;5(215):215ra172;Glienke et al. Front Pharmacol (2015) 6:21;Kakarla & Gottschalk 52 Cancer J (2014) 20(2):151-5;Riddell et al. Cancer J (2014) 20(2):141-4;Pegram et al. Cancer J (2014) 20(2):127-33;Cheadle et al. Immunol Rev (2014) 257(1):91-106;Barrett et al. Annu Rev Med (2014) 65:333-47; Sadelain et al. Cancer Discov (2013) 3(4):388-98; Cartellieri et al., J Biomed Biotechnol (2010) 956304; the disclosures of which are incorporated herein by reference in their entireties. The term "bispecific or tandem CAR" refers to a CAR that contains a secondary CAR binding domain that can either amplify or inhibit the activity of a primary CAR.The term "inhibitory chimeric antigen receptor" or "iCAR" is used interchangeably herein to refer to a CAR in which the binding iCAR uses dual antigen targeting to stop the activation of an active CAR through the binding of a second inhibitory receptor with an inhibitory signaling domain of the secondary CAR binding domain, resulting in the inhibition of primary CAR activation. Inhibitory CARs (iCARs) are designed to control the activity of CAR-T cells through the activation of the signaling module of the inhibitory receptor. This approach combines the activity of two CARs, one of which generates a dominant-negative signal that limits the response of CAR-T cells activated by the activating receptor. Upon binding to a specific antigen expressed only by normal tissues, the iCAR can turn off the response of the countervailing activator CAR. In this way, iCAR-T cells can distinguish cancer cells from healthy cells and reversibly block the function of transduced T cells in an antigen-selective manner. The CTLA-4 or PD-1 intracellular domains in the iCAR induce inhibitory signals on T lymphocytes, resulting in less cytokine production, less efficient target cell lysis, and altered lymphocyte motility. The term "tandem CAR" or "TanCAR" refers to a CAR that mediates bispecific activation of T cells through the binding of two chimeric receptors designed to deliver stimulatory or costimulatory signals in response to the independent binding of two different tumor-associated antigens.

[0273] Typically, chimeric antigen receptor T cells (CAR-T cells) are T cells that have been recombinantly modified by transduction with an expression vector encoding a CAR, substantially in accordance with the teachings above.

[0274] In some embodiments, the engineered T cells are allogeneic with respect to the individual being treated. Graham et al. (2018) Cell 7(10) E155. In some embodiments, the allogeneic engineered T cells are fully HLA-matched. However, not all patients have a fully matched donor, and a cell product that is suitable for all patients regardless of HLA type provides an alternative.

[0275] Since the cell product may consist of the subject's own T cells, the population of cells to be administered to the subject is necessarily variable. Therefore, identifying the optimal concentration of CAR-T cells is optimized by the caregiver according to the needs of the subject being treated and monitored by conventional laboratory tests. Additionally, since CAR-T cell agents are variable, the response to such agents may vary, thus involving ongoing monitoring and management of treatment-related toxicity, which is managed with a course of pharmacological immunosuppression or B cell depletion before administration of CAR-T cell treatment. Typically, at least 1×10 6 Cells / kg were administered and at least 1 × 10 7 Cells / kg, at least 1 x 10 8 Cells / kg, at least 1 x 10 9 Cells / kg, at least 1 x 10 10 Cells / kg, or more, is usually limited by the number of T cells obtained during collection. The engineered cells can be infused into a subject in any physiologically acceptable medium by any convenient route of administration, usually intravenously, although they may also be introduced by other routes where the cells may find a suitable site for proliferation.

[0276] When the T cells used in the practice of the present invention are allogeneic T cells, such cells can be modified to reduce graft-versus-host disease.For example, the engineered cells of the present invention can be TCRαβ receptor knockout achieved by gene editing techniques.TCRαβ is a heterodimer, and both α and β chains need to be present in order for it to be expressed.A single gene encodes the α chain (TRAC), whereas there are two genes that encode the β chain, so the TRAC locus KO is deleted for this purpose.A number of different approaches have been used to achieve this deletion, such as CRISPR / Cas9; meganuclease; engineered I-CreI homing endonuclease, etc. See, e.g., Eyquem et al. (2017) Nature 543:113-117, in which the TRAC coding sequence is replaced by the CAR coding sequence; and Georgiadis et al. (2018) Mol. Ther. 26:1215-1227, in which clustered regularly interspaced short palindromic repeats (CRISPR) / Cas9-mediated TRAC disruption and CAR expression are coupled without directly integrating the CAR into the TRAC locus. Alternative strategies to prevent GVHD engineer T cells to express inhibitors of TCRαβ signaling, for example, using a truncated form of CD3ζ as a TCR inhibitory molecule.

[0277] Chemokine and cytokine agents as cofactors In some embodiments, the binding protein is administered in combination with additional cytokines, including, but not limited to, IL2, IL7, IL12, IL15 (see U.S. Patent No. 10,398,761, issued September 13, 2019), and IL18, including their respective analogs and variants.

[0278] Activation-induced cell death inhibitors In some embodiments, the binding protein is administered in combination with one or more cofactors that inhibit activation-induced cell death (AICD). AICD is a form of programmed cell death that results from the interaction of Fas receptor (e.g., Fas, CD95) with Fas ligand (e.g., FasL, CD95 ligand) and helps maintain peripheral immune tolerance. AICD effector cells express FasL, and apoptosis is induced in cells that express the Fas receptor. Activation-induced cell death is a negative regulator of activated T lymphocytes that results from repeated stimulation of their T cell receptor. Examples of agents that inhibit AICD that may be used in combination with the binding proteins described herein include, but are not limited to, cyclosporine A (Shih, et al., (1989) Nature 339:625-626), IL16 and analogs (including rhIL16, Idziorek, et al., (1998) Clinical and Experimental Immunology 112:84-91), TGFb1 (Genesteir, et al., (1999) J Exp Med189(2): 231-239), and vitamin E (Li-Weber, et al., (2002) J Clin Investigation 110(5):681-690).

[0279] physical method In some embodiments, the cofactor is an anti-neoplastic physical method, including, but not limited to, radiation therapy, cryotherapy, hyperthermia, surgery, laser ablation, and proton beam therapy.

[0280] immune disease The present disclosure further provides methods of treating a subject suffering from a disease, disorder, or condition by administration of a therapeutically effective amount of an IL10Rα / IL2Rγ binding protein (or a nucleic acid encoding an IL10Rα / IL2Rγ binding protein, including a recombinant virus encoding an IL10Rα / IL2Rγ binding protein) of the present disclosure. Disorders amenable to treatment with an IL10Rα / IL2Rγ binding protein (including pharma- ceutically acceptable formulations comprising an IL10Rα / IL2Rγ binding protein, and / or a nucleic acid molecule encoding such an IL10Rα / IL2Rγ binding protein, including a recombinant virus encoding such an IL10Rα / IL2Rγ binding protein) include viral infections (e.g., AIDS, influenza, chronic HCV, chronic viral hepatitis B, C, or D), Helicobacter pylori infection, HTLV, organ rejection, graft-versus-host disease, autoimmune thyroid disease, multiple sclerosis, allergies, asthma, Alzheimer's disease, and chronic kidney disease (CKD), asthma, and chronic myeloma. Neurodegenerative diseases including Immersion disease, Systemic Lupus Erythematosus (SLE), Autoinflammatory diseases, Inflammatory Bowel Disease (IBD), Crohn's disease, Diabetes including type 1 or type 2 diabetes, Inflammation, Autoimmune diseases, Atopic diseases, Paraneoplastic autoimmune diseases, Cartilage inflammation, Arthritis, Rheumatoid arthritis, Juvenile arthritis, Juvenile rheumatoid arthritis, Juvenile rheumatoid arthritis, Polyarticular juvenile rheumatoid arthritis, Systemic juvenile rheumatoid arthritis, Juvenile ankylosing spondylitis, Juvenile enteropathic arthritis, Juvenile reactive arthritis, Juvenile Reiter's syndrome, Seronegative enthesopathy syndrome (SEA syndrome) Inflammatory or autoimmune diseases include, but are not limited to, juvenile dermatomyositis, juvenile psoriatic arthritis, juvenile scleroderma, juvenile systemic lupus erythematosus, juvenile vasculitis, small joint rheumatoid arthritis, polyarticular rheumatoid arthritis, systemic rheumatoid arthritis, ankylosing spondylitis, enteropathic arthritis, reactive arthritis, Reiter's syndrome, and SEA syndrome (seronegative, enthesopathy, arthropathy syndrome). In certain embodiments, the method does not cause anemia.

[0281] Other examples of proliferative and / or differentiative disorders suitable for treatment with the IL10Rα / IL2Rγ binding proteins of the present disclosure (including IL10Rα / IL2Rγ binding proteins and / or pharma- ceutically acceptable formulations comprising nucleic acid molecules encoding them, including recombinant viruses encoding such IL10Rα / IL2Rγ binding proteins) include, but are not limited to, skin disorders. Skin disorders may involve abnormal activity of a cell or group or layer of cells in the dermal, epidermal, or hypodermal layers, or an abnormality at the dermal-epidermal junction. For example, the skin disorder may involve abnormal activity of keratinocytes (e.g., hyperproliferative basal keratinocytes and immediately suprabasal keratinocytes), melanocytes, Langerhans cells, Merkel cells, immune cells, and other cells found in one or more of the epidermal layers, such as the basal layer (stratum germinativum), the spinous layer, the granular layer, the lucid layer, or the stratum corneum. In other embodiments, the disorder may involve abnormal activity of dermal cells, such as dermal endothelium, fibroblasts, immune cells (e.g., mast cells or macrophages), found in the dermal layers, such as the papillary layer or the reticular layer.

