Genetically engineered interleukin-10 and its fusion protein

Abstract

The present disclosure provides IL-10 variant proteins, fusion proteins comprising IL-10 variant proteins and polypeptides, and uses thereof. The disclosure also provides methods for producing same and pharmaceutical compositions comprising same.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 385,130, filed November 28, 2022, the contents of which are incorporated herein by reference in their entirety.

[0002] Sequence Listing Description The contents of the electronic sequence listing (size: 39,626 bytes; and creation date: November 23, 2023) with file name 23P0592-seq list.xml are incorporated herein by reference in their entirety.

[0003] FIELD OF THE INVENTION The present disclosure relates to IL-10 variant proteins, fusion proteins, and uses thereof. The present disclosure further relates to methods of producing IL-10 variant proteins and fusion proteins. [Background technology]

[0004] IL-10 is an important immunoregulatory cytokine with therapeutic potential. Binding of IL-10 to the IL-10 receptor leads to activation of three STAT transcription factors: STAT1, STAT3, and STAT5. The IL-10 / STAT3 axis contributes to anti-inflammatory and immune tolerance. 1 IL-10 exerts antitumor activity through activation of intratumoral cytotoxic CD8+ T cells 2,3 IL-10 binds to the IL-10 receptor on tumor-resident CD8+ T cells and can directly activate and expand tumor-resident CD8+ T cells. 4 Administration of PEGylated IL-10 (pegylodecakin) induced systemic immune activation in cancer patients, including elevated granzyme B and IFNγ levels and sustained expansion of activated CD8+ T cells both in the blood and tumor. 5 .

[0005] Systemic IL-10 therapy has been investigated in clinical trials, but efficacy has been elusive due to its short half-life and systemic toxicity. Various approaches have been investigated to prevent systemic toxicity, including oral administration of IL-10 recombinant protein or fusion of IL-10 to a targeting antibody. 6,7,8 Local delivery of IL-10 could also be achieved by local administration of plasmid DNA, viruses, mRNA, or using IL-10-transduced host cells, such as tumor-infiltrating lymphocytes (TILs), Tregs, or chimeric antigen receptor (CAR) T cells that home to the tumor site. Genetically engineered TILs or CAR T cells expressing IL-10 could provide autocrine signaling to enhance T cell effector function and proliferation. For these approaches, IL-10 variant proteins with relatively strong CD8 stimulatory activity are required.

[0006] Attempts have been made to generate IL-10 variant proteins with higher binding affinity to the IL-10 receptor using a yeast display system. 9,10,11 Because wild-type IL-10 cannot be readily expressed on the yeast surface, a genetically engineered monomeric IL-10 variant protein (M1) was used in yeast display to identify IL-10 variant proteins with enhanced IL-10 receptor binding. These variants contain multiple substitutions and exhibit enhanced STAT3 activation in some cell types but not others. For example, the IL-10 variant protein Super 10, which contains four amino acid substitutions (N18Y, N92Q, T100D, and R104W), induced enhanced STAT1 activity but not STAT3 activity in CD4+ or CD8+ T cells. 11 Additionally, the ability of Super 10 to induce granzyme B secretion in CD8+ T cells was significantly reduced when compared with wild-type IL-10. Different screening strategies for IL-10 variant proteins will likely affect the results. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] WO2021181091A1 [Patent Document 2] WO2021243057A1 [Patent Document 3] PCT / US22 / 77660 [Patent Document 4] WO2021231741 Summary of the Invention [Problem to be solved by the invention]

[0008] The present disclosure relates to novel IL-10 variant proteins and IL-10 variant fusion molecules with altered CD8 stimulatory activity. IL-10 variant proteins can be administered directly to tumor sites, e.g., via plasmids, viruses, or RNA, resulting in localized expression of the IL-10 variant proteins in tumor tissue to avoid systemic toxicity. IL-10 variant proteins can also be administered using IL-10 variant-transduced host cells. In addition, fusion proteins containing IL-10 variant proteins, e.g., IL-10-Fc or antibody-IL-10, can be used in the preparation of biologics. [Means for solving the problem]

[0009] To achieve the above-mentioned objectives, one embodiment of the present disclosure provided herein is an IL-10 variant protein comprising: (1) a single substitution of an amino acid at position 18 compared to that of wild-type IL-10; (2) a first substitution of an amino acid at position 18 and a second substitution of an amino acid at position 104 or 107 compared to that of wild-type IL-10; or (3) a first substitution of an amino acid at position 18, a second substitution of an amino acid at position 104, and a third substitution of an amino acid at position 107 compared to that of wild-type IL-10.

[0010] Preferably, wild-type IL-10 comprises an amino acid sequence having at least 80%, preferably at least 90%, at least 95%, at least 98%, more preferably at least 99% identity to SEQ ID NO:2.

[0011] Preferably, the substitution of the amino acid at position 18 comprises N18A, N18D, N18I, N18Y, N18M, N18F, N18L, N18W, N18K or N18R.

[0012] Preferably, the substitution of the amino acid at position 104 comprises R104Q.

[0013] Preferably, the substitution of the amino acid at position 107 comprises R107A, R107E, R107Q or R107D.

[0014] Preferably, the IL-10 variant protein is selected from the group consisting of N18A / R104Q, N18A / R107A, N18A / R107E, N18A / R107Q, N18A / R107D, N18D / R104Q, N18D / R107A, N18D / R107E, N18D / R107Q, N18D / R107D, N18M / R104Q, N18M / R107A, N18M / R107E, N18M / R107Q, N18M / R107D, N18F / R104Q, N18F / R107A, N18F / R107E, N18M / R107Q, N18M / R107D, 07E, N18F / R107Q, N18F / R107D, N18L / R104Q, N18L / R107A, N18L / R107E, N18L / R107Q, N18L / R107D, N18A / R104Q / R107A, N18A / R104Q / R107E, N18A / R104Q / R107Q, N18A / R104Q / R107D, N18D / R104Q / R107A, N18D / R104Q / R107E, N18D / R104Q / R107Q, N18D / R104Q / R107D, N 18M / R104Q / R107A, N18M / R104Q / R107E, N18M / R104Q / R107Q, N18M / R104Q / R107D, N18F / R104Q / R107A, N18F / R104Q / R107E, N18F / R10 4Q / R107Q, N18F / R104Q / R107D, N18L / R104Q / R107A, N18L / R104Q / R107E, N18L / R104Q / R107Q, N18L / R104Q / R107D, N18W / R104Q / R107 A, N18W / R104Q / R107A, N18W / R104Q / R107Q, N18W / R104Q / R107D, N18K / R104Q / R107A, N18K / R104Q / R107E, N18K / R104Q / R107Q, N18K / R104Q / R107D, N18R / R104Q / R107A, N18R / R104Q / R107E, N18R / R104Q / R107Q, and N18R / R104Q / R107D.

[0015] Preferably, the IL-10 variant protein is a monomer or a dimer.

[0016] Preferably, the IL-10 variant protein further comprises a signal peptide.

[0017] Preferably, the signal peptide comprises the amino acid sequence of SEQ ID NO:25.

