Il-2 variant polypeptide and use thereof

Abstract

Provided are an IL-2 variant polypeptide and a use thereof.

Description

IL-2 variant peptides and their uses Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to interleukin-2 (IL-2) protein engineering and immune regulation. Background Technology

[0002] IL-2, discovered in 1976, is a T-cell growth factor that plays a role in maintaining the long-term growth of T cells in vitro. IL-2 can bind to specific high-affinity receptors on activated mouse and human T cells. IL-2 is mainly produced by CD4+ T cells and dendritic cells, while the IL-2 receptor is expressed on CD4+ T cells, CD8+ T cells, and other cell populations such as NK cells. Therefore, antigens can trigger and amplify specific immune responses through the interaction between IL-2 and its receptor.

[0003] The IL-2 receptor consists of three subunits: the α chain (IL-2Rα, encoded by the gene IL2RA, also known as CD25), the β chain (IL-2Rβ, encoded by the gene IL2RB, also known as CD122), and the IL-2Rγ (encoded by the gene IL2RG, also known as CD132). IL-2 can bind to three different IL-2 receptors with varying affinities: a low-affinity receptor containing only IL-2Rα (Kd approximately 10). -8 M), intermediate affinity receptors containing IL-2Rβ and IL-2Rγ (Kd approximately 10). -9 M), and high-affinity receptors containing IL-2Rα, IL-2Rβ, and IL-2Rγ (Kd approximately 10). -11 M). The β / γ chains are responsible for signal transduction (JAK–STAT pathway, especially STAT5), while the α chain mainly enhances ligand binding affinity and promotes ligand "assembly" in the receptor complex, thereby determining the "dose threshold" and sensitivity of different immune cells to IL-2. Regulatory T cells (Tregs) consistently and highly express IL-2Rα, preferentially assemble high-affinity αβγ receptors on their surface, and their survival, proliferation, and inhibitory functions are highly dependent on IL-2–STAT5 signaling; resting CD8+... + T cells and NK cells express more medium-affinity (βγ) receptors. Activation of lymphocytes can induce CD25 upregulation, converting medium-affinity receptors into high-affinity receptors. Overexpression of IL-2Rα in activated T cells results in approximately ten times the number of low-affinity receptors compared to high-affinity receptors. Therefore, under low-dose administration or stimulation with engineered IL-2 that is α-receptor biased, Tregs can be more selectively expanded, thereby correcting the Treg / effective T cell (Teff) imbalance and restoring peripheral tolerance.

[0004] Upon binding to the IL-2 receptor with intermediate-affinity IL-2Rβγ or high-affinity IL-2Rαβγ, IL-2 induces phosphorylation and activation of STAT5 protein. Activated STAT5 protein then translocates to the nucleus, regulating the transcription of numerous IL-2 target genes, thereby influencing T cell proliferation, enhancing the cytotoxic activity of natural killer (NK) cells and lymphokine-activated killer (LAK) cells, and playing a crucial role in the generation of helper T cells 9 (TH9) and the differentiation of regulatory T cells (Treg cells). The inhibitory function and homeostasis of Treg cells also depend on the stable expression and epigenetic consolidation of the transcription factor FOXP3; IL-2-STAT5 signaling directly drives FOXP3 transcription and maintains its lineage stability. Therefore, IL-2 plays a vital role in immune cell differentiation and the maintenance of immune cell function, thus influencing the dominant cell types in infection, autoimmune diseases, and cancer environments.

[0005] IL-2 is clinically used to treat tumors. Currently, high-dose recombinant IL-2 protein Aldesleukin has been approved by the FDA for the treatment of melanoma and renal cell carcinoma. However, the use of high-dose IL-2 has led to very serious side effects, including capillary leak syndrome, hypotension, fever, and diarrhea. These side effects are caused by the activation of excessive peripheral T cells and NK cells induced by high-dose IL-2. Therefore, reducing IL-2 activity, especially developing drugs with low IL-2 activity, can help alleviate the toxic side effects of IL-2 protein in tumor treatment. Furthermore, the production of recombinant IL-2 protein Aldesleukin is very unstable, resulting in very low yields. Therefore, research and development aimed at improving the production and stability of recombinant IL-2 protein is also essential.

[0006] Furthermore, multiple clinical studies have demonstrated that low-dose IL-2 can selectively expand peripheral Tregs in the human body and is associated with clinical improvement (e.g., evidence in indications such as NCT00529035 chronic graft-versus-host disease, NCT01353833 type 1 diabetes, and NCT02465580 systemic lupus erythematosus). However, the clinical application of wild-type IL-2 is limited by the safety-efficacy trade-off: once the dose or exposure exceeds the threshold, it can simultaneously activate Teff and NK cells, offsetting or counteracting the Treg-mediated therapeutic effect, resulting in a narrow therapeutic window. To address these limitations, in recent years, various engineered IL-2 molecules with "receptor preference + pharmacokinetic optimization" (such as NKTR-358, AMG592, RG7835, etc.) have emerged, striving to reduce stimulation of effector cells while maintaining Treg selectivity; however, except for a few molecules, the magnitude / sustainability of Treg expansion and clinical translation of most candidates are still limited by factors such as dose-safety trade-offs, tissue penetration, and exposure control. Furthermore, it is crucial to recognize the negative regulatory role of the inflammatory microenvironment on Treg amplification and functional maintenance. During disease activity, pro-inflammatory cytokines such as IL-6, TNF-α, IL-1β, and IFN-γ are significantly upregulated. These upregulations can weaken the stable expression of FOXP3 and the inhibitory function of Tregs by activating STAT3, inhibiting STAT5, and disrupting metabolic and epigenetic homeostasis. Simultaneously, inflammation-related metabolic alterations limit Treg energy metabolism and growth signaling, and insufficient local IL-2 supply and its competitive utilization further exacerbate the suppression of Treg amplification and impaired function. Therefore, in the context of inflammation, the use of IL-2 or its variants alone often results in insufficient Treg amplification. Combining anti-inflammatory factors (such as drugs antagonizing the IL-6 pathway) with Treg-amplifying IL-2 or its engineered variants can alleviate inflammatory interference while utilizing receptor-preferred IL-2 variants to achieve low-threshold, highly effective stimulation of Tregs. This produces a synergistic effect at both the tissue and peripheral levels, more effectively restoring the Treg / Teff balance and achieving systemic regulation of autoimmune and inflammation-related diseases.

[0007] This invention engineered the IL-2 receptor binding interface and molecular conformation to obtain IL-2 variant peptides with improved druggability and targeted immune effects, and constructed fusion proteins, dimers, and immune conjugates accordingly. These variants can achieve receptor-biased regulation according to therapeutic needs: one type of IL-2 variant peptide has a high selective activation capacity for Tregs, significantly reducing non-specific stimulation of Teff and NK cells, achieving stable, controllable, and safe peripheral and tissue Treg expansion, and maintaining FOXP3-dependent Treg lineage homeostasis and immunomodulatory function; another type can be used to enhance anti-tumor immune responses. For inflammatory / autoimmune indications characterized by Treg imbalance, the Treg-biased variant can be used in combination with antibodies antagonizing IL-6 / IL-6R, TNF-α / TNFR, or other inflammation-related pathways, or modularly assembled within the same molecule, to alleviate the adverse effects of inflammation on stable FOXP3 expression, further enhancing Treg amplification and inhibition functions, and providing a new, translatable therapeutic strategy for autoimmune and inflammatory diseases related to Treg imbalance. For oncology indications, the effector cell-biased variant can be used alone or in combination with existing antitumor therapies (e.g., immune checkpoint inhibitors or cell therapy), or assembled within the same molecule, thereby achieving regulation and clinical translation of immune effects in different disease scenarios. Summary of the Invention

[0008] This invention belongs to the field of immunotherapy and immunomodulation technology, and particularly relates to a functional IL-2 variant polypeptide obtained through structural design and molecular engineering, and its application in the selective amplification of Tregs. The IL-2 variant polypeptide of this invention exhibits good physicochemical stability, improved druggability, and manufacturability, facilitating large-scale purification and formulation development, and significantly improves pharmacodynamic and pharmacokinetic properties compared to wild-type IL-2. Based on this, the IL-2 variant of this invention, through amino acid substitution and / or point mutation at specific sites, finely regulates its binding characteristics with the IL-2Rα and IL-2Rβγ receptor complexes, thereby specifically amplifying Treg cells, significantly reducing non-specific activation of peripheral effector lymphocytes, improving the safety margin, and reducing systemic immune-related toxicities. This IL-2 variant can efficiently induce Tregs (CD4+) within a large dosing window. + CD25 + FoxP3 +Activation of pSTAT5 signaling in Tregs exhibits high receptor selectivity and Treg amplification, providing a novel pharmacological strategy for achieving immune tolerance reconstitution and treating autoimmune diseases. Building upon this, the present invention also proposes an optional extended implementation scheme: combining the Treg-biased IL-2 variant peptide with anti-inflammatory factors (e.g., drugs antagonizing the IL-6 pathway) or constructing immune conjugates to mitigate the adverse effects of inflammation on stable FOXP3 expression, thereby further enhancing Treg amplification and immunosuppressive functions. This optional scheme can further improve the therapeutic efficacy of IL-2 variants in inflammatory disease states, providing a more clinically translatable treatment strategy for diseases characterized by Treg dysfunction or immune imbalance, such as type 1 diabetes, systemic lupus erythematosus, inflammatory bowel disease, rheumatoid arthritis, multiple sclerosis, myasthenia gravis, psoriasis, and autoimmune hepatitis.

[0009] Specifically, this relates to IL-2 variant peptides with improved receptor selectivity and druggability, as well as immunomodulatory proteins (including but not limited to fusion proteins, dimers, and immune conjugates) containing such variants, their encoding nucleic acids, vectors, and host cells; and to pharmaceutical compositions containing the above molecules, methods for their preparation and purification, and their application in oncology and immune-related diseases. In some embodiments, the IL-2 variant is Treg-biased, capable of selectively expanding regulatory T cells (Tregs) in tissues / organs and peripheral tissues without significantly activating effector T cells, and can be used in combination with anti-inflammatory drugs (e.g., anti-IL-6 / IL-6R or anti-TNF-α / TNFR antibodies) to broaden the therapeutic window and reduce systemic inflammatory burden; other embodiments cover the application of IL-2 monomeric peptides for immune activation and anti-tumor therapy.

[0010] On one hand, the present invention provides an innovative modification of IL-2 peptides to obtain functional IL-2 variant peptides, which are easy to produce and purify, and have improved pharmacodynamic properties, thereby enabling safer and more effective treatment or prevention of diseases such as infections, tumors, and autoimmune diseases caused by the suppression of immune cell function.

[0011] On one hand, after extensive research, this invention has unexpectedly developed a class of IL-2 variant peptides, which are modified from wild-type human IL-2 peptide (SEQ ID NO:1) and possess one of the following characteristics:

[0012] 1. It has the characteristics of high yield and / or easy purification in mammalian cells.

[0013] 2. Reduces binding activity with IL-2Rβγ or IL-2Rαβγ.

[0014] 3. Reduce or eliminate binding activity with IL-2Rα.

[0015] The following is a detailed description of the present invention.

[0016] In a first aspect, the present invention provides an IL-2 variant polypeptide comprising replacing the peptide segment QSKNFHLRPR at positions 74-83 of the IL-2 polypeptide with another polypeptide fragment, the IL-2 variant polypeptide having superior yield and purity in mammalian cells and / or being easier to purify.

[0017] In some preferred embodiments, the other polypeptide fragments that replace peptides 74-83 are STGDFD (SEQ ID NO:2).

[0018] In some preferred embodiments, the other polypeptide fragments that replace peptides 74-83 are TGFFD (SEQ ID NO:3).

[0019] In some preferred embodiments, the other polypeptide fragments that replace peptides 74-83 are TGDFDL (SEQ ID NO:4).

[0020] In some preferred embodiments, the other polypeptide fragments that replace peptides 74-83 are ADGSLH (SEQ ID NO:5).

[0021] In some preferred embodiments, the other polypeptide fragments that are replaced by peptides 74-83 are GEASLH (SEQ ID NO:6).

[0022] In some preferred embodiments, the other polypeptide fragments that replace peptides 74-83 are GEGSLH (SEQ ID NO:7).

[0023] In other embodiments, the IL-2 variant peptide 74-83 is replaced while introducing a C125 site mutation, which in some preferred embodiments is S.

[0024] In some specific embodiments, the amino acid sequence of the IL-2 variant peptide is selected from: STGDFD / C125S (SEQ ID NO:9), TGDFD / C125S (SEQ ID NO:10), TGDFDL / C125S (SEQ ID NO:11), ADGSLH / C125S (SEQ ID NO:12), GEASLH / C125S (SEQ ID NO:13), and GEGSLH / C125S (SEQ ID NO:14).

[0025] In other embodiments, the IL-2 variant peptide provided by the present invention comprises replacing the peptide fragment QSKNFHLRPR at positions 74-83 of the wild-type human IL-2 peptide (SEQ ID NO:1) with other peptide fragments, while introducing site mutations at the IL-2-IL-2Rα binding interface or the IL-2-IL-2Rβγ binding interface, or / and introducing mutations to alter the glycosylation modification of the IL-2 variant peptide, or / and reduce or eliminate its IL-2Rα binding activity, or / and reduce its IL-2Rβγ binding activity.

[0026] In some preferred embodiments, the IL-2 variant peptide site 74-83 is replaced while introducing a C125S site mutation, and a T3 site mutation, which in some preferred embodiments is A.

[0027] In some preferred embodiments, the IL-2 variant peptide site 74-83 is replaced while introducing a C125S site mutation, and a mutation at the N88 site, which in some preferred embodiments is mutated to R or D.

[0028] In some preferred embodiments, the IL-2 variant peptide site 74-83 is replaced while introducing a mutation at the C125S site and a mutation at the S130 site, which in some preferred embodiments is mutated to R.

[0029] In some preferred embodiments, the IL-2 variant peptide site 74-83 is replaced while introducing a C125S site mutation, and a K35 site mutation, which in some preferred embodiments is a D mutation.

[0030] In some preferred embodiments, the IL-2 variant peptide site 74-83 is replaced while introducing a C125S site mutation, and a mutation at site F42 is introduced, which in some preferred embodiments is mutated to A, L, or Y.

[0031] In some preferred embodiments, the IL-2 variant peptide site 74-83 is replaced while introducing a C125S site mutation, and a D20 site mutation, which in some preferred embodiments is a T or Y or R or H or S mutation.

[0032] In some preferred embodiments, the IL-2 variant peptide site 74-83 is replaced while introducing a C125S site mutation, and a mutation at the E61 site, which in some preferred embodiments is mutated to R or Q.

[0033] In some preferred embodiments, the IL-2 variant peptide site 74-83 is replaced while introducing a C125S site mutation, and a mutation at the E62 site, which in some preferred embodiments is mutated to Q.

[0034] In some preferred embodiments, the IL-2 variant peptide site 74-83 is replaced while introducing a C125S site mutation, and a K63 site mutation, which in some preferred embodiments is mutated to D.

[0035] In some preferred embodiments, the IL-2 variant peptide site 74-83 is replaced while introducing a C125S site mutation, and a K64 site mutation, which in some preferred embodiments is mutated to D.

[0036] In some preferred embodiments, the IL-2 variant peptide site 74-83 is replaced while introducing a C125S site mutation, and a mutation is introduced at the E68 site, which in some preferred embodiments is mutated to K.

[0037] In some preferred embodiments, the IL-2 variant peptide site 74-83 is replaced while introducing a C125S site mutation, and a mutation at R38 site is introduced, which in some preferred embodiments is mutated to D.

[0038] In some specific embodiments, the amino acid sequence of the IL-2 variant peptide is selected from: TGDFD / T3A / C125S (SEQ ID NO:15), TGDFD / N88R / C125S (SEQ ID NO:16), TGDFD / N88D / C125S (SEQ ID NO:17), TGDFD / C125S / S130R (SEQ ID NO:18), TGDFD / K35D / C125S (SEQ ID NO:19), TGDFD / F42A / C125S (SEQ ID NO:20), TGDFD / F42L / C125S (SEQ ID NO:21), TGDFD / F42Y / C125S (SEQ ID NO:22), TGDFD / D20T / C125S (SEQ ID NO:23), TGDFD / D20Y / C125S (SEQ ID NO:24). NO:24), TGDFD / D20R / C125S (SEQ ID NO:25), TGDFD / D20H / C125S (SEQ ID NO:26), TGDFD / E61R / C125S (SEQ ID NO:27), TGDFD / K64D / C125S (SEQ ID NO:28), TGDFD / R38D / C125S (SEQ ID NO:29), TGDFD / D20S / C125S (SEQ ID NO:125), TGDFD / K63D / C125S (SEQ ID NO:126), TGDFD / E68K / C125S (SEQ ID NO:127), TGDFD / E61Q / C125S (SEQ ID NO:128), TGDFD / E62Q / C125S (SEQ ID NO:129).

[0039] In some preferred embodiments, the IL-2 variant peptide sites 74-83 are replaced while introducing mutations at T3, N88, C125 and S130 sites. In some preferred embodiments, the T3 site is mutated to A, the N88 site is mutated to R or D, the C125 site is mutated to S and the S130 site is mutated to R.