[0282] Examples of skin disorders include psoriasis, psoriatic arthritis, dermatitis (eczema), e.g., exfoliative dermatitis or atopic dermatitis, pityriasis rubra pilaris, pityriasis rosea, parapsoriasis, pityriasis lichenoidis, lichen planus, lichen nitidus, ichthyosa-like dermatoses, keratodermas, skin diseases, alopecia areata, pyoderma gangrenosum, vitiligo, pemphigoid (e.g., ophthalmic cicatricial pemphigoid or bullous pemphigoid), urticaria, porokeratosis, hyperproliferation and inflammation of epithelium-associated cells lining the joint capsule. The skin disorders include rheumatoid arthritis with psoriasis, dermatitis such as seborrheic dermatitis and sunburn, keratosis such as seborrheic keratosis, senile keratosis, actinic keratosis, actin-induced keratosis, and follicular keratosis, acne vulgaris, keloids and prevention against keloid formation, birthmarks, human papillomavirus (HPV) infections such as warts, condyloma, or warts including condyloma acuminata, and genital warts, leukoplakia, lichen planus, and keratitis. The skin disorder can be dermatitis, for example atopic dermatitis or allergic dermatitis, or psoriasis.

[0283] The compositions of the present disclosure (including IL10Rα / IL2Rγ binding proteins and / or pharma- ceutically acceptable formulations containing nucleic acid molecules encoding them, including recombinant viruses encoding such IL10Rα / IL2Rγ binding proteins) can also be administered to patients suffering from (or likely to suffer from) psoriasis or psoriatic disorders. The term "psoriasis" is intended to have its medical meaning, i.e., a disease that primarily affects the skin, producing raised, thickened, scaly, non-scarring lesions. The lesions are usually sharply demarcated erythematous papules covered with overlapping shiny scales. The scales are typically silvery or slightly opaque. Nail involvement is frequent, resulting in pitting, peeling, thickening, and discoloration of the nails. Psoriasis is sometimes accompanied by arthritis, which can be disabling. Hyperproliferation of keratinocytes, along with epidermal inflammation and reduced differentiation of keratinocytes, is the primary feature of psoriatic epidermal hyperplasia. Several mechanisms have been proposed to explain the keratinocyte hyperproliferation that characterizes psoriasis. Impaired cell-mediated immunity has also been implicated in the pathogenesis of psoriasis. Examples of psoriatic disorders include chronic constant psoriasis, plaque psoriasis, moderate to severe plaque psoriasis, psoriasis vulgaris, eruptive psoriasis, erythrodermic psoriasis, generalized pustular psoriasis, annular pustular psoriasis, or localized pustular psoriasis.

[0284] Combination of IL10Rα / IL2Rγ binding proteins with additional therapeutic agents for autoimmune diseases: The present disclosure provides for the use of an IL10Rα / IL2Rγ binding protein of the present disclosure in combination with one or more additional active agents ("cofactors") in the treatment of autoimmune diseases. As used herein, the term "cofactor" includes agents that can be administered or introduced separately, e.g., formulated separately for separate administration (e.g., can be provided in a kit), and / or therapies that can be administered or introduced in combination with an IL10Rα / IL2Rγ binding protein.

[0285] As used herein, the term "in combination with" when used in reference to administration of multiple agents to a subject refers to administration of a first agent and at least one additional (i.e., second, third, fourth, fifth, etc.) agent to a subject. For purposes of the present invention, one agent (e.g., an IL10Rα / IL2Rγ binding protein) is considered to be administered in combination with a second agent (e.g., a therapeutic autoimmune antibody such as Humira®) if the biological effect resulting from administration of the first agent persists in the subject upon administration of the second agent such that the therapeutic effects of the first and second agents overlap. For example, therapeutic antibodies are sometimes administered by IV infusion every two weeks (e.g., adalimumab in the treatment of Crohn's disease), while an IL10Rα / IL2Rγ binding protein of the present disclosure may be administered more frequently, e.g., daily, BID, or weekly. However, administration of a first agent (e.g., etanercept) provides a therapeutic effect over an extended period of time, and administration of a second agent (e.g., IL10Rα / IL2Rγ binding protein) provides its therapeutic effect while the therapeutic effect of the first agent continues, and thus, even if the first agent is administered at a time significantly separated (e.g., days or weeks) from the time of administration of the second agent, the second agent is considered to be administered in combination with the first agent. In one embodiment, an agent is considered to be administered in combination with a second agent if the first and second agents are administered in parallel (within 30 minutes of each other), simultaneously, or sequentially. In some embodiments, a first agent is considered to be administered "concurrently" with a second agent if the first and second agents are administered within about 24 hours of each other, preferably within about 12 hours of each other, preferably within about 6 hours of each other, preferably within about 2 hours of each other, or preferably within about 30 minutes of each other. The term "in combination with" is also understood to apply to the situation where a first agent and a second agent are co-formulated in a single pharma- ceutically acceptable formulation and the co-formulation is administered to a subject. In certain embodiments, the IL10Rα / IL2Rγ binding protein and cofactor are administered or applied sequentially, e.g., one agent is administered before one or more other agents.In other embodiments, the IL10Rα / IL2Rγ binding protein and the cofactor are administered in parallel, e.g., two or more agents are administered at or about the same time; the two or more agents may be in two or more separate formulations or may be combined in a single formulation (i.e., a co-formulation). Whether the agents are administered sequentially or in parallel, they are considered to be administered in combination for purposes of this disclosure.

[0286] In some embodiments, the cofactor is one or more agents selected from the group consisting of corticosteroids (including but not limited to prednisone, budesonide, prednilisone), Janus kinase inhibitors (including but not limited to tofacitinib (Xeljanz®)), calcineurin inhibitors (including but not limited to cyclosporine and tacrolimus), mTor inhibitors (including but not limited to sirolimus and everolimus), IMDH inhibitors (including but not limited to azathioprine, leflunomide, and mycophenolate), biologics such as abatacept (Orencia®) or etanercept (Enbrel®), and therapeutic antibodies. Examples of therapeutic antibodies that may be administered as cofactors in combination with the IL10Rα / IL2Rγ binding proteins of the present disclosure in treating autoimmune diseases include anti-CD25 antibodies (e.g., daclizumab and basiliximab), anti-VLA-4 antibodies (e.g., natalizumab), anti-CD52 antibodies (e.g., alemtuzumab), anti-CD20 antibodies (e.g., rituximab, ocrelizumab), anti-TNF antibodies (e.g., infliximab and adalimumab), anti-IL6R antibodies (e.g., tocilizumab), anti-TNFα antibodies (e.g., adalimumab (Humira®), golimumab, and infliximab). ximab), anti-integrin-α4β7 antibodies (e.g., vedolizumab), anti-IL17a antibodies (e.g., brodalumab or secukinumab), anti-IL4Rα antibodies (e.g., dupilumab), anti-RANKL antibodies, IL6R antibodies, anti-IL1β antibodies (e.g., canakinumab), anti-CD11a antibodies (e.g., efalizumab), anti-CD3 antibodies (e.g., muramonab), anti-IL5 antibodies (e.g., mepolizumab, reslizumab), anti-BLyS antibodies (e.g., belimumab); and anti-IL12 / IL23 antibodies (e.g., ustekinumab).

[0287] Many therapeutic antibodies have been approved for clinical use against autoimmune diseases. Examples of antibodies approved by the U.S. Food and Drug Administration (FDA) for use in treating autoimmune diseases in subjects suffering from autoimmune diseases that can be administered as cofactors in combination with the IL10Rα / IL2Rγ binding proteins of the present disclosure (and optionally additional cofactors) for the treatment of the indicated autoimmune diseases are provided in Table 6.

[0288] (Table 6) TIFF2024504923000039.tif238169

[0289] The aforementioned antibodies useful as cofactors in the practice of the methods of the disclosure can be administered alone or in the form of any antibody drug conjugate (ADC) comprising an antibody, a linker, and one or more drugs (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 drugs) or in a modified (e.g., PEGylated) form.

[0290] In some embodiments, the cofactor is a vaccine. The IL10Rα / IL2Rγ binding proteins of the invention can be administered to a subject in combination with a vaccine as an adjuvant to enhance the immune response to the vaccine, according to the teachings of U.S. Patent No. 5,800,819, issued Sep. 1, 1998 to Doyle et al. Examples of vaccines that may be combined with the IL10Rα / IL2Rγ binding proteins of the invention include HSV vaccines, Bordetella pertussis, Escherichia coli vaccines, pneumococcal vaccines, including multivalent pneumococcal vaccines such as Prevnar® 13, diphtheria, tetanus, and whooping cough vaccines (including combination vaccines such as Pediatrix® and Pentacel®), chickenpox vaccines, Haemophilus influenzae type B vaccines, human papilloma virus vaccines such as Gardasil®, polio vaccines, leptospirosis vaccines, combined respiratory vaccines, Moraxella vaccines, and live attenuated or killed virus vaccine products such as bovine respiratory disease vaccine (RSV), Fluzone® and Quadravlent®. These include multivalent human influenza vaccines such as Fluzone®, feline leukemia vaccines, transmissible gastroenteritis vaccines, COVID-19 vaccines, as well as rabies vaccines.