[0018] In another aspect of the present disclosure, there is provided herein a fusion protein comprising: (1) a polypeptide comprising an antibody or fragment thereof, an antagonist, a ligand of a receptor or target protein, a half-life extending moiety, or a protein trap; and (2) an IL-10 variant protein as described above fused to the polypeptide.

[0019] Preferably, the polypeptide is fused to the IL-10 variant protein via a linker.

[0020] Preferably, the linker comprises the amino acid sequence of SEQ ID NO: 19-24.

[0021] Preferably, the IL-10 variant protein is fused to the N-terminus or C-terminus of the polypeptide.

[0022] Preferably, the IL-10 variant protein is a monomer or a dimer.

[0023] Preferably, the fusion protein further comprises the amino acid sequence of SEQ ID NO:18.

[0024] Preferably, the antibody comprises an anti-PD-L1 antibody.

[0025] Preferably, IL-10 variant proteins: An amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95% identity with SEQ ID NO: 14 and Anti-PD-L1 antibodies: YP7G IgG1, comprising a heavy chain amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95% identity to SEQ ID NO: 26 and a light chain amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95% identity to SEQ ID NO: 27; YP7G IgG4 comprising a heavy chain amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95% identity to SEQ ID NO: 28 and a light chain amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95% identity to SEQ ID NO: 29; or avelumab comprising a heavy chain amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95% identity to SEQ ID NO: 30 and a light chain amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95% identity to SEQ ID NO: 31. Includes.

[0026] Preferably, (1) the antibody is a human, humanized, or chimeric antibody; (2) the antibody is a full-length antibody of class IgG, optionally wherein the class IgG antibody has an isotype selected from IgG1, IgG2, IgG3, and IgG4; (3) the antibody comprises an Fc region variant, optionally an Fc region variant that alters effector function and / or a variant that alters antibody half-life; (4) the antibody is optionally an antibody fragment selected from the group consisting of F(ab')2, Fab', Fab, Fv, single domain antibody (VHH), and scFv; (5) the antibody comprises an immunoconjugate, optionally wherein the immunoconjugate comprises a therapeutic agent for the treatment of a CSF1R-mediated, PDL1-mediated, PD1-mediated, or VEGF-mediated disease or condition; or (6) the antibody is a multispecific antibody, optionally a bispecific antibody.

[0027] In another aspect of the present disclosure, there is provided herein an isolated polynucleotide or vector encoding the above-mentioned IL-10 variant protein or the above-mentioned fusion protein.

[0028] In another aspect of the disclosure, provided herein is an isolated host cell comprising the isolated polynucleotide or the vector described above.

[0029] In another aspect of the present disclosure, provided herein is a method for producing an IL-10 variant protein or a fusion protein, comprising culturing the host cell described above, such that the IL-10 variant protein or fusion protein is produced.

[0030] In another aspect of the present disclosure, there is provided herein a pharmaceutical composition comprising the above-mentioned IL-10 variant protein, the above-mentioned fusion protein, or the above-mentioned isolated polynucleotide or the above-mentioned vector, optionally together with a pharmaceutically acceptable carrier, diluent or excipient.

[0031] In another aspect of the present disclosure, there is provided herein use of an IL-10 variant protein as described above, a fusion protein as described above, or an isolated polynucleotide or vector as described above for the manufacture of a medicament. [Brief explanation of the drawings]

[0032] [Figure 1]

[0023] Figure 1 depicts STAT3 activity induced by IL-10 variant proteins in a cell-based screen using HeLa IL-10R1-STAT3 luciferase reporter cells. HeLa IL-10R1-STAT3 reporter cells were transfected with plasmids expressing wild-type IL-10 or its variants. Luciferase signals were measured 48 hours after transfection. Data shown are the percentage change in relative light units (RLU) compared to wild-type IL-10 plasmid transfection. [Figure 2]Figure 1 depicts dose-dependent activation of STAT3 by IL-10-containing culture supernatant in HeLa IL-10R1-STAT3 reporter cells. IL-10-containing culture supernatant was collected from wild-type HeLa cells transfected with increasing amounts of wild-type IL-10 or variant-encoding plasmids (0-60 ng / well). Luciferase signals were measured 16 hours after addition of IL-10-containing culture supernatant to HeLa IL-10R1-STAT3 reporter cells. [Figure 3] Figure 1 depicts the ability of N18A and N18D to better enhance granzyme B secretion from CD8+ T cells compared to wild-type IL-10, Super 10, and R5A11. IL-10-containing culture supernatant collected from HeLa cells transfected with IL-10 plasmid was used to treat CD8+ T cells. [Figure 4] Figure 1 depicts that purified recombinant IL-10 variant proteins N18A and N18D exhibited higher STAT3 activation activity in HeLa IL-10R1-STAT3 reporter cells compared to wild-type (WT) IL-10. All data are presented as fold change in relative luminescence units (RLU) compared to WT and are the mean ± SD of the indicated number of independent biological replicates, **p≦0.01, ***p≦0.001. [Figure 5] FIG. 1 depicts that purified IL-10 variant proteins, including N18A, N18F, N18I, N18L and N18Y, induced higher granzyme B secretion from CD8+ T cells when compared to wild-type IL-10. [Figure 6] 1 depicts the purification yield of IL-10-Fc fusion proteins. Addition of R104Q or R104Q / R107A substitutions to the IL-10(N18 variant)-Fc fusion protein increased the protein purification yield. [Figure 7]Figure 1 depicts the results of STAT3 activation in HeLa IL-10R1-STAT3 reporter cells induced by IL-10-Fc fusion proteins. (A) IL-10(N18I)-Fc and IL-10(N18I / R104Q)-Fc induced higher STAT3 activation compared to wild-type IL-10(WT)-Fc fusion protein. (B) IL-10(N18Y)-Fc, IL-10(N18Y / R104Q), and IL-10(N18I / R104Q / R107A)-Fc induced higher STAT3 activation compared to wild-type IL-10(WT)-Fc fusion protein. [Figure 8] Figure 8A depicts the results of STAT3 activation in HeLa IL-10R1-STAT3 reporter cells induced by avelumab-IL-10 (wild-type), avelumab-IL-10 (R107A), and avelumab-IL-10 (N18A / R107A) fusion proteins. The addition of the N18A substitution to avelumab-IL-10 (R107A) resulted in higher granzyme B secretion from CD8+ T cells. Figure 8B depicts the purification yield of avelumab-IL-10 fusion protein. Data are presented as mean ± SD, *P<0.05 (n=3). [Figure 9]

[0023] Figure 1 depicts the results of dose-dependent granzyme B secretion from CD8+ T cells treated with YP7G-IL-10(R107A) and YP7G-IL-10(N18A / R107A) fusion proteins in (A) IgG1 and (B) IgG4 formats. Addition of the N18A substitution to YP7G-IL-10(R107A) resulted in higher granzyme B secretion from CD8+ T cells. DETAILED DESCRIPTION OF THE INVENTION

[0033] These and other aspects of the present disclosure will now be described in more detail with respect to other embodiments described herein. It should be understood that the present invention may be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

[0034] The nomenclature used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used in the description of the invention and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise.

[0035] As used herein, the terms "comprise," "including," "including," "including," "have," "having," "contain," "containing," or any other variation thereof, are intended to cover a non-exclusive inclusion subject to any limitations expressly stated. For example, a composition, mixture, process, or method that includes a list of elements is not necessarily limited to only those elements and may include other elements not expressly listed or inherent in such composition, mixture, process, or method.