[0040] In some specific embodiments, the amino acid sequence of the IL-2 variant peptide is selected from: STGDFD / T3A / N88R / C125S / S130R (SEQ ID NO:30), TGDFD / T3A / N88R / C125S / S130R (SEQ ID NO:31), TGDFDL / T3A / N88R / C125S / S130R (SEQ ID NO:32), ADGSLH / T3A / N88R / C125S / S130R (SEQ ID NO:33), GEASLH / T3A / N88R / C125S / S130R (SEQ ID NO:34), and GEASLH / T3A / N88R / C125S / S130R (SEQ ID NO:35).

[0041] In some preferred embodiments, the IL-2 variant peptide sites 74-83 are replaced while introducing mutations at T3, K35, N88, C125, and S130 sites. In some preferred embodiments, the T3 site is mutated to A, the K35 site to D, the N88 site to R or D, the C125 site to S, and the S130 site to R.

[0042] In some specific embodiments, the amino acid sequence of the IL-2 variant peptide is selected from: STGDFD / T3A / K35D / N88R / C125S / S130R (SEQ ID NO:36), TGDFD / T3A / K35D / N88R / C125S / S130R (SEQ ID NO:37), TGDFDL / T3A / K35D / N88R / C125S / S130R (SEQ ID NO:38), ADGSLH / T3A / K35D / N88R / C125S / S130R (SEQ ID NO:39), GEASLH / T3A / K35D / N88R / C125S / S130R (SEQ ID NO:40), GEASLH / T3A / K35D / N88R / C125S / S130R (SEQ ID NO:40). NO:41), GEGSLH / T3A / K35D / N88D / C125S / S130R (SEQ ID NO:42).

[0043] In some preferred embodiments, the IL-2 variant peptide site 74-83 is replaced while introducing mutations at T3, D20, K35, C125 and S130 sites. In some preferred embodiments, the T3 site is mutated to A, the D20 site is mutated to T or Y or R or H or S, the K35 site is mutated to D, the C125 site is mutated to S and the S130 site is mutated to R.

[0044] In some specific embodiments, the amino acid sequence of the IL-2 variant peptide is selected from:

[0045] TGDFD / T3A / D20T / K35D / C125S / S130R(SEQ ID NO:43),

[0046] TGDFD / T3A / D20Y / K35D / C125S / S130R(SEQ ID NO:44),

[0047] TGDFD / T3A / D20R / K35D / C125S / S130R(SEQ ID NO:45),

[0048] TGDFD / T3A / D20H / K35D / C125S / S130R(SEQ ID NO:46),

[0049] TGDFD / T3A / D20S / K35D / C125S / S130R (SEQ ID NO: 47).

[0050] In some preferred embodiments, the IL-2 variant peptide sites 74-83 are replaced while introducing mutations at T3, F42, N88, C125, and S130 sites. In some preferred embodiments, the T3 site is mutated to A, the F42 site is mutated to L or Y or A, the N88 site is mutated to D or R, the C125 site is mutated to S, and the S130 site is mutated to R.

[0051] In some specific embodiments, the amino acid sequence of the IL-2 variant peptide is selected from:

[0052] TGDFD / T3A / F42L / N88R / C125S / S130R(SEQ ID NO:48)、

[0053] TGDFD / T3A / F42Y / N88R / C125S / S130R(SEQ ID NO:49),

[0054] TGDFD / T3A / F42A / N88R / C125S / S130R (SEQ ID NO: 50).

[0055] In some preferred embodiments, the IL-2 variant peptide sites 74-83 are replaced while introducing mutations at sites T3, E61, N88, C125, and S130. In some preferred embodiments, the T3 site is mutated to A, the E61 site to R, the N88 site to D or R, the C125 site to S, and the S130 site to R.

[0056] In some specific embodiments, the amino acid sequence of the IL-2 variant peptide is selected from:

[0057] TGDFD / T3A / E61R / N88R / C125S / S130R (SEQ ID NO: 51).

[0058] In some preferred embodiments, the IL-2 variant peptide sites 74-83 are replaced while introducing mutations at T3, K64, N88, C125, and S130 sites. In some preferred embodiments, the T3 site is mutated to A, the K64 site to D, the N88 site to D or R, the C125 site to S, and the S130 site to R.

[0059] In some specific embodiments, the amino acid sequence of the IL-2 variant peptide is selected from:

[0060] TGDFD / T3A / K64D / N88R / C125S / S130R(SEQ ID NO:52),

[0061] TGDFD / T3A / K64D / N88D / C125S / S130R (SEQ ID NO: 130).

[0062] In some preferred embodiments, the IL-2 variant peptide sites 74-83 are replaced while introducing mutations at T3, R38, N88, C125, and S130 sites. In some preferred embodiments, the T3 site is mutated to A, the R38 site to D, the N88 site to D or R, the C125 site to S, and the S130 site to R.

[0063] In some specific embodiments, the amino acid sequence of the IL-2 variant peptide is selected from:

[0064] TGDFD / T3A / R38D / N88R / C125S / S130R (SEQ ID NO: 53).

[0065] In a second aspect, the present invention provides an IL-2 variant polypeptide immunoconjugate comprising a first domain and an Fc domain, wherein the first domain is an IL-2 variant polypeptide, and the Fc domain comprises a homodimerized Fc domain or a heterodimerized Fc domain.

[0066] In one embodiment of the IL-2 variant polypeptide immunoconjugate provided by the present invention, a homodimeric Fc domain or a heterodimeric Fc domain is included. The Fc domain may be an Fc domain of human immunoglobulin IgG1, IgG2, IgG3, or IgG4 having a native or variant sequence; or it may be an Fc domain of mouse immunoglobulin IgG1, IgG2a, IgG2b, or IgG3. In a preferred embodiment, the Fc domain includes substitutions selected from L234A, L235A, P329G, S354C, T366W, T366S, L368A, Y349C, Y407V, H435R, and Y436F (according to the EU numbering system). In a more preferred embodiment, the Fc domain comprises a knocks-in-holes (KIH) structure: a “knob” mutation T366W is set in the CH3 domain of one heavy chain, and a “hole” mutation T366S / L368A / Y407V is set in the CH3 domain of the paired heavy chains; optionally, electrostatic pairing engineering and / or engineered disulfide bond stabilization (such as Y349C / S354C) may be combined to suppress homodimerization and polymer formation. In one embodiment, the Fc domain is derived from the human immunoglobulin Fc domain shown in SEQ ID NO:59, 60, 61, 62; in one embodiment, the Fc domain is derived from the mouse immunoglobulin Fc domain shown in SEQ ID NO:63, 64, 65, 66; in one embodiment, the Fc domain is selected from the human IgG1 Fc domain, and the human IgG1 Fc domain is introduced with a combination of L234A and L235A mutations (SEQ ID NO:67) (labeled "Fc LALA" herein) or a combination of L234A, L235A and P329G mutations (SEQ ID NO:68) (labeled "Fc LALAPG" herein) to eliminate Fc-mediated ADCC or CDC activity.

[0067] In one embodiment of the IL-2 variant polypeptide immunoconjugate provided by the present invention, the first domain and the Fc domain are directly or indirectly connected through a linker / hinge region.

[0068] In some embodiments, the indirect link may comprise a linker peptide. For example, the linker peptide comprises one, two, three, four, five, six, or more units selected from GGGGS, GGGS, GGS, and / or GS. For example, the linker peptide may comprise an amino acid sequence selected from the group shown below: SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, and SEQ ID NO: 58.

[0069] In some embodiments, the IL-2 variant peptide may be linked to the N-terminus and / or C-terminus of the dimerized Fc domain.

[0070] In some specific embodiments, the IL-2 variant peptide immunoconjugate comprises two identical heavy chains, said heavy chains comprising structures selected from the following:

[0071] 1) A heavy chain comprising an IL-2 variant polypeptide-adaptor peptide-Fc heavy chain from the N-terminus to the C-terminus; wherein the adaptor peptide may or may not be present;

[0072] 2) A heavy chain comprising, from the N-terminus to the C-terminus, the structure of an Fc-adaptor peptide-IL-2 variant polypeptide; wherein the adaptor peptide may or may not be present.

[0073] In this process, the two heavy chains containing the Fc heavy chain are homodimerized through the Fc domain.

[0074] In this application, unless otherwise specified, "heavy chain" refers to a peptide chain containing an Fc heavy chain, wherein "Fc heavy chain" refers to a peptide chain containing a hinge region of the heavy chain constant region and CH2 and CH3 and / or CH4 regions. The "Fc domain" described in the invention may include only one Fc heavy chain, or it may include a structure formed by the dimerization of two Fc heavy chains.

[0075] In another embodiment, the IL-2 variant peptide immunoconjugate comprises two distinct heavy chains, said heavy chains comprising structures selected from the following:

[0076] 1) A first heavy chain comprising an IL-2 variant polypeptide-adaptor peptide-Fc heavy chain structure from the N-terminus to the C-terminus; a second heavy chain being an immunoglobulin Fc heavy chain; wherein the adaptor peptide may or may not be present;

[0077] 2) A first heavy chain comprising an Fc heavy chain-adaptor peptide-IL-2 variant polypeptide structure from the N-terminus to the C-terminus; a second heavy chain being an immunoglobulin Fc heavy chain; wherein the adaptor peptide may or may not be present.

[0078] The two heavy chains containing the Fc heavy chain are heterodimerized by Fc, for example, through the mortar and pestle structure of the Fc domain.

[0079] In some embodiments, the linker peptide between the two different domains is absent, and the two different domains are connected by chemical bonds or hinge regions.

[0080] In some embodiments, a linker peptide is present, and indirect linkage comprises linking via the linker peptide. For example, the linker peptide comprises one, two, three, four, five, six, or more units selected from GGGGS, GGGS, GGS, and / or GS. For example, the linker peptide may comprise an amino acid sequence selected from the group shown below: SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, and SEQ ID NO: 58.

[0081] In some specific embodiments, the IL-2 variant polypeptide immunoconjugate comprises a first heavy chain consisting of SEQ ID NO:131 or thereof, and a second heavy chain consisting of SEQ ID NO:133 or thereof; or comprises a first heavy chain consisting of SEQ ID NO:132 or thereof, and a second heavy chain consisting of SEQ ID NO:133 or thereof.

[0082] In a third aspect, the IL-2 variant polypeptide immunoconjugate comprises a first domain, a second domain, and / or an Fc domain, wherein the first domain is an IL-2 variant polypeptide as described in the first aspect of the present invention, the second domain is an antibody or its antigen-binding fragment or fusion protein, and the Fc domain is an Fc domain having the same definition as the Fc domain in the second aspect of the present invention.

[0083] In some implementations, the antigen-binding fragment of the second domain may be selected from Fab, Fab', Fv, F(ab)2, F(ab')2, scFv, VHH, di-scFv and / or dAb.

[0084] In some embodiments, the second domain can selectively target PD-1, PD-L1, PD-L2, OX40, OX40L, 4-1BB, 4-1BBL, ICOS, ICOSL, TIGIT, CTLA-4, LAG-3, TIM-3, CD3, CD2, CD40, CD40L, CD47, CD6, CD19, CD20, CD22, CD38, CD52, CD79a, CD79b, CD123, C D33, CD30, CD138, BCMA, GPRC5D, CCR8, CCR4, CCR5, NKG2A, Nkp36, VEGF, VEGFR, HGF, EGFR, HER2, HER-3, H ER-4, PSMA, CEA, Trop-2, EpCAM, DLL1, MUC1, MUC2, MUC3, MUC4, MUC5AC, MUC5B, MUC7, IL-1β, IL-1R, IL-1 Ra, IL-4, IL-4Rα, IL-5, IL-5R, IL-6, IL-6R, IL-7, IL-7Rα, IL-12p35, IL-13, IL-15, IL-15Rα, IL-17A, I L-17F, IL-17RA, IL-17RB, IL-18, IL-18R, IL-21, IL-21R, IL-22, IL-22R, IL-23p19, IL-12 / 23p40, IL-2 5. IL-31, IL-31RA, IL-33(ST2), IL-36R, TSLP, TNF-α, TNFR, IFN-α, IFN-β, IFN-γ, IFNAR1, IFNAR2, IFN-γR, GM-CSF, GM-CSFRα, C3, C5, C5a, C5aR1, α4β7 integrin, α4 ​​integrin, αEβ7, β7, ICAM-1, VCAM-1, MAdCAM-1, and TL1A.

[0085] In some embodiments, the first and second domains of the IL-2 variant polypeptide immunoconjugate, or the first, second, and Fc domains, can be directly or indirectly connected via a linker peptide / hinge region.

[0086] For example, the IL-2 variant polypeptide immunoconjugate provided by the present invention comprises a first domain and a second domain, and comprises three chains with the following structure:

[0087] 1) The first heavy chain contains the structure of VH-CH1-Fc heavy chain from N-terminus to C-terminus; the second heavy chain contains the structure of IL-2 variant polypeptide-adaptor peptide-Fc heavy chain from N-terminus to C-terminus; the light chain contains the structure of VL-CL from N-terminus to C-terminus.

[0088] 2) The first heavy chain contains the structure of VH-CH1-Fc heavy chain from N-terminus to C-terminus; the second heavy chain contains the structure of Fc heavy chain-adaptor peptide-IL-2 variant polypeptide from N-terminus to C-terminus; the light chain contains the structure of VL-CL from N-terminus to C-terminus.

[0089] 3) The first heavy chain contains the structure of IL-2 variant polypeptide-adaptor peptide-VH-CH1-Fc heavy chain from N-terminus to C-terminus; the second heavy chain contains the Fc heavy chain from N-terminus to C-terminus; and the light chain contains the structure of VL-CL from N-terminus to C-terminus.

[0090] 4) The first heavy chain contains the structure of VH-CH1-Fc heavy chain-adaptor peptide-IL-2 variant polypeptide from N-terminus to C-terminus; the second heavy chain contains the Fc heavy chain from N-terminus to C-terminus; and the light chain contains the structure of VL-CL from N-terminus to C-terminus.

[0091] 5) The first heavy chain contains the structure of VH-CH1-Fc heavy chain from N-terminus to C-terminus; the second heavy chain contains the Fc heavy chain from N-terminus to C-terminus; the light chain contains the structure of IL-2 variant polypeptide-adaptor peptide-VL-CL from N-terminus to C-terminus.

[0092] 6) The first heavy chain contains the structure of the VH-CH1-Fc heavy chain from the N-terminus to the C-terminus; the second heavy chain contains the Fc heavy chain from the N-terminus to the C-terminus; and the light chain contains the structure of the VL-CL-adaptor peptide-IL-2 variant polypeptide from the N-terminus to the C-terminus.

[0093] The adaptor peptide may or may not be present.

[0094] In this process, the two heavy chains containing the Fc heavy chain are heterodimerized through the Fc domain. CH1 represents the CH1 domain of the immunoglobulin heavy chain, and CL represents the CL domain of the immunoglobulin light chain. VH-CH1 and VL-CL pair up to form Fab, and the second domain is in the form of Fab.

[0095] In some embodiments, the linker peptide between the two different domains is absent, and the two different domains are connected by chemical bonds or hinge regions.

[0096] In some embodiments, a linker peptide is present, and indirect linkage comprises linking via the linker peptide. For example, the linker peptide comprises one, two, three, four, five, six, or more units selected from GGGGS, GGGS, GGS, and / or GS. For example, the linker peptide may comprise an amino acid sequence selected from the group shown below: SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, and SEQ ID NO: 58.

[0097] In a preferred embodiment, adjacent domains are connected by a linker peptide, and preferably, the first domain is connected to Fc by a linker peptide.

[0098] In a preferred embodiment, adjacent domains are connected by a linker peptide, and preferably, the first domain and the second domain are connected by a linker peptide.

[0099] In a preferred embodiment, adjacent domains are connected by a linker peptide, and preferably, the first domain is connected to Fc via a linker peptide / hinge region.

[0100] In a preferred embodiment, adjacent domains are connected by a linker peptide, and preferably, the second domain is connected to Fc via a linker peptide / hinge region.

[0101] In some preferred embodiments, the second domain is an antibody or antigen-binding fragment of IL-6 or IL-6R, wherein the antibody / fragment may be selected from tocilizumab, sarilumab, satralizumab, siltuximab, and their functionally equivalent fragments / biosimilars.

[0102] In some preferred embodiments, the second domain is an antibody / antigen-binding fragment or fusion protein targeting TNF-α or its receptor, wherein the antibody / fragment may be selected from adalimumab, infliximab, golimumab, certolizumab pegol, etanercept, and their functionally equivalent fragments / biosimilars.

[0103] In some specific embodiments, the IL-2 variant polypeptide immunoconjugate comprises a first heavy chain consisting of SEQ ID NO:134 or thereof, a second heavy chain consisting of SEQ ID NO:131 or thereof, and a light chain consisting of SEQ ID NO:135 or thereof.

[0104] In some specific embodiments, the IL-2 variant polypeptide immunoconjugate comprises a first heavy chain consisting of SEQ ID NO:134 or thereof, a second heavy chain consisting of SEQ ID NO:132 or thereof, and a light chain consisting of SEQ ID NO:135 or thereof.

[0105] In some specific embodiments, the IL-2 variant polypeptide immunoconjugate comprises a first heavy chain consisting of SEQ ID NO:136 or therefrom, a second heavy chain consisting of SEQ ID NO:131 or therefrom, and a light chain consisting of SEQ ID NO:137 or therefrom.