[0291] Selective activation IL10 stimulates macrophages (e.g., monocytes) as well as T cells (e.g., CD4 + T cells and CD8 +It is known that IL10Rα and IL2Rγ are active against T cells. In some embodiments, the methods provided herein use the binding proteins of the present disclosure that bind IL10Rα and IL2Rγ, resulting in selective activation of T cells relative to activation of macrophages. Macrophages are a cell type that express both IL10Rα and IL10Rβ receptors, but if activated too strongly, can cause side effects such as anemia. Selective activation of T cells relative to macrophages is beneficial because L10-activated macrophages can phagocytose aged red blood cells, which manifests as anemia in patients receiving IL10. Binding proteins as described herein that provide selective substantial activation of T cells while providing minimal activation of macrophages can result in molecules that produce lower side effects, such as anemia, compared to the natural IL10 ligand. Other problems and toxicities associated with IL10 activation are described, for example, in Fioranelli and Grazia, J Integr Cardiol 1(1):2-6, 2014. Such problems can be circumvented by using the binding proteins of the present disclosure that specifically bind to IL10Rα and IL2Rγ.

[0292] In some embodiments, provided herein are methods of selectively inducing an activity in one or more of a first cell type over one or more of a second cell type by contacting a population of cells comprising both a first cell type and a second cell type with an IL10Rα / IL2Rγ binding protein described herein. In certain embodiments, the first cell type is a CD4 + T cells, CD8 + In another embodiment, the first cell type is a CD4 T cell, a B cell, and / or a NK cell, and the second cell type is a monocyte. + T cells and / or CD8 +and the second cell type is a NK cell, a B cell, and / or a monocyte. In certain embodiments, the activity of the first cell type induced by IL10Rα / IL2Rγ is at least 1.2-fold greater than the activity of the second cell type.

[0293] Dosage The dosage, toxicity, and therapeutic efficacy of such binding protein or nucleic acid compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. Data obtained from cell culture assays and animal studies can be used in formulating a range of dosages for use in humans. The dosage of such compounds is preferably within a range of circulating concentrations that include the ED50 with minimal acceptable toxicity. Dosages can vary within this range depending on the dosage form used and the route of administration utilized. For any compound used in the methods of the invention, a therapeutically effective dose can be estimated initially from cell culture assays. Doses can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.

[0294] As defined herein, a therapeutically effective amount (i.e., an effective dosage) of a subject binding protein depends on the polypeptide selected. For example, a single dose amount ranging from approximately 0.001 to 0.1 mg per kg of patient body weight can be administered; in some embodiments, about 0.005, 0.01, 0.05 mg / kg can be administered.

[0295] In some embodiments, a pharma- ceutically acceptable form of the binding protein of the present disclosure is administered to a subject according to a "low-dose" treatment protocol as described in U.S. Patent Nos. 9,669,071 and 10,293,028B2 to Klatzman et al., the entire teachings of which are incorporated herein by reference. Additional low-dose protocols are described in Smith, KA (1993) Blood 81(6):1414-1423; He, et al., (2016) Nature Medicine 22(9):991-993.

[0296] In some embodiments of the present disclosure, methods and compositions are provided for the treatment and / or prevention of a neoplastic disease, disorder, or condition in a subject by administration to the subject of a therapeutically effective amount of a binding protein of the present disclosure, wherein the serum concentration is at or above an effective concentration of the binding protein sufficient to promote proliferation of CD3-activated primary human T cells for such binding protein for a majority of the period (i.e., greater than about 50%, alternatively greater than about 60%, alternatively greater than about 70%, alternatively greater than about 80%, alternatively greater than about 90%) of the period (e.g., at least 24 hours, alternatively at least 48 hours, alternatively at least 72 hours, alternatively at least 96 hours, alternatively at least 120 hours, alternatively at least 144 hours, alternatively at least 7 days, alternatively at least 10 days, alternatively at least 12 days, alternatively at least 14 days, alternatively at least 28 days, alternatively at least 45 days, alternatively at least 60 days, or more). 10 PRO The above is an alternative to EC 20 PRO The above is an alternative to EC 30 PRO The above is an alternative to EC 40 PRO That’s all, EC 50 PRO The above is an alternative to EC 60 PRO or greater), but below the effective concentration for such binding protein, at a serum concentration of such binding protein sufficient to induce T cell activation (e.g., EC100 PRO Below, alternatively EC 90 PRO Below, alternatively EC 80 PRO Below, alternatively EC 70 PRO Below, EC 60 PRO Below, alternatively EC 50 PRO (see below).

[0297] In some embodiments of the present disclosure, methods and compositions are provided for the treatment and / or prevention of a neoplastic disease, disorder, or condition in a subject by administration to the subject of a therapeutically effective amount of a binding protein described herein sufficient to maintain a serum concentration of the binding protein for greater than about 50%, alternatively greater than about 60%, alternatively greater than about 70%, alternatively greater than about 80%, alternatively greater than about 90% for at least 24 hours, alternatively at least 96 hours, alternatively at least 120 hours, alternatively at least 144 hours, alternatively at least 7 days, alternatively at least 10 days, alternatively at least 12 days, alternatively at least 14 days, alternatively at least 28 days, alternatively at least 45 days, alternatively at least 60 days, or longer.

[0298] In some embodiments of the disclosure, methods and compositions are provided for the treatment and / or prevention of a neoplastic disease, disorder, or condition in a subject by administration to the subject of a therapeutically effective amount of a binding protein sufficient to maintain a serum concentration of the binding protein at or above the effective concentration for more than about 50%, alternatively greater than about 60%, alternatively greater than about 70%, alternatively greater than about 80%, alternatively greater than about 90% of a period of at least 24 hours, alternatively at least 96 hours, alternatively at least 120 hours, alternatively at least 144 hours, alternatively at least 7 days, alternatively at least 10 days, alternatively at least 12 days, alternatively at least 14 days, alternatively at least 28 days, alternatively at least 45 days, alternatively at least 60 days, or longer.

[0299] According to another aspect, a method is provided for stimulating the immune system of an animal by administering a binding protein of the present disclosure. The method is useful for treating disease states in which the host immune response is deficient. In treating a subject, a therapeutically effective dose of the compound (i.e., active ingredient) is administered. A therapeutically effective dose refers to an amount of the active ingredient that results in amelioration of symptoms or prolongation of survival of the subject. The effective dose varies with the characteristics of the binding protein administered, the physical characteristics of the subject being treated, the nature of the disease or condition, and the like. A single administration can range from about 50,000 IU / kg to about 1,000,000 IU / kg or more, more typically about 600,000 IU / kg. This may be repeated several times a day (e.g., 2-3 times per day) for several days (e.g., about 3-5 consecutive days), and then repeated one or more times after a period of rest (e.g., about 7-14 days). Thus, an effective dose may include only a single administration, or multiple administrations over a period of time (eg, about 20-30 individual administrations of about 600,000 IU / kg each given over a period of about 10-20 days).

[0300] The composition can be administered from one or more times per day to one or more times per week, including once every other day. One skilled in the art will recognize that certain factors, including but not limited to the severity of the disease or disorder, previous treatments, the overall health and / or age of the subject, and other diseases present, can affect the dosage and timing required to effectively treat the subject. Furthermore, treatment of a subject with a therapeutically effective amount of the binding protein can include a single treatment or can include a series of treatments. In one embodiment, the composition is administered every 8 hours for 5 days, followed by a rest period of 2-14 days, e.g., 9 days, followed by administration every 8 hours for an additional 5 days. In another embodiment, the composition is administered every other day for a period of at least 6 days, optionally at least 10 days, optionally at least 14 days, optionally at least 30 days, optionally at least 60 days.

[0301] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[0302] Compounds that exhibit toxic side effects may be used, but care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to non-infected cells, thereby reducing side effects. The toxicity and therapeutic efficacy of the binding protein can be determined by standard pharmaceutical procedures in cell culture or experimental animals. Cell culture assays and animal studies can be used to determine the LD 50 (the dose that is lethal to 50% of the population) and ED 50 The dose that is therapeutically effective in 50% of the population can be determined. The dose ratio between toxic and therapeutic effects is the therapeutic index, which is defined as the LC 50 / EC 50 The therapeutic index can be expressed as a ratio of ED 0 to ED 1. Binding proteins that exhibit large therapeutic indices are preferred. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosages suitable for use in humans. The dosage of such variants is preferably within the range of ED 0 to ED 1 with little or no toxicity. 50 The dosage may vary within this range depending on a variety of factors, such as the dosage form employed, the route of administration utilized, the condition of the subject, etc.

[0303] The therapeutically effective dose was first determined from the EC 50 The dose can then be estimated by determining the EC 50 The dosage form can be formulated in animal models to achieve a circulating plasma concentration range that includes. Such information can be used to more accurately determine the dose that is useful in humans. Levels in plasma can be measured, for example, by HPLC. The exact formulation, route of administration, and dosage can be selected by the individual physician in consideration of the patient's condition.

[0304] The attending physician of a patient treated with the binding proteins of the present disclosure will know how and when to terminate, interrupt, or adjust administration due to toxicity, organ dysfunction, and the like. Conversely, the attending physician will also know to adjust treatment to higher levels if the clinical response is not adequate (barring toxicity). The size of the dose administered in managing the disorder of interest will vary with the severity of the condition being treated, the route of administration, and the like. The severity of the condition can be evaluated, in part, for example, by standard prognostic evaluation methods. Furthermore, the dose and perhaps the frequency of administration will also vary according to the age, weight, and response of the individual patient. EXAMPLES

[0305] Example 1 - pSTAT3 Flow Cytometry Assay PBMCs were purified from healthy non-smoking donor blood collected in a Leukoreduction system chamber using the human Miltenyi MACSprep PBMC isolation kit. Purified PBMCs (500,000 cells per well) were left unstimulated or stimulated with 100 nM concentration of WT IL10 or 84 anti-IL10R1 / IL2Rγ V H H 2Cells were stimulated with one of the following antibodies for 20 minutes at 37°C. Cells were fixed with Fix Buffer I (available from BD Biosciences, San Jose CA as catalog number 557870) for 15 minutes at 37°C. Cells were then washed and permeabilized with chilled Perm Buffer III (available from BD Biosciences, catalog number 558050) overnight at -20°C. Cells were washed to remove the permeabilization buffer and blocked with Human TruStain FcX (available from BioLegend, San Diego CA as catalog number 422301) and mouse serum for 5 minutes at room temperature. Cells were then treated with an antibody cocktail (Table 7 below) for 1 hour at room temperature. After antibody staining, cells were washed, fixed and measured on a Cytek® Aurora Spectral flow cytometer (available from Cytek Biosciences, Fremont CA). Data was analyzed using FlowJo software (commercially available from Becton Dickinson Corp, Franklin Lakes, NJ). Different cell lineages were gated using their lineage markers, and the geometric mean fluorescence intensity of pSTAT3 expression was calculated on FlowJo.