[0036] As used herein, the term "about" indicates that a value includes, for example, the inherent variation of error for the measuring device, the method utilized to determine the value, or variation between study subjects. Typically, the term is intended to encompass approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% or less, depending on the context.

[0037] Use of the term "or" in the claims is used to mean "and / or" unless expressly indicated to mean only alternatives or that the alternatives are mutually exclusive. However, the present disclosure supports a definition that means only alternatives and "and / or."

[0038] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications, patent disclosures, patents, and other references cited herein are incorporated by reference in their entirety for the teachings relevant to the sentence and / or paragraph in which the reference is presented.

[0039] The following representative examples illustrate various features and embodiments of the present disclosure, which are intended to be illustrative and not limiting. Those skilled in the art will readily appreciate that the specific examples are merely exemplary of the invention as more fully described in the claims that follow. It should be understood that all embodiments and features described in this application are interchangeable and combinable with all embodiments contained therein.

[0040] The object of the present disclosure is to provide IL-10 variant proteins comprising: (1) a single substitution of an amino acid at position 18 compared to the amino acid of wild-type IL-10; (2) a first substitution of an amino acid at position 18 and a second substitution of an amino acid at position 104 or 107 compared to the amino acid of wild-type IL-10; or (3) a first substitution of an amino acid at position 18, a second substitution of an amino acid at position 104, and a third substitution of an amino acid at position 107 compared to the amino acid of wild-type IL-10.

[0041] In embodiments, wild-type IL-10 comprises an amino acid sequence having at least 80%, preferably at least 90%, at least 95%, at least 98%, more preferably at least 99% identity to SEQ ID NO:2.

[0042] The term "IL-10 variant protein" refers to wild-type IL-10 having one, two, three, or more amino acid substitutions within its sequence. The substitution numbering for IL-10 variant proteins is based on the wild-type IL-10 sequence, e.g., SEQ ID NO: 2. The term "wild-type IL-10" refers to naturally occurring IL-10 that does not contain artificial mutations. Wild-type IL-10 from various organisms includes the following sequences referenced by NCBI accession numbers: 1INR_A (human), NP_001129092.2 (chimpanzee), XP_004028338.1 (gorilla), and ABI63906.1 (rhesus macaque), XP_046539742.1 (zebra), XP_044614621.1 (donkey), XP_006094927.1 (small brown bat), and XP_058394138.1 (rhinoceros).

[0043] In embodiments, the amino acid substitution at position 18 comprises N18A, N18D, N18I, N18Y, N18M, N18F, N18L, N18W, N18K, or N18R. The amino acid substitution at position 104 comprises R104Q. The amino acid substitution at position 107 comprises R107A, R107E, R107Q, or R107D.

[0044] In embodiments, the IL-10 variant protein is selected from the group consisting of N18A / R104Q, N18A / R107A, N18A / R107E, N18A / R107Q, N18A / R107D, N18D / R104Q, N18D / R107A, N18D / R107E, N18D / R107Q, N18D / R107D, N18M / R104Q, N18M / R107A, N18M / R107E, N18M / R107Q, N18M / R107D, N18F / R104Q, N18F / R107A, N18F / R1 07E, N18F / R107Q, N18F / R107D, N18L / R104Q, N18L / R107A, N18L / R107E, N18L / R107Q, N18L / R107D, N18A / R104Q / R107A, N18A / R104Q / R107E, N18A / R104Q / R107Q, N18A / R104Q / R107D, N18D / R104Q / R107A, N18D / R104Q / R107E, N18D / R104Q / R107Q, N18D / R104Q / R107D, N 18M / R104Q / R107A, N18M / R104Q / R107E, N18M / R104Q / R107Q, N18M / R104Q / R107D, N18F / R104Q / R107A, N18F / R104Q / R107E, N18F / R10 4Q / R107Q, N18F / R104Q / R107D, N18L / R104Q / R107A, N18L / R104Q / R107E, N18L / R104Q / R107Q, N18L / R104Q / R107D, N18W / R104Q / R107 A, N18W / R104Q / R107A, N18W / R104Q / R107Q, N18W / R104Q / R107D, N18K / R104Q / R107A, N18K / R104Q / R107E, N18K / R104Q / R107Q, N18K / R104Q / R107D, N18R / R104Q / R107A, N18R / R104Q / R107E, N18R / R104Q / R107Q, and N18R / R104Q / R107D.

[0045] The term "substitution" refers to the replacement of one or more amino acids with other amino acids in a protein sequence. For example, N18A denotes that the original amino acid, asparagine (N), at position 18 in a protein sequence is replaced with alanine (A). Similarly, R107D denotes that the original amino acid, arginine, at position 107 in the sequence is replaced with aspartic acid (D). The substitution designation of (original amino acid) (position) (replaced amino acid) may be consistent with that commonly used in the art.

[0046] In embodiments, the IL-10 variant protein further comprises a signal peptide. Preferably, the signal peptide comprises the amino acid sequence of SEQ ID NO:25.

[0047] The term "signal peptide" refers to a short sequence typically linked to the N-terminus of a protein and responsible for directing the protein to its correct intracellular destination, such as the endoplasmic reticulum, Golgi apparatus, or plasma membrane. Signal peptides are typically 10 to 30 amino acids long and contain three domains: (1) an N-terminal domain consisting of positively charged amino acids that binds to a transport protein. The transport protein aids in transport of the signal peptide and the protein across the membrane; (2) a hydrophobic core domain that forms a stable α-helical structure that aids in insertion of the signal peptide into the membrane; and (3) a C-terminal domain that contains a recognition site for signal peptidase, an enzyme that cleaves the signal peptide from the protein after it has crossed the membrane. Signal peptides are crucial for improving protein production because they aid in transport and localization of proteins after translation. Preferably, the signal peptide has the amino acid sequence MYRMQLLSCIALSLALVTNS (SEQ ID NO: 25).

[0048] The present disclosure also provides (1) a polypeptide comprising an antibody or fragment thereof, an antagonist, a ligand for a receptor or target protein, a half-life extending moiety, or a protein trap; and (2) a fusion protein comprising the above-described IL-10 variant protein fused to the polypeptide.

[0049] The term "antibody" refers to a molecule comprising one or more polypeptide chains that specifically binds to or is immunologically reactive with a particular antigen. Exemplary antibodies of the present disclosure include monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, antibody fusions (e.g., fusion proteins), multispecific antibodies (i.e., bispecific antibodies), monovalent antibodies (e.g., single-arm antibodies), multivalent antibodies, antigen-binding fragments (e.g., Fab', F(ab')2, Fab, Fv, rIgG, and scFv fragments), and synthetic antibodies (or antibody mimetics).

[0050] The term "full length antibody" refers to an antibody having a structure substantially similar to a native antibody structure or having a heavy chain including an Fc region as defined herein.