[0106] In some specific embodiments, the IL-2 variant polypeptide immunoconjugate comprises a first heavy chain consisting of SEQ ID NO:136 or therefrom, a second heavy chain consisting of SEQ ID NO:132 or therefrom, and a light chain consisting of SEQ ID NO:137 or therefrom.

[0107] In some specific embodiments, the IL-2 variant polypeptide immunoconjugate comprises a first heavy chain consisting of SEQ ID NO:146 or therefrom, a second heavy chain consisting of SEQ ID NO:133 or therefrom, and a light chain consisting of SEQ ID NO:135 or therefrom.

[0108] In some specific embodiments, the IL-2 variant polypeptide immunoconjugate comprises a first heavy chain consisting of SEQ ID NO:134 or thereof, a second heavy chain consisting of SEQ ID NO:147 or thereof, and a light chain consisting of SEQ ID NO:148 or thereof.

[0109] For example, the IL-2 variant polypeptide immunoconjugate provided by the present invention comprises a first domain and a second domain, and comprises two chains with the following structure:

[0110] 1) The first heavy chain contains the second domain antigen-binding domain - Fc heavy chain from the N-terminus to the C-terminus; the second heavy chain contains the IL-2 variant polypeptide - adaptor peptide - Fc heavy chain from the N-terminus to the C-terminus.

[0111] 2) The first heavy chain contains the second domain antigen-binding domain - Fc heavy chain structure from the N-terminus to the C-terminus; the second heavy chain contains the Fc heavy chain - adaptor peptide - IL-2 variant polypeptide structure from the N-terminus to the C-terminus.

[0112] 3) The first heavy chain contains the structure of IL-2 variant polypeptide-adaptor peptide-second domain antigen-binding domain-Fc heavy chain from N-terminus to C-terminus; the second heavy chain contains the Fc heavy chain from N-terminus to C-terminus.

[0113] 4) The first heavy chain contains the structure of the second domain antigen-binding domain-Fc heavy chain-adaptor peptide-IL-2 variant polypeptide from the N-terminus to the C-terminus; the second heavy chain contains the Fc heavy chain from the N-terminus to the C-terminus.

[0114] In this process, the two heavy chains containing the Fc heavy chain are heterodimerized by the Fc domain, wherein the second domain antigen-binding domain is in the form of VHH or ScFv.

[0115] In some embodiments, the linker peptide between the two different domains is absent, and the two different domains are connected by chemical bonds or hinge regions.

[0116] In some embodiments, a linker peptide is present, and indirect linkage comprises linking via the linker peptide. For example, the linker peptide comprises one, two, three, four, five, six, or more units selected from GGGGS, GGGS, GGS, and / or GS. For example, the linker peptide may comprise an amino acid sequence selected from the group shown below: SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, and SEQ ID NO: 58.

[0117] In a preferred embodiment, adjacent domains are connected by a linker peptide, and preferably, the first domain is connected to Fc by a linker peptide.

[0118] In a preferred embodiment, adjacent domains are connected by a linker peptide, and preferably, the first domain and the second domain are connected by a linker peptide.

[0119] In a preferred embodiment, adjacent domains are connected by a linker peptide, and preferably, the first domain is connected to Fc via a linker peptide / hinge region.

[0120] In a preferred embodiment, adjacent domains are connected by a linker peptide, and preferably, the second domain is connected to Fc via a linker peptide / hinge region.

[0121] In some preferred embodiments, the second domain is an antibody or antigen-binding fragment of IL-6 or IL-6R, wherein the antibody / fragment may be selected from tocilizumab, sarilumab, satralizumab, siltuximab, and their functionally equivalent fragments / biosimilars.

[0122] In some preferred embodiments, the second domain is an antibody / antigen-binding fragment or fusion protein targeting TNF-α or its receptor, wherein the antibody / fragment may be selected from adalimumab, infliximab, golimumab, certolizumab pegol, etanercept, and their functionally equivalent fragments / biosimilars.

[0123] In some specific embodiments, the IL-2 variant polypeptide immunoconjugate comprises a first heavy chain consisting of SEQ ID NO:151 or thereof, and a second heavy chain consisting of SEQ ID NO:131 or thereof.

[0124] In some specific embodiments, the IL-2 variant polypeptide immunoconjugate comprises a first heavy chain consisting of SEQ ID NO:151 or thereof, and a second heavy chain consisting of SEQ ID NO:132 or thereof.

[0125] In a fourth aspect, the present invention provides a multifunctional IL-2 variant polypeptide immunoconjugate, the IL-2 variant polypeptide immunoconjugate comprising a first domain, an Fc domain, a second domain, and a third domain of the IL-2 variant polypeptide as described in the first aspect of the present invention, wherein the second domain comprises an antibody or an antigen-binding fragment thereof, and wherein the third domain comprises an antibody or an antigen-binding fragment thereof, or the third domain is a functional protein or an active fragment thereof.

[0126] In some embodiments, the Fc domain has the same definition as the Fc domain in the second aspect of the present invention.

[0127] In some embodiments, the third domain is an antibody or antigen-binding fragment that may be the same as or different from the second domain.

[0128] For example, in some embodiments, the antigen-binding fragment of the second domain and the antigen-binding fragment of the third domain can each independently be Fab, Fab', Fv, F(ab)2, F(ab')2, scFv, VHH, di-scFv, or dAb.

[0129] For example, in some embodiments, the second and third domains can each independently and selectively target PD-1, PD-L1, PD-L2, OX40, OX40L, 4-1BB, 4-1BBL, ICOS, ICOSL, TIGIT, CTLA-4, LAG-3, TIM-3, CD3, CD2, CD40, CD40L, CD47, CD6, CD19, CD20, CD22, CD38, CD52, CD79a, C D79b, CD123, CD33, CD30, CD138, BCMA, GPRC5D, CCR8, CCR4, CCR5, NKG2A, Nkp36, VEGF, VEGFR, HGF, EGFR, HE R2, HER-3, HER-4, PSMA, CEA, Trop-2, EpCAM, DLL1, MUC1, MUC2, MUC3, MUC4, MUC5AC, MUC5B, MUC7, IL-1β, IL -1R, IL-1Ra, IL-4, IL-4Rα, IL-5, IL-5R, IL-6, IL-6R, IL-7, IL-7Rα, IL-12p35, IL-13, IL-15, IL-15Rα, IL -17A, IL-17F, IL-17RA, IL-17RB, IL-18, IL-18R, IL-21, IL-21R, IL-22, IL-22R, IL-23p19, IL-12 / 23p40, IL-25, IL-31, IL-31RA, IL-33(ST2), IL-36R, TSLP, TNF-α, TNFR, IFN-α, IFN-β, IFN-γ, IFNAR1, IFNAR2, IFN-γR, GM-CSF, GM-CSFRα, C3, C5, C5a, C5aR1, α4β7 integrin, α4 ​​integrin, αEβ7, β7, ICAM-1, VCAM-1, MAdCAM-1, and TL1A.

[0130] In some embodiments, the second domain is an antibody or its antigen-binding fragment or its functional fragment as described above, and the third domain is a functional protein or its active fragment.

[0131] For example, in some embodiments, the functional protein of the third domain or its active fragment is selected from: sIL-6R, sgp130, CD80, CD86, CD83, LAG3, ICOSL, OX40L, CD40, CD2, TACI, BR3, TGFBR, VEGFR, IL-1Ra, IL-15, IL-7, IL-4, and / or IL-12.

[0132] In some embodiments, the IL-2 variant peptide immunoconjugate includes a first domain, a second domain, and a third domain, wherein the second and third domains have the same antigen-binding domain. The IL-2 variant peptide immunoconjugate comprises two identical heavy chains; or it comprises two identical heavy chains and two identical light chains. The heavy and light chains comprise structures selected from the following:

[0133] 1) A first heavy chain, comprising an IL-2 variant polypeptide-adaptor peptide-antigen-binding domain-Fc heavy chain structure from the N-terminus to the C-terminus; and a light chain, comprising a VL-CL structure from the N-terminus to the C-terminus; wherein the light chain may or may not be present; for example, the light chain is present when the antigen-binding domain is Fab; and absent when the antigen-binding domain is ScFv or VHH.

[0134] 2) A first heavy chain, comprising an antigen-binding domain-Fc heavy chain-adaptor peptide-IL-2 variant polypeptide structure from the N-terminus to the C-terminus; and a light chain, comprising a VL-CL structure from the N-terminus to the C-terminus; wherein the light chain may or may not be present; for example, the light chain is present when the antigen-binding domain is Fab; and absent when the antigen-binding domain is ScFv or VHH.

[0135] 3) The first heavy chain contains an antigen-binding domain -Fc heavy chain structure from the N-terminus to the C-terminus; the light chain contains an IL-2 variant polypeptide - adaptor peptide -VL-CL structure from the N-terminus to the C-terminus.

[0136] 4) The first heavy chain contains the antigen-binding domain - Fc heavy chain structure from the N-terminus to the C-terminus; the light chain contains the VL-CL-adaptor peptide - IL-2 variant polypeptide structure from the N-terminus to the C-terminus.

[0137] In this process, two heavy chains containing the Fc heavy chain are homodimerized through the Fc domain. The antigen-binding domain can be in the form of VH-CH1, ScFv, or VHH, where CH1 represents the CH1 domain of the immunoglobulin heavy chain and CL represents the CL domain of the immunoglobulin light chain. VH-CH1 and VL-CL pair up to form Fab.

[0138] The adaptor peptide may or may not be present.

[0139] In some embodiments, the linker peptide between the two different domains is absent, and the two different domains are connected by chemical bonds or hinge regions.

[0140] In some embodiments, a linker peptide is present, and indirect linkage comprises linking via the linker peptide. For example, the linker peptide comprises one, two, three, four, five, six, or more units selected from GGGGS, GGGS, GGS, and / or GS. For example, the linker peptide may comprise an amino acid sequence selected from the group shown below: SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, and SEQ ID NO: 58.

[0141] In some preferred embodiments, both the second and third domains are antigen-binding fragments that bind to IL-6, said antigen-binding fragments being selected from siltuximab or its functionally equivalent fragments / biosimilars.

[0142] In some preferred embodiments, both the second and third domains are antigen-binding fragments that bind to TNF-α, and the antigen-binding fragments are selected from adalimumab or its functionally equivalent fragments / biosimilars.

[0143] In some preferred embodiments, both the second and third domains are selected from adalimumab, and the IL-2 variant polypeptide immunoconjugate comprises a first heavy chain consisting of SEQ ID NO:138 or thereof, and a light chain consisting of SEQ ID NO:135 or thereof; or comprises a first heavy chain consisting of SEQ ID NO:140 or thereof, and a light chain consisting of SEQ ID NO:135 or thereof; or comprises a first heavy chain consisting of SEQ ID NO:142 or thereof, and a light chain consisting of SEQ ID NO:135 or thereof; or comprises a first heavy chain consisting of SEQ ID NO:144 or thereof, and a light chain consisting of SEQ ID NO:135 or thereof.

[0144] In some preferred embodiments, both the second and third domains are selected from siltuximab, and the IL-2 variant polypeptide immunoconjugate comprises a first heavy chain consisting of SEQ ID NO:139 or thereof, and a light chain consisting of SEQ ID NO:137 or thereof; or comprises a first heavy chain consisting of SEQ ID NO:141 or thereof, and a light chain consisting of SEQ ID NO:137 or thereof; or comprises a first heavy chain consisting of SEQ ID NO:143 or thereof, and a light chain consisting of SEQ ID NO:137 or thereof; or comprises a first heavy chain consisting of SEQ ID NO:145 or thereof, and a light chain consisting of SEQ ID NO:137 or thereof; or comprises a first heavy chain consisting of SEQ ID NO:150 or thereof, and a light chain consisting of SEQ ID NO:149 or thereof.

[0145] In some embodiments, the IL-2 variant peptide immunoconjugate of the present invention includes a first domain, a second domain, and a third domain, wherein the second and third domains have the same antigen-binding domain. The IL-2 variant peptide immunoconjugate comprises two different heavy chains, or comprises two different heavy chains and two identical light chains, wherein the heavy and light chains comprise structures selected from the following:

[0146] 1) A first heavy chain, which contains a second-domain antigen-binding domain-a third-domain antigen-binding domain-Fc heavy chain from the N-terminus to the C-terminus; a second heavy chain, which contains an IL-2 variant peptide-adaptor peptide-Fc heavy chain from the N-terminus to the C-terminus; and a light chain, which contains a VL-CL structure from the N-terminus to the C-terminus; wherein the light chain may or may not be present; for example, when the antigen-binding domain is Fab, the light chain is present; for example, when the antigen-binding domain is ScFv or VHH, the light chain is absent.

[0147] 2) A first heavy chain, which contains a second-domain antigen-binding domain-a third-domain antigen-binding domain-Fc heavy chain from the N-terminus to the C-terminus; a second heavy chain, which contains an Fc heavy chain-adaptor peptide-IL-2 variant polypeptide from the N-terminus to the C-terminus; and a light chain, which contains a VL-CL structure from the N-terminus to the C-terminus; wherein the light chain may or may not be present; for example, when the antigen-binding domain is Fab, the light chain is present; for example, when the antigen-binding domain is ScFv or VHH, the light chain is absent.

[0148] 3) The first heavy chain contains an IL-2 variant polypeptide-adaptor peptide-second domain antigen-binding domain-third domain antigen-binding domain-Fc heavy chain structure from the N-terminus to the C-terminus; the second heavy chain contains an Fc heavy chain from the N-terminus to the C-terminus; and the light chain contains a VL-CL structure from the N-terminus to the C-terminus. The light chain may or may not be present. For example, the light chain is present when the antigen-binding domain is Fab; and the light chain is absent when the antigen-binding domain is ScFv or VHH.

[0149] 4) A first heavy chain, which contains a second antigen-binding domain-a third antigen-binding domain-Fc heavy chain structure from the N-terminus to the C-terminus; a second heavy chain, which contains an Fc heavy chain-adaptor peptide-IL-2 variant polypeptide structure from the N-terminus to the C-terminus; and a light chain, which contains a VL-CL structure from the N-terminus to the C-terminus; wherein the light chain may or may not be present; for example, when the antigen-binding domain is Fab, the light chain is present; for example, when the antigen-binding domain is ScFv or VHH, the light chain is absent.

[0150] 5) A first heavy chain containing a second domain antigen-binding domain - Fc heavy chain from the N-terminus to the C-terminus; a second heavy chain containing an IL-2 variant polypeptide - adaptor peptide - third domain antigen-binding domain - Fc heavy chain from the N-terminus to the C-terminus; a light chain containing a VL-CL structure from the N-terminus to the C-terminus; wherein the light chain may or may not be present; for example, when the antigen-binding domain is Fab, the light chain is present; for example, when the antigen-binding domain is ScFv or VHH, the light chain is absent.

[0151] 6) A first heavy chain containing a second structural domain antigen-binding domain - Fc heavy chain from the N-terminus to the C-terminus; a second heavy chain containing a third structural domain antigen-binding domain - Fc heavy chain - adaptor peptide - IL-2 variant polypeptide from the N-terminus to the C-terminus; a light chain containing a VL-CL structure from the N-terminus to the C-terminus; wherein the light chain may or may not be present; for example, when the antigen-binding domain is Fab, the light chain is present; for example, when the antigen-binding domain is ScFv or VHH, the light chain is absent.

[0152] In this process, two heavy chains containing the Fc heavy chain are heterodimerized by the Fc domain. The antigen-binding domain can be in the form of VH-CH1, ScFv, and / or VHH, where CH1 represents the CH1 domain of the immunoglobulin heavy chain and CL represents the CL domain of the immunoglobulin light chain. VH-CH1 and VL-CL pair up to form Fab.

[0153] The adaptor peptide may or may not be present.

[0154] In some embodiments, the linker peptide between the two different domains is absent, and the two different domains are connected by chemical bonds or hinge regions.

[0155] In some embodiments, a linker peptide is present, and indirect linkage comprises linking via the linker peptide. For example, the linker peptide comprises one, two, three, four, five, six, or more units selected from GGGGS, GGGS, GGS, and / or GS. For example, the linker peptide may comprise an amino acid sequence selected from the group shown below: SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, and SEQ ID NO: 58.

[0156] In some preferred embodiments, the second and / or third domains are selected from antibody / antigen binding fragments or fusion proteins that target IL-6 / IL-6R or TNF-α / TNFR (e.g., functional equivalents or biosimilars of tocilizumab, sarilumab, satralizumab, siltuximab, adalimumab, infliximab, golimumab, etanercept).

[0157] In some preferred embodiments, both the second and third domains are selected from adalimumab, and the IL-2 variant polypeptide immunoconjugate comprises a first heavy chain consisting of SEQ ID NO:155 or thereof, a second heavy chain consisting of SEQ ID NO:131 or thereof, and a light chain consisting of SEQ ID NO:135 or thereof; or comprises a first heavy chain consisting of SEQ ID NO:155 or thereof, a second heavy chain consisting of SEQ ID NO:132 or thereof, and a light chain consisting of SEQ ID NO:135 or thereof; or comprises a first heavy chain consisting of SEQ ID NO:134 or thereof, a second heavy chain consisting of SEQ ID NO:146 or thereof, and a light chain consisting of SEQ ID NO:135 or thereof.

