Human interleukin-2 variants and uses thereof

Abstract

Human interleukin-2 variants and uses thereof are provided. Specifically, IL-2 variants (or derivatives thereof) are provided that have lost or reduced affinity with the high affinity receptor IL-2Rα / β / γ and maintain affinity with the intermediate affinity receptor IL-2Rβ / γ. Also provided are recombinant / fusion proteins comprising the IL-2 variants and human antibody fragments. The activity of the IL-2 variants is improved, approaching that of wild-type human IL-2.

Description

[Technical field]

[0001] This application claims priority to a patent application filed on July 30, 2021 (application number 2021108684095) and a patent application filed on June 1, 2022 (2022106214259).

[0002] The present disclosure relates to human interleukin-2 (IL-2) variants or derivatives thereof having one or more amino acid mutations. The IL-2 variants or derivatives thereof have lost or reduced affinity with the high affinity receptor (IL-2Rα / β / γ) and maintain affinity with the intermediate affinity receptor (IL-2Rβ / γ). The present disclosure also relates to immunoconjugates comprising the human IL-2 variants, encoding polynucleotides, vectors, host cells, pharmaceutical compositions, methods of preparation, methods of treatment and uses thereof. Technical Background

[0003] Human interleukin-2 (IL-2) is also known as T cell growth factor (TCGF). The IL-2 gene is located on chromosome 4 (4q27) and contains a total sequence of about 7 kb. IL-2 is composed of about 133 amino acids and has a molecular weight of about 15 kD. Doris Morgan et al., in 1976 and 1977, respectively, discovered that culture medium of activated T cells could promote the proliferation of T cells. The stimulatory factor in the culture medium was purified and identified as IL-2.

[0004] The first in vitro cell experiments showed that T cells could secrete IL-2 and express IL-2 receptor (IL-2R) on the cell surface after being activated by TCR and CD28. The binding of IL-2 to IL-2R could cause T cell proliferation and effects on T cells. IL-2 is a molecule that plays a central role in T cell immune responses. In vivo experiments showed that after knocking out IL-2 or its receptor, animals developed autoimmunity. IL-2 activates not only effector cells (such as T cells and NK cells) but also regulatory T cells, thereby suppressing excessive immunity against self.

[0005] IL-2 acts through IL-2R. IL-2R consists of three subunits: IL-2Rα (i.e., CD25), IL-2Rβ (i.e., CD122), and IL-2Rγ (i.e., CD132). The three subunits can form three forms of receptors, with the high affinity receptor comprising all three subunits of IL-2Rα / β / γ, the medium affinity receptor comprising IL-2Rβ / γ, and the low affinity receptor comprising IL-2Rα. IL-2Rβ and IL-2Rγ are required for the activation of downstream signaling pathways by IL-2. When IL-2 simultaneously binds to IL-2Rβ and IL-2Rγ, the two receptor subunits form a heterodimer to phosphorylate intracellular STAT5, which enters the nucleus, thereby causing the transcription and expression of the corresponding genes. IL-2Rα is not required for signal transduction, but can facilitate the binding of IL-2 to IL-2Rβ and IL-2Rγ.

[0006] IL-2Rγ is expressed in all immune cells. IL-2Rβ is expressed in CD8+ T cells, NK cells, and regulatory T cells, and its expression level increases after T cell activation. IL-2Rα is persistently highly expressed in regulatory T cells and transiently expressed in activated CD8+ T cells, after which its expression level is downregulated.

[0007] IL-2 is primarily synthesized by activated T cells, especially CD4+ helper T cells. It stimulates T cell proliferation and differentiation, induces the production of cytotoxic T lymphocytes (CTLs) and differentiation of peripheral blood lymphocytes into cytotoxic cells and lymphokine-activated killer (LAK) cells, promotes the expression of cytokines and cytolytic molecules by T cells, promotes B cell proliferation and differentiation and immunoglobulin synthesis by B cells, and stimulates the production, proliferation, and activation of natural killer (NK) cells. The ability of IL-2 to expand lymphocyte populations in vivo and improve the effector functions of these cells translates into its antitumor effects, making IL-2 immunotherapy a treatment option for certain patients with metastatic cancer. High-dose IL-2 is currently approved for the treatment of metastatic renal cell carcinoma and malignant melanoma.

[0008] The IL-2 variants disclosed in WO 2009 / 135615 have a mutation at position 20, 88, or 126. The IL-2 variants disclosed in WO 2012 / 062228 have a mutation at at least one of positions 38, 42, 45, 62, 68, or 88. The IL-2 variants disclosed in US 8906356 have a mutation at positions 91 and 126. The IL-2 variants disclosed in US 9732134 have a mutation at at least one of positions 15, 16, 22, 84, 88, or 95. The IL-2 variants disclosed in US 7803361 and US 8124066 contain the mutation R38W. Summary of the Invention

[0009] The present disclosure relates to IL-2 variants (or derivatives thereof) having one or more amino acid mutations, conjugates thereof, as well as methods of use and preparation of the IL-2 variants.

[0010] (IL-2 variants or derivatives thereof) In a first aspect, the disclosure provides an IL-2 variant (or a derivative thereof) that comprises one or more amino acid mutations compared to mature wild-type human IL-2 (SEQ ID No. 1).

[0011] "Interleukin-2" or "IL-2" refers to any naturally occurring IL-2 from any mammal, e.g., primates such as humans, and rodents such as mice and rats. The term includes both unprocessed IL-2 and any processed IL-2 from cells. The term also includes naturally occurring IL-2 variants, e.g., splice variants or allelic variants.

[0012] "Wild-type human IL-2" is identical to an IL-2 variant, except that at each amino acid position of the IL-2 variant, it has an amino acid of wild-type IL-2 type. The term "wild-type" is intended to encompass one or more naturally occurring amino acid mutations that do not affect the ability to bind to the IL-2 receptor.