[0051] The term "antibody fragment" refers to a portion of a full-length antibody that is capable of binding to the same antigen as the full-length antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; monovalent or single-arm antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0052] The term "antagonist" refers to a molecule that binds to a receptor and prevents the receptor from binding to its ligand. Antagonists can be classified into two main types: competitive antagonists and non-competitive antagonists. Competitive antagonists bind to the same binding site as the receptor and prevent the receptor from binding to its ligand. Non-competitive antagonists bind to a different binding site on the receptor, thereby altering the activity of the receptor by changing its structure. Exemplary antagonists of the disclosure include Bevacizumab, Ranibizumab, Brolucizumab, Sevacizumab, Varisacumab, Nabicixizumab, Vulinacimab, Olinvacimab, Alacizumab, Icrucumab, Aflibercept, Conbercept, or an anti-PD-L1 IgG1 antibody fragment (atezolizumab, avelumab, or durvalumab).

[0053] The term "half-life extending moiety" refers to a molecule that can increase the half-life of a protein. The moiety may be capable of maintaining the stability or enzymatic activity of the fusion protein. Exemplary half-life extending moieties of the present disclosure include XTEN protein polymers. 12 , GlycoTAIL and FlexiTAIL 13 , albumin binding domain 14 , PAS conversion 15 , ELP conversion 16 , HAP conversion 17 , or gelatin-like protein 18 Examples include:

[0054] The term "protein trap" refers to a molecule that allows for the identification of proteins of interest based on their unique subcellular localization, without the need for specific antibodies for each protein. Protein traps tag proteins with an epitope, and then specific antibodies to the epitope can be used to localize the tagged protein. This allows for the identification of proteins that are localized to specific subcellular compartments, such as the nucleus, cytoplasm, or membrane. Exemplary protein traps of the present disclosure include the epitope tag developed by Sineshchekova et al. 19 , or GFP tag 20 Examples include:

[0055] In embodiments, the IL-10 variant protein may be fused to the polypeptide via a linker.

[0056] The term "peptide linker" or "linker" refers to a peptide of 1 to 42 amino acids that can increase protein stability or folding, protein expression, improve biological activity, or allow targeting to a protein. A wide variety of polypeptide linkers are known in the art and can be used in the compositions and methods of the present disclosure, including, but not limited to, A(EAAAK)4ALEA(EAAAK)4A, (GGGGS) n=1、2、4 ,PAPAP,AEAAAAKEAAAKA,(AP) n=5~17 In preferred embodiments, peptide linkers that can be used in the present disclosure are listed below.

[0057] [Table 1]

[0058] In embodiments, the IL-10 variant protein is fused to the N-terminus or C-terminus of the polypeptide.

[0059] The terms "N-terminus" and "C-terminus" refer to the two ends of a protein. The N-terminus is the end of the chain containing a free amino group, while the C-terminus is the end of the chain containing a free carboxyl group. Proteins are typically described in the art from N-terminus to C-terminus.

[0060] In embodiments, the IL-10 variant protein is a monomer or a dimer.

[0061] The terms "monomer" or "dimer" refer to the structure of a protein. While a monomer refers to a single protein molecule, a dimer refers to a protein molecule composed of two monomer units held together by weak or non-covalent interactions, such as hydrogen bonds or hydrophobic interactions. A dimeric protein may be either a homodimer or a heterodimer. A homodimer is composed of two identical monomer units, while a heterodimer is composed of two different monomer units. In the present disclosure, IL-10 is typically a homodimer in living organisms. The dimeric structure of IL-10 allows it to bind to two receptors, which enhances its signaling ability. Although IL-10 can exist in a monomeric form, the monomer has low activity.

[0062] In embodiments, the fusion protein further comprises the amino acid sequence of SEQ ID NO:18.

[0063] In embodiments, the antibody comprises an anti-PD-L1 antibody.

[0064] The term "anti-PD-L1 antibody" refers to an antibody that binds to PD-L1 with sufficient affinity such that the antibody is useful as a therapeutic and / or diagnostic agent for targeting PD-L1.

[0065] In embodiments, the IL-10 variant protein comprises an amino acid sequence at least 80%, preferably at least 90%, more preferably at least 95% identical to SEQ ID NO: 14; and the anti-PD-L1 antibody comprises: YP7G IgG1 comprising a heavy chain amino acid sequence at least 80%, preferably at least 90%, more preferably at least 95% identical to SEQ ID NO: 26, and a light chain amino acid sequence at least 80%, preferably at least 90%, more preferably at least 95% identical to SEQ ID NO: 27; YP7G IgG4 comprising a heavy chain amino acid sequence at least 80%, preferably at least 90%, more preferably at least 95% identical to SEQ ID NO: 28, and a light chain amino acid sequence at least 80%, preferably at least 90%, more preferably at least 95% identical to SEQ ID NO: 29; or avelumab comprising a heavy chain amino acid sequence at least 80%, preferably at least 90%, more preferably at least 95% identical to SEQ ID NO: 30, and a light chain amino acid sequence at least 80%, preferably at least 90%, more preferably at least 95% identical to SEQ ID NO: 31.

[0066] In embodiments, (1) the antibody is a human, humanized, or chimeric antibody; (2) the antibody is a full-length antibody of class IgG, optionally wherein the class IgG antibody has an isotype selected from IgG1, IgG2, IgG3, and IgG4; (3) the antibody comprises an Fc region variant, optionally an Fc region variant that alters effector function and / or a variant that alters antibody half-life; (4) the antibody is optionally an antibody fragment selected from the group consisting of F(ab')2, Fab', Fab, Fv, single domain antibody (VHH), and scFv; (5) the antibody comprises an immunoconjugate, optionally wherein the immunoconjugate comprises a therapeutic agent for the treatment of a CSF1R-mediated, PDL1-mediated, PD1-mediated, or VEGF-mediated disease or condition; or (6) the antibody is a multispecific antibody, optionally a bispecific antibody.

[0067] The term "human antibody" refers to an antibody that possesses an amino acid sequence that corresponds to that of an antibody produced by a human or human cell, or derived from a non-human source that utilizes the human antibody repertoire or other human antibody coding sequences. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues.

[0068] The term "humanized antibody" refers to a chimeric antibody comprising amino acid sequences derived from non-human HVRs and human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs correspond to those of a non-human antibody and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that has been humanized.

[0069] The term "chimeric antibody" refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or organism, and the remainder of the heavy and / or light chain is derived from a different source or organism.

[0070] The term "hypervariable region" or "HVR" refers to each of the regions of an antibody variable domain that are hypervariable in sequence and / or form structurally defined loops ("hypervariable loops"). Typically, native antibodies contain six HVRs; heavy chain variable domain V H Three regions (HVR-H1, HVR-H2, HVR-H3) in the light chain variable domain V L Each HVR generally comprises four chains with three HVRs (HVR-L1, HVR-L2, and HVR-L3). HVRs generally comprise amino acid residues from the hypervariable loops and / or from the "complementarity-determining regions" (CDRs).