[0158] In some preferred embodiments, both the second and third domains are selected from siltuximab, and the IL-2 variant polypeptide immunoconjugate comprises a first heavy chain consisting of SEQ ID NO:136 or thereof, a second heavy chain consisting of SEQ ID NO:153 or thereof, and a light chain consisting of SEQ ID NO:137 or thereof.

[0159] In other embodiments, the multifunctional IL-2 variant peptide immunoconjugate of the present invention includes a first domain, a second domain, and a third domain, wherein the second domain and the third domain are different antibody or antigen-binding fragments, and comprises two or three chains with the following structure:

[0160] 1) A first heavy chain, which contains a second-domain antigen-binding domain-a third-domain antigen-binding domain-Fc heavy chain from the N-terminus to the C-terminus; a second heavy chain, which contains an IL-2 variant peptide-adaptor peptide-Fc heavy chain from the N-terminus to the C-terminus; and a light chain, which contains a VL-CL structure from the N-terminus to the C-terminus; wherein the light chain may or may not be present; for example, when the antigen-binding domain is Fab, the light chain is present; for example, when the antigen-binding domain is ScFv or VHH, the light chain is absent.

[0161] 2) A first heavy chain, which contains a second-domain antigen-binding domain-a third-domain antigen-binding domain-Fc heavy chain from the N-terminus to the C-terminus; a second heavy chain, which contains an Fc heavy chain-adaptor peptide-IL-2 variant polypeptide from the N-terminus to the C-terminus; and a light chain, which contains a VL-CL structure from the N-terminus to the C-terminus; wherein the light chain may or may not be present; for example, when the antigen-binding domain is Fab, the light chain is present; for example, when the antigen-binding domain is ScFv or VHH, the light chain is absent.

[0162] 3) The first heavy chain contains an IL-2 variant polypeptide-adaptor peptide-second domain antigen-binding domain-third domain antigen-binding domain-Fc heavy chain structure from the N-terminus to the C-terminus; the second heavy chain contains an Fc heavy chain from the N-terminus to the C-terminus; and the light chain contains a VL-CL structure from the N-terminus to the C-terminus. The light chain may or may not be present. For example, the light chain is present when the antigen-binding domain is Fab; and the light chain is absent when the antigen-binding domain is ScFv or VHH.

[0163] 4) A first heavy chain comprising, from N-terminus to C-terminus, a second antigen-binding domain-a third antigen-binding domain-Fc heavy chain-adaptor peptide-IL-2 variant polypeptide; a second heavy chain comprising, from N-terminus to C-terminus, an Fc heavy chain; and a light chain comprising, from N-terminus to C-terminus, a VL-CL structure; wherein the light chain may or may not be present; for example, when the antigen-binding domain is Fab, the light chain is present; for example, when the antigen-binding domain is ScFv or VHH, the light chain is absent.

[0164] 5) A first heavy chain containing a second domain antigen-binding domain - Fc heavy chain from the N-terminus to the C-terminus; a second heavy chain containing an IL-2 variant polypeptide - adaptor peptide - third domain antigen-binding domain - Fc heavy chain from the N-terminus to the C-terminus; a light chain containing a VL-CL structure from the N-terminus to the C-terminus; wherein the light chain may or may not be present; for example, when the antigen-binding domain is Fab, the light chain is present; for example, when the antigen-binding domain is ScFv or VHH, the light chain is absent.

[0165] 6) A first heavy chain containing a second structural domain antigen-binding domain - Fc heavy chain from the N-terminus to the C-terminus; a second heavy chain containing a third structural domain antigen-binding domain - Fc heavy chain - adaptor peptide - IL-2 variant polypeptide from the N-terminus to the C-terminus; a light chain containing a VL-CL structure from the N-terminus to the C-terminus; wherein the light chain may or may not be present; for example, when the antigen-binding domain is Fab, the light chain is present; for example, when the antigen-binding domain is ScFv or VHH, the light chain is absent.

[0166] 7) A first heavy chain containing a second structural domain antigen-binding domain - Fc heavy chain from the N-terminus to the C-terminus; a second heavy chain containing an IL-2 variant polypeptide - adaptor peptide - Fc heavy chain - third structural domain antigen-binding domain from the N-terminus to the C-terminus; a light chain containing a VL-CL structure from the N-terminus to the C-terminus; wherein the light chain may be present or absent; for example, when the antigen-binding domain is Fab, the light chain is present; for example, when the antigen-binding domain is ScFv or VHH, the light chain is absent.

[0167] 8) A first heavy chain comprising a second antigen-binding domain-Fc heavy chain-a third antigen-binding domain from the N-terminus to the C-terminus; a second heavy chain comprising an IL-2 variant polypeptide-adaptor peptide-Fc heavy chain from the N-terminus to the C-terminus; and a light chain comprising a VL-CL structure from the N-terminus to the C-terminus; wherein the light chain may or may not be present; for example, the light chain is present when the antigen-binding domain is Fab; and the light chain is absent when the antigen-binding domain is ScFv or VHH.

[0168] 9) A first heavy chain comprising a second antigen-binding domain-Fc heavy chain-third antigen-binding domain structure from the N-terminus to the C-terminus; a second heavy chain comprising an Fc heavy chain-adaptor peptide-IL-2 variant polypeptide structure from the N-terminus to the C-terminus; and a light chain comprising a VL-CL structure from the N-terminus to the C-terminus; wherein the light chain may be present or absent; for example, the light chain is present when the antigen-binding domain is Fab; and the light chain is absent when the antigen-binding domain is ScFv or VHH.

[0169] 10) The first heavy chain contains a structure from the N-terminus to the C-terminus consisting of a second domain antigen-binding domain-a third domain antigen-binding domain-Fc heavy chain; the second heavy chain contains an Fc heavy chain from the N-terminus to the C-terminus; and the light chain contains a structure from the N-terminus to the C-terminus consisting of an IL-2 variant polypeptide-adaptor peptide-VL-CL.

[0170] 11) The first heavy chain, which contains the second domain antigen-binding domain-third domain antigen-binding domain-Fc heavy chain from the N-terminus to the C-terminus; the second heavy chain, which contains the Fc heavy chain from the N-terminus to the C-terminus; and the light chain, which contains the VL-CL-adaptor peptide-IL-2 variant polypeptide from the N-terminus to the C-terminus.

[0171] 12) The first heavy chain contains the second domain antigen-binding domain - Fc heavy chain from the N-terminus to the C-terminus; the second heavy chain contains the third domain antigen-binding domain - Fc heavy chain from the N-terminus to the C-terminus; the light chain contains the IL-2 variant polypeptide - adaptor peptide - VL-CL from the N-terminus to the C-terminus.

[0172] 13) The first heavy chain contains the second domain antigen-binding domain - Fc heavy chain from the N-terminus to the C-terminus; the second heavy chain contains the third domain antigen-binding domain - Fc heavy chain from the N-terminus to the C-terminus; the light chain contains the VL-CL-promoter peptide - IL-2 variant polypeptide from the N-terminus to the C-terminus.

[0173] 14) The first heavy chain contains the second structural domain antigen-binding domain - Fc heavy chain structure from the N-terminus to the C-terminus; the second heavy chain contains the Fc heavy chain - third structural domain antigen-binding domain structure from the N-terminus to the C-terminus; the light chain contains the IL-2 variant polypeptide - adaptor peptide - VL-CL structure from the N-terminus to the C-terminus.

[0174] 15) The first heavy chain contains the second domain antigen-binding domain - Fc heavy chain structure from the N-terminus to the C-terminus; the second heavy chain contains the Fc heavy chain - third domain antigen-binding domain structure from the N-terminus to the C-terminus; the light chain contains the VL-CL-adaptor peptide - IL-2 variant polypeptide structure from the N-terminus to the C-terminus.

[0175] 16) The first heavy chain contains a second domain antigen-binding domain-Fc heavy chain-third domain antigen-binding domain from the N-terminus to the C-terminus; the second heavy chain contains an Fc heavy chain from the N-terminus to the C-terminus; and the light chain contains an IL-2 variant polypeptide-adaptor peptide-VL-CL from the N-terminus to the C-terminus.

[0176] 17) The first heavy chain contains a second domain antigen-binding domain-Fc heavy chain-third domain antigen-binding domain from the N-terminus to the C-terminus; the second heavy chain contains an Fc heavy chain from the N-terminus to the C-terminus; and the light chain contains a VL-CL-adaptor peptide-IL-2 variant polypeptide from the N-terminus to the C-terminus.

[0177] The two heavy chains containing the Fc heavy chain are heterodimerized by the Fc domain. The antigen-binding domains of the second and third domains can be in the form of VH-CH1, ScFv and / or VHH, where CH1 represents the CH1 domain of the immunoglobulin heavy chain and CL represents the CL domain of the immunoglobulin light chain. VH-CH1 and VL-CL pair up to form Fab. The second and third domains bind different and independent antigens.

[0178] The adaptor peptide may or may not be present.

[0179] In some embodiments, the linker peptide between the two different domains is absent, and the two different domains are connected by chemical bonds or hinge regions.

[0180] In some embodiments, a linker peptide is present, and indirect linkage comprises linking via the linker peptide. For example, the linker peptide comprises one, two, three, four, five, six, or more units selected from GGGGS, GGGS, GGS, and / or GS. For example, the linker peptide may comprise an amino acid sequence selected from the group shown below: SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, and SEQ ID NO: 58.

[0181] In some preferred embodiments, the second and / or third domains are selected from antibody / antigen binding fragments or fusion proteins that target IL-6 / IL-6R or TNF-α / TNFR (e.g., functional equivalents or biosimilars of tocilizumab, sarilumab, satralizumab, siltuximab, adalimumab, infliximab, golimumab, etanercept).

[0182] The connection sites and orientations between the domains can be located at the N-terminus or C-terminus of any adjacent domain, and the IL-2 variant polypeptide can be directly or via a linker peptide linked to the antibody heavy chain or light chain or the immunoglobulin Fc segment.

[0183] In some specific embodiments, the second domain is selected from Siltuximab, and the third domain is selected from adalimumab; the IL-2 variant polypeptide immunoconjugate comprises a first heavy chain consisting of SEQ ID NO:151 or thereof, a second heavy chain consisting of SEQ ID NO:146 or thereof, and a light chain consisting of SEQ ID NO:135 or thereof.

[0184] In some specific embodiments, the second domain is selected from Siltuximab, and the third domain is selected from adalimumab; the IL-2 variant polypeptide immunoconjugate comprises a first heavy chain consisting of SEQ ID NO:152 or thereof, a second heavy chain consisting of SEQ ID NO:151 or thereof, and a light chain consisting of SEQ ID NO:148 or thereof.

[0185] In some specific embodiments, the second domain is selected from Siltuximab, and the third domain is selected from adalimumab; the IL-2 variant polypeptide immunoconjugate comprises a first heavy chain consisting of SEQ ID NO:154 or thereof, a second heavy chain consisting of SEQ ID NO:131 or thereof, and a light chain consisting of SEQ ID NO:135 or thereof.

[0186] In some specific embodiments, the second domain is selected from Siltuximab, and the third domain is selected from adalimumab; the IL-2 variant polypeptide immunoconjugate comprises a first heavy chain consisting of SEQ ID NO:154 or thereof, a second heavy chain consisting of SEQ ID NO:132 or thereof, and a light chain consisting of SEQ ID NO:135 or thereof.

[0187] To limit systemic inflammation and broaden the safety window, this invention further provides a combination strategy: the IL-2 variant fusion molecule (or its pharmaceutical composition) is administered on the same day or sequentially with anti-IL-6 / anti-IL-6R / anti-TNF-α / anti-TNFR antibodies or their antigen-binding fragments, preferably via subcutaneous or intravenous administration. This combination can maintain the activation and amplification of Tregs while inhibiting systemic inflammation and adverse events mediated by IL-6 and TNF-α. The combination can be a fixed combination (co-formulation of the same formulation or lyophilized reconstruction in the same vial) or a non-fixed combination (separate formulations, combined administration), and the dose ratio and order of administration can be determined based on the indications, baseline inflammatory markers, and pharmacokinetic parameters.

[0188] In suitable implementations, available antibodies (commercially available or functionally equivalent fragments thereof) for human indications include, but are not limited to: (i) IL-6 / IL-6R: tocilizumab, sarilumab, satralizumab, siltuximab; (ii) TNF axis: adalimumab, infliximab, golimumab, certolizumab pegol, etanercept. In mouse models, scientifically available homologous antibodies or equivalents may be used, such as anti-mouse IL-6 (e.g., clone MP5-20F3), anti-mouse IL-6R (e.g., clone MR16-1), and anti-mouse TNF-α (e.g., clones XT3.11, MP6-XT22). Alleles and subtypes compatible with the target species are preferred to avoid Fc-dependent interference and ensure readout consistency. The above examples are merely illustrative of accessibility and equivalence and do not constitute limitations.

[0189] In another aspect, the present invention relates to a polynucleotide encoding the aforementioned variant polypeptide or a variant thereof, and a vector containing the polynucleotide. Vectors that can be used for insertion are well known in the art, including but not limited to cloning vectors and expression vectors; in one embodiment, the vector is a plasmid.

[0190] In another aspect, the present invention also relates to host cells comprising the aforementioned polynucleotides or carriers. Such host cells include, but are not limited to, eukaryotic cells such as animal cells (e.g., mammalian cells, such as mouse cells, human cells, etc.), yeast cells, insect cells, and plant cells; particularly preferably, the host cell of the present invention is a mammalian CHO cell.

[0191] In another aspect, the present invention provides a composition comprising the aforementioned IL-2 variant polypeptide, the multifunctional IL-2 variant polypeptide immunoconjugate, the immunoconjugate, a corresponding nucleotide molecule or carrier, and a pharmaceutically acceptable loading agent. The pharmaceutical composition may further comprise, in combination with, an anti-IL-6 / IL-6R / TNF-α / TNFR or other anti-inflammatory-related antibody or its antigen-binding fragment, and may be formulated as an injectable solution, a lyophilized powder for injection, or a pre-filled syringe formulation, etc.

[0192] The present invention provides a method for preparing and purifying the above-mentioned variant immunoconjugate, comprising culturing the host cells to express the IL-2 variant polypeptide immunoconjugate, and purifying the variant immunoconjugate.

[0193] The present invention provides a method for regulating the activity of antigen-presenting cells, comprising contacting the antigen-presenting cells with the IL-2 variant polypeptide immunoconjugate to stimulate their activation.

[0194] This invention provides a time window and method for inducing and amplifying Tregs in mice. Based on pharmacodynamic readouts of the IL-2 variant peptide or its fusion complex (including but not limited to changes in the proportion and absolute count of Tregs in peripheral blood or lymphoid tissue), suitable observation and sampling timelines for single or repeated dosing are determined. In non-human primate models, this invention further provides pharmacokinetic (PK) characteristics and corresponding Treg amplification time windows and methods.

[0195] The pharmaceutical compositions of the present invention can be administered by methods known in the art, such as, but not limited to, injection. Preferably, the formulations of the present invention are administered subcutaneously; in some preferred embodiments, the pharmaceutical compositions of the present invention are administered in unit doses.

[0196] In another aspect, the present invention provides the use of the IL-2 variant peptide of the present invention or its immunoconjugates, compositions, polynucleotides, carriers, or host cells to prepare medicaments for the prevention, improvement, and / or treatment of tumors, cancers, autoimmune diseases, and inflammation-related diseases. The tumors include solid tumors and hematologic malignancies; the inflammation-related diseases may include, but are not limited to, rheumatoid arthritis, systemic lupus erythematosus, psoriasis / psoriatic arthritis, inflammatory bowel disease (such as ulcerative colitis, Crohn's disease), multiple sclerosis, myasthenia gravis, Sjögren's syndrome, atopic dermatitis, alopecia areata, thyroid autoimmune diseases, and transplant-related immune imbalances (such as graft-versus-host disease, transplant rejection).

[0197] On the other hand, the IL-2 variant peptide and its fusion complex can also be used for other indications of immune homeostasis regulation, including but not limited to intervention of post-infection immune imbalance; the above uses do not constitute a limitation on the scope of the present invention.

[0198] On the other hand, the IL-2 variant peptides and their immunoconjugates disclosed in this invention have anti-infective effects, including but not limited to infections caused by bacteria, viruses and parasites.

[0199] The following detailed embodiments and examples illustrate the implementation of the present invention, but do not limit the scope of protection of the present invention; all equivalent substitutions or modifications made by those skilled in the art without departing from the spirit and substance of the present invention should be covered within the scope of the present invention. Attached Figure Description

[0200] The specific features of the invention involved in this application are shown in the appended claims. The features and advantages of the invention can be better understood by referring to the exemplary embodiments and drawings described in detail below. A brief description of the drawings is as follows:

[0201] Figures 1A and 1B show the SDS-PAGE images of the purified IL-2 variant polypeptide complex after expression in this application.

[0202] Figures 2A and 2B show the SEC-HPLC chromatograms of the IL-2 variant polypeptide complex after expression and purification in this application.

[0203] Figure 3 shows the binding of the IL-2 variant peptide complex to human IL-2Rα and IL-2Rβ / γ proteins.

[0204] Figure 4 shows the binding of the IL-2 variant peptide complex to unactivated primary human T cells and the expression of CD25 on primary T cells.