[0013] "Mature wild-type human IL-2" refers to the mature form of wild-type human IL-2. Unprocessed human IL-2 further includes an N-terminal signal peptide of about 20 amino acids, which is not present in the mature IL-2 molecule. Exemplary amino acid sequences of mature wild-type human IL-2 include, but are not limited to, the segment 21-153 of, for example, INSDC database accession number CAA25742.1, Swiss-Prot P60568.1, and Genbank NP_000577.2. In some embodiments, mature wild-type human IL-2 is the amino acid sequence shown in SEQ ID No. 1. Please note that for recombinant expression purposes, an additional methionine M is required at the N-terminus. However, one of skill in the art will understand that all sequences with or without M at the first position are included within the scope of "mature wild-type human IL-2" in this application.

[0014] In some embodiments, the amino acid mutations performed in the present application result in a reduction in the length of the region on IL-2 that binds to IL-2Rα (amino acid positions from about 26 to about 47) compared to mature wild-type human IL-2.

[0015] In some embodiments, amino acid mutations include substitutions, deletions (or truncations), insertions (or additions), modifications, and any combination thereof. For example, to alter the binding properties of IL-2, one amino acid (e.g., an amino acid with a different structure and / or chemical properties) may be used to replace another amino acid. Amino acid substitutions include unnatural amino acids and the 20 standard amino acids. Amino acid mutations may be generated using methods known in the art, including site-directed mutagenesis, PCR, gene synthesis, chemical modification, and the like.

[0016] In some embodiments, an IL-2 variant (or derivative thereof) according to the present disclosure has reduced affinity for IL-2Rα and unchanged or increased affinity for IL-2Rβ and / or IL-2Rγ.

[0017] "Affinity" refers to the strength of the sum of non-covalent interactions between the binding site of a molecule and its ligand. Unless otherwise specified, "affinity" herein refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair, e.g., a receptor and a ligand. Affinity is usually measured using the dissociation constant (K D ) (K 解離 and K. 結合 Affinity can be measured by methods common in the art, including those described herein.

[0018] In some embodiments, an IL-2 variant (or derivative thereof) according to the present disclosure has reduced affinity with the high affinity receptor (IL-2Rα / β / γ) but maintains or increases affinity with the intermediate affinity receptor (IL-2Rβ / γ).

[0019] In some embodiments, "high affinity IL-2 receptor" refers to the heterotrimeric form of the IL-2 receptor, consisting of a receptor gamma subunit (also known as cytokine receptor common subunit gamma, γc, or CD132), a receptor beta subunit (also known as CD122 or p70), and a receptor alpha subunit (also known as CD25 or p55).

[0020] In some embodiments, "intermediate affinity IL-2 receptor" refers to an IL-2 receptor that contains only the gamma and beta subunits, but not the alpha subunit (see Olejniczak and Kasprzak, MedSci Monit 14, RA179-189, 2008).

[0021] In some embodiments, an IL-2 variant (or derivative thereof) according to the present disclosure has reduced activation of regulatory T cells (Tregs) and / or unaffected or increased activation of immune effector cells (e.g., T cells, NK cells).

[0022] In some embodiments of an IL-2 variant (or derivative thereof) according to the present disclosure, the mutation is: Tyrosine (Y) at position 31, Lysine (K) at position 32, Asparagine (N) at position 33, Proline (P) at position 34, Lysine (K) at position 35, Leucine (L) at position 36, Threonine (T) at position 37, Arginine (R) at position 38, Methionine (M) at position 39, Leucine (L) at position 40, threonine (T) at position 41, phenylalanine (F) at position 42, Mutations of lysine (K) at position 43, phenylalanine (F) at position 44, and tyrosine (Y) at position 45; Cysteine ​​(C) at position 125, It occurs in one or more of the following positions (consecutive or non-consecutive) (counting from position 1 of SEQ ID No. 1).

[0023] In some embodiments of an IL-2 variant (or derivative thereof) according to the present disclosure, the mutations occur at any one or more of positions 31-32 and 125 (contiguous or non-contiguous).

[0024] In some embodiments of an IL-2 variant (or derivative thereof) according to the present disclosure, the mutations occur at any one or more of positions 35-41 and 125 (contiguous or non-contiguous).

[0025] In some embodiments of an IL-2 variant (or derivative thereof) according to the present disclosure, the mutations occur at any one or more of positions 43-45 and 125 (contiguous or non-contiguous).

[0026] In some embodiments of an IL-2 variant (or derivative thereof) according to the present disclosure, the mutations occur at any one or more of positions 31-45 and 125 (contiguous or non-contiguous).

[0027] In some embodiments of an IL-2 variant (or derivative thereof) according to the present disclosure, the mutations occur at any one or more of positions 31-32, 35-41, and 125 (contiguous or non-contiguous).

[0028] In some embodiments of an IL-2 variant (or derivative thereof) according to the present disclosure, the mutations occur at any one or more of positions 35-41, 43-45, and 125 (contiguous or non-contiguous).

[0029] In some embodiments of an IL-2 variant (or derivative thereof) according to the present disclosure, the mutations occur at any one or more of positions 31-43 and 125 (contiguous or non-contiguous).

[0030] In some other embodiments, when the native sequence described in public databases or known literature is used as the wild-type mature IL-2, the positions of the amino acid mutations are counted from amino acid A at position 1.

[0031] In some particular embodiments, Amino acids at positions 31 to 45 mutated to Gly-Gly-Asn-Pro-Met-His-Gly-Leu-Asp-Gly-Phe-Gly (SEQ ID No. 11), The amino acid at positions 31-32 mutated to Gly-Gly, The amino acids at positions 35 to 41 mutated to Met-His-Gly-Leu-Asp-Gly (SEQ ID No. 12), Amino acids at positions 35-43 mutated to Met-Gly-Gly-Leu-Gly-Gly (SEQ ID No. 13), Amino acids at positions 35 to 41 mutated to Gly-Gly-Gly-Gly-Gly-Gly (SEQ ID No. 14), The amino acids at positions 43-45 were mutated to Gly; The amino acid at position 125 was mutated to Ala, The present invention provides IL-2 variants comprising any one of the above mutations or a combination thereof.