[0306] Table 7. Antibody cocktail TIFF2024504923000040.tif66160

[0307] Example 2. Screening: 84 anti-IL10Rα / IL2Rγ V H H 2 We screened for pSTAT3 activity in various cell populations that make up PBMCs (Figures 2A-2E). + Anti-IL10Rα / IL2Rγ V showed at least a 50% increase in pSTAT3 induction over unstimulated controls in T cells H H 2 were considered as "hits." Screening yielded 12 hits (see Table 8 below). These 12 anti-IL10Rα / IL2Rγ V H H2 compared with unstimulated controls, CD4 + T cells and CD8 + We demonstrated nominal pSTAT3 activation in B cells and NK cells, while retaining robust pSTAT3 activation in T cells, and showed minimal pSTAT3 induction in monocytes.

[0308] (Table 8) pSTAT3 MFI TIFF2024504923000041.tif149160

[0309] Example 3: Dose-response experiment: The four anti-IL10Rα / IL2Rγ V antibodies with the highest levels of activity were identified from the initial screen based on pSTAT3 induction (Example 2). H H 2 Proteins, B cells, CD4 + T cells, NK cells, CD8 + The PBMCs were further tested in dose-response experiments on B cells, CD4 T cells, and monocytes, as detailed in Example 1. + T cells, NK cells, CD8 + T cells and monocytes were left unstimulated or stimulated with wild-type human IL10 (wt hIL10) or four anti-IL10Rα / IL2Rγ V H H 2 Proteins were stimulated over a range of concentrations from 0.0001 nM to 100 nM in 10-fold dilutions with one of the following: (DR395 (DR229-DR239), DR441 (DR236-DR231), DR471 (DR241-DR231), and DR465 (DR240-DR231)). A table summarizing pSTAT3 MFI in dose-response experiments in various cell lines is shown below.

[0310] (Table 9A) TIFF2024504923000042.tif52166

[0311] (Table 9B) TIFF2024504923000043.tif52167

[0312] (Table 9C) TIFF2024504923000044.tif52166

[0313] (Table 9D) TIFF2024504923000045.tif51163

[0314] (Table 9E) TIFF2024504923000046.tif51163

[0315] As can be seen from the data above and from Figures 3A-3E, the anti-IL10Rα / IL2Rγ V H H 2 The protein demonstrated a dose-dependent induction of pSTAT3 and CD4 + T cells and CD8 + The most extensive induction was seen in T cells, followed by NK cells and B cells. Monocytes showed very minimal induction of pSTAT3, especially compared to the level of pSTAT3 induction seen with WT IL10. The foregoing demonstrates the ability of the disclosed IL10Rα / IL2Rγ binding proteins to provide selective cell type activation that retains the desirable property of stimulating T cells while having minimal effects on monocytes.

[0316] Example 4-V H H generation Camels were allowed to acclimate in the research facility for at least 7 days prior to immunization. Antigen was diluted in 1x PBS (approximately 1 mg total antigen). Antigen quality was assessed by SDS-PAGE to confirm purity (e.g., >80%). First, 10 mL of CFA (followed by 6 rounds with IFA) was added to the mortar, then 10 mL of antigen in 1x PBS was slowly added to the mortar and crushed with a pestle. The antigen and CFA / IFA were crushed until the components appeared milky white and difficult to disperse. Camels were injected subcutaneously at least 6 sites on the body with antigen emulsified in CFA, with approximately 2 mL injected at each site (10 mL total per camel). Stronger immune responses were generated by injecting larger volumes at more sites. Immunizations were performed 7 times, 1 week (7 days) apart. To avoid leakage of the emulsion, the needle was inserted into the subcutaneous space for 10-15 seconds after each injection. Alternatively, gently pulling the syringe plunger also prevented leakage.Blood samples were collected 3 days after the seventh immunization.

[0317] After immunization, a library was constructed. Briefly, RNA was extracted from blood and transcribed into cDNA. H The H region was obtained through a two-step PCR that fragmented approximately 400 bp. The PCR product and the pMECS phagemid vector were digested with Pst I and Not I, and then ligated into pMECS / Nb recombinant. After ligation, the product was transformed into E. coli TG1 cells by electroporation. The transformants were then enriched in growth medium and plated. Finally, the library size was estimated by counting the number of colonies.

[0318] After library construction, biopanning of the library was performed to screen candidates against the antigen. Phage display technology was applied in this procedure. Positive colonies were identified by PE-ELISA.

[0319] Example 5 - Recombinant production and purification Codon-optimized DNA inserts were cloned into modified pcDNA3.4 (Genscript) for small-scale expression in HEK293 cells in 24-well plates. Binding molecules were purified substantially according to the following procedure: 96×4 mL of supernatant in 4×24-well blocks were rearranged into 4×96-well, 1 mL blocks using a Hamilton Star automation system. PhyNexus micropipette tips (Biotage, San Jose CA) holding 80 μL of Ni-Excel IMAC resin (Cytiva) were equilibrated with wash buffer: PBS pH 7.4, 30 mM imidazole. PhyNexus tips were immersed and cycled through 14 cycles of 1 mL pipetting across all 4×96-well blocks. PhyNexus tips were washed in 2×1 mL blocks holding wash buffer. PhyNexus tips were eluted in 3×0.36 mL blocks holding elution buffer: PBS pH 7.4, 400 mM imidazole. PhyNexus chips were regenerated in 3×1 mL blocks of 0.5 M sodium hydroxide.

[0320] Purified protein eluates were quantified using a Biacore® T200 essentially according to the following procedure: 10 uL of the initial 96 x 0.36 mL eluate was transferred to a Biacore® 96-well microplate and diluted to 60 uL in HBS-EP+ buffer (10 mM Hepes pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.05% Tween 20). Each of the 96 samples was injected onto a CM5 series S chip previously functionalized with an anti-histidine capture antibody (Cytiva): injections are performed at 5 μL / min for 18 seconds. Capture levels were recorded 60 seconds after a buffer wash. To eliminate cell-to-cell surface variability, 10 uL of known V HA standard curve of H concentrations (270, 90, 30, 10, 3.3, 1.1 μg / mL) was acquired on each of the four Biacore chip flow cells. 96 captures were interpolated to the standard curve using a non-linear model including specific and non-specific one-site binding. Concentrations in the first elution block ranged from 12 to 452 μg / mL, corresponding to 4 to 149 μg. SDS-PAGE analysis of five randomly chosen samples was performed to confirm that the molecular weight of the eluate corresponded to the expected value (approximately 30 kDa).

[0321] Protein concentration was normalized using a Hamilton Star automated system, essentially following the procedure below: Concentration values ​​were imported into an Excel spreadsheet, where pipetting volumes were calculated to perform a dilution to 50 μg / mL in 0.22 mL. The spreadsheet was imported into the Hamilton Star method, dedicated to performing dilution pipetting, using the initial elution block and elution buffer as diluent. The final normalized plate was sterile filtered using a 0.22 μm filter plate (Corning).

[0322] Example 6. Evaluation of the binding affinity of IL10Ra / IL2Rg dimers via SPR All experiments were performed on a Biacore T200 instrument (Cytiva) equipped with Protein A or CAP biotin chips in 10 mM Hepes, 150 mM NaCl, 0.05% (v / v) polysorbate 20 (PS20), and 3 mM EDTA (HBS-EP+ buffer). For experiments on Protein A chips, Fc fusion ligand was flowed at 5 μl / min for various times ranging from 18 to 300 s to reach the capture loads listed in the table below. After ligand capture, injections of a two-fold dilution series of analyte, typically including at least five concentrations between 1 μM and 1 nM, were performed in either high performance or single cycle kinetics mode. Surface regeneration was achieved by flowing 10 mM glycine-HCl, pH 1.5 (60 s, 50 μL / min). Buffer-subtracted sensograms were processed with Biacore T200 Evaluation Software and globally fitted with a 1:1 Langmuir binding model (bulk shift set to zero) to obtain kinetic and affinity constants (k a , k d , K D ) was extracted. MAX <100 RU indicates a surface density compatible with kinetic analysis. Experiments on CAP chips were performed as described above with an additional capture step of Biotin CAPture reagent (10 sec, 40 uL / min) performed prior to capture of the biotinylated ligand. Calculated Rmax was generated using the formula Rmax = Load (RU) x Ligand valency x (Molecular weight of analyte / Molecular weight of ligand). Surface activity was defined as the ratio of experimental Rmax / calculated Rmax. Results of these experiments are provided below under Sample Information and Experimental Results.