[0205] Figure 5 shows the binding of the IL-2 variant peptide complex to activated primary human CD4T and CD8T cells and the expression of CD25 on T cells.

[0206] Figure 6 shows the STAT5 phosphorylation of primary T cells purified by IL-2 variant polypeptide complex induced by this application.

[0207] Figure 7 shows the phosphorylation of STAT5 in T cells of fresh human PBMCs induced by the IL-2 variant peptide complex.

[0208] Figure 8 shows the phosphorylation of STAT5 in T cells of fresh cynomolgus monkey PBMCs induced by the IL-2 variant peptide complex.

[0209] Figure 9 shows the phosphorylation of STAT5 in spleen T cells of BALB / c mice induced by the IL-2 variant peptide complex.

[0210] Figure 10 shows the results of Treg amplification in BALB / c mice induced by the IL-2 variant peptide complex.

[0211] Figure 11 shows the results of Treg amplification in BALB / c mice induced by different doses of IL-2 variant peptide complex.

[0212] Figure 12 shows the results of Treg amplification in BALB / c mice induced by different doses of IL-2 variant peptide complex in combination with anti-IL-6 antibody / anti-TNF-α antibody.

[0213] Figure 13A shows the ear thickness results of the IL-2 variant peptide complex in an MC903-induced dermatitis mouse model.

[0214] Figure 13B shows the results of IL-2 variant peptide complex inhibiting MC903-induced dermatitis in mouse plasma IgE.

[0215] Figure 14A shows the PK results of the IL-2 variant peptide complex in cynomolgus monkeys.

[0216] Figure 14B shows the PK results of the anti-IL-6 / IL-2 variant peptide immunoconjugate in cynomolgus monkeys.

[0217] Figure 15 shows the results of Treg amplification in cynomolgus monkeys induced by the 2mpk IL-2 variant peptide complex.

[0218] Figure 16 shows the results of Treg amplification in cynomolgus monkeys induced by a 10mpk anti-IL-6 / IL-2 variant peptide immunoconjugate. Detailed Implementation

[0219] Unless otherwise defined, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. In case of conflict, the definitions in this specification shall prevail.

[0220] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the invention. While only certain exemplary materials and methods are described herein, many similar or equivalent methods and materials may be used in practice with the present disclosure.

[0221] Unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” used herein include their plural forms. The open-ended expression “comprising / including” means that it may also contain elements or steps not specifically listed, and also covers situations where it consists solely of said elements or steps. The term “about,” unless otherwise specified, means a variation of no more than ±5% of the target value; where tolerances are given separately in a particular embodiment, those tolerances shall prevail.

[0222] The term "stimulation of immune cell activation" refers to the regulation of immune cell proliferation and activation by the variant polypeptides and their immune conjugates disclosed in this invention. It can also be extended to any other immune cell regulator that, after treating immune cells, affects the expression of immune cells, including but not limited to cell proliferation markers such as Ki67, secretion of granzyme, and upregulation of cytokines such as IFNg or TNFα. The immune cells can be extended to T lymphocyte lines and primary T lymphocytes, NK cell lines and primary NK cells, etc.

[0223] The term "antitumor activity" refers to any biological activity that reduces or prevents the proliferation or viability of tumor cells in vivo and / or in vitro. In one embodiment, the antitumor activity is the antitumor effect of the variant peptides and their immunoconjugates described in this invention.

[0224] The term "IL-2" refers to a mature polypeptide or fragment thereof that exhibits at least approximately 85% amino acid identity with the protein encoded by the NCBI Gene ID:3558 gene and possesses IL-2R binding activity with a complex protein composed of the IL-2Rα subunit (encoded by the NCBI Gene ID:3558 gene) and / or the IL-2Rβ subunit (encoded by the NCBI Gene ID:3558 gene) and / or the IL-2Rγ subunit (encoded by the NCBI Gene ID:3558 gene), thereby promoting STAT5 phosphorylation and mediating the activation or proliferation of immune cells. Unless otherwise stated, human IL-2 may refer to a protein that exhibits at least approximately 85% amino acid identity with the protein encoded by NCBI Gene ID:3558.

[0225] The terms “wild-type IL-2” or “WT IL-2” refer to a recombinant protein with a natural amino acid sequence identical to human interleukin-2, without engineered mutations affecting receptor binding or signal transduction. Unless otherwise stated, wild-type IL-2 (WT IL-2) may refer to a monomeric / non-fusion form of recombinant human IL-2 or a dimer-like molecule formed by the fusion of WT IL-2 with the Fc domain of immunoglobulin. In the examples described herein, WT IL-2 may be used as a negative / baseline control or a mechanism control. The terms “IL-2 variant peptide,” “IL-2 variant,” “IL-2 mutant,” or “IL-2 mutant peptide” are used interchangeably to refer to a protein / peptide obtained by substitution, deletion, or insertion (including segment substitution) at one or more amino acid sites relative to the IL-2 protein, retaining some or all of the functional properties of IL-2 (such as binding to IL-2Rβγ, induction of immune cell activation / proliferation), whose activity may be selectively attenuated or redirected depending on the intended use. Non-limiting examples include substitutions in the aa74–83 region (e.g., replacing QSKNFHLRPR with TGDFD, STGDFD, TGDFDL, etc.), which can occur in combination with point mutations. The term "amino acid mutation" indicates that a specific site is replaced by another natural amino acid; for example, T3A indicates that the third amino acid is replaced by A instead of T.

[0226] "IL-2 receptor (IL-2R)" and "IL-2Rα / IL-2Rβ / IL-2Rγ" refer to the CD25(α), CD122(β), and CD132(γ) subunits, respectively; α alone has low affinity, βγ dimer has medium affinity, and αβγ trimer has high affinity. Unless otherwise stated, "receptor binding capacity / affinity" can be characterized by biophysical methods such as BLI / SPR using KD / kon / koff / response or equivalent indices.

[0227] The terms "IL-2 variant peptide fusion complex / IL-2 variant peptide complex / multifunctional IL-2 variant peptide immunoconjugate" are used interchangeably in this document, referring to fusion proteins composed of one or more IL-2 variant peptides or their domains covalently linked with other functional domains (such as antigen-binding domains, Fc domains, etc.) (which may contain linkers). When containing heterologous modules related to immune regulation or targeting (such as anti-IL-6, anti-TNF-α binding domains), they can be called immunoconjugates. Adjacent domains can be directly covalently fused or indirectly linked through adaptor peptides; the linker site can be located at the N-terminus or C-terminus of adjacent domains.

[0228] The activation and proliferation of NK and T cells include, but are not limited to, the upregulation of NK and T cell expression of proliferation-related genes, cytokine secretion, and enhanced killing ability of target cells.

[0229] The activation and proliferation-related genes include, but are not limited to, CD69, CD25, CD122, Ki67, CD44, KLRG1, T-bet, and Myc.

[0230] The cytokines include, but are not limited to, TNFα, IFNγ, IL-2, IL-6, IL-10, GzmB, and Perforin.

[0231] The target cell killing includes, but is not limited to, the killing effects of NK and T cells on tumor cells, infected cells, and foreign cells.

[0232] The terms "peptide" and "protein" are used interchangeably in this document, both referring to linear or subsequently modified chain macromolecules formed by amino acid residues linked by peptide bonds. Amino acid abbreviations use the one-letter / three-letter system known in the art (e.g., A / Ala, R / Arg, N / Asn, etc.). The terms "N-terminus / C-terminus" refer to the ends of the polypeptide chain containing the free amino / carboxyl groups, respectively.

[0233] The proteins, peptides, and / or amino acid sequences involved in this application should also be understood to include at least the following range: variants or homologs that have the same or similar functions as the said protein or peptide.

[0234] In this application, the "variant" can be a protein or polypeptide whose amino acid sequence has been substituted, deleted, or added with one or more amino acids. For example, the functional variant may comprise a protein or polypeptide that has undergone amino acid alteration by substitution, deletion, and / or insertion of at least one, such as 1-30, 1-20, or 1-10, or even 1, 2, 3, 4, or 5 amino acids. The functional variant may substantially retain the biological properties of the protein or polypeptide prior to the alteration (e.g., substitution, deletion, or addition). For example, the functional variant may retain at least 0.1%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the biological activity (e.g., antigen-binding capacity) of some aspect of the protein or polypeptide prior to the alteration. For example, the substitution may be a conservative substitution, and in certain specific cases, the variant protein or polypeptide may completely lose some aspects of the biological properties of the protein and / or polypeptide.

[0235] The term "homology" refers to a protein or polypeptide that has a high sequence identity with the target sequence within a specified comparison window and exhibits the same or similar function under the conditions defined in this invention. Unless otherwise stated, "high sequence identity" generally means at least about 85%, preferably ≥90%, and more preferably ≥95% (identity is calculated using a recognized algorithm such as BLAST / FASTA / ALIGN, allowing for reasonable gaps to be introduced to obtain the best alignment). The calculation of "sequence identity" involves aligning two sequences within a set comparison window, counting the number of matching positions, dividing by the window size, and multiplying by 100 to obtain the percentage. Identity assessment can be performed using known software such as BLAST, ALIGN, and MegAlign, with their default or optimized parameters. Those skilled in the art can select an appropriate algorithm as needed to achieve the maximum alignment of full-length or partial sequences.

[0236] Normally, in a polypeptide chain, an amino group is linked to another carboxyl group to form a chain. However, at the two ends of a protein, there are amino acid residues that have not formed peptide bonds, namely the polypeptide chain end carrying a free amino group and the polypeptide chain end carrying a carboxyl group, respectively. In this application, the term "N-terminus" generally refers to the end of the polypeptide chain where the amino acid residue carries a free amino group. In this application, the term "C-terminus" generally refers to the end of the polypeptide chain where the amino acid residue carries a free carboxyl group.

[0237] The term "immunoglobulin" refers to an antibody comprising an amino acid fragment that specifically binds to an antigen and a constant region fragment. The antigen-specific binding region is the segment in the immunoglobulin that determines key differences; it can also be called an antigen-binding domain, an "epitope," or an "antigen determinant." The antigen-binding domain typically consists of the antibody heavy chain variable region (VH) and the antibody light chain variable region (VL). However, it does not necessarily include both. The antigen-binding domain of the antibodies disclosed in this invention is not limited to the conventional VH and VL domains, but also includes the antigen-binding domains contained in any other type of antibody, but not limited to, recombinant antibodies, single-domain antibodies, heavy chain antibodies, chimeric antibodies, bispecific antibodies, and other unconventional antibodies and combinations thereof. The constant region refers to the common structural region of the immunoglobulin, comprising the antibody light chain constant region and the heavy chain constant region.

[0238] The term "immunoglobulin Fc domain" refers to the Fc fragment formed from one Fc fragment and two identical Fab fragments in the traditional antibody IgG after hydrolysis with papain. This Fc fragment contains the CH2, CH3, and hinge regions of the antibody heavy chain. The traditional Fc fragment binds to Fc fragment receptors, mediating related biological effects. Site-specific mutations can alter its binding ability to the corresponding target receptors, thus affecting its biological function. The immunoglobulin Fc domain disclosed in this invention includes, but is not limited to, traditional Fc fragments and any other forms of Fc mutants. It can be a natural or engineered variant of human IgG1 / IgG2 / IgG3 / IgG4 or mouse-like variants, and may include effectorless mutations (such as LALA or LALAPG) and / or assembly engineering such as KIH (e.g., CH3 region T366W and paired T366S / L368A / Y407V). The aforementioned engineering is used for assembly and stability regulation, without altering the domain definition and counting. The following provides an exemplary Fc domain of human immunoglobulin IgG1:

[0239] EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK, and the Fc domain of human immunoglobulin IgG4:

[0240] In this invention, "direct linking" means linking directly through a bond, rather than through a linker peptide; "indirect linking" means linking through a linker peptide.

[0241] The term "linker peptide," also called "connector," refers to a short peptide chain with conformational flexibility, formed by the combination of amino acid Gly (G) and Ser (S) residues, wherein the ratio of the number of Gly amino acids to the number of Ser amino acids is guaranteed to be ≥1. The linker peptides disclosed in this invention can be extended to any short peptide with this characteristic, for example, including 1, 2, 3, 4, 5, 6, or more units selected from GGGGS and / or GGGS. Exemplary linker peptide amino acid sequences are provided below, including but not limited to GGGGS (SEQ ID NO:54), GGGGSGGGGS (SEQ ID NO:55), GGGGSGGGGSGGGGS (SEQ ID NO:56), GGGGSGGGGSGGGGSGGGS (SEQ ID NO:57), and GGGGSGGGS (SEQ ID NO:58).

[0242] This invention unexpectedly reveals that IL-2 variant peptides, formed by creatively mutating wild-type IL-2, and immunoconjugates containing these IL-2 variant peptides, can be expressed in large quantities and correctly in mammalian cells. This avoids the large-scale aggregation and abnormal assembly that occurs during the expression of natural IL-2 protein or Aldesleukin protein, and is easy to mass-produce and purify, thus ensuring an effective and continuous supply of related products. Furthermore, by introducing specific mutations, the activity of IL-2 in stimulating T and NK cells can be reduced, and the proliferation of Tregs can be specifically induced without significantly affecting the proliferation of non-Treg CD4+ T and CD8+ T cells, thereby reducing or eliminating potential side effects induced by IL-2 stimulation.

[0243] In this application, the term "nucleic acid molecule" generally refers to any length of isolated nucleotide, deoxyribonucleotide, or ribonucleotide or analogue, isolated from its natural environment or synthesized artificially.

[0244] In this application, the term "immunoconjugate" generally refers to a polypeptide molecule conjugated with one or more heterologous molecules (including but not limited to cytotoxins).

[0245] The term "vector" refers to a nucleic acid vector (such as a plasmid, viral vector, etc.) that can be used to insert and express the target polynucleotide. "Host cell" refers to the cell used to express the protein / fusion protein, including but not limited to mammalian cells (such as CHO, HEK293), yeast, insect or plant cells.

[0246] The term "amino acid identity" can refer to the maximum correspondence obtained when amino acid sequences are compared and aligned (with gaps introduced if necessary). The numerical value of percentage identity can be determined using sequence comparison software, algorithms, or visually. For the comparison of the identity of two or more or mixed tandem amino acids of the second binding domain, the percentage identity with any subunit of the second binding domain should be calculated by comparing each subunit with it. Various algorithms and software, such as, but not limited to, NCBI BLAST software, are known in the art for obtaining amino acid sequence alignments.

[0247] In this application, the statement "the IL-2 variant peptide site 74-83 is replaced" refers to the amino acid residues from amino acid position 73 onwards (i.e., starting from amino acid position 74) up to amino acid position 84. For example, the IL-2 variant peptide site 74-83 is replaced with TGDFD, and the IL-2 variant site 74-83 peptide, i.e., the QSKNFHLRPR peptide, in wild-type or with other site mutations, is completely replaced by TGDFD.

[0248] In this application, the term "amino acid mutation" refers to the substitution of an amino acid position by another natural amino acid. For example, a mutation of the T amino acid at position 3 to an A amino acid is denoted as T3A, which is a variant polypeptide obtained by changing the amino acid T at position 3 of the mature wild-type IL-2 polypeptide to amino acid A.

[0249] The term "regulatory T cells (Tregs)" usually refers to CD4+ cells. + CD25 + FOXP3 + Cell population.

[0250] The terms "control," "control molecule," and "positive control" used in this application refer to the disclosed Treg-biased IL-2 variant fusion proteins RG7835 (melredableukin alfa, RO7049665) and efavaleukin alfa (AMG 592), both of which are engineered IL-2 variants fused with immunoglobulin domains, designed to selectively activate the IL-2Rαβγ receptor and amplify Tregs, serving as reference controls for the molecules of this invention under similar mechanisms.

[0251] The term "Treg-biased IL-2 control molecule" as used in this specification includes, but is not limited to, RG7835 and AMG 592. These names are used only for identification of source and mechanism and do not constitute a limitation on their specific preparation process, quality standards, or clinical indications.

[0252] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings and examples. However, those skilled in the art will understand that the following drawings and examples are for illustrative purposes only and are not intended to limit the scope of the invention. Various objects and advantages of the present invention will become apparent to those skilled in the art from the following detailed description of the drawings and preferred embodiments.