[0032] In some specific embodiments, the IL-2 variant is (1) a YK to GG mutation at positions 31-32, a KLTRMLT to MHGLDG mutation at positions 35-41, a KFY to G mutation at positions 43-45, and a C to A mutation at position 125, or (2) mutations of amino acids at positions 31 to 45 to GGNPMHGLDGFG and mutation of amino acid at position 125 to A; is selected from the group consisting of The mutant sequence is SEQ ID No. 2 or 15. When containing the mutation, the IL-2 mutant has reduced affinity for IL-2Rα while maintaining or increasing affinity for IL-2Rβ / γ.

[0033] In some specific embodiments, the IL-2 variant is a mutation at positions 35-41 of KLTRMLT to MHGLDG and a mutation at position 125 of the amino acid to A, the mutant sequence being SEQ ID No. 3; When the IL-2 mutant comprises a mutation selected from the group consisting of:

[0034] In some specific embodiments, the IL-2 variant is a YK to GG mutation at positions 31-32 and an amino acid at position 125 to A, the mutant sequence being SEQ ID No. 4; When the IL-2 mutant comprises a mutation selected from the group consisting of:

[0035] In some specific embodiments, the IL-2 variant is a mutation of KFY to G at positions 43-45 and an amino acid mutation of A at position 125, the mutant sequence being SEQ ID No. 5; When the IL-2 mutant comprises a mutation selected from the group consisting of:

[0036] In some specific embodiments, the IL-2 variant is (1) YK to GG mutation at positions 31-32, (2) a mutation at positions 35-41 of KLTRMLT to MHGLDG; (3) a mutation of the amino acid at position 125 to A; and the mutant sequence is SEQ ID No. 6. When the IL-2 mutant comprises a mutation selected from the group consisting of:

[0037] In some specific embodiments, the IL-2 variant is (1) a mutation at positions 35-41 from KLTRMLT to MHGLDG; (2) a mutation of KFY to G at positions 43–45; (3) a mutation of the amino acid at position 125 to A; and the mutant sequence is SEQ ID No. 7. When the IL-2 mutant comprises a mutation selected from the group consisting of:

[0038] In some specific embodiments, the IL-2 variant is (1) YK to GG mutation at positions 31-32, (2) a mutation of KFY to G at positions 43–45; (3) a mutation of the amino acid at position 125 to A; and the mutant sequence is SEQ ID No. 8. When the IL-2 mutant comprises a mutation selected from the group consisting of:

[0039] In some specific embodiments, the IL-2 variant is a mutation at positions 35-43 of KLTRMLTFK to MGGLGG and a mutation at position 125 of the amino acid to A, the mutant sequence being SEQ ID No. 9; When the IL-2 mutant comprises a mutation selected from the group consisting of:

[0040] In some specific embodiments, the IL-2 variant is a mutation of KLTRMLT to GGGGGG at positions 35 to 41, a mutation of amino acids 43 to 45 to G, and a mutation of amino acid 125 to A, the mutant sequence being SEQ ID No. 10; When the IL-2 mutant comprises a mutation selected from the group consisting of:

[0041] In some specific embodiments, the amino acid sequence of the IL-2 variant (or a derivative thereof) is selected from SEQ ID Nos. 2-10.

[0042] [Table 1-1] [Table 1-2] [Table 1-3]

[0043] Naturally expressed human IL-2 has a total of 153 amino acids, with amino acids at positions 1-20 being the signal peptide. This has a total of 133 amino acids (i.e., mature human IL-2) after cleavage of the signal peptide, corresponding to positions 1-133 of SEQ ID No. 1 in the present disclosure. For purposes of recombinant expression, artificially produced IL-2 has an additional methionine at the first position (corresponding to the start codon AUG).

[0044] JPEG0007618848000004.jpg40166

[0045] In some embodiments, the IL-2 variant is in monomeric form.

[0046] In a second aspect, the present disclosure provides derivatives of IL-2 variants, including IL-2 variants of the present disclosure, having a modification selected from the group consisting of PEGylation, glycosylation, conjugation to albumin, conjugation to Fc, hydroxyethylation, and de-O-glycosylation.

[0047] In some embodiments, derivatives of IL-2 variants include full length or parts of the IL-2 variants of the present disclosure, or functional derivatives thereof, functional fragments, bioactive peptides, fusion proteins, isoforms or salts thereof further obtained based on the IL-2 variants of the present disclosure, such as fusion proteins comprising the IL-2 variants, dimers, trimers, or multimers of the IL-2 variants, various modified forms of the IL-2 variants (e.g., after PEGylation, glycosylation, conjugation to albumin, conjugation to Fc, hydroxyethylation, de-O-glycosylation, etc.).

[0048] In some embodiments, the IL-2 variant derivative is PEGylated (which may be expressed as PEG-IL-2).

[0049] In some other embodiments, the PEG-IL-2 variant comprises a methoxy-PEG-aldehyde (mPEG-ALD) linker. In certain embodiments, the average molecular weight of the PEG is about 5 kD to about 50 kD, particularly 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 kD, or about 5 kD to about 40 kD, or about 10 kD to about 30 kD, or between about 10 kD and about 30 kD, or between about 15 kD and about 30 kD, about 20 kD.

[0050] In certain embodiments, the mPEG-ALD linker comprises a PEG molecule having an average molecular weight selected from the group consisting of about 5 kDa, about 10 kDa, about 12 kDa and about 20 kDa. In some embodiments, the aldehyde group of the mPEG-ALD can be acetaldehyde, propionaldehyde, or butyraldehyde, etc.

[0051] In one embodiment, the IL-2 variant derivative has an increased serum half-life compared to mature wild-type human IL-2.

[0052] In some embodiments, the derivative of the IL-2 variant is an IL-2 variant conjugated to an antibody or antigen-binding fragment thereof. In some embodiments, the derivative of the IL-2 variant is an IL-2 variant conjugated to an Fc fragment of a human antibody. In a particular embodiment, the derivative of the IL-2 variant is an IL-2 variant conjugated at its C-terminus to an Fc fragment of a human antibody (i.e., the IL-2 variant according to the present application is conjugated to the N-terminus of the Fc fragment of a human antibody).