[0323] Example 7: Dose-response experiments with Fc versions: Two of the VHHs and their Fc versions were tested in dose-response curves as described above. PBMCs were left unstimulated or stimulated with WT IL10 or one of the two IL10R1 / 2y VHHs [DR395 (DR229-DR239), DR465 (DR240-DR231)] or the Fc versions of the VHHs [DR992 (H1, DR240-DR231), DR995 (A2, DR229-DR239)] at concentrations ranging from 0.1 pM to 100 nM for 20 min at 37° C. as detailed in Example 1. Staining and analysis were performed as described in Example 1. The results are provided in the accompanying drawings in Figures 9A-E. The Fc molecules H1-Fc [DR992, DR240-DR231)] and A2-Fc [DR995 (DR229-DR239)] were more potent than their non-Fc counterparts H1 [DR465 (DR240-DR231)] and A2 [DR395 (DR229-DR239)] in inducing pSTAT3 signaling in CD4 T cells, CD8 T cells, B cells, and NK cells. pSTAT3 signaling in monocytes was still very minimal compared to the level of pSTAT3 induction seen with WT IL10.

[0324] Example 8: Monocyte functional assay: Human monocytes were purified from human PBMCs using CD14 microbeads (Miltenyi Biotech 130-050-201). Purified monocytes were seeded at 100,000 cells per well in 96-well flat-bottom plates and treated with IL10 at concentrations ranging from 0.1 pM to 100 nM in complete RPMI medium [RPMI containing 10% FBS and 1× penicillin / streptomycin (Gibco, Cat. No. 15-140-122)] for 48 hours at 37°C. After 48 hours of treatment, plates were spun down at 400 g for 5 minutes and supernatants were collected. Supernatants were tested in a Meso Scale discovery assay (Meso Scale Discovery, Cat. No. K151A9H) to measure levels of cytokines IL1b, IL6, IL8, and TNFa. Results of these studies are provided in Figures 10A-10D of the accompanying drawings. IL10R1 / 2y VHHs H1 [DR465 (DR240-DR231)] and A2 [DR395 (DR229-DR239)] did not inhibit LPS-induced secretion of IL1b, IL6, TNFa, and IL8, which correlated with the lack of induction of pSTAT3 signaling in monocytes. The Fc versions of the molecules were slightly more potent than their non-Fc counterparts in inhibiting LPS-induced IL1b secretion and TNFa production, but did not cause complete inhibition even at higher concentrations of stimuli.

[0325] Example 9: CD8 blast function assay: Human CD8 T cells were purified from human PBMCs by negative selection using a CD8+ T cell isolation kit (Milteyi Biotec 130-096-495).The isolated CD8 T cells were then activated for 3 days using a human CD8 T cell activation / expansion kit (Miltenyi Biotec 130-091-441). Day 3 CD8 T cell blasts were then treated with IL10 at concentrations ranging from 0.1 pM to 100 nM in Yssel's medium [containing 0.25% w / v human albumin (Sigma, Cat. No. A9080), 1x ITS-X (human) (Gibco, Cat. No. 51500056), 30 mg / L transferrin (Roche, Cat. No. 10652202001), 2 mg / L PA BioXtra (Sigma, Cat. No. P5585), 1x LA-OA-albumin (Sigma, Cat. No. L9655), 1x penicillin / streptomycin (Gibco, Cat. No. 15-140-122), 1% human serum (Gemini, Cat. No. 507533011) (IMDM, Gibco, Cat. No. 122440-053)] for 72 hours at 37°C. During the last 5 hours of incubation, cells were treated with 1:1000 monensin (eBiosciences, Cat. No. 00-4505-51). After incubation, cells were washed with PBS and stained with Zombie NIR fixable viability dye (Biolegend, Cat. No. 423105) for 15 minutes at 4°C in the dark. Cells were washed twice with premade FACS buffer (PBS+2% FBS) and then fixed in IC fixation buffer (Invitrogen 00-8222) for 20 minutes at room temperature. Cells were then spun down and permeabilized with 1× permeabilization buffer (Invitrogen 00-833) for 5 minutes.

[0326] Cells were resuspended in permeabilization buffer, briefly blocked with 1:10 Human TruStain FcX Fc Block (Biolegend, Cat. No. 422302), and then stained with anti-Granzyme A antibody (Biolegend, Cat. No. 507206) and anti-Granzyme B antibody (BD 562462) for 1 hour at room temperature in the dark. Cells were then washed twice with FACS buffer and resuspended in FACS buffer containing 1% PFA (Electron Microscopy Sciences, Cat. No. 15710) for at least 10 minutes at room temperature in the dark, before being acquired on a Cytek Aurora Spectral flow cytometer. Data was analyzed using FlowJo software. The results of these studies are provided in Figures 11A and 11B of the accompanying drawings.

[0327] The IL10R1 / 2y VHHs H1 [DR465 (DR240-DR231)] and A2 [DR395 (DR229-DR239)] induced granzyme A production at higher stimuli and were weak inducers of granzyme B production. Addition of Fc to the molecule enhanced its activity, with H1-Fc and A2-Fc being as potent as WT in inducing granzyme A production and also more potent in inducing granzyme B production.

[0328] Table 10: Anti-human IL10Ra sdAb CDRs TIFF2024504923000047.tif204170TIFF2024504923000048.tif39170

[0329] Table 11. Human anti-IL2Rg sdAb CDRs TIFF2024504923000049.tif192170

[0330] Table 12. Mouse anti-IL2Rg sdAb CDRs TIFF2024504923000050.tif135164

[0331] Table 13: Human anti-IL10Ra VHH amino acid sequences TIFF2024504923000051.tif232169TIFF2024504923000052.tif246169TIFF2024504923000053.tif35169

[0332] Table 14. Human anti-IL2Rg VHH amino acid sequences TIFF2024504923000054.tif238155TIFF2024504923000055.tif242155TIFF2024504923000056.tif30155

[0333] Table 15. Mouse anti-IL2Rg VHH amino acid sequences TIFF2024504923000057.tif219170TIFF2024504923000058.tif30170

[0334] Table 16: Anti-IL10Ra sdAb VHH DNA sequences TIFF2024504923000059.tif224142TIFF2024504923000060.tif243142TIFF2024504923 000061.tif243142TIFF2024504923000062.tif243142TIFF2024504923000063.tif95142

[0335] Table 17: Anti-IL2Rg VHH DNA sequences TIFF2024504923000064.tif229144TIFF2024504923000065.tif245144TIFF2024504923000066.tif245144 TIFF2024504923000067.tif245144TIFF2024504923000068.tif241144TIFF2024504923000069.tif107144

[0336] Table 18. Mouse anti-IL2Rg VHH DNA sequences TIFF2024504923000070.tif239160TIFF2024504923000071.tif243160TIFF2024504923000072.tif243160TIFF2024504923000073.tif41160

[0337] Table 19. Binding of anti-hIL10Ra / hIL2Rg dual VHH to human and cynomolgus IL2Rg-Fc TIFF2024504923000074.tif216166TIFF2024504923000075.tif143166

[0338] Table 20. Effect of linker length of IL10Ra / IL10Rg VHH dimers on pSTAT3 induction in CD8 T cells TIFF2024504923000076.tif79160TIFF2024504923000077.tif233160TIFF2024504923000078.tif233160TIFF2024504923000079.tif40160

[0339] Table 21. Effect of linker length of IL10Ra / IL10Rg VHH dimers on pSTAT3 induction in CD4 T cells TIFF2024504923000080.tif166160TIFF2024504923000081.tif233160TIFF2024504923000082.tif175160

[0340] Table 22. Effect of linker length of IL10Ra / IL10Rg VHH dimers on pSTAT3 induction in monocytes TIFF2024504923000083.tif31160TIFF2024504923000084.tif233160TIFF2024504923000085.tif233160TIFF2024504923000086.tif89160

[0341] Table 23. Activity of VHH dimers on IFNγ secretion from CD8 T cells TIFF2024504923000087.tif98160TIFF2024504923000088.tif233160TIFF2024504923000089.tif243160

[0342] Table 24. Activity of VHH dimers on Granzyme A secretion from CD8 T cells TIFF2024504923000090.tif175160TIFF2024504923000091.tif233160TIFF2024504923000092.tif166160

[0343] Table 25. Activity of VHH dimers on Granzyme B secretion from CD8 T cells TIFF2024504923000093.tif40160TIFF2024504923000094.tif233160TIFF2024504923000095.tif233160TIFF2024504923000096.tif79160

[0344] Table 26. Activity of VHH dimers on IL-9 secretion from CD8 T cells TIFF2024504923000097.tif127160TIFF2024504923000098.tif233160TIFF2024504923000099.tif214160

[0345] Table 27. Activity of VHH dimers on IL-1β secretion from LPS-treated monocytes TIFF2024504923000100.tif233160TIFF2024504923000101.tif233160TIFF2024504923000102.tif176160

[0346] Table 28. Activity of VHH dimers on IL-6 secretion from LPS-treated monocytes TIFF2024504923000103.tif31160TIFF2024504923000104.tif233160TIFF2024504923000105.tif233160TIFF2024504923000106.tif156160

[0347] Table 29. Activity of VHH dimers on TNF-α secretion in LPS-treated monocytes TIFF2024504923000107.tif60160TIFF2024504923000108.tif233160TIFF2024504923000109.tif233160TIFF2024504923000110.tif127160

[0348] Table 30. Activity of VHH dimers on IL-8 secretion from LPS-treated monocytes TIFF2024504923000111.tif69160TIFF2024504923000112.tif233160TIFF2024504923000113.tif233160TIFF2024504923000114.tif118160

[0349] Nucleic acid sequence TIFF2024504923000115.tif101160TIFF2024504923000116.tif242160TIFF2024504923000117.tif238160TIFF2024504923000118.tif237160TIFF2024504923000119.tif242160TIFF2024504923000120.tif232160TIFF2024504923000121.tif242160TIFF2024504923000122.tif242159TIFF2024504923000123.tif242160TIFF2024504923000124.tif242160TIFF2024504923000125.tif242159TIFF2024504923000126.tif242159TIFF2024504923000127.tif242160TIFF2024504923000128.tif242160TIFF2024504923000129.tif242159TIFF2024504923000130.tif242159TIFF2024504923000131.tif238159TIFF2024504923000132.tif237159TIFF2024504923000133.tif237160TIFF2024504923000134.tif242159TIFF2024504923000135.tif242159TIFF2024504923000136.tif242160TIFF2024504923000137.tif242160TIFF2024504923000138.tif242160TIFF2024504923000139.tif242160TIFF2024504923000140.tif242160TIFF2024504923000141.tif242160TIFF2024504923000142.tif242160TIFF2024504923000143.tif238160TIFF2024504923000144.tif237160TIFF2024504923000145.tif242160TIFF2024504923000146.tif232160TIFF2024504923000147.tif242160TIFF2024504923000148.tif179160.