[0253] Example 1: Expression and purification of polypeptide complexes

[0254] IL-2 peptide Aldesleukin (IL-2 C125S, SEQ ID NO:8), and IL-2 peptide variants based on Aldesleukin with other mutations and / or substitutions of peptides 74-83, were constructed into pcDNA3.1 plasmids and expressed in ExpiCHO cells via (G4S)2 linker sequences linked to IgG1 Fc LALA (L234A / L235A), or with anti-IL-6 or TNF-α antibody peptides or peptide complexes. Specific information is as follows:

[0255] Table 1. Expression information of IL-2 variant peptide complexes

[0256] Table 2. Expression yield and purity of IL-2 variant peptide complexes

[0257] To further identify the screened variant peptides, it is necessary to express the immunoconjugate of this application in mammalian cells. Therefore, an expression plasmid vector for the peptide complex was first constructed. Approximately 24 hours before plasmid transfection, ExpiCHO cells were passaged to a cell density of approximately 3-4 × 10⁻⁴ cells / year. 6 cells / ml. When the cell density is 6 × 10⁶ 6 When the cell / ml concentration is >95%, take 25ug of the expression vector containing the mixed heavy and light chains and use ExpiFectamine. TM25 ml of ExpiCHO cells were transfected with CHO and cultured at 37°C, 130 rpm, and 8% CO2 for 7 days. The cell culture product was centrifuged, and the supernatant was collected. After filtration through a 0.45 MCE filter, the target peptide complex was purified using a Protein Acolumn and collected. The complex was then concentrated by centrifugation using a Vivaspin 20 Centrifugal Concentrator at 10K. Quantification was performed using the A280 method with a NanoDrop 2000. Antibody purity was determined by SDS-PAGE or SEC-HPLC. Figure 1 shows the results of SDS-PAGE detection of the peptide complex expression, and Figure 2 shows the results of SEC-HPLC detection. In this application, neither the substitution of peptide 74-83 in the IL-2 variant peptide complex with a specific peptide nor the introduction of a specific mutation in the substitution of peptide 74-83 with a specific peptide did produce significantly higher polymers compared to the wild-type IL-2 peptide complex, and both yielded higher amounts and purity. Similarly, the peptide complex composed of the IL-2 variant peptide and the anti-IL-6 / TNF-α antibody also did not produce significantly higher polymers. The results are shown in Table 2 and Figures 1A, 1B, 2A, and 2B.

[0258] Example 2-1: Octet R8 molecular interaction detection of affinity for human IL-2Rα and IL-2Rβγ

[0259] In one specific embodiment, the present invention discloses the binding affinity of the IL-2 variant peptide complex to human IL-2Rα and IL-2Rβγ.

[0260] The Protein A probe was immersed in PBST for 10 min; the IL-2 variant polypeptide complex antibody was prepared to a final concentration of 5 μg / mL; human IL-2Rα and IL-2Rβγ proteins were prepared at a concentration of 500 nM (diluent: PBST). Using PBST as the diluent, the proteins were serially diluted 2-fold in a 96-well plate to obtain 8 concentration gradients, with the 8th concentration being 0 nM (PBST); after establishing the operating method, the required reagents and antibodies were added sequentially to the prepared equilibration buffer, binding buffer, and dissociation buffer according to the operating method for detection. The detection was performed, and the data were analyzed and exported using Octet R8 system analysis software. Table 3 shows the KD and Response values ​​of the immunoconjugates of this application with human IL-2Rα (Acro, ILA-H52H9) and IL-2Rβγ (Acro, ILG-H5283). After the 74-83 peptide fragment of the IL-2 variant polypeptide complex of this application was replaced by a specific peptide fragment, the binding affinity with IL-2Rα and / or IL-2Rβγ did not change significantly (WT IL2, i.e., AN_CyL2_001, had a low response level, mainly due to the low purity and low level of active ingredients after the protein was expressed and produced). After the 74-83 peptide fragment was replaced by a specific peptide fragment and a specific mutation was introduced, its binding affinity with human IL-2Rα and / or IL-2Rβγ was significantly lower than that of the IL-2 variant polypeptide complex AN_CyL2_003 with wild-type IL2 activity. Furthermore, the present invention introduces specific mutant combinations into the IL-2 variant polypeptide complex to reduce or eliminate the binding affinity of IL-2Rα and / or IL-2Rβγ to varying degrees.

[0261] Table 3. Binding affinity values ​​of IL-2 variant peptide complexes to human IL-2Rα and IL-2Rβγ

[0262] Example 2-2: ELISA binding of IL-2 polypeptide complex to human IL-2R protein

[0263] Human IL-2Rα protein (Sinobio, 10165-H08H) or IL-2Rβ / γ complex (ACROBiosystems, ILG-H5283) was prepared at 1 μg / mL, and 100 μL was coated per well of a 96-well ELISA plate. The plate was incubated overnight at 4°C, washed three times with PBST, blocked with 2% BSA at room temperature for 1 h, washed three times with PBST, and then the target peptide complex was added at an initial concentration of 10 μg / mL. The plate was then serially diluted 1:5 at 100 μL / well and incubated at room temperature for 1 h. After washing three times with PBST, HRP-labeled anti-human IgG Fc secondary antibody (Thermo Fisher, A18823, 1:10,000 dilution) was added and incubated at room temperature for 30 min. The plate was washed three times with PBST. TMB chromogenic buffer (100 μL / well) was added and reacted until the reaction reached a suitable temperature. The reaction was terminated with stop solution, and the OD was read at 450 nm. The control sample AN_CyL2_003 is a fusion protein of wild-type human IL-2 and human Fc. The results are shown in Figure 3: Compared with AN_CyL2_003, the test sample showed significantly reduced binding to IL-2Rα and significantly reduced binding to IL-2Rβ / γ.

[0264] Example 3: Binding of IL-2 polypeptide complex to primary cells

[0265] Example 3-1: Binding of IL-2 peptide complex to fresh human PBMC cells

[0266] In one specific embodiment, the present invention evaluates the binding affinity of the IL-2 variant peptide complex to fresh human PBMCs: fresh (unactivated) PBMCs from the Myosin donor DL252112 were collected, counted, and then... 5 Cells were seeded per well in a 96-well V-bottom plate, and PBS + 2% FBS was used as staining / dilution / washing buffer throughout the process. Human Fc (BD, 564220) was added first to block for 10–15 min; then V450 live / dead dye, BV786 anti-human CD8 (BD, 563823), APC-Cy7 anti-human CD4 (biolegend, 317450), and FITC anti-human CD25 (eBioscience, 11-0257-42) were added sequentially and incubated at room temperature for 30 min each; after washing twice, 100 μL of the target antibody (starting at 2 μM, 10 concentrations in total) was added in a 4-fold gradient and incubated for 30 min; after washing twice more, PE-labeled anti-human Fc secondary antibody (BioLegend, 410708) was added and incubated at room temperature for 30 min; after a final wash of 2–3 times, the cells were resuspended in 200 μL buffer for analysis. The staining, dilution, and washing were performed separately on CD4 cells. + CD8 + Subgroups and CD4 +The geometric mean fluorescence intensity (GeoMFI) of the PE channels was read from the CD25^hi (Treg) population. The results (see Figure 4) showed that in unactivated fresh PBMCs, CD4 Tconv (CD4+CD25-) and CD8 T cells expressed almost no CD25, while Treg cells significantly expressed CD25. No significant binding signals were detected in Tconv and CD8 cells for any of the test antibodies, while AN_CyL2_003 and AN_CyL2_082 showed significant binding in the Treg population.

[0267] Example 3-2: Binding of the IL-2 peptide complex to activated human PBMC cells

[0268] α-human CD3 (Invitrogen, 16-0037-81) and α-human CD28 (Invitrogen, 16-0289-85) were each prepared at 1 μg / mL, and 100 μL / well was used to coat 6-well plates. The plates were incubated overnight at 4°C. The next day, the plates were washed once with PBS, and PBMCs (SaiLi Biotechnology, XW0102090W) were added and seeded at the standard density. Activation was completed by incubation at 37°C and 5% CO2 for 24 h. After collection and counting, the cells were sputtered at 5 × 10⁻⁶ cells / well. 4 Seeds were inoculated per well in a 96-well V-bottom plate. PBS + 2% FBS was used as the staining / dilution / washing buffer throughout the process. First, human Fc blocking agent (BD, 564220) was added and incubated at room temperature for 10–15 min. Then, V450 live / dead dye, BV786 anti-human CD8 (BD, 563823), and APC-Cy7 anti-human CD4 (BioLegend, 317450) were added sequentially, and the plates were incubated at room temperature in the dark for 30 min. After two washes, the target peptide complex was added at an initial concentration of 1 μM, serially diluted 4-fold (10 concentration points), 100 μL / well, and incubated at room temperature for 30 min. After two more washes, Alexa Fluor-labeled anti-human Fc secondary antibody (BioLegend, 410714) was added and incubated at room temperature for 30 min. A final 2–3 washes were performed, and the plates were resuspended in 200 μL buffer before data collection. The results showed (see Figure 5) that in CD3 / CD28 activated human PBMCs, the EC50 of AN_CyL2_060 and AN_CyL2_082 binding on CD4 and CD8 cells were both in the tens of nanomolar range.

[0269] Example 4: IL-2 variant peptide complex stimulates STAT5 phosphorylation in T cells

[0270] Example 4-1: IL-2 peptide complex stimulates phosphorylation of STAT5 in human T cells

[0271] In one specific embodiment, this invention discloses the phosphorylation of STAT5 in human T cells by an IL-2 variant peptide complex. PBMC cells (Miaoshun Biotechnology, PB050C-W) were rapidly resuscitated in a 37°C water bath after being harvested from liquid nitrogen; the cells were then purified using a Stemcell T cell purification kit (EasySep). TM Human T Cell Isolation Kit (17951) was used to purify T cells from PBMCs. The purified T cells were then stimulated overnight with anti-CD3 (Thermo, OKT3) and anti-CD28 (Thermo, CD28.2) to induce upregulation of IL-2Rα and IL-2Rβ expression. Subsequently, the T cells were starved for 6 hours in a low-serum culture medium of 2% FBS + 1640. After stimulation for 15 minutes with a specified concentration of IL-2 variant peptide complex or recombinant IL-2 protein (Sino Biological: 11848-HNAH1-E), they were used with BD... TM Cells were treated with Phosflow Fix Buffer I (BD:557870) at room temperature for 1 h, followed by washing twice with Perm / Wash Buffer I. Then, PE anti-STAT5 Phospho (Tyr694) Antibody (Biolegend: 936904) was added and stained at room temperature for 30 min before flow cytometry analysis. As shown in Figure 6, the phosphorylation ability of STAT5 induced by the IL-2 variant peptide complex 74-83 peptide segment was not significantly changed after the specific peptide segment was replaced. However, the phosphorylation ability of STAT5 induced by the specific peptide segment 74-83 peptide segment was significantly reduced after the specific peptide segment was replaced and a specific mutation was introduced.

[0272] Example 4-2: IL-2 peptide complex stimulates STAT5 phosphorylation in fresh human PBMC cells

[0273] In one specific embodiment, this invention discloses the stimulation of STAT5 phosphorylation in fresh human PBMCs by an IL-2 variant peptide complex. Fresh human PBMCs (SaiLi Bio, xw0120399w) were starved overnight in 2% FBS + RPMI 1640 medium with low serum. The next day, the IL-2 variant peptide complex was added and incubated for 15 min at a preset concentration. Subsequently, BD... TMPhosflow Fix Buffer I (BD, 557870) was used for fixation at room temperature for 1 hour. After washing with Perm / Wash Buffer I, PE-labeled anti-p-STAT5 (Tyr694) antibody (BioLegend, 936904) was added, and staining was performed at room temperature in the dark for 30 minutes before instrumental analysis. Tregs (CD4+) were distinguished by CD4 / CD25 ratio. + CD25 + ) and Tconv(CD4 + CD25 - Simultaneously analyze CD8 + Subpopulations. GeoMFI of p-STAT5 was read from each subpopulation. The results (see Figure 7) showed that AN_CyL2_060 and AN_CyL2_082 selectively induced STAT5 phosphorylation in Treg cells in unactivated fresh PBMCs, while inhibiting Tconv and CD8+ phosphorylation. + The cell's p-STAT5 level was not significantly affected, demonstrating excellent Treg-induced selectivity.

[0274] Example 4-3: IL-2 peptide complex stimulates STAT5 phosphorylation in fresh cynomolgus monkey PBMC cells

[0275] In one specific embodiment, this invention discloses the stimulation of STAT5 phosphorylation in fresh cynomolgus monkey PBMCs by an IL-2 variant peptide complex. Fresh monkey-derived PBMCs (Shanghai Xihua) were placed in 2% FBS + RPMI 1640 medium with low serum and starved overnight. The next day, the IL-2 variant peptide complex was added and incubated for 15 min at a preset concentration. Subsequently, BD... TM Phosflow Fix Buffer I (BD, 557870) was used for fixation at room temperature for 1 hour. After washing with Perm / Wash Buffer I, PE-labeled anti-p-STAT5 (Tyr694) antibody (BioLegend, 936904) was added, and staining was performed at room temperature in the dark for 30 minutes before instrumental analysis. Tregs (CD4+) were distinguished by CD4 / CD25 ratio. + CD25 + ) and Tconv(CD4 + CD25 - Simultaneously analyze CD8 + Subgroups. GeoMFI of p-STAT5 was read from each subgroup. The results (see Figure 8) show that AN_CyL2_060 and AN_CyL2_082 also preferentially activate Treg p-STAT5 in unactivated cynomolgus monkey PBMCs, only inhibiting Tconv and CD8 at high doses. + Cells showed a slight upregulation.

[0276] Example 4-4: IL-2 peptide complex stimulates phosphorylation of STAT5 in fresh mouse spleen T cells

[0277] Single-cell suspensions were prepared from the spleens of BALB / c wild-type mice, treated with schizoaflavins, and then starved overnight in 2% FBS–RPMI 1640 low-serum medium. The next day, the target IL-2 variant peptide complex was added and incubated for 15 min at the preset concentration. Subsequently, BD... TM Phosflow Fix Buffer I (BD, 557870) was used for fixation at room temperature for 1 hour. After washing with Perm / Wash Buffer I, PE-labeled anti-p-STAT5 (Tyr694) antibody (Invitrogen, 12-9010-42) was added, and staining was performed at room temperature in the dark for 30 minutes before detection. Detection was then performed on CD4+. + With CD8 + The positivity rate of p-STAT5 was read in the subsets. The results (see Figure 9) showed that AN_CyL2_060 and AN_CyL2_082 selectively increased CD4+ in unactivated mouse spleen-derived T cells. + The positivity rate of p-STAT5 in cells, while that for CD8 + The effect on cells was not significant.

[0278] Example 5: In vivo efficacy evaluation of the IL-2 peptide complex (mouse Treg amplification)

[0279] To verify the in vivo amplification effect of the IL-2 variant peptide complex on Tregs, SPF-grade BALB / c wild-type mice (purchased from Shanghai Nanfang Model Biotechnology) were used. The test drug was AN_CyL2_060, and the control was wild-type human IL-2 (SinoBio, batch number GMP-11848-HNAE). Three mice were used in each group. The dosage and administration method are shown in Table 4.

[0280] Table 4. Grouping, Dosage, and Method of In Vivo Experiments

[0281] Blood was collected from the tail vein on days 2, 5, and 7 after drug administration. 50 μL of whole blood was added to 5 μL of magnetic beads for counting and then treated with Foxp3 / transcription factor fixation / permeabilization buffer (Thermofisher, 00-5523-00) and stained with AF647 anti-mouse Foxp3. Treg was defined as CD4+. + CD25^hiFoxp3 + Simultaneously record CD4 + CD25 - (Tconv) and CD8 +Subpopulations. The absolute cell count (cells / μL) of each subpopulation in peripheral blood was calculated using counting beads and normalized to the pre-drug (Day 0) values ​​to obtain the Treg fold-change to Day 0. The primary evaluation endpoints were the absolute Treg count and its relative Tconv and CD8 count on days 2–7 post-drug administration. + The ratio of cells.

[0282] Under the conditions of this experiment, significant Treg amplification was observed on day 5 after a single dose of AN_CyL2_060 (0.5-1 mg / kg, SC / IP), with a fold increase of >50 from baseline (Day 0); no significant difference was observed between subcutaneous and intraperitoneal administration. The control WT IL-2 showed only a mild proliferation-inducing effect under the same conditions (see Figure 10). These data support the efficacy potential of AN_CyL2_060 in in vivo to induce Treg amplification and the feasibility of multiple administration routes.

[0283] To further compare the in vivo effects of AN_CyL2_060 with the control, RG7835 (MCE, HY-P99728) and AMG592 (MCE, HY-P99877) were selected as positive reference antibodies. AN_CyL2_060 was administered subcutaneously as a single dose in SPF-grade BALB / c mice at three dosage levels: 0.1, 0.3, and 1 mg / kg. Peripheral blood was collected from mice on day 5 post-administration, processed with erythrocytosis, and analyzed by flow cytometry to calculate Treg and CD8+ levels. + The absolute cell count (cells / μL) was measured, and the Treg / CD8 ratio was assessed. Results (see Figure 11) showed that AN_CyL2_060 dose-dependently increased both the absolute Treg count and the Treg / CD8 ratio in the range of 0.1–1 mg / kg, and its Treg induction effect was significantly higher than that of the controls RG-7835 and AMG-592. These data indicate that AN_CyL2_060 has a superior Treg amplification effect in vivo compared to the controls.

[0284] Example 6: In vivo efficacy evaluation of the combination of IL-2 peptide complex and anti-inflammatory related antibody (mouse Treg amplification)

[0285] To assess whether limiting the rise of inflammatory mediators in vivo helps amplify the Treg amplification effect, SPF-grade BALB / c wild-type mice (purchased from Shanghai Southern Model Biotechnology) were randomly assigned to groups, with the day of administration recorded as Day 0. The test drug was the IL-2 peptide complex AN_CyL2_060, in combination with either an anti-mouse IL-6 antibody (Selleck, A2118) or an anti-mouse TNF-α antibody (BioXCell, BE0058). The administration regimen, dosage, and route are shown in Table 5; n = 2 per group.