[0053] In some cases, the derivative of the IL-2 variant is in a dimeric form, either homodimer or heterodimer. As an example, modifications are introduced into the Fc fragment to promote heterodimerization. In some embodiments, the Fc fragment comprises a knob-into-hole (KIH) structure, which includes introducing a knob structure into the interface of the first Fc fragment and introducing a hole structure into the interface of the second Fc fragment, and vice versa. This allows the knob structure to localize within the hole structure, promoting the formation of heterodimers and suppressing the formation of homodimers. The knob structure is constructed by replacing small amino acid side chains from the interface of the first Fc fragment with larger side chains such as tyrosine or tryptophan, while the pore structure is constructed by replacing large amino acid side chains with smaller amino acid side chains such as alanine or threonine at the interface of the second Fc fragment.

[0054] (zygote) In a third aspect, the present disclosure provides a conjugate comprising a first component and a second component, the first component being directly conjugated (or indirectly conjugated via a linker) to the second component, the first component being an IL-2 variant (or derivative thereof) of the present disclosure, and the second component being not IL-2 or an IL-2 variant (or derivative thereof).

[0055] In some embodiments, the IL-2 variant (or derivative thereof) is conjugated to at least one second moiety.

[0056] In some embodiments, the IL-2 variant (or derivative thereof) and the second moiety form a fusion protein via a peptide bond.

[0057] In some specific embodiments, the IL-2 variant (or derivative thereof) is attached (or indirectly attached via a linker) to the carboxyl terminus of the second component.

[0058] In some specific embodiments, the IL-2 variant (or derivative thereof) is conjugated (or indirectly conjugated via a linker) to the amino terminus of the second component.

[0059] In some embodiments, the second component is an antigen-binding component, which refers to a polypeptide molecule that specifically binds to an epitope.

[0060] In some embodiments, the antigen binding moiety can direct the moiety attached thereto (e.g., the IL-2, variants or derivatives thereof of the present disclosure) to a target site (e.g., a tumor cell or tumor). The antigen binding moiety can be an antibody or an antigen-binding fragment.

[0061] Antibody is used in the broadest sense herein and includes various antibody structures as long as it exhibits antigen binding activity.Antibody includes, but is not limited to, monoclonal antibody, polyclonal antibody, multispecific antibody (such as bispecific antibody) and antigen binding fragment.Antibody can include mouse antibody, human antibody, humanized antibody, chimeric antibody and camel antibody.

[0062] In some particular embodiments, the antibody or antigen-binding fragment thereof is selected from the group consisting of a polypeptide complex comprising an antibody heavy chain variable region and an antibody light chain variable region, a Fab, an Fv, an sFv, an F(ab')2, a linear antibody, a single chain antibody, an scFv, an sdAb, an sdFv, a nanobody, a peptibody, a domain antibody, and a multispecific antibody (bispecific antibody, diabody, triabody and tetrabody, tandem di-scFv, tandem tri-scFv).

[0063] In some embodiments, when the IL-2 variant (or its derivative) is linked to two or more antigen-binding moieties, each antigen-binding moiety can be independently selected from an antibody and an antigen-binding fragment. For example, the first antigen-binding moiety can be a Fab molecule and the second antigen-binding moiety can be an scFv molecule. For example, the first antigen-binding moiety can be an scFv molecule, while the second antigen-binding moiety is also an scFv molecule. In some embodiments, the first and second antigen-binding moieties are independently directed against different antigens or the same antigen. In some embodiments, the antibody or antigen-binding fragment thereof targets a tumor antigen.

[0064] In some embodiments, the tumor antigen is, for example, a member of the MAGE family (e.g., MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, MAGE-Xp2, MAGE-B2, MAGE-Xp3, MAGE-B3, MAGE-Xp4, MAGE-B4, MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5), the GAGE ​​family (e.g., GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9), MART-1 / These include, but are not limited to, Melan-A, gp75, gp100, DPPIV, ADAbp, cyclophilin, CEA, CAP-1, CAP-2, etv6, aml1, PSA (PSA-1, PSA-2 and PSA-3), PSMA, T-cell receptor, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, tyrosinase, p53, MUC family, HER2 / neu, p21ras, RCAS1, α-fetoprotein, E-cadherin, α-catenin, β-catenin, γ-catenin, p120ctn, PRAME, NY-ESO-1, cdc27, SSX-1, SSX-2, SSX-1, SSX-4, SSX-5, SCP-1, CT-7.

[0065] Pharmaceutical Composition In a fourth aspect, the present disclosure provides a pharmaceutical composition comprising an IL-2 variant (or derivative thereof) or conjugate according to the present disclosure, and optionally a pharma- ceutical acceptable diluent, carrier or excipient. The pharmaceutical composition may be a lyophilized formulation or an injectable solution.

[0066] In some particular embodiments, a unit dose of the pharmaceutical composition may contain from 0.01 wt% to 99 wt% of the IL-2 variant (or derivative thereof) or conjugate. Alternatively, the amount of IL-2 variant (or derivative thereof) or conjugate contained in a unit dose of the pharmaceutical composition is 0.1 to 2000 mg (e.g., 1 to 1000 mg; 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 5, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 mg).

[0067] (Polynucleotide) In a fifth aspect, the present disclosure provides a polynucleotide encoding an IL-2 variant (or a derivative thereof) of the present disclosure.

[0068] Those skilled in the art will appreciate that nucleotide sequences encoding the same amino acid sequence may differ. Due to codon degeneracy and codon bias in different hosts, different nucleotide sequences may encode the same amino acid sequence, and all of these sequences are within the scope of the present disclosure.

[0069] (Expression Vector) In a sixth aspect, the present invention provides an expression vector comprising a polynucleotide according to the present invention. The expression vector may be a eukaryotic expression vector, a prokaryotic expression vector or a shuttle expression vector.

[0070] (host cell) In a seventh aspect, the present disclosure provides a host cell comprising an expression vector according to the present disclosure, or expressing an IL-2 variant according to the present disclosure, or expressing a conjugate according to the present disclosure.

[0071] In some embodiments, a host cell contains (e.g., is transformed or transfected with) an expression vector that includes a polynucleotide according to the disclosure.

[0072] In some embodiments, the host cell is a prokaryotic or eukaryotic cell.