[0071] The term "FR" refers to variable domain residues other than HVR residues. The FR of a variable domain typically consists of four domains: FR1, FR2, FR3, and FR4. Thus, the HVR and FR sequences typically appear in the following order in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0072] The term "class" of an antibody refers to the type of constant domain or constant region carried by its heavy chain. There are five major antibody classes: IgA, IgD, IgE, IgG, and IgM, and several of these are further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0073] The term "Fc region" refers to a dimeric complex comprising the C-terminal polypeptide sequence of an immunoglobulin heavy chain, where the C-terminal polypeptide sequence is obtainable by papain digestion of an intact antibody. The Fc region can comprise a native or variant Fc sequence. Although the boundaries of the Fc sequence of an immunoglobulin heavy chain can vary, the human IgG heavy chain Fc sequence is usually defined to stretch from an amino acid residue at about position Cys226, or from about position Pro230, to the carboxyl terminus of the Fc sequence. However, the C-terminal lysine (Lys447) of the Fc sequence may or may not be present. The Fc sequence of an immunoglobulin generally comprises two constant domains, a CH2 domain and a CH3 domain, and optionally a CH4 domain.

[0074] The term "Fc region variant" refers to an Fc region sequence (eg, a human IgG1, IgG2, IgG3, or IgG4 Fc region) that comprises an amino acid substitution at one or more amino acid residue positions.

[0075] The term "F(ab')2" refers to a pair of Fab fragments that are generally covalently linked near their carboxy termini by hinge cysteines between them. Other chemical couplings of antibody fragments are also known in the art.

[0076] The term "Fab" refers to a V-unit linked by an intermolecular disulfide bond. H , CH1 and V L , C L The term "antibody fragment" refers to an antibody fragment containing a region.

[0077] The term "Fab'" refers to an antibody fragment containing a Fab fragment having a free sulfhydryl group on CH1. The Fab' can be alkylated or conjugated to an enzyme, toxin, or other protein of interest.

[0078] The term "Fv" refers to an antibody fragment that contains a complete antigen-recognition and binding site. This region consists of a dimer of one heavy- and one light-chain variable domain in tight association, which may be of a covalent nature, for example, in an scFv. Fvs are formed by the interaction of three HVRs from each variable domain to form a V H -V L It is in this configuration that defines an antigen-binding site on the surface of the dimer. Collectively, the six HVRs, or a subset thereof, confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three HVRs specific for an antigen) has the ability to recognize and bind antigen, although typically with lower affinity than the entire binding site.

[0079] The term "single domain antibody (VHH)" refers to a single domain antibody comprising a variable domain (VHH) of the heavy chain. H ) While a canonical antibody contains two heavy chains and two light chains, a single domain antibody has only one heavy chain.

[0080] The term "single chain Fv" or "scFv" refers to the V of an antibody. H Domain and V L"Fv" refers to an antibody fragment comprising V domains, wherein these domains are present in a single polypeptide chain. Generally, an Fv polypeptide comprises a V domain that enables the scFv to form the desired antigen-binding structure. H Domains and V L It further comprises a polypeptide linker between the domains.

[0081] The term "multispecific antibody" refers to an antibody having at least two different binding sites, each with binding specificities for different antigens. Optionally, the term "bispecific antibody" refers to an antibody having two different binding sites.

[0082] The present disclosure also provides an isolated polynucleotide or vector encoding the above-mentioned IL-10 variant protein or the above-mentioned fusion protein.

[0083] The present disclosure also provides an isolated host cell comprising the above-described isolated polynucleotide or the above-described vector.

[0084] The term "polynucleotide" refers to a biological polymer composed of nucleotide monomers covalently linked in a chain. Examples of polynucleotides include DNA (deoxyribonucleic acid), RNA (ribonucleic acid), mRNA (messenger RNA), and analogs of DNA or RNA produced using nucleotide analogs. Polynucleotides may have an open reading frame encoding an antigenic polypeptide and can be used as ribonucleic acid vaccines (NAVs). Once delivered to the body, including by injection, inhalation, or oral administration, polynucleotides may be taken up by cells and translated into antigenic polypeptides, which are then presented to the immune system to generate an immune response. Exemplary polynucleotides as NAVs of the present disclosure include IL-10 variant proteins and fusion proteins thereof.

[0085] The term "vector" refers to a molecule that carries genetic material (e.g., a polynucleotide) into another cell and can be used for protein expression, gene therapy, vaccine development, and genetic engineering. Vectors can be delivered to the body as NAVs. The present disclosure provides one or more vectors that encode antigenic polypeptides. Exemplary vectors of the present disclosure include adenovirus, retrovirus, poxvirus, adeno-associated virus, baculovirus, herpes simplex virus, pcDNA3.4, pET, pCMV, pUC19, pBR322, pUC18, pOET, or pMV261.

[0086] The term "isolated host cell" refers to a cell, such as E. coli, that may be capable of producing a protein encoded by a polynucleotide or vector. For example, recombinant production of an IL-10 variant protein or a fusion protein thereof is accomplished by isolating a nucleic acid encoding the IL-10 variant protein or a fusion protein thereof. The nucleic acid is then inserted into one or more vectors for further cloning and / or expression in the isolated host cell. Suitable isolated host cells and culture methods for cloning or expressing protein-encoding vectors are well known in the art and include prokaryotic or eukaryotic cells. After expression, the protein can be isolated from the cell paste in a soluble fraction and further purified.

[0087] The present disclosure also provides a method for producing an IL-10 variant protein or fusion protein, comprising culturing the host cell described above so that the IL-10 variant protein or fusion protein is produced.

[0088] The present disclosure also provides a pharmaceutical composition comprising the above-described IL-10 variant protein, the above-described fusion protein, or the above-described isolated polynucleotide or the above-described vector, optionally together with a pharmaceutically acceptable carrier, diluent, or excipient.

[0089] The term "pharmaceutical composition" refers to a preparation in a form that allows the biological activity of the active ingredient to be effective and that does not contain additional ingredients that are toxic to the subject to which the composition is administered. A pharmaceutical composition may contain one or more active agents. For example, a pharmaceutical composition may contain an anti-PD-L1 antibody as the only active agent in the formulation, or it may contain an anti-PD-L1 antibody and one or more additional active agents, such as an immune stimulatory agent such as IL-10, or an inhibitor of an immune checkpoint molecule.

[0090] The term "pharmaceutically acceptable carrier" means an ingredient in a pharmaceutical composition, other than an active ingredient, that is nontoxic to a subject to which it is administered. Pharmaceutically acceptable carriers include buffers, excipients, stabilizers, or preservatives.

[0091] The present disclosure also provides the use of the above-mentioned IL-10 variant protein, the above-mentioned fusion protein, or the above-mentioned isolated polynucleotide or the above-mentioned vector for the manufacture of a medicament. [Example]

[0092] The following examples are provided merely to illustrate the present disclosure and not to limit its scope in any way.