[0286] Table 5-1 Grouping, Dosage, and Method of In Vivo Experiments

[0287] Table 5-2 Grouping, Dosage, and Method of In Vivo Experiments

[0288] Peripheral blood was collected on day 5 after drug administration, processed with erythropoietin, and analyzed by flow cytometry to calculate the absolute number of Treg cells (cells / μL). Compared with AN_CyL2_060 monotherapy, its combination with anti-IL-6 or anti-TNF-α further increased the absolute number of peripheral blood Tregs (Figure 12). Under the conditions of this experiment, the number of Tregs in the combination group was significantly higher than that of monotherapy, suggesting that administering a Treg-biased IL-2 variant while inhibiting systemic inflammatory mediators can achieve better in vivo Treg amplification.

[0289] Example 7: In vivo efficacy evaluation of the IL-2 peptide complex in an MC903-induced BALB / c mouse atopic dermatitis model

[0290] To evaluate the regulatory effects of the IL-2 variant peptide complex of this invention on epithelial-driven type II inflammatory responses and systemic biomarkers (such as total IgE), an MC903-induced atopic dermatitis model was established and its efficacy was evaluated. Six- to eight-week-old SPF-grade BALB / c mice (purchased from Shanghai Southern Model Biological Institute) were randomly divided into groups (n=6). The model was established by continuous topical application of MC903 (2 nmol / day) to one ear for approximately 9-11 days. Simultaneously, AN_CyL2_060 (0.5 mg / kg, BIW) or an equal volume of carrier was administered subcutaneously. Drug administration and modeling began concurrently; ear thickness changes were recorded at fixed time windows. Plasma was collected on day 11, and total IgE in mice was measured using a commercial mouse total IgE ELISA kit (BioLegend, 432404) according to the manufacturer's instructions. The results showed that MC903 significantly induced an increase in ear thickness, while AN_CyL2_060 did not significantly alleviate the local swelling (Figure 13A); at the same time, MC903 significantly increased total IgE, and treatment with AN_CyL2_060 could reverse this increasing trend (Figure 13B).

[0291] Example 8: Pharmacokinetic evaluation of IL-2 variant peptide complexes and their immunoconjugates in cynomolgus monkeys.

[0292] Healthy male cynomolgus macaques (approximately 6 kg; Shanghai Xihua) were selected and divided into single animal groups. They were administered the following drugs subcutaneously: AN_CyL2_060 and AN_CyL2_082, 2 mg / kg; and AN_CyL2_096, 10 mg / kg. Blood samples were collected intravenously before administration, at 4 h and 24 h post-administration, and on Day 5 and Day 7. Plasma was separated for later use. The plasma concentration of the target antibody was measured as follows: An ELISA plate was coated with 1 μg / mL anti-human IL-2 capture antibody (R&D, MAB2021), blocked with PBST / 2% BSA for 1 h, and then incubated with appropriately diluted monkey plasma for 1 h. After washing, HRP-labeled goat anti-human IgG Fc detection reagent (SouthernBiotech, 2049-05, which undergoes heterogeneous cross-adsorption and does not recognize monkey anti-antibodies) was added and incubated for 30 min. TMB color development was performed, and the absorbance was read at 450 nm after termination. The plasma concentration was calculated based on a reference standard curve prepared in the same batch, and the data was processed and plotted using GraphPad Prism8. As shown in Figures 14A and 14B, both AN_CyL2_060 and AN_CyL2_096 achieved measurable and sustained systemic exposure in cynomolgus monkeys, demonstrating favorable pharmacokinetic characteristics.

[0293] Example 9: Efficacy evaluation of IL-2 variant peptide complexes and their immunoconjugates in cynomolgus monkeys.

[0294] Healthy male cynomolgus macaques (approximately 6 kg; Shanghai Xihua) were selected and divided into single groups. A single subcutaneous injection of the following drugs was administered: AN_CyL2_060 and AN_CyL2_082, 2 mg / kg each; and AN_CyL2_096, 10 mg / kg. Peripheral venous blood was collected before administration (Day 0) and on Days 5, 7, 12, and 22 (approximately 4 mL each time; AN_CyL2_096 was collected until Day 7). After PBMC isolation, Fc blockade was performed, and T cell markers were stained on the surface and treated using a Foxp3 transcription factor fixation / permeabilization system (Thermo Fisher, 00-5523-00), followed by staining with APC-labeled Foxp3 antibody. Tregs were defined as CD4+. + CD25^hiFoxp3 + Simultaneously record CD4 + CD25-(Tconv) and CD8 +Subpopulations. Using live peripheral lymphocytes as the parent population, the proportion of each subpopulation was calculated, and the fold change in percentage of Treg was obtained by normalization to Day 0. At the same time, the ratios of Treg / Tconv and Treg / CD8 were calculated.

[0295] Under the conditions of this experiment, single administration of AN_CyL2_060 and AN_CyL2_082 resulted in peak Treg amplification on Day 7, representing approximately 30-fold amplification from baseline, and maintained at approximately 10-fold levels until Day 22 (Figure 15). The Treg / Tconv and Treg / CD8T ratios approached or exceeded 2-fold at the peak on Day 7. AN_CyL2_096 (10 mg / kg) also showed significant Treg amplification within the observation window up to Day 7 (Figure 16). These data suggest that the IL-2 variant peptide complex and its immunoconjugate of this invention can achieve significant and sustained Treg amplification in non-human primates.

Claims

1. An IL-2 variant polypeptide having a peptide segment from 74 to 83 replaced with TGDFD, STGDFD, TGDFDL, ADGSLH, GEASLH, or GEGSLH relative to the wild-type IL-2 polypeptide sequence of SEQ ID NO:

1.

2. The IL-2 variant polypeptide of claim 1, comprising an amino acid mutation at position C125; preferably, the mutation is C125S.

3. The IL-2 variant polypeptide of any of the preceding claims, comprising an amino acid mutation at position K64; preferably, the mutation is K64D.

4. The IL-2 variant polypeptide of any of the preceding claims, comprising an amino acid mutation at one or more positions selected from T3, N88, S130, K35, D20, F42, E61, R38, K43, E68; preferably, the IL-2 variant polypeptide comprises one or more amino acid mutations selected from T3A, N88R / D, S130R, K35D, D20T / Y / R / H / S, F42L / Y / A, E61R / Q, R38D, K43D, E68K.

5. The IL-2 variant polypeptide of any of the preceding claims, comprising amino acid mutations at positions C125 and K64; preferably, the IL-2 variant polypeptide comprises amino acid mutations C125S and K64D.

6. The IL-2 variant polypeptide of any of the preceding claims, comprising amino acid mutations at positions T3, K64, N88, and C125; preferably, the IL-2 variant polypeptide comprises amino acid mutations T3A, K64D, N88D / R, and C125S; preferably, the IL-2 variant polypeptide further comprises an amino acid mutation at position S130, and more preferably, the IL-2 variant polypeptide further comprises an amino acid mutation S130R.

7. The IL-2 variant polypeptide of any of the preceding claims, comprising an amino sequence selected from TGDFD / K64D / C125S (SEQ ID NO: 28), TGDFD / T3A / K64D / N88R / C125S / S130R (SEQ ID NO: 52), TGDFD / T3A / K64D / N88D / C125S / S130R (SEQ ID NO: 130).

8. An IL-2 variant polypeptide immunoconjugate comprising a first domain of an IL-2 variant polypeptide of any of claims 1-7, and an immunoglobulin Fc domain, the IL-2 variant polypeptide and the immunoglobulin Fc domain being linked by or without a linker peptide.

9. The IL-2 variant polypeptide immunoconjugate of claim 8, wherein, The anti-immunoglobulin Fc fragment is selected from the group consisting of human IgGl Fc domain (SEQ ID NO: 59), human IgG2 Fc domain (SEQ ID NO: 60), human IgG3 Fc domain (SEQ ID NO: 61), human IgG4 Fc domain (SEQ ID NO: 62), mouse IgGl Fc domain (SEQ ID NO: 63), mouse IgG2a Fc domain (SEQ ID NO: 64), mouse IgG2b Fc domain (SEQ ID NO: 65), or mouse IgG3 Fc domain (SEQ ID NO: 66).

10. The IL-2 variant polypeptide immunoconjugate of any of claims 8-9, wherein, The Fc domain is selected from the group consisting of human IgGl Fc domain, and incorporates a combination of L234A and L235A mutations (SEQ ID NO: 67) or a combination of L234A, L235A and P329G mutations (SEQ ID NO: 68) in the human IgGl Fc domain.

11. The IL-2 variant polypeptide immunoconjugate of any of claims 8-10, wherein, The IL-2 variant polypeptide is linked to the N-terminus of the Fc domain with or without a linker peptide; or the IL-2 variant polypeptide is linked to the C-terminus of the Fc domain with or without a linker peptide.

12. The IL-2 variant polypeptide immunoconjugate of any of claims 8-11, wherein, The IL-2 variant polypeptide immunoconjugate comprises two identical heavy chains selected from the group consisting of: (1) a heavy chain comprising the structure of IL-2 variant polypeptide-linker peptide-Fc heavy chain from N-terminus to C-terminus; wherein the linker peptide can be present or absent; or (2) a heavy chain comprising the structure of Fc heavy chain-linker peptide-IL-2 variant polypeptide from N-terminus to C-terminus; wherein the linker peptide can be present or absent.

13. The IL-2 variant polypeptide immunoconjugate of any of claims 8-11, wherein, The IL-2 variant polypeptide immunoconjugate comprises two different heavy chains selected from the group consisting of: (1) a first heavy chain comprising the structure of IL-2 variant polypeptide-linker peptide-Fc heavy chain from N-terminus to C-terminus; and a second heavy chain being an immunoglobulin Fc heavy chain; wherein the linker peptide can be present or absent; or (2) a first heavy chain comprising the structure of Fc heavy chain-linker peptide-IL-2 variant polyeptide from N-terminus to C-terminus; and a second heavy chain being an immunoglobin Fc heavy chain; wherein the linker peptide can be present or absent.

14. The IL-2 variant polypeptide immunoconjugate of any of claims 8-13, wherein, When the linker peptide is absent between two different domains, the two different domains are linked by a chemical bond or a hinge region.

15. The IL-2 variant polypeptide immunoconjugate of any of claims 8-13, wherein, When the linker peptide is present, the linker peptide comprises 1, 2, 3, 4, 5, 6 or more fragments selected from the group consisting of GGGGS, GGGS, GGS, GS.

16. The IL-2 variant polypeptide immunoconjugate of claim 15, wherein, The linker peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57 and SEQ ID NO:

58.

17. The IL-2 variant polypeptide immunoconjugate of claims 11-18, comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 95, SEQ ID NO:

132.

18. An IL-2 variant polypeptide immunoconjugate comprising a first domain of an IL-2 variant polypeptide as set forth in any one of claims 1-7, an immunoglobulin Fc domain, and a second domain, wherein, The second domain comprises an antibody or antigen binding fragment thereof; preferably the second domain is linked to the Fc domain with or without a linker peptide, the IL-2 variant polypeptide is linked to the Fc domain or the second domain with or without a linker peptide.

19. The IL-2 variant polypeptide immunoconjugate of claim 18, wherein, The immunoglobulin Fc domain is as claimed in claim 9 or claim 10.

20. The IL-2 variant polypeptide immunoconjugate of any of claims 18-19, wherein, The second domain is an antigen binding fragment selected from the group consisting of Fab, Fab’, Fv, F(ab)2, F(ab’)2, scFv, VHH, di-scFv and / or dAb.

21. The IL-2 variant polypeptide immunoconjugate of any of claims 18-20, wherein, The antibody or antigen binding fragment thereof specifically targets one or more antigens selected from the group consisting of PD-1, PD-L1, PD-L2, OX40, OX40L, 4-1BB, 4-1BBL, ICOS, ICOSL, TIGIT, CTLA-4, LAG-3, TIM-3, CD3, CD2, CD40, CD40L, CD47, CD6, CD19, CD20, CD22, CD38, CD52, CD79a, CD79b, CD123, CD33, CD30, CD138, BCMA, GPRC5D, CCR8, CCR4, CCR5, NKG2A, Nkp36, VEGF, VEGFR, HGF, EGFR, HER2, HER-3, HER-4, PSMA, CEA, Trop-2, EpCAM, DLL1, MUC1, MUC2, MUC3, MUC4, MUC5AC, MUC5B, MUC7, IL-1β, IL-1R, IL-1Ra, IL-4, IL-4Rα, IL-5, IL-5R, IL-6, IL-6R, IL-7, IL-7Rα, IL-12p35, IL-13, IL-15, IL-15Rα, IL-17A, IL-17F, IL-17RA, IL-17RB, IL-18, IL-18R, IL-21, IL-21R, IL-22, IL-22R, IL-23p19, IL-12 / 23p40, IL-25, IL-31, IL-31RA, IL-33 (ST2), IL-36R, TSLP, TNF-α, TNFR, IFN-α, IFN-β, IFN-γ, IFNAR1, IFNAR2, IFN-γR, GM-CSF, GM-CSFRα, C3, C5, C5a, C5aR1, α4β7 integrin, α4 integrin, αEβ7, β7, ICAM-1, VCAM-1, MAdCAM-1 and TL1A.

22. The IL-2 variant polypeptide immunoconjugate of any of claims 18-21, wherein, The second domain comprises an antibody or antigen-binding fragment thereof that binds IL-6 or IL-6R; preferably the antibody or antigen-binding fragment thereof is selected from the group consisting of tocilizumab, sarilumab, satralizumab, siltuximab antibody or antigen-binding fragment thereof or functionally equivalent fragment thereof or biosimilar thereof.

23. The IL-2 variant polypeptide immunoconjugate of any of claims 18-21, wherein, The second domain comprises an antibody or antigen-binding fragment thereof that binds TNF-a or a receptor thereof; preferably the antibody or antigen-binding fragment thereof is selected from the group consisting of adalimumab, infliximab, golimumab, certolizumab pegol, etanercept antibody or antigen-binding fragment thereof or functionally equivalent fragment thereof or biosimilar thereof.

24. The IL-2 variant polypeptide immunoconjugate of any of claims 18-20, wherein, The IL-2 variant polypeptide immunoconjugate comprises three peptide chains selected from: (1) a first heavy chain comprising the structure VH-CH1-Fc heavy chain from N- to C-terminus; a second heavy chain comprising the structure IL-2 variant polypeptide-linker peptide-Fc heavy chain from N- to C-terminus; a light chain comprising the structure VL-CL from N- to C-terminus; or the three peptide chains are selected from: (2) a first heavy chain comprising the structure VH-CH1-Fc heavy chain from N- to C-terminus; a second heavy chain comprising the structure Fc heavy chain-linker peptide-IL-2 variant polypeptide from N- to C-terminus; a light chain comprising the structure VL-CL from N- to C-terminus; or the three peptide chains are selected from: (3) a first heavy chain comprising the structure IL-2 variant polypeptide-linker peptide-VH-CH1-Fc heavy chain from N- to C-terminus; a second heavy chain comprising the structure Fc heavy chain from N- to C-terminus; a light chain comprising the structure VL-CL from N- to C-terminus; or the three peptide chains are selected from: (4) a first heavy chain comprising the structure VH-CH1-Fc heavy chain-linker peptide-IL-2 variant polypeptide from N- to C-terminus; a second heavy chain comprising the structure Fc heavy chain from N- to C-terminus; a light chain comprising the structure VL-CL from N- to C-terminus; or the three peptide chains are selected from: (5) a first heavy chain comprising the structure VH-CH1-Fc heavy chain from N-terminus to C-terminus; a second heavy chain comprising the structure Fc heavy chain from N-terminal to C-terminal; a light chain comprising the structure IL-2 variant polypeptide-linker peptide-VL-CL from N- to C-terminus; or the three peptide chains are selected from (6) a first heavy chain comprising the structure VH-CH1-Fc heavy chain from N- terminus to C-terminus; a second heavy chain comprising the structure Fc heavy chain from 25. The IL-2 variant polypeptide immunoconjugate of claim 24, wherein, The linker peptide can be present or absent.

26. The IL-2 variant polypeptide immunoconjugate of any of claims 18-20, wherein, The IL-2 variant polypeptide immunoconjugate comprises two peptide chains selected from: (1) a first heavy chain comprising the structure of a second domain antigen binding domain-Fc heavy chain from N-terminus to C-terminus; a second heavy chain comprising the structure of an IL-2 variant polypeptide-linker peptide-Fc heavy chain from N-terminus to C-terminus; or the two peptide chains are selected from: (2) a first heavy chain comprising the structure of a second domain antigen binding domain-Fc heavy chain from N-terminus to C-terminus; a second heavy chain comprising the structure of a Fc heavy chain-linker peptide-IL-2 variant polypeptide from N-terminus to C-terminus; or the two peptide chains are selected from: (3) a first heavy chain comprising the structure of an IL-2 variant polypeptide-linker peptide-second domain antigen binding domain-Fc heavy chain from N-terminus to C-terminus; a second heavy chain comprising the structure of a Fc heavy chain; or the two peptide chains are selected from: (4) a first heavy chain comprising the structure of a second domain antigen binding domain-Fc heavy chain-linker peptide-IL-2 variant polypeptide from N-terminus to C-terminus; a second heavy chain comprising the structure of a Fc heavy chain.