[0073] In some embodiments, the host cell is a bacteria, yeast, or mammalian cell, particularly Pichia pastoris or Saccharomyces cerevisiae.

[0074] In some particular embodiments, the host cell is a prokaryotic microorganism, such as E. coli.

[0075] In some particular embodiments, the host cell is a eukaryotic cell.

[0076] In some embodiments, host cells such as plant cells and insect cells that express glycosylated peptides are used.Vertebrate cells can also be used as host cells, for example, mammalian cell lines in suspension culture, monkey kidney CV1 cell line (COS-7), human embryonic kidney cell line (293 or 293T cell), baby hamster kidney cell (BHK), mouse Sertoli cell (TM4 cell), monkey kidney cell (CV1), VERO-76, human cervical cancer cell (HELA), dog kidney cell (MDCK), buffalo rat hepatocyte (BRL3A), human lung cell (W138), human hepatocyte (Hep G2), mouse breast tumor cell (MMT060562), MRC5 cell, FS4 cell, CHO cell, myeloma cell line (YO, NS0, P3X63, Sp2 / 0, etc.).

[0077] (Methods of Use and Treatment) In an eighth aspect, the present disclosure provides the use of an IL-2 variant (or derivative thereof), conjugate or pharmaceutical composition according to the present disclosure in the manufacture of a medicament.

[0078] In some embodiments there is provided the use of an IL-2 variant (or a derivative thereof) in the treatment of proliferative and immune disorders, modulating T cell mediated immune responses, and stimulating the immune system of an individual.

[0079] In some embodiments, the proliferative disease may be a tumor or cancer (eg, a metastatic tumor or cancer), or may be a solid tumor.

[0080] In some embodiments, the disclosed IL-2 variants (or derivatives thereof), conjugates, or pharmaceutical compositions may be used to treat and stimulate the host's immune system (particularly to enhance the cellular immune response).

[0081] In some embodiments, the enhanced cellular immune response includes improved T cell function, improved B cell function, restored lymphocyte function, increased expression of IL-2 receptors, increased T cell responsiveness, increased activity of IL-2 receptors, increased expression of natural killer cells or lymphokine-activated killer (LAK) cells. Increased activity of These may include, but are not limited to:

[0082] In some embodiments, the IL-2 variants (or derivatives thereof), conjugates, or pharmaceutical compositions of the present disclosure are used to treat a proliferative condition, such as cancer, non-limiting examples of which include bladder cancer, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, colon cancer, colorectal cancer, rectal cancer, gastric cancer, prostate cancer, blood cancer, skin cancer, squamous cell carcinoma, bone cancer, and kidney cancer.

[0083] In some embodiments, the IL-2 variants (or derivatives thereof), conjugates, or pharmaceutical compositions of the disclosure are used to treat neoplasms located in the abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testes, ovaries, thymus, thyroid), eyes, head and neck, nervous system (central and peripheral), lymphatic system, pelvis, skin, soft tissue, spleen, chest, genitourinary system.

[0084] In some embodiments, the IL-2 variants (or derivatives thereof), conjugates, or pharmaceutical compositions of the present disclosure are used to treat a precancerous condition or cancer metastasis, the cancer being selected from the group consisting of renal cell carcinoma, skin cancer, lung cancer, colorectal cancer, breast cancer, brain cancer, and head and neck cancer.

[0085] In some embodiments, the IL-2 variants (or derivatives thereof), conjugates, or pharmaceutical compositions of the present disclosure are used to treat hypergammaglobulinemia, lymphoproliferative conditions, paraproteinemia, purpura, sarcoidosis, Sézary syndrome, Waldenstrom's macroglobulinemia, Gaucher's disease, histiocytosis.

[0086] In some embodiments, the disclosed IL-2 variants (or derivatives thereof), conjugates, or pharmaceutical compositions are used to treat autoimmune diseases, transplant rejection, post-traumatic immune responses, and infectious diseases.

[0087] In some embodiments, the autoimmune disease may be selected from the group consisting of type I diabetes, rheumatoid arthritis, multiple sclerosis, chronic gastritis, Crohn's disease, Graves' disease, Bechterew's disease, psoriasis, myasthenia gravis, autoimmune hepatitis, APECED, Churg-Strauss syndrome, ulcerative colitis, glomerulonephritis, Guillain-Barré syndrome, Hashimoto's thyroiditis, lichen sclerosus, systemic lupus erythematosus, PANDAS, rheumatic fever, sarcoidosis, Sjogren's syndrome, stiff man syndrome, scleroderma, Wegener's granulomatosis, vitiligo, autoimmune enteropathy, Goodpasture's syndrome, dermatomyositis, polymyositis, autoimmune allergy, and asthma.

[0088] In some embodiments, the IL-2 variant (or a derivative thereof) may be used in combination with an immunosuppressant.

[0089] In some embodiments, the immunosuppressant is selected from the group consisting of glucocorticoids, azathioprine, cyclosporine A, mycophenolate mofetil, tacrolimus, anti-CD3 antibodies, anti-CD25 antibodies, anti-TNF-α antibodies, methotrexate, cyclosporine, sirolimus, everolimus, fingolimod, and cyclophosphamide.

[0090] In some embodiments, a therapeutically effective amount of an IL-2 variant (or derivative thereof), conjugate, or pharmaceutical composition of the present disclosure is administered to the subject.

[0091] In some embodiments, the subject in need, such as a patient or individual, is typically a mammal, such as a human.

[0092] In some embodiments, an IL-2 variant (or derivative thereof), conjugate, or pharmaceutical composition of the disclosure is administered to a subject at least twice a day, at least once a day, at least once every 48 hours, at least once every 72 hours, at least once a week, at least once every two weeks, at least once a month, at least once every two months, or at least once every three months.

[0093] In some embodiments, the IL-2 variant (or derivative thereof), conjugate, or pharmaceutical composition of the present disclosure is administered via any route, for example, by parenteral injection (e.g., subcutaneous or intravenous injection). [Brief description of the drawings]

[0094] [Figure 1] 1 is a curve of the interaction between wild-type human IL-2, IL-2 mutants or derivatives thereof and the IL-2Rα receptor. [Diagram 2] 1 is a curve of the interaction between wild-type human IL-2, IL-2 mutants or derivatives thereof and the IL-2Rβ / γ receptor. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0095] The following examples are included to further illustrate the present disclosure, but these examples are not intended to limit the scope of the disclosure.