[0093] Example 1: Design of IL-10 variant proteins The sequences for exemplary IL-10 wild-type (WT) and variants containing substitutions are depicted below. Note that the substitution numbering for the IL-10 monomer is based on the mature IL-10 sequence, i.e., SEQ ID NO: 2. IL-10 precursor sequence (SEQ ID NO: 1)

[0094] [ka]

[0095] IL-10 mature sequence (SEQ ID NO: 2)

[0096] [ka]

[0097] IL-10 N18A (SEQ ID NO: 3)

[0098] [ka]

[0099] IL-10 N18D (SEQ ID NO: 4)

[0100] [ka]

[0101] IL-10 N18M (SEQ ID NO: 5)

[0102] [ka]

[0103] IL-10 N18F (SEQ ID NO: 6)

[0104] [ka]

[0105] IL-10 N18L (SEQ ID NO: 7)

[0106] [ka]

[0107] IL-10 N18V (SEQ ID NO: 8)

[0108] [ka]

[0109] IL-10 N18I (SEQ ID NO: 9)

[0110] [ka]

[0111] IL-10 N18Y (SEQ ID NO: 10)

[0112] [ka]

[0113] IL-10 N18W (SEQ ID NO: 11)

[0114] [ka]

[0115] IL-10 N18K (SEQ ID NO: 12)

[0116] [ka]

[0117] IL-10 N18R (SEQ ID NO: 13)

[0118] [ka]

[0119] IL-10 N18A / R107A (SEQ ID NO: 14)

[0120] [ka]

[0121] IL-10 N18I / R104Q (SEQ ID NO: 15)

[0122] [ka]

[0123] IL-10 N18Y / R104Q (SEQ ID NO: 16)

[0124] [ka]

[0125] IL-10 N18Y / R104Q / R107A (SEQ ID NO: 17)

[0126] [ka]

[0127] IL-10 N18A-Fc (SEQ ID NO: 18)

[0128] [ka]

[0129] The IL-10 sequence is in italics, the linker is underlined, and the human IgG Fc is in bold.

[0130] In embodiments, the IL-10 variant proteins of the disclosure do not comprise at least one amino acid substitution at positions 92 and / or 99. In preferred embodiments, the IL-10 variant proteins of the disclosure do not comprise the group of amino acid substitutions at positions 18, 92, and 99. In preferred embodiments, the IL-10 variant proteins of the disclosure do not comprise the group of amino acid substitutions at positions 18, 92, 99, and 111.

[0131] In another embodiment, the IL-10 variant proteins of the present disclosure do not comprise at least one amino acid substitution at positions 14, 21, 24, 25, 28, 74, 90, 92, 96, and / or 100. In a preferred embodiment, the IL-10 variant proteins of the present disclosure do not comprise the group of amino acid substitutions at positions 18, 21, 25, 92, 96, 100, and 104. In a preferred embodiment, the IL-10 variant proteins of the present disclosure do not comprise the group of amino acid substitutions at positions 14, 18, 21, 24, 25, 28, 74, 90, 92, 96, 100, and 104.

[0132] Example 2: Screening for IL-10 variant proteins by transient transfection of a stable reporter cell line with a plasmid encoding the IL-10 variant. Local delivery of IL-10 using mRNA, viruses, DNA vectors, or administration of host cells genetically engineered to express IL-10 has the potential to avoid the adverse effects induced by systemic IL-10 administration. IL-10 may be expressed and secreted by cells at the disease site or by IL-10-transduced host cells that migrate to the disease site. Secreted IL-10 in the target environment may act in an autocrine or paracrine manner and activate cells expressing the IL-10 receptor, which is composed of IL-10R1 and IL-10R2. Screening assays using phage display or yeast display primarily identify IL-10 variant proteins based on their binding ability to the IL-10 receptor, rather than activation of specific signaling pathways. Changes in binding affinity to the IL-10 receptor do not necessarily translate to changes in specific activity toward target cells due to the complexity of receptor signaling. A cell-based assay was established to simulate IL-10 expression and function at the site of action, which would be able to identify IL-10 variant proteins with either altered activity, altered expression levels, or both.

[0133] To establish a cell-based screening assay, HeLa cells were chosen because they can be easily transfected (for cell-based screening). HeLa cells express endogenous IL-10R2 but not IL-10R1. Therefore, we generated HeLa IL-10R1-STAT3 luciferase reporter cells, which stably express human IL-10R1 and the STAT3 Firefly luciferase reporter gene under the transcriptional control of STAT3. STAT3 Firefly luciferase reporter lentivirus was purchased from Cellomics Technology (PLV-10065-50). Lentivirus encoding IL-10R1 (NM_001558) was generated using the transfer vector pLAS5w.Phyg and standard methods. Puromycin and hygromycin were used to select HeLa cells expressing both the IL-10R1 and STAT3 reporter genes. The resulting stable HeLa IL-10R1-STAT3 cells were used for subsequent screening and activity studies. Transfection of HeLa IL-10R1-STAT3 cells with an IL-10 expression plasmid resulted in IL-10 protein secretion, which could then bind to cell surface IL-10 receptors (IL-10R1 / IL-10R2), thus leading to the induction of STAT3-dependent luciferase activity.

[0134] The expression vector pcDNA3.4 containing the IL-2 signal peptide (MYRMQLLSCIALSLALVTNS, SEQ ID NO: 25) followed by the sequence of IL-10 wild type (SEQ ID NO: 2) or its variants (e.g., SEQ ID NOs: 3 to 17) was constructed by GenScript services.

[0135] HeLa IL-10R1-STAT3 cells were grown in DMEM medium supplemented with 10% FBS and 1% Pen / Strep at 37°C with 5% CO. One day before transfection, HeLa IL-10R1-STAT3 cells were added at 2 × 10 4Cells / well were seeded into 96-well solid white flat-bottom TC-treated plates in 100 μL of growth medium. Transient transfection of HeLa IL-10R1-STAT3 cells with 1.25 ng / well of pcDNA3.4 plasmid expressing wild-type IL-10 or variants was performed using PolyJet transfection reagent (SignaGen Laboratories: SL100688). After 6 hours, the medium was removed and replaced with fresh medium. Forty-eight hours after transfection, luciferase activity was determined using the ONE-Glo™ Luciferase Assay System (Promega: E6120) according to the manufacturer's instructions. The relative light units (RLU) of luciferase activity obtained with wild-type IL-10 plasmid transfection were set as the basal value. The STAT3-activating activity of the IL-10 variant proteins was expressed as a percentage increase or decrease relative to basal activity. Amino acid substitutions of asparagine (N18) at position 18 of IL-10 have different effects on STAT3 activation. Substitution of N18 with the amino acids alanine (N18A), aspartic acid (N18D), leucine (N18L), methionine (N18M), valine (N18V), and tyrosine (Y) resulted in increased STAT3 activity, whereas substitution of N18 with lysine (N18K), arginine (N18R), and tryptophan (W) resulted in significantly reduced STAT3 activation (Fig. 1).

[0136] To confirm that secreted IL-10 variant proteins have the same effect on STAT3 activation when acting paracrinely, the same HeLa IL-10R1-STAT3 reporter cells were used, but the source of IL-10 protein was replaced by culture supernatant collected from transfection of wild-type IL-10 or variants into wild-type HeLa cells. IL-10-containing culture supernatant was first generated as follows: Wild-type HeLa cells were cultured at 3 × 10 4Cells / well were seeded into solid-bottom 96-well microplates in 100 μL of growth medium. HeLa cells were transfected with various amounts of plasmids expressing wild-type IL-10 or variants (ranging from 60 ng / well to 0.0275 ng / well, prepared using 3-fold dilutions) using Polyjet according to the supplier's protocol. Supernatants containing secreted IL-10 protein were then collected 48 hours later and subsequently added to cultures of HeLa IL-10R1-STAT3 reporter cells. After an additional 16 hours of culture, luciferase activity was determined using the ONE-Glo™ Luciferase Assay System according to the manufacturer's instructions. Culture supernatants from transfection of the IL-10 variant proteins N18A and N18D induced higher STAT3 activation compared to wild-type IL-10 across a wide range of plasmid amounts used for transfection (Figure 2).