27. The IL-2 variant polypeptide immunoconjugate of claim 26, wherein, The antigen binding domain is a VHH or a ScFv.

28. The IL-2 variant polypeptide immunoconjugate of claim 26 or 27, wherein, The linker peptide can be present or absent.

29. The IL-2 variant polypeptide immunoconjugate of any of claims 18-28, wherein, When the linker peptide is absent between two different domains, the two different domains are connected by a chemical bond or a hinge region.

30. The IL-2 variant polypeptide immunoconjugate of any of claims 18-28, wherein, When the linker peptide is present, the linker peptide comprises 1, 2, 3, 4, 5, 6 or more fragments selected from GGGGS, GGGS, GGS, GS.

31. The IL-2 variant polypeptide immunoconjugate of claim 30, wherein, The linker peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57 and SEQ ID NO:

58.

32. An IL-2 variant polypeptide immunoconjugate comprising a first domain of an IL-2 variant polypeptide as set forth in any one of claims 1-20, an immunoglobulin Fc domain, a second domain, and a third domain, wherein, The second domain comprises an antibody or an antigen binding fragment thereof, wherein the third domain comprises an antibody or an antigen binding fragment thereof, or the third domain comprises a functional protein or an active fragment thereof.

33. The IL-2 variant polypeptide immunoconjugate of claim 32, wherein, The immunoglobulin Fc domain is as described in claim 9 or claim 10.

34. The IL-2 variant polypeptide immunoconjugate of any of claims 32-33, wherein, the second and third domains are each independently an antibody or antigen-binding fragment thereof that binds IL-6 or IL-6R; preferably the antibody or antigen-binding fragment thereof is selected from the group consisting of tocilizumab, sarilumab, satralizumab, siltuximab antibody or antigen-binding fragment thereof or functionally equivalent fragment thereof or biosimilar thereof.

35. The IL-2 variant polypeptide immunoconjugate of any of claims 32-34, wherein, the second and third domains are each independently an antibody or antigen-binding fragment thereof that binds IL-6 or IL-6R; preferably the antibody or antigen-binding fragment thereof is selected from the group consisting of tocilizumab, sarilumab, satralizumab, siltuximab antibody or antigen-binding fragment thereof or functionally equivalent fragment thereof or biosimilar thereof.

36. The IL-2 variant polypeptide immunoconjugate of any of claims 32-34, wherein, the second and third domains are each independently an antibody or antigen-binding fragment thereof that binds TNF-a or a receptor thereof; preferably the antibody or antigen-binding fragment thereof is selected from the group consisting of adalimumab, infliximab, golimumab, certolizumab pegol, etanercept antibody or antigen-binding fragment thereof or functionally equivalent fragment thereof or biosimilar thereof.

37. The IL-2 variant polypeptide immunoconjugate of any of claims 32-33, wherein, The second domain is an antibody or antigen-binding fragment thereof or functional fragment thereof of claims 34-36, and the third domain is a functional protein or active fragment thereof.

38. The IL-2 variant polypeptide immunoconjugate of claim 37, wherein, The third domain comprises a functional protein or active fragment thereof selected from the group consisting of sIL-6R, sgp130, CD80, CD86, CD83, LAG3, ICOSL, OX40L, CD40, CD2, TACI, BR3, TGFBR, VEGFR, IL-1Ra, IL-15, IL-7, IL-4, and / or IL-12.

39. The IL-2 variant polypeptide immunoconjugate of any of claims 32-38, wherein, The second domain and the third domain have the same antigen-binding domain, and the IL-2 variant polypeptide fusion polypeptide complex comprises two identical heavy chains, or two identical heavy chains and two identical light chains; preferably, the heavy chain and light chain have a structure selected from the group consisting of: (1) a first heavy chain comprising, from N-terminus to C-terminus, the structure of IL-2 variant polypeptide-linker peptide-antigen binding domain-Fc heavy chain; a light chain comprising, from N-terminus to C-terminus, the structure of VL-CL; wherein the light chain can be present or absent; or the heavy chain and light chain have a structure selected from the group consisting of: (2) a first heavy chain comprising, from N-terminus to C-terminus, the structure of antigen binding domain-Fc heavy chain-linker peptide-IL-2 variant polypeptide; a light chain comprising, from N-terminus to C-terminus, the structure of VL-CL; wherein the light chain can be present or absent; or the heavy chain and light chain have a structure selected from the group consisting of: (3) a first heavy chain comprising, from N-terminus to C-terminus, the structure of antigen binding domain-Fc heavy chain; a light chain comprising, from N-terminus to C-terminus, the structure of IL-2 variant polypeptide-linker peptide-VL-CL; or the heavy chain and light chain have a structure selected from the group consisting of: (4) a first heavy chain comprising, from N-terminus to C-terminus, the structure of antigen binding domain-Fc heavy chain; a light chain comprising, from N-terminus to C-terminus, the structure of VL-CL-linker peptide-IL-2 variant polypeptide.

40. The IL-2 variant polypeptide immunoconjugate of any of claims 32-38, wherein, The second and third domains have the same antigen-binding domain, and the IL-2 variant peptide fusion peptide complex comprises two distinct heavy chains, or two distinct heavy chains and two identical light chains; preferably, the heavy and light chains have structures selected from: (1) a first heavy chain comprising a structure from the N-terminus to the C-terminus containing the second domain antigen-binding domain-the third domain antigen-binding domain-Fc heavy chain; a second heavy chain comprising a structure from the N-terminus to the C-terminus containing the IL-2 variant peptide-adaptor peptide-Fc heavy chain; and a light chain comprising a structure from the N-terminus to the C-terminus containing the VL-CL; wherein the light chain may or may not be present; or the heavy and light chains have structures selected from: (2) The first heavy chain comprises a structure from the N-terminus to the C-terminus containing a second domain antigen-binding domain-a third domain antigen-binding domain-Fc heavy chain; the second heavy chain comprises a structure from the N-terminus to the C-terminus containing an Fc heavy chain-adaptor peptide-IL-2 variant polypeptide; and the light chain comprises a structure from the N-terminus to the C-terminus containing a VL-CL; wherein the light chain may be present or absent; or the heavy chain and the light chain have a structure selected from the following: (3) the first heavy chain comprises a structure from the N-terminus to the C-terminus containing an IL-2 variant polypeptide-adaptor peptide-a second domain antigen-binding domain-a third domain antigen-binding domain-Fc heavy chain; the second heavy chain comprises an Fc heavy chain from the N-terminus to the C-terminus; and the light chain comprises a structure from the N-terminus to the C-terminus containing a VL-CL; The light chain may or may not be present; or the heavy chain and light chain may have a structure selected from the following: (4) a first heavy chain comprising a second domain antigen-binding domain-third domain antigen-binding domain-Fc heavy chain from the N-terminus to the C-terminus; a second heavy chain comprising an Fc heavy chain-adaptor peptide-IL-2 variant polypeptide from the N-terminus to the C-terminus; and a light chain comprising a VL-CL structure from the N-terminus to the C-terminus; wherein the light chain may or may not be present; or the heavy chain and light chain may have a structure selected from the following: (5) a first heavy chain comprising a second domain antigen-binding domain-Fc heavy chain from the N-terminus to the C-terminus; and a second heavy chain comprising an Fc heavy chain-adaptor peptide-IL-2 variant polypeptide from the N-terminus to the C-terminus. The structure comprises an IL-2 variant polypeptide-adaptor peptide-third domain antigen-binding domain-Fc heavy chain from the N-terminus to the C-terminus; and a light chain comprising a VL-CL structure from the N-terminus to the C-terminus; wherein the light chain may be present or absent; or the heavy chain and light chain have a structure selected from the following: (6) a first heavy chain comprising a second domain antigen-binding domain-Fc heavy chain from the N-terminus to the C-terminus; a second heavy chain comprising a third domain antigen-binding domain-Fc heavy chain-adaptor peptide-IL-2 variant polypeptide from the N-terminus to the C-terminus; and a light chain comprising a VL-CL structure from the N-terminus to the C-terminus; wherein the light chain may be present or absent.

41. The IL-2 variant polypeptide immunoconjugate of any of claims 32-38, wherein, The second and third domains are different antibodies or antigen binding fragments; preferably, the IL-2 variant polypeptide immunoconjugate comprises two or three peptide chains selected from the following structures: (1) a first heavy chain comprising the structure second domain antigen binding domain-third domain antigen binding domain-Fc heavy chain from N- to C-terminus; a second heavy chain comprising the structure IL-2 variant polypeptide-linker peptide-Fc heavy chain from N- to C-terminus; a light chain comprising the structure VL-CL from N- to C-terminus; wherein the light chain can be present or absent; or the IL-2 variant polypeptide immunoconjugate comprises two or three peptide chains selected from the following structures: (2) a first heavy chain comprising the structure second domain antigen binding domain-third domain antigen binding domain-Fc heavy chain from N- to C-terminus; a second heavy chain comprising the structure Fc heavy chain-linker peptide-IL-2 variant polypeptide from N- to C-terminus; a light chain comprising the structure VL-CL from N- to C-terminus; wherein the light chain can be present or absent; or the IL-2 variant polypeptide immunoconjugate comprises two or three peptide chains selected from the following structures: (3) a first heavy chain comprising the structure IL-2 variant polypeptide-linker peptide-second domain antigen binding domain-third domain antigen binding domain-Fc heavy chain from N- to C-terminus; a second heavy chain comprising the structure Fc heavy chain; a light chain comprising the structure VL-CL from N- to C-terminus; wherein the light chain can be present or absent; or the IL-2 variant polypeptide immunoconjugate comprises two or three peptide chains selected from the following structures: (4) a first heavy chain comprising the structure second domain antigen binding domain-third domain antigen binding domain-Fc heavy chain-linker peptide-IL-2 variant polypeptide from N- to C-terminus; a second heavy chain comprising the structure Fc heavy chain; a light chain comprising the structure VL-CL from N- to C-terminus; wherein the light chain cana second heavy chain comprising the structure IL-2 variant polypeptide - linker peptide - Fc heavy chain - third domain antigen binding domain from N- to C-terminus; a light chain comprising the structure VL-CL from N- to C-terminus; wherein the light chain can be present or absent; or the IL-2 variant polypeptide immunoconjugate comprises two or three peptide chains selected from the group consisting of: (8) a first heavy chain comprising the structure second domain antigen binding domain - Fc heavy chain - third domain antigen binding domain from N- to C-terminus; a second heavy chain comprising the structure IL-2 variant polypeptide - linker peptide - Fc heavy chain from N- to C-terminus; a light chain comprising the structure VL-CL from N- to C-terminus; wherein the light chain can be present or absent; or the IL-2 variant polypeptide immunoconjugate comprises two or three peptide chains selected from the group consisting of: (9) a first heavy chain comprising the structure second domain antigen binding domain - Fc heavy chain - third domain antigen binding domain from N- to C-terminus; a second heavy chain comprising the structure Fc heavy chain - linker peptide - IL-2 variant polypeptide from N- to C-terminus; a light chain comprising the structure VL-CL from N- to C-terminus; wherein the light chain can be present or absent; or the IL-2 variant polypeptide immunoconjugate comprises two or three peptide chains selected from the group consisting of: (10) a first heavy chain comprising the structure second domain antigen binding domain - third domain antigen binding domain - Fc heavy chain from N- to C-terminus; a second heavy chain comprising the structure Fc heavy chain from N- to C-terminus; a light chain comprising the structure IL-2 variant polypeptide - linker peptide - VL-CL from N- to C-terminus; or the IL-2 variant polypeptide immunoconjugate comprises two or three chains selected from the group consisting of: (11) a first heavy chain comprising the structure second domain antigen binding domain - third domain antigen binding domain- Fc heavy chain from N- to C-terminus; a second heavy chain comprising thethe IL-2 variant polypeptide immunoconjugate comprises two or three peptide chains selected from the group consisting of: (15) a first heavy chain comprising the structure second domain antigen binding domain - Fc heavy chain - third domain antigen binding domain from N- to C-terminus; a second heavy chain comprising the structure Fc heavy chain from N- to C-terminus; a light chain comprising the structure VL - CL - linker peptide - IL-2 variant polypeptide from N- to C-terminus or the IL-2 variant polypeptide immunoconjugate comprises two or three peptide chains selected from the group consisting of: (16) a first heavy chain comprising the structure second domain antigen binding domain - Fc heavy chain - third domain antigen binding domain from N- to C-terminus; a second heavy chain comprising the structure Fc heavy chain from N- to C-terminus; a light chain comprising the structure IL-2 variant polypeptide - linker peptide - VL - CL from N- to C-terminus or the IL-2 variant polypeptide immunoconjugate comprises two or three peptide chains selected from the group consisting of: (17) a first heavy chain comprising the structure second domain antigen binding domain - Fc heavy chain - third domain antigen binding domain from N- to C-terminus; a second heavy chain comprising the structure Fc heavy chain from N- to C-terminus; a light chain comprising the structure VL - CL - linker peptide - IL-2 variant polypeptide from N- to C-terminus.

42. The IL-2 variant polypeptide immunoconjugate of any of claims 39-41, wherein, The adaptor peptide may or may not be present.

43. The IL-2 variant polypeptide immunoconjugate of claim 42, wherein, When the linker peptide between two different structural domains is absent, the two different structural domains are connected by chemical bonds or hinge regions.

44. The IL-2 variant polypeptide immunoconjugate of claim 42, wherein, When the adaptor peptide is present, the adaptor peptide comprises one, two, three, four, five, six or more fragments selected from GGGGS, GGGS, GGS, GS.

45. The IL-2 variant polypeptide immunoconjugate of claim 44, wherein, The adaptor peptide comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57 and SEQ ID NO:

58.

46. The IL-2 variant polypeptide immunoconjugate of any of claims 39-41, wherein, The antigen-binding domain is VH-CH1, VHH, and / or ScFv.

47. The IL-2 variant polypeptide immunoconjugate according to any one of claims 18-45, comprising an amino acid sequence selected from the group consisting of: SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:148, SEQ ID NO:149, SEQ ID NO:

153.

48. An immunoconjugate comprising an IL-2 variant polypeptide as described in any one of claims 1-7, and / or an IL-2 variant polypeptide immunoconjugate as described in any one of claims 8-47.

49. A nucleic acid molecule encoding an IL-2 variant polypeptide as described in any one of claims 1-7, and / or an IL-2 variant polypeptide immunoconjugate as described in any one of claims 8-47.

50. A vector comprising the nucleic acid molecule of claim 49.

51. A cell comprising and / or expressing an IL-2 variant polypeptide as described in any one of claims 1-7, and / or an IL-2 variant polypeptide immunoconjugate as described in any one of claims 8-47, an immunoconjugate as described in claim 48, a nucleic acid molecule as described in claim 49, and / or a vector as described in claim 50.

52. A composition comprising an IL-2 variant polypeptide as described in any one of claims 1-7, and / or an IL-2 variant polypeptide immunoconjugate as described in any one of claims 8-47, an immunoconjugate as described in claim 48, a nucleic acid molecule as described in claim 49, and / or a carrier as described in claim 50, and / or a cell as described in claim 51, and optionally a pharmaceutically acceptable carrier.

53. A method for preparing an IL-2 variant polypeptide as described in any one of claims 1-7, and / or an IL-2 variant polypeptide immunoconjugate as described in any one of claims 8-47, comprising culturing cells according to claim 51 under conditions that enable the fusion polypeptide to be expressed.

54. A method for inhibiting tumor or tumor cell growth and / or proliferation or for treating autoimmune and inflammation-related diseases, comprising administering an effective amount of an IL-2 variant polypeptide as described in any one of claims 1-7, and / or an IL-2 variant polypeptide immunoconjugate as described in any one of claims 8-47, an immunoconjugate as described in claim 48, a nucleic acid molecule as described in claim 49, and / or a carrier as described in claim 50, and / or a cell as described in claim 51, and / or a composition as described in claim 52.

55. The use of the IL-2 variant peptide of any one of claims 1-7, and / or the IL-2 variant peptide immunoconjugate of any one of claims 8-47, the immunoconjugate of claim 48, the nucleic acid molecule of claim 49, and / or the carrier of claim 50, and / or the cell of claim 51, and / or the composition of claim 52 in the preparation of a medicament, wherein the medicament is used for the prevention, improvement and / or treatment of tumors, cancers, autoimmune and inflammation-related diseases.

56. The method or use according to claims 54-55, wherein the tumor comprises a solid tumor and / or a hematoma.

57. The use or method according to claim 56, wherein the inflammation-related diseases may include rheumatoid arthritis, systemic lupus erythematosus, psoriasis / psoriatic arthritis, inflammatory bowel disease (such as ulcerative colitis, Crohn's disease), multiple sclerosis, myasthenia gravis, Sjögren's syndrome, atopic dermatitis, alopecia areata, thyroid autoimmune diseases, and transplant-related immune imbalances (such as graft-versus-host disease, transplant rejection).

58. A combination therapy comprising administering to a patient in need an effective amount of an IL-2 variant peptide as described in any one of claims 1-7, and / or an IL-2 variant peptide immunoconjugate as described in any one of claims 8-47, an immunoconjugate as described in claim 48, a nucleic acid molecule as described in claim 49, and / or a carrier as described in claim 50, and / or a cell as described in claim 51, and / or a composition as described in claim 52, and an anti-IL-6 antibody, and / or an anti-TNF-α antibody.

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