[0096] Example 1. Recombinant Expression and Preparation of Wild-Type Human IL-2 and IL-2 Mutants or Derivatives Synthesis, expression and purification of IL-2 variant nucleic acid sequences.

[0097] 1. In this example, wild-type human IL-2 and IL-2 mutants (IL-2-1 to IL-2-9) were expressed separately, purified and prepared using the HPC4 tag at the C-terminus of the molecule. IL-2-10 is a mutant of IL-2-1 with a human IgG1 Fc sequence added to the C-terminus, which was then purified and prepared.

[0098] The whole gene was synthesized, and the coding sequence of each protein was subcloned into the expression vector pcDNA3.1 by double enzyme digestion. The final expression vector was confirmed for its accuracy by enzyme digestion and sequencing, and finally transfected into DH5α clonal strain. Plasmids suitable for transfection were extracted by plasmid extraction kit and transfected into mammalian HEK293 cells using transfection reagent for transient expression, and each protein was purified by affinity chromatography.

[0099] 2. Plasmid Transformation of DH5α 80-100 ng of expression plasmid containing the coding sequence was pipetted into previously prepared DH5α competent cells. Competent cells were transformed by heat shock after addition of the plasmid.

[0100] The transformed competent cells were added to LB liquid medium, placed in a shaker at 37°C, and cultured at 200 rpm for about 30 minutes. The cultured competent cells were removed from the shaker, and a portion of the suspension was pipetted onto a plate containing ampicillin, which was then placed in an incubator and cultured overnight at 37°C.

[0101] A single clone was selected from a fresh culture plate and cultured in 2-5 mL of LB medium at 37°C and 200 rpm for 8 hours. The culture was inoculated into 200 mL of LB medium at a ratio of 1 / 500 and cultured at 37°C and 200 rpm for 16 hours. The culture suspension was collected and centrifuged to remove the supernatant.

[0102] 3. Thawing and Passaging HEK293 Cells The medium was pre-warmed to 37°C by setting the water bath at 37°C.

[0103] The HEK293 cells were removed from the liquid nitrogen tank and immediately placed in a 37°C water bath and gently shaken to rapidly thaw (approximately 1 minute).

[0104] The outer wall of the tube was sterilized with 75% ethanol and the tube was placed in a biosafety cabinet. The cells were transferred to a 15 mL centrifuge tube containing 10 mL of medium and centrifuged at 800 rpm for 5 min. After centrifugation, the supernatant was removed and the cells were resuspended in a small amount of fresh medium and then transferred to a culture flask. Fresh medium was added and the flask was gently shaken to uniformly disperse the cells, and cell samples were collected for counting and viability detection. The density was 3 × 10 5 ~4×10 5 The cells / mL were controlled and viability was >95%.

[0105] The cells were placed in an incubator at 37°C, 110 rpm, and 5% CO2. 6 After cell culture for 2-3 days until a density of 1000 cells / mL was reached, the cells were passaged by replenishing with fresh medium. The density and viability of the cells were monitored.

[0106] 4. Plasmid Transfection of HEK293 Cells One day before transfection, HEK293 cells were cultured at 1 × 10 6 The cells were cultured in 1 L suspension at an inoculation density of 1 × 10 cells / mL and incubated in an incubator at 37 °C, 110 rpm, and 5% CO2. On the day of transfection, the cell density was increased to 1 × 10 6 cells / mL~1.5×10 6 Controlled in cells / mL.

[0107] DNA transfection reagent mixture: DNA and transfection reagent were added to the transfection buffer, mixed well, and incubated at 37°C. The DNA transfection reagent mixture was added to the cells to be transfected, placed in an incubator, and incubated at 37°C, 110 rpm, and 5% CO2. Approximately 4-6 days after transfection, the cell culture was removed from the incubator and centrifuged to collect the supernatant and cells.

[0108] 5. Protein Purification After 5 days of incubation, the cell culture fluid was collected and centrifuged, and the supernatant was collected and filtered through a 0.22 μm filter. The sample was dialyzed at 4°C against 1×PBS, pH 7.4 buffer. After dialysis, the sample was subjected to affinity purification using medium conjugated with HPC4 antibody, and further purified by gel filtration chromatography using superdex200 to obtain highly pure target protein.

[0109] Example 2. Determination of the affinity of wild-type human IL-2 and IL-2 mutants or derivatives thereof to IL-2Rα The binding characteristics of wild-type human IL-2 and its IL-2 mutants or derivatives (IL-2-1 to IL-2-10) obtained in Example 1 to IL-2Rα were detected by the Octet platform. The Octet platform detects and analyzes interactions between biomolecules based on biolayer interferometry (BLI).

[0110] 1. Laboratory equipment, reagents and consumables Octet® Octet RED96e System (Pall Fortebio Corp, Menlo Park, Calif.); Analysis buffer: SD buffer (PBS(pH7.4)+0.01% BSA+0.02% Tween20), Acetate buffer (10 mM, pH 4.0, 5.0, 6.0), AR2G (Fortebio, Catalog No. 18-5092), 96-well plates (Greiner Bio-One part number 655209).

[0111] 2. The experiment was carried out with reference to the experimental plan below. The entire experiment was completed under the conditions of 30°C, 1000 rpm, 220 μl / well. The proteins used in the experiment were IL2-Rα-his and IL-2 mutants or their derivatives obtained by transient expression and affinity purification in HEK293.

[0112] The sample analyzed was IL-2Rα, the dilution buffer was SD buffer, and the concentrations of the analytes were 500 nM, 240 nM, 120 nM, 60 nM, 30 nM, 15 nM (and an additional concentration of 1000 nM for some variants).

[0113] 3. Experimental Data and Processing The experimental data were processed using Data Analysis Software 9.0. A 1:1 model, i.e., one wild-type human IL-2 or IL-2 variants (specifically IL-2-1 to IL-2-10) bound to one IL-2Rα, was selected for fitting. The method of global fitting was applied, i.e., six concentrations were analyzed as a group. The fitting results are shown in Figure 1.