[0137] (Example 3: CD8+T stimulation activity) Granzyme B plays an important role in the antitumor activity of CD8+ T cells, and IL-10 can improve granzyme B production both in vitro and in vivo through STAT3 activation. The effect of amino acid substitution at N18 on granzyme B secretion was investigated. CD8+ T cells were isolated from PBMCs using Ficoll-Paque PLUS (Cytiva: 17144003) and purified using CD8 microbeads (Miltenyi Biotec: 130-045-201). After 72 hours of stimulation with T cell TransAct (Miltenyi Biotec: 130-111-160) in AIM-V medium (Thermo Fisher Scientific: 12055083), activated CD8+ T cells were rested for 4 hours. Culture supernatants collected from wild-type HeLa cells transfected with wild-type IL-10 or variants (as previously described) were added to CD8+ T cell cultures. After an additional 72 hours of culture at 37°C in a CO2 incubator, the culture supernatants of IL-10-treated CD8+ T cells were collected, and the concentration of granzyme B was determined by ELISA (R&D Systems: DY2906-05) according to the manufacturer's instructions. Treatment of CD8+ T cells with culture supernatants containing N18A and N18D induced greater granzyme B secretion from CD8+ T cells compared with culture supernatants containing wild-type IL-10 or the IL-10 variant proteins R5A11 or Super 10 (Figure 3). R5A11 (WO2021181091A1) and Super 10 (WO2021243057A1) are IL-10 variant proteins with higher binding affinity to the IL-10 receptor.

[0138] In summary, a single amino acid substitution at N18 of IL-10 was able to alter IL-10 activity.

[0139] Example 4: STAT3 activation by purified IL-10 variant proteins The altered IL-10 activity in culture supernatants containing IL-10 variant proteins could be due to changes in protein expression levels, protein activity, or both. The ability of purified IL-10 variant proteins to activate STAT3 was examined. Wild-type IL-10 and the N18 variant were cloned into pcDNA3.4 with an N-terminal IL-2 signal peptide and a C-terminal (G4S) x 3 linker plus a polyhistidine tag (6 x His) for purification. Proteins were produced by transient transfection in F293 cells according to the manufacturer's protocol (ThermoFisher FreeStyle 293 Expression System) and subsequently purified by standard Ni-NTA chromatography. The purity of the purified proteins was confirmed by SDS-PAGE and Coomassie Blue. The activity of purified wild-type IL-10 and variants was assessed in HeLa IL-10R1-STAT3 cells. HeLa IL-10R1-STAT3 cells were cultured at 5 x 10 ng / ml. 4 Cells / well were seeded into white solid-bottom 96-well microplates in 100 μL of growth medium. The cells were then incubated at 37°C in a CO2 incubator for an additional 6 hours or overnight to allow cells to adhere to the plate. Subsequently, the cells were stimulated with 8 ng / mL or 24 ng / mL of His-tagged wild-type IL-10 or variants. After an additional 18 hours of culture, STAT3 luciferase activity was determined using the ONE-Glo™ Luciferase Assay System (Promega E6120) according to the manufacturer's instructions. As shown in Figure 4, purified IL-10 variant proteins N18A and N18D exhibited higher STAT3 activation activity compared to wild-type IL-10.

[0140] Example 5: IL-10 Fc fusion protein An amino acid substitution at position 18 was introduced into the IL-10-Fc fusion protein by standard mutagenesis techniques. The protein was produced by transient transfection in CHO-S cells and purified by a two-step purification process including protein A chromatography and SEC chromatography. CD8+ T cell treatment and granzyme B measurement were performed as described in Example 2. As shown in Figure 5, IL-10-Fc fusion proteins containing the IL-10 variant proteins N18A, N18F, N18I, N18L, and N18Y exhibited higher CD8+ T cell stimulatory activity.

[0141] Example 6: Additional Genetic Manipulations In a patent application (PCT / US22 / 77660) owned by the inventors, the inventors demonstrated that substitution of arginine at amino acids 104, 107, or both of IL-10 (specifically R104Q, R107A, or a combination) improves the purification yield of IL-10 fusion proteins. If the properties of improved manufacturability and enhanced activity could be combined into one molecule, this would benefit the development of IL-10 fusion biopharmaceuticals. In exemplary experiments, the N18 substitution was introduced into IL-10(R104Q)-Fc and IL-10(R104Q / R107A)-Fc fusion proteins by standard mutagenesis techniques. The proteins were produced by transient transfection in CHO-S cells and purified by a two-step purification process involving Protein A and SEC chromatography. When the N18A substitution was introduced into the IL-10(R104Q)-Fc fusion protein, the resulting IL-10(N18A / R104Q)-Fc fusion protein had better purification yields than the IL-10(WT)-Fc or IL-10(N18A)-Fc fusion proteins (Figure 6A). When the N18I substitution was introduced into the IL-10(R104Q)-Fc and IL-10(R104Q / R107A)-Fc fusion proteins, the resulting IL-10(N18I / R104Q)-Fc and IL-10(N18I / R104Q / R107A)-Fc fusion proteins had better purification yields than the IL-10(WT)-Fc or IL-10(N18I)-Fc fusion proteins (Figure 6B). When the N18Y substitution was introduced into IL-10(R104Q)-Fc and IL-10(R104Q / R107A)-Fc fusion proteins, the resulting IL-10(N18Y / R104Q)-Fc and IL-10(N18Y / R104Q / R107A)-Fc fusion proteins had better purification yields than IL-10(WT)-Fc or IL-10(N18Y)-Fc fusion proteins (Figure 6C). The activity of the novel variants was evaluated using HeLa IL-10R1-STAT3 cells. The activity of IL-10(N18I / R104Q)-Fc was comparable to that of IL-10(N18I)-Fc and better than that of IL-10(WT)-Fc (Figure 7A).The activity of IL-10(N18Y / R104Q)-Fc or IL-10(N18Y / R104Q / R107A)-Fc was comparable to that of IL-10(N18Y)-Fc and better than that of IL-10(WT)-Fc (Figure 7B).

[0142] The benefit of combining these substitutions was also tested in an antibody-IL-10 fusion format. The N18A substitution was introduced into an antibody-IL-10 fusion protein using standard mutagenesis techniques. Details of exemplary antibody-IL-10 fusion proteins, including YP7G-IL-10(R107A) and avelumab-IL-10(R107A), can be found in WO2021231741 and PCT / US22 / 77660. The protein was produced by transient transfection in CHO-S cells and purified using a two-step purification process involving Protein A and SEC chromatography. Introduction of the N18A substitution into avelumab-IL-10(R107A) significantly enhanced STAT3 activation while maintaining the improved yield achieved by the R107A substitution (Figure 8). Addition of the N18A substitution to YP7G-IL-10(R107A) in either the IgG1 (HC: SEQ ID NO: 26; LC: SEQ ID NO: 27) or IgG4 (HC: SEQ ID NO: 28; LC: SEQ ID NO: 29) formats also significantly enhanced granzyme B secretion from CD8+ T cells (Figure 9).