[0350] It is understood that the embodiments described herein are for illustrative purposes only, and that various modifications or changes in light thereof will be suggested to those skilled in the art and are intended to be included within the spirit and scope of this application and the scope of the appended claims. Sequences of sequence accession numbers cited herein are incorporated herein by reference.

Claims

1. Anti-IL10Rα V H H antibody and anti-IL2Rγ V H IL10Rα / IL2Rγ binding proteins that specifically bind IL10Rα and IL2Rγ, including H antibodies, (A) The anti-IL10Rα V H the H antibody comprises a complementarity determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:264, a CDR2 comprising the amino acid sequence of SEQ ID NO:2, and a CDR3 comprising the amino acid sequence of SEQ ID NO:3; and The anti-IL2Rγ V H H antibody, i) CDR1 comprising the amino acid sequence of SEQ ID NO:25 or SEQ ID NO:270, CDR2 comprising the amino acid sequence of SEQ ID NO:26, and CDR3 comprising the amino acid sequence of SEQ ID NO:27; ii) a CDR1 comprising the amino acid sequence of SEQ ID NO:29 or SEQ ID NO:271, a CDR2 comprising the amino acid sequence of SEQ ID NO:30, and a CDR3 comprising the amino acid sequence of SEQ ID NO:31; iii) CDR1 comprising the amino acid sequence of SEQ ID NO:33 or SEQ ID NO:272, CDR2 comprising the amino acid sequence of SEQ ID NO:34, and CDR3 comprising the amino acid sequence of SEQ ID NO:35; iv) CDR1 comprising the amino acid sequence of SEQ ID NO:37 or SEQ ID NO:273, CDR2 comprising the amino acid sequence of SEQ ID NO:38, and CDR3 comprising the amino acid sequence of SEQ ID NO:39; v) CDR1 comprising the amino acid sequence of SEQ ID NO:41 or SEQ ID NO:274, CDR2 comprising the amino acid sequence of SEQ ID NO:42, and CDR3 comprising the amino acid sequence of SEQ ID NO:43; or vi) CDR1 comprising the amino acid sequence of SEQ ID NO:45 or SEQ ID NO:275, CDR2 comprising the amino acid sequence of SEQ ID NO:46, and CDR3 comprising the amino acid sequence of SEQ ID NO:47 contains; or (B) The anti-IL10Rα V H the H antibody comprises a complementarity determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:265, a CDR2 comprising the amino acid sequence of SEQ ID NO:6, and a CDR3 comprising the amino acid sequence of SEQ ID NO:7; and The anti-IL2Rγ V H H antibody, i) CDR1 comprising the amino acid sequence of SEQ ID NO:25 or SEQ ID NO:270, CDR2 comprising the amino acid sequence of SEQ ID NO:26, and CDR3 comprising the amino acid sequence of SEQ ID NO:27; ii) a CDR1 comprising the amino acid sequence of SEQ ID NO:29 or SEQ ID NO:271, a CDR2 comprising the amino acid sequence of SEQ ID NO:30, and a CDR3 comprising the amino acid sequence of SEQ ID NO:31; iii) CDR1 comprising the amino acid sequence of SEQ ID NO:33 or SEQ ID NO:272, CDR2 comprising the amino acid sequence of SEQ ID NO:34, and CDR3 comprising the amino acid sequence of SEQ ID NO:35; iv) CDR1 comprising the amino acid sequence of SEQ ID NO:37 or SEQ ID NO:273, CDR2 comprising the amino acid sequence of SEQ ID NO:38, and CDR3 comprising the amino acid sequence of SEQ ID NO:39; v) CDR1 comprising the amino acid sequence of SEQ ID NO:41 or SEQ ID NO:274, CDR2 comprising the amino acid sequence of SEQ ID NO:42, and CDR3 comprising the amino acid sequence of SEQ ID NO:43; or vi) CDR1 comprising the amino acid sequence of SEQ ID NO:45 or SEQ ID NO:275, CDR2 comprising the amino acid sequence of SEQ ID NO:46, and CDR3 comprising the amino acid sequence of SEQ ID NO:47 contains; or (C) The anti-IL10Rα V H the H antibody comprises a complementarity determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:266, a CDR2 comprising the amino acid sequence of SEQ ID NO:10, and a CDR3 comprising the amino acid sequence of SEQ ID NO:11; and The anti-IL2Rγ V H H antibody, i) CDR1 comprising the amino acid sequence of SEQ ID NO:25 or SEQ ID NO:270, CDR2 comprising the amino acid sequence of SEQ ID NO:26, and CDR3 comprising the amino acid sequence of SEQ ID NO:27; ii) a CDR1 comprising the amino acid sequence of SEQ ID NO:29 or SEQ ID NO:271, a CDR2 comprising the amino acid sequence of SEQ ID NO:30, and a CDR3 comprising the amino acid sequence of SEQ ID NO:31; iii) CDR1 comprising the amino acid sequence of SEQ ID NO:33 or SEQ ID NO:272, CDR2 comprising the amino acid sequence of SEQ ID NO:34, and CDR3 comprising the amino acid sequence of SEQ ID NO:35; iv) CDR1 comprising the amino acid sequence of SEQ ID NO:37 or SEQ ID NO:273, CDR2 comprising the amino acid sequence of SEQ ID NO:38, and CDR3 comprising the amino acid sequence of SEQ ID NO:39; v) CDR1 comprising the amino acid sequence of SEQ ID NO:41 or SEQ ID NO:274, CDR2 comprising the amino acid sequence of SEQ ID NO:42, and CDR3 comprising the amino acid sequence of SEQ ID NO:43; or vi) CDR1 comprising the amino acid sequence of SEQ ID NO:45 or SEQ ID NO:275, CDR2 comprising the amino acid sequence of SEQ ID NO:46, and CDR3 comprising the amino acid sequence of SEQ ID NO:47 contains; or (D) The anti-IL10Rα V H the H antibody comprises a complementarity determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO:13 or SEQ ID NO:267, a CDR2 comprising the amino acid sequence of SEQ ID NO:14, and a CDR3 comprising the amino acid sequence of SEQ ID NO:15; and The anti-IL2Rγ V H H antibody, i) CDR1 comprising the amino acid sequence of SEQ ID NO:25 or SEQ ID NO:270, CDR2 comprising the amino acid sequence of SEQ ID NO:26, and CDR3 comprising the amino acid sequence of SEQ ID NO:27; ii) a CDR1 comprising the amino acid sequence of SEQ ID NO:29 or SEQ ID NO:271, a CDR2 comprising the amino acid sequence of SEQ ID NO:30, and a CDR3 comprising the amino acid sequence of SEQ ID NO:31; iii) CDR1 comprising the amino acid sequence of SEQ ID NO:33 or SEQ ID NO:272, CDR2 comprising the amino acid sequence of SEQ ID NO:34, and CDR3 comprising the amino acid sequence of SEQ ID NO:35; iv) CDR1 comprising the amino acid sequence of SEQ ID NO:37 or SEQ ID NO:273, CDR2 comprising the amino acid sequence of SEQ ID NO:38, and CDR3 comprising the amino acid sequence of SEQ ID NO:39; v) CDR1 comprising the amino acid sequence of SEQ ID NO:41 or SEQ ID NO:274, CDR2 comprising the amino acid sequence of SEQ ID NO:42, and CDR3 comprising the amino acid sequence of SEQ ID NO:43; or vi) CDR1 comprising the amino acid sequence of SEQ ID NO:45 or SEQ ID NO:275, CDR2 comprising the amino acid sequence of SEQ ID NO:46, and CDR3 comprising the amino acid sequence of SEQ ID NO:47 contains; or (E) The anti-IL10Rα V H the H antibody comprises a complementarity determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO:17 or SEQ ID NO:268, a CDR2 comprising the amino acid sequence of SEQ ID NO:18, and a CDR3 comprising the amino acid sequence of SEQ ID NO:19; and The anti-IL2Rγ V H H antibody, i) CDR1 comprising the amino acid sequence of SEQ ID NO:25 or SEQ ID NO:270, CDR2 comprising the amino acid sequence of SEQ ID NO:26, and CDR3 comprising the amino acid sequence of SEQ ID NO:27; ii) a CDR1 comprising the amino acid sequence of SEQ ID NO:29 or SEQ ID NO:271, a CDR2 comprising the amino acid sequence of SEQ ID NO:30, and a CDR3 comprising the amino acid sequence of SEQ ID NO:31; iii) CDR1 comprising the amino acid sequence of SEQ ID NO:33 or SEQ ID NO:272, CDR2 comprising the amino acid sequence of SEQ ID NO:34, and CDR3 comprising the amino acid sequence of SEQ ID NO:35; iv) CDR1 comprising the amino acid sequence of SEQ ID NO:37 or SEQ ID NO:273, CDR2 comprising the amino acid sequence of SEQ ID NO:38, and CDR3 comprising the amino acid sequence of SEQ ID NO:39; v) CDR1 comprising the amino acid sequence of SEQ ID NO:41 or SEQ ID NO:274, CDR2 comprising the amino acid sequence of SEQ ID NO:42, and CDR3 comprising the amino acid sequence of SEQ ID NO:43; or vi) CDR1 comprising the amino acid sequence of SEQ ID NO:45 or SEQ ID NO:275, CDR2 comprising the amino acid sequence of SEQ ID NO:46, and CDR3 comprising the amino acid sequence of SEQ ID NO:47 contains; or (F) The anti-IL10Rα V H the H antibody comprises a complementarity determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO:21 or SEQ ID NO:269, a CDR2 comprising the amino acid sequence of SEQ ID NO:22, and a CDR3 comprising the amino acid sequence of SEQ ID NO:23; and The anti-IL2Rγ V H H antibody, i) CDR1 comprising the amino acid sequence of SEQ ID NO:25 or SEQ ID NO:270, CDR2 comprising the amino acid sequence of SEQ ID NO:26, and CDR3 comprising the amino acid sequence of SEQ ID NO:27; ii) a CDR1 comprising the amino acid sequence of SEQ ID NO:29 or SEQ ID NO:271, a CDR2 comprising the amino acid sequence of SEQ ID NO:30, and a CDR3 comprising the amino acid sequence of SEQ ID NO:31; iii) CDR1 comprising the amino acid sequence of SEQ ID NO:33 or SEQ ID NO:272, CDR2 comprising the amino acid sequence of SEQ ID NO:34, and CDR3 comprising the amino acid sequence of SEQ ID NO:35; iv) CDR1 comprising the amino acid sequence of SEQ ID NO:37 or SEQ ID NO:273, CDR2 comprising the amino acid sequence of SEQ ID NO:38, and CDR3 comprising the amino acid sequence of SEQ ID NO:39; v) CDR1 comprising the amino acid sequence of SEQ ID NO:41 or SEQ ID NO:274, CDR2 comprising the amino acid sequence of SEQ ID NO:42, and CDR3 comprising the amino acid sequence of SEQ ID NO:43; or vi) CDR1 comprising the amino acid sequence of SEQ ID NO:45 or SEQ ID NO:275, CDR2 comprising the amino acid sequence of SEQ ID NO:46, and CDR3 comprising the amino acid sequence of SEQ ID NO:47 Including, The IL10Rα / IL2Rγ binding protein.