[0114] The experimental data were fitted to a 1:1 binding model to obtain affinity Kd values ​​for wild-type human IL-2 or IL2 mutants or derivatives with the receptor IL-2Rα.

[0115] The binding data for wild-type human IL-2 and IL-2 mutants or derivatives thereof to IL-2Rα are shown in FIG.

[0116] The results showed that IL-2-1, IL-2-6, IL-2-8, IL-2-9, and IL-2-10 did not bind to IL-2Rα, and the interactions between IL-2-2, IL-2-3, IL-2-4, IL-2-5, IL-2-7 and IL-2Rα were reduced or maintained to some extent.

[0117] This suggested the following: (1) Mutation of amino acids at positions 31-32 to GG did not significantly affect the binding of IL-2 to IL-2Rα. (2) Mutation of amino acids 43-45 to G had a certain effect on the binding of IL-2 to IL-2Rα. (3) Mutation of KLTRMLT to MHGLDG at positions 35 to 41 did not significantly affect the binding of IL-2 to IL-2Rα. (4) The combination of the above three mutations significantly altered the protein structure, significantly reducing the binding of IL-2 to IL-2Rα, but no binding between the two was observed under experimental conditions.

[0118] [Table 2]

[0119] Example 3. Determination of the affinity of wild-type human IL-2 and IL-2 mutants or derivatives thereof to IL-2Rβ / γ The affinity of wild-type human IL-2 and its mutants or derivatives in Example 1 to IL-2Rβ / γ was detected by experiments.

[0120] 1. Laboratory equipment, reagents and consumables Octet® Octet RED96e System (Pall Fortebio Corp, Menlo Park, Calif.); Analysis buffer: SD buffer (PBS(pH7.4)+0.01% BSA+0.02% Tween20), Acetate buffer (10 mM, pH 4.0, 5.0, 6.0), AR2G (Fortebio, Catalog No. 18-5092), 96-well plates (Greiner Bio-One part number 655209).

[0121] For preparation of IL-2Rβ / γ-Fc heterodimer, IL-2Rβ and IL-2Rγ subunits were cloned and fused to Fc hole and Fc knob, respectively. IL-2Rβ-Fc-hole and IL-2Rγ-Fc-knob were co-transfected into HEK293 cells. The heterodimer was purified using Protein A and molecular sieves Superdex 200.

[0122] 2. The entire experiment was completed under the following conditions: 30°C, 1000 rpm, 220 μl / well.

[0123] The analyte samples were IL-2Rβ / γ heterodimers and the dilution buffer was SD buffer. The analyte concentrations were 750 nM, 500 nM, 250 nM, 125 nM, 62.5 nM, and 31.3 nM.

[0124] 3. Experimental Data and Processing The experimental data were processed using Data Analysis Software 9.0. A 1:1 model, i.e., one wild-type human IL-2 or IL-2 mutants or derivatives (IL-2-1 to IL-2-10) bound to one IL-2Rβ / γ, was selected for fitting. The method of global fitting was applied, i.e., six concentrations were analyzed as a group.

[0125] The data obtained from the experiments were fitted to a 1:1 binding model to obtain affinity Kd values ​​of wild-type human IL-2 or IL2 mutants or their derivatives with IL-2Rβ / γ, and the binding data are shown in Figure 2 and Table 3.

[0126] The results showed that the interaction between IL-2 mutants or their derivatives (IL-2-1 to IL-2-10) and IL-2Rβ / γ was not altered.

[0127] [Table 3]

[0128] Example 4. CTLL-2 cell proliferation activity assay for wild-type IL-2, IL-2 mutants or their derivatives The biological activity of IL-2 was detected by following the proliferation rate of the dependent cell line CTLL-2 under different IL-2 concentrations.

[0129] The cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum and 1% of two antibiotics (penicillin-streptomycin solution) in an incubator at 37°C and 5% CO2. The cells in the logarithmic growth phase were trypsinized and counted under a microscope to obtain 1 × 10 4 ~5×10 4 Cell suspensions were prepared at 1 × 10 cells / ml. 100 μl of the suspension was transferred to three identical wells of a 96-well culture plate, three per plate for each cell type. 3 ~5×10 3 Pipette in cells / well and use 100 μl of medium as a blank control. Plates were incubated overnight at 37° C.

[0130] Groups with different concentrations were set up: blank, 0.001 nM, 0.01 nM, 0.1 nM, 1 nM, and 10 nM.

[0131] At 0 and 24 hours, Cell Counting Kit-8 (CCK-8) and serum-free minimal essential medium were mixed at a volume ratio of 1:10, added to the test wells at 100 μL per well, and incubated in an incubator with 5% CO2 for 1 hour.

[0132] The absorbance at a wavelength of 490 nm was measured using a microplate reader, and the values ​​for each plate were recorded.

[0133] The results were as shown in Table 4. All of the IL-2 mutants or their derivatives had CTLL-2 cell proliferation activity, indicating that the mutations had no significant effect on the signaling function of the IL-2Rβ / γ receptor subunit complex. The proliferation activity of IL-2-1, IL-2-6, IL-2-8, and IL-2-9 mutants on CTLL-2 cells was significantly lower than that of the positive control. The positive control did not bind to the IL-2Rα receptor, but the positive control bound to the IL-2Rα receptor, thereby enhancing the binding to IL-2Rβ / γ, and had a higher proliferation activity on CTLL-2 cells.

[0134] IL-2-10 was a recombinant protein formed by connecting the C-terminus of the IL-2-1 mutant to the N-terminus of the Fc fragment (defined in this application as a derivative of the IL-2 mutant). It had a molecular weight sufficient to enhance binding to IL-2Rβ / γ, and thus maintained the same proliferation activity as wild-type human IL-2 on CTLL-2 cells. The data are shown in Table 4.