[0143] References The references listed below and referred to herein are incorporated herein by reference unless the specification expressly provides otherwise. (References) 1. SARAIVA, Margarida; VIEIRA, Paulo; O'GARRA, Anne. Biology and therapeutic potential of interleukin-10. Journal of Experimental Medicine, 2019, 217.1: e20190418. 2. MUMM, John B., et al. IL-10 elicits IFNγ-dependent tumor immune surveillance. Cancer cell, 2011, 20.6: 781-796. 3. FUJII, Shin-ichiro, et al. Interleukin-10 promotes the maintenance of antitumor CD8+ T-cell effector function in situ. Blood, The Journal of the American Society of Hematology, 2001, 98.7: 2143-2151. 4. EMMERICH, Jan, et al. IL-10 directly activates and expands tumor-resident CD8+ T cells without de novo infiltration from secondary lymphoid organs. Cancer research, 2012, 72.14: 3570-3581.

Claims

1. (1) a single amino acid substitution at position 18 compared to that of wild-type IL-10; (2) a first substitution of an amino acid at position 18 and a second substitution of an amino acid at position 104 or 107 relative to the amino acid of wild-type IL-10; or (3) a first substitution of an amino acid at position 18, a second substitution of an amino acid at position 104, and a third substitution of an amino acid at position 107, compared to the amino acid of wild-type IL-10; IL-10 variant proteins comprising:

2. 2. The IL-10 variant protein of claim 1, wherein wild-type IL-10 comprises an amino acid sequence having at least 80%, preferably at least 90%, at least 95%, at least 98%, more preferably at least 99% identity to SEQ ID NO:

2.

3. 2. The IL-10 variant protein of claim 1, wherein the amino acid substitution at position 18 comprises N18A, N18D, N18I, N18Y, N18M, N18F, N18L, N18W, N18K or N18R.

4. 2. The IL-10 variant protein of claim 1, wherein the amino acid substitution at position 104 comprises R104Q.

5. 2. The IL-10 variant protein of claim 1, wherein the amino acid substitution at position 107 comprises R107A, R107E, R107Q or R107D.

6. N18A / R104Q, N18A / R107A, N18A / R107E, N18A / R107Q, N18A / R107D, N18D / R104Q, N18D / R107A, N18D / R107E, N18D / R107Q, N18D / R107 D, N18M / R104Q, N18M / R107A, N18M / R107E, N18M / R107Q, N18M / R107D, N18F / R104Q, N18F / R107A, N18F / R107E, N18F / R107Q, N18F / R10 7D, N18L / R104Q, N18L / R107A, N18L / R107E, N18L / R107Q, N18L / R107D, N18A / R104Q / R107A, N18A / R104Q / R107E, N18A / R104Q / R107Q, N18A / R104Q / R107D, N18D / R104Q / R107A, N18D / R104Q / R107E, N18D / R104Q / R107Q, N18D / R104Q / R107D, N18M / R104Q / R107A, N18M / R1 04Q / R107E, N18M / R104Q / R107Q, N18M / R104Q / R107D, N18F / R104Q / R107A, N18F / R104Q / R107E, N18F / R104Q / R107Q, N18F / R104Q / R1 07D, N18L / R104Q / R107A, N18L / R104Q / R107E, N18L / R104Q / R107Q, N18L / R104Q / R107D, N18W / R104Q / R107A, N18W / R104Q / R107E, N18 2. The IL-10 variant protein of claim 1, comprising at least one substitution selected from the group consisting of W / R104Q / R107Q, N18W / R104Q / R107D, N18K / R104Q / R107A, N18K / R104Q / R107E, N18K / R104Q / R107Q, N18K / R104Q / R107D, N18R / R104Q / R107A, N18R / R104Q / R107E, N18R / R104Q / R107Q, and N18R / R104Q / R107D.

7. The IL-10 variant protein of claim 1, which is a monomer or a dimer.

8. The IL-10 variant protein of claim 1 , further comprising a signal peptide.

9. 9. The IL-10 variant protein of claim 8, wherein the signal peptide comprises the amino acid sequence of SEQ ID NO:

25.

10. (1) a polypeptide, including an antibody or fragment thereof, an antagonist, a ligand for a receptor or target protein, a half-life extending moiety, or a protein trap; and (2) The IL-10 variant protein according to claim 1 fused to a polypeptide. A fusion protein comprising:

11. The fusion protein of claim 10, wherein the polypeptide is fused to the IL-10 variant protein via a linker.

12. The fusion protein of claim 11, wherein the linker comprises an amino acid sequence of SEQ ID NO: 19 to 24.

13. The fusion protein of claim 10, wherein the IL-10 variant protein is fused to the N-terminus or C-terminus of the polypeptide.

14. The fusion protein of claim 10, wherein the IL-10 variant protein is a monomer or a dimer.

15. The fusion protein of claim 10, further comprising the amino acid sequence of SEQ ID NO:

18.

16. 11. The fusion protein of claim 10, wherein the antibody comprises an anti-PD-L1 antibody.

17. IL-10 variant proteins: An amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95% identity with SEQ ID NO: 14 and Anti-PD-L1 antibodies: YP7G IgG1, comprising a heavy chain amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95% identity to SEQ ID NO: 26 and a light chain amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95% identity to SEQ ID NO: 27; YP7G IgG4 comprising a heavy chain amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95% identity to SEQ ID NO: 28 and a light chain amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95% identity to SEQ ID NO: 29; or avelumab comprising a heavy chain amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95% identity to SEQ ID NO: 30 and a light chain amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95% identity to SEQ ID NO:

31.

17. The fusion protein of claim 16, comprising:

18. (1) whether the antibody is human, humanized, or chimeric; (2) the antibody is a full-length antibody of class IgG, optionally the class IgG antibody has an isotype selected from IgG1, IgG2, IgG3, and IgG4; (3) whether the antibody comprises an Fc region variant, optionally an Fc region variant that alters effector function and / or a variant that alters antibody half-life; (4) The antibody is an antibody fragment, optionally selected from the group consisting of F(ab')2, Fab', Fab, Fv, single domain antibody (VHH), and scFv; (5) the antibody comprises an immunoconjugate, and optionally, the immunoconjugate comprises a therapeutic agent for the treatment of a CSF1R-mediated, PDL1-mediated, PD1-mediated, or VEGF-mediated disease or condition; or (6) The antibody is a multispecific antibody, optionally a bispecific antibody; The fusion protein of claim 10.

19. 19. An isolated polynucleotide or vector encoding an IL-10 variant protein according to any one of claims 1 to 9 or a fusion protein according to any one of claims 10 to 18.

20. 20. An isolated host cell comprising the isolated polynucleotide or vector of claim 19.

21. 21. A method for producing an IL-10 variant protein or fusion protein, comprising culturing the host cell of claim 20 so that the IL-10 variant protein or fusion protein is produced.

22. A pharmaceutical composition comprising an IL-10 variant protein according to any one of claims 1 to 9, a fusion protein according to any one of claims 10 to 18, or an isolated polynucleotide or vector according to claim 19, optionally together with a pharmaceutically acceptable carrier, diluent or excipient.

23. 20. Use of an IL-10 variant protein according to any one of claims 1 to 9, a fusion protein according to any one of claims 10 to 18 or an isolated polynucleotide or vector according to claim 19 for the manufacture of a medicament.

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