2. Anti-IL10Rα V H The anti-IL2Rγ V H The IL10Rα / IL2Rγ binding protein of claim 1, wherein the H antibody is at the C-terminus.

3. 3. The IL10Rα / IL2Rγ binding protein of claim 2, comprising a sequence having at least 90% identity to any one of SEQ ID NOs:49-59.

4. Anti-IL2Rγ V H H antibody at the N-terminus, and H The IL10Rα / IL2Rγ binding protein of claim 1, wherein the H antibody is at the C-terminus.

5. 5. The IL10Rα / IL2Rγ binding protein of claim 4, comprising at least 90% identity to any one of SEQ ID NOs:60 and 61.

6. Anti-IL10Rα V H H antibody and the anti-IL2Rγ V H 6. The IL10Rα / IL2Rγ binding protein of any one of claims 1 to 5, wherein the H antibody is linked to the IL10Rα / IL2Rγ binding protein by a peptide linker.

7. The IL10Rα / IL2Rγ binding protein of claim 6, wherein the peptide linker comprises 1 to 50 amino acids.

8. 8. The IL10Rα / IL2Rγ binding protein of any one of claims 1 to 7, which is conjugated to an Fc polypeptide or an Fc domain.

9. The IL10Rα / IL2Rγ binding protein of claim 8, comprising SEQ ID NO:556 or SEQ ID NO:

558.

10. 9. The IL10Rα / IL2Rγ binding protein of any one of claims 1 to 8, which is PEGylated.

11. Anti-IL10Rα V H H antibody and anti-IL2Rγ V H An IL10Rα / IL2Rγ binding protein that specifically binds IL10Rα and IL2Rγ, comprising an H antibody, wherein the IL10Rα / IL2Rγ binding protein is linked to an Fc polypeptide or Fc domain from IgG1, IgG2, IgG3, or IgG4.