[0135] [Table 4]

Claims

1. A human interleukin-2 (IL-2) variant comprising an amino acid mutation compared to the mature wild-type human IL-2 shown in SEQ ID No. 1, said mutation comprising: Mutation of amino acid residues at positions 31 to 45 of the mature wild-type human IL-2 to the sequence shown in SEQ ID No. 11; Mutation of amino acid residues at positions 35 to 41 of the mature wild-type human IL-2 to the sequence shown in SEQ ID No. 12 or 14; Mutation of amino acid residues at positions 35 to 43 of the mature wild-type human IL-2 to the sequence shown in SEQ ID No. 13; a mutation of the amino acid residues at positions 31-32 of the mature wild-type human IL-2 to GG; and Mutation of amino acid residues at positions 43-45 of the mature wild-type human IL-2 to G. IL-2 mutants selected from the group consisting of:

2. The IL-2 mutant is SEQ ID Nos. 2 to 10 and 15, and SEQ ID Nos. 2 to 10 and 15 with M added to the N-terminus 2. The IL-2 variant of claim 1, which is as set forth in a sequence selected from the group consisting of:

3. The IL-2 variant, as compared to the mature wild-type human IL-2, has a reduced affinity for IL-2Rα, and have maintained or increased affinity for IL-2Rβ / γ; The IL-2 mutant of claim 1.

4. PEGylation, glycosylation, conjugation to albumin, conjugation to Fc, hydroxyethylation, and de-O-glycosylation having a modification selected from the group consisting of: A derivative of an IL-2 mutant comprising the IL-2 mutant according to any one of claims 1 to 3.

5. The conjugation to Fc refers to the conjugation of human IgG1 Fc to the C-terminus of the IL-2 mutant, and The PEGylation refers to the attachment of PEG at the N-terminus of the IL-2 variant. A derivative of the IL-2 mutant according to claim 4.

6. The PEG has a molecular weight of 5 kD to 80 kD. A derivative of the IL-2 mutant according to claim 5.

7. The PEG has a molecular weight of 10 kD to 20 kD. A derivative of the IL-2 mutant according to claim 5.

8. A first component; and A second component; and A conjugate comprising: The first component is directly or indirectly joined via a linker to the second component; and The first component is an IL-2 variant according to any one of claims 1 to 3. zygote.

9. The conjugate of claim 8, wherein the second component is an antibody or an antigen-binding fragment thereof.

10. An IL-2 variant according to any one of claims 1 to 3, and Pharmaceutically acceptable diluents, carriers or excipients A pharmaceutical composition comprising:

11. A nucleic acid molecule encoding an IL-2 mutant according to any one of claims 1 to 3.

12. An expression vector comprising the nucleic acid molecule of claim 11.

13. A host cell comprising the expression vector of claim 12.

14. An IL-2 mutant according to any one of claims 1 to 3 for use in the prevention or treatment of a disease selected from the group consisting of proliferative diseases, metastasis of proliferative diseases, and immune diseases.

15. The IL-2 mutant of claim 14, wherein the proliferative disease is a tumor or cancer.

16. The IL-2 variant of claim 15, wherein the tumor or cancer is selected from the group consisting of epithelial cell carcinoma, endothelial cell carcinoma, squamous cell carcinoma, papillomavirus carcinoma, adenocarcinoma, sarcoma, and teratoma.

17. The IL-2 variant of claim 15, wherein the tumor or cancer is selected from the group consisting of melanoma, lymphoma, bladder cancer, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, colon cancer, colorectal cancer, rectal cancer, gastric cancer, prostate cancer, blood cancer, skin cancer, squamous cell carcinoma, bone cancer and renal cancer.

18. The IL-2 variant of claim 15, wherein the tumor or cancer is located in the abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, adrenal glands, parathyroid glands, pituitary gland, testes, ovaries, thymus, thyroid gland, eye, head and neck, central nervous system, peripheral nervous system, lymphatic system, pelvis, skin, soft tissue, spleen, chest or genitourinary system.

19. The IL-2 mutant of claim 14, wherein the immune disease is selected from the group consisting of autoimmune diseases, autoimmune reactions after organ transplantation, post-traumatic immune responses and infectious diseases.

20. The IL-2 variant of claim 19, wherein the immune disease is selected from the group consisting of systemic lupus erythematosus, diabetes, eczema, rheumatoid arthritis, multiple sclerosis, chronic gastritis, Crohn's disease, Graves' disease, Bechterew's disease, psoriasis, myasthenia gravis, autoimmune hepatitis, APECED, Churg-Strauss syndrome, ulcerative colitis, glomerulonephritis, Guillain-Barré syndrome, Hashimoto's thyroiditis, lichen sclerosus, PANDAS, rheumatic fever, sarcoidosis, Sjogren's syndrome, stiff man syndrome, scleroderma, Wegener's granulomatosis, vitiligo, autoimmune enteropathy, Goodpasture's syndrome, dermatomyositis, polymyositis, autoimmune allergy, and asthma.

21. The IL-2 mutant of claim 20, wherein the immune disease is selected from the group consisting of hypergammaglobulinemia, lymphoproliferative disorders, paraproteinemia, purpura, sarcoidosis, Sezary syndrome, Waldenstrom's macroglobulinemia, Gaucher's disease, and histiocytosis.

22. An IL-2 mutant according to any one of claims 1 to 3 for use in enhancing a cellular immune response.

23. The IL-2 variant of claim 22, wherein the enhanced cellular immune response is selected from the group consisting of improved T cell function, improved B cell function, restored lymphocyte function, increased expression of IL-2 receptors, increased T cell responsiveness, and increased activity of natural killer cells or lymphokine-activated killer (LAK) cells.

24. An IL-2 variant as described in claim 14 for use in combination with an immunosuppressant.

25. The IL-2 variant of claim 24, wherein the immunosuppressant is selected from the group consisting of glucocorticoids, azathioprine, cyclosporine A, mycophenolate mofetil, tacrolimus, anti-CD3 antibodies, anti-CD25 antibodies, anti-TNF-α antibodies, methotrexate, cyclosporine, sirolimus, everolimus, fingolimod and cyclophosphamide.

26. Use of an IL-2 mutant according to any one of claims 1 to 3 in the manufacture of a medicament for the prevention or treatment of a disease selected from the group consisting of proliferative diseases, metastasis of proliferative diseases and immune diseases.

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