12. A first polypeptide of formula #1: Anti-IL10Rα V H H antibody-L1 a -UH1-Fc1[1] and a second polypeptide of formula #2: Anti-IL2Rγ V H H antibody-L2 b -UH2-Fc2[2] 1. A heterodimeric IL10Rα binding protein / IL2Rγ binding protein pair comprising: During the ceremony, L1 and L2 are GSA linkers and a and b are independently selected from 0 (absent) or 1 (present); - UH1 and UH2 are each an upper hinge domain of a human immunoglobulin independently selected from the group consisting of IgG1, IgG2, IgG3, and IgG4 upper hinges, optionally containing the amino acid substitution C220S (EU numbering); Fc1 is a polypeptide comprising the lower hinge domain, the CH2 domain, and the CH3 domain of a human immunoglobulin selected from the group consisting of IgG1, IgG2, IgG3, and IgG4, and comprises one or more amino acid substitutions that promote heterodimerization with Fc2; and - FC2 is a polypeptide comprising the lower hinge domain, the CH2 domain, and the CH3 domain of a human immunoglobulin selected from the group consisting of IgG1, IgG2, IgG3, and IgG4, and comprises one or more amino acid substitutions that promote heterodimerization with Fc1; and The polypeptide of formula 1 and the polypeptide of formula 2 are linked by at least one interchain disulfide bond; and (A) The anti-IL10Rα V H the H antibody comprises a complementarity determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:264, a CDR2 comprising the amino acid sequence of SEQ ID NO:2, and a CDR3 comprising the amino acid sequence of SEQ ID NO:3; and The anti-IL2Rγ V H H antibody, i) CDR1 comprising the amino acid sequence of SEQ ID NO:25 or SEQ ID NO:270, CDR2 comprising the amino acid sequence of SEQ ID NO:26, and CDR3 comprising the amino acid sequence of SEQ ID NO:27; ii) a CDR1 comprising the amino acid sequence of SEQ ID NO:29 or SEQ ID NO:271, a CDR2 comprising the amino acid sequence of SEQ ID NO:30, and a CDR3 comprising the amino acid sequence of SEQ ID NO:31; iii) CDR1 comprising the amino acid sequence of SEQ ID NO:33 or SEQ ID NO:272, CDR2 comprising the amino acid sequence of SEQ ID NO:34, and CDR3 comprising the amino acid sequence of SEQ ID NO:35; iv) CDR1 comprising the amino acid sequence of SEQ ID NO:37 or SEQ ID NO:273, CDR2 comprising the amino acid sequence of SEQ ID NO:38, and CDR3 comprising the amino acid sequence of SEQ ID NO:39; v) CDR1 comprising the amino acid sequence of SEQ ID NO:41 or SEQ ID NO:274, CDR2 comprising the amino acid sequence of SEQ ID NO:42, and CDR3 comprising the amino acid sequence of SEQ ID NO:43; or vi) CDR1 comprising the amino acid sequence of SEQ ID NO:45 or SEQ ID NO:275, CDR2 comprising the amino acid sequence of SEQ ID NO:46, and CDR3 comprising the amino acid sequence of SEQ ID NO:47 contains; or (B) The anti-IL10Rα V H the H antibody comprises a complementarity determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:265, a CDR2 comprising the amino acid sequence of SEQ ID NO:6, and a CDR3 comprising the amino acid sequence of SEQ ID NO:7; and The anti-IL2Rγ V H H antibody, i) CDR1 comprising the amino acid sequence of SEQ ID NO:25 or SEQ ID NO:270, CDR2 comprising the amino acid sequence of SEQ ID NO:26, and CDR3 comprising the amino acid sequence of SEQ ID NO:27; ii) a CDR1 comprising the amino acid sequence of SEQ ID NO:29 or SEQ ID NO:271, a CDR2 comprising the amino acid sequence of SEQ ID NO:30, and a CDR3 comprising the amino acid sequence of SEQ ID NO:31; iii) CDR1 comprising the amino acid sequence of SEQ ID NO:33 or SEQ ID NO:272, CDR2 comprising the amino acid sequence of SEQ ID NO:34, and CDR3 comprising the amino acid sequence of SEQ ID NO:35; iv) CDR1 comprising the amino acid sequence of SEQ ID NO:37 or SEQ ID NO:273, CDR2 comprising the amino acid sequence of SEQ ID NO:38, and CDR3 comprising the amino acid sequence of SEQ ID NO:39; v) CDR1 comprising the amino acid sequence of SEQ ID NO:41 or SEQ ID NO:274, CDR2 comprising the amino acid sequence of SEQ ID NO:42, and CDR3 comprising the amino acid sequence of SEQ ID NO:43; or vi) CDR1 comprising the amino acid sequence of SEQ ID NO:45 or SEQ ID NO:275, CDR2 comprising the amino acid sequence of SEQ ID NO:46, and CDR3 comprising the amino acid sequence of SEQ ID NO:47 contains; or (C) The anti-IL10Rα V H the H antibody comprises a complementarity determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:266, a CDR2 comprising the amino acid sequence of SEQ ID NO:10, and a CDR3 comprising the amino acid sequence of SEQ ID NO:11; and The anti-IL2Rγ V H H antibody, i) CDR1 comprising the amino acid sequence of SEQ ID NO:25 or SEQ ID NO:270, CDR2 comprising the amino acid sequence of SEQ ID NO:26, and CDR3 comprising the amino acid sequence of SEQ ID NO:27; ii) a CDR1 comprising the amino acid sequence of SEQ ID NO:29 or SEQ ID NO:271, a CDR2 comprising the amino acid sequence of SEQ ID NO:30, and a CDR3 comprising the amino acid sequence of SEQ ID NO:31; iii) CDR1 comprising the amino acid sequence of SEQ ID NO:33 or SEQ ID NO:272, CDR2 comprising the amino acid sequence of SEQ ID NO:34, and CDR3 comprising the amino acid sequence of SEQ ID NO:35; iv) CDR1 comprising the amino acid sequence of SEQ ID NO:37 or SEQ ID NO:273, CDR2 comprising the amino acid sequence of SEQ ID NO:38, and CDR3 comprising the amino acid sequence of SEQ ID NO:39; v) CDR1 comprising the amino acid sequence of SEQ ID NO:41 or SEQ ID NO:274, CDR2 comprising the amino acid sequence of SEQ ID NO:42, and CDR3 comprising the amino acid sequence of SEQ ID NO:43; or vi) CDR1 comprising the amino acid sequence of SEQ ID NO:45 or SEQ ID NO:275, CDR2 comprising the amino acid sequence of SEQ ID NO:46, and CDR3 comprising the amino acid sequence of SEQ ID NO:47 contains; or (D) The anti-IL10Rα V H the H antibody comprises a complementarity determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO:13 or SEQ ID NO:267, a CDR2 comprising the amino acid sequence of SEQ ID NO:14, and a CDR3 comprising the amino acid sequence of SEQ ID NO:15; and The anti-IL2Rγ V H H antibody, i) CDR1 comprising the amino acid sequence of SEQ ID NO:25 or SEQ ID NO:270, CDR2 comprising the amino acid sequence of SEQ ID NO:26, and CDR3 comprising the amino acid sequence of SEQ ID NO:27; ii) a CDR1 comprising the amino acid sequence of SEQ ID NO:29 or SEQ ID NO:271, a CDR2 comprising the amino acid sequence of SEQ ID NO:30, and a CDR3 comprising the amino acid sequence of SEQ ID NO:31; iii) CDR1 comprising the amino acid sequence of SEQ ID NO:33 or SEQ ID NO:272, CDR2 comprising the amino acid sequence of SEQ ID NO:34, and CDR3 comprising the amino acid sequence of SEQ ID NO:35; iv) CDR1 comprising the amino acid sequence of SEQ ID NO:37 or SEQ ID NO:273, CDR2 comprising the amino acid sequence of SEQ ID NO:38, and CDR3 comprising the amino acid sequence of SEQ ID NO:39; v) CDR1 comprising the amino acid sequence of SEQ ID NO:41 or SEQ ID NO:274, CDR2 comprising the amino acid sequence of SEQ ID NO:42, and CDR3 comprising the amino acid sequence of SEQ ID NO:43; or vi) CDR1 comprising the amino acid sequence of SEQ ID NO:45 or SEQ ID NO:275, CDR2 comprising the amino acid sequence of SEQ ID NO:46, and CDR3 comprising the amino acid sequence of SEQ ID NO:47 contains; or (E) The anti-IL10Rα V H the H antibody comprises a complementarity determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO:17 or SEQ ID NO:268, a CDR2 comprising the amino acid sequence of SEQ ID NO:18, and a CDR3 comprising the amino acid sequence of SEQ ID NO:19; and The anti-IL2Rγ V H H antibody, i) CDR1 comprising the amino acid sequence of SEQ ID NO:25 or SEQ ID NO:270, CDR2 comprising the amino acid sequence of SEQ ID NO:26, and CDR3 comprising the amino acid sequence of SEQ ID NO:27; ii) a CDR1 comprising the amino acid sequence of SEQ ID NO:29 or SEQ ID NO:271, a CDR2 comprising the amino acid sequence of SEQ ID NO:30, and a CDR3 comprising the amino acid sequence of SEQ ID NO:31; iii) CDR1 comprising the amino acid sequence of SEQ ID NO:33 or SEQ ID NO:272, CDR2 comprising the amino acid sequence of SEQ ID NO:34, and CDR3 comprising the amino acid sequence of SEQ ID NO:35; iv) CDR1 comprising the amino acid sequence of SEQ ID NO:37 or SEQ ID NO:273, CDR2 comprising the amino acid sequence of SEQ ID NO:38, and CDR3 comprising the amino acid sequence of SEQ ID NO:39; v) CDR1 comprising the amino acid sequence of SEQ ID NO:41 or SEQ ID NO:274, CDR2 comprising the amino acid sequence of SEQ ID NO:42, and CDR3 comprising the amino acid sequence of SEQ ID NO:43; or vi) CDR1 comprising the amino acid sequence of SEQ ID NO:45 or SEQ ID NO:275, CDR2 comprising the amino acid sequence of SEQ ID NO:46, and CDR3 comprising the amino acid sequence of SEQ ID NO:47 contains; or (F) The anti-IL10Rα V H the H antibody comprises a complementarity determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO:21 or SEQ ID NO:269, a CDR2 comprising the amino acid sequence of SEQ ID NO:22, and a CDR3 comprising the amino acid sequence of SEQ ID NO:23; and The anti-IL2Rγ V H H antibody, i) CDR1 comprising the amino acid sequence of SEQ ID NO:25 or SEQ ID NO:270, CDR2 comprising the amino acid sequence of SEQ ID NO:26, and CDR3 comprising the amino acid sequence of SEQ ID NO:27; ii) a CDR1 comprising the amino acid sequence of SEQ ID NO:29 or SEQ ID NO:271, a CDR2 comprising the amino acid sequence of SEQ ID NO:30, and a CDR3 comprising the amino acid sequence of SEQ ID NO:31; iii) CDR1 comprising the amino acid sequence of SEQ ID NO:33 or SEQ ID NO:272, CDR2 comprising the amino acid sequence of SEQ ID NO:34, and CDR3 comprising the amino acid sequence of SEQ ID NO:35; iv) CDR1 comprising the amino acid sequence of SEQ ID NO:37 or SEQ ID NO:273, CDR2 comprising the amino acid sequence of SEQ ID NO:38, and CDR3 comprising the amino acid sequence of SEQ ID NO:39; v) CDR1 comprising the amino acid sequence of SEQ ID NO:41 or SEQ ID NO:274, CDR2 comprising the amino acid sequence of SEQ ID NO:42, and CDR3 comprising the amino acid sequence of SEQ ID NO:43; or vi) CDR1 comprising the amino acid sequence of SEQ ID NO:45 or SEQ ID NO:275, CDR2 comprising the amino acid sequence of SEQ ID NO:46, and CDR3 comprising the amino acid sequence of SEQ ID NO:47 contains; or (G) The anti-IL10Rα V H H antibody が, a CDR1 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to any CDR1 sequence in a row of Table 10, or having 0, 1, 2, or 3 amino acid changes, optionally conservative amino acid changes, compared to said sequence; a CDR2 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to any CDR2 sequence in a row of Table 10, or having 0, 1, 2, or 3 amino acid changes, optionally conservative amino acid changes, compared to said sequence; and A CDR3 sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to any of the CDR3 sequences listed in Table 10, or having 0, 1, 2, or 3 amino acid changes, optionally conservative amino acid changes, compared to the sequence. and The anti-IL2Rγ V H H antibody, a CDR1 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to any CDR1 sequence listed in Table 11 or Table 12, or having 0, 1, 2, or 3 amino acid changes, optionally conservative amino acid changes, compared to said sequence; a CDR2 having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to any CDR2 sequence listed in Table 11 or Table 12, or having 0, 1, 2, or 3 amino acid changes, optionally conservative amino acid changes, compared to said sequence; and A CDR3 sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to any CDR3 sequence listed in Table 11 or Table 12, or having 0, 1, 2, or 3 amino acid changes, optionally conservative amino acid changes, compared to said sequence. Including, The heterodimeric IL10Rα-binding protein / IL2Rγ-binding protein pair.

13. 13. An isolated nucleic acid encoding an IL10Rα / IL2Rγ binding protein according to any one of claims 1 to 10, or a heterodimeric IL10Rα binding protein / IL2Rγ binding protein pair according to claim 12.

14. An expression vector comprising the nucleic acid of claim 13.

15. 15. An isolated host cell comprising the vector of claim 14.

16. A pharmaceutical composition comprising an IL10Rα / IL2Rγ binding protein according to any one of claims 1 to 10, or a heterodimeric IL10Rα binding protein / IL2Rγ binding protein pair according to claim 12, and a pharma- ceutically acceptable carrier.

17. A pharmaceutical composition comprising an IL10Rα / IL2Rγ binding protein according to any one of claims 1 to 10 or a heterodimeric IL10Rα binding protein / IL2Rγ binding protein pair according to claim 12, or a pharmaceutical composition according to claim 16, for treating a neoplastic disease in a subject in need thereof.

18. A pharmaceutical composition comprising the IL10Rα / IL2Rγ binding protein of any one of claims 1 to 10 or the heterodimeric IL10Rα binding protein / IL2Rγ binding protein pair of claim 12, or the pharmaceutical composition of claim 16, for treating an autoimmune or inflammatory disease, disorder or condition in a subject in need thereof.

19. 13. A pharmaceutical composition comprising the IL10Rα / IL2Rγ binding protein of any one of claims 1 to 10 or the heterodimeric IL10Rα binding protein / IL2Rγ binding protein pair of claim 12 for use in a method of selectively inducing an activity in one or more of a first cell type over one or more of a second cell type, the method comprising contacting a population of cells comprising both the first cell type and the second cell type with the IL10Rα / IL2Rγ binding protein or the heterodimeric IL10Rα binding protein / IL2Rγ binding protein pair, thereby selectively inducing an activity in one or more of the first cell types over one or more of the second cell types.

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