WO2023005680A1 - 一种人白细胞介素2变体及其用途 - Google Patents

一种人白细胞介素2变体及其用途 Download PDF

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WO2023005680A1
WO2023005680A1 PCT/CN2022/105789 CN2022105789W WO2023005680A1 WO 2023005680 A1 WO2023005680 A1 WO 2023005680A1 CN 2022105789 W CN2022105789 W CN 2022105789W WO 2023005680 A1 WO2023005680 A1 WO 2023005680A1
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variant
cells
affinity
mutated
seq
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French (fr)
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董明晖
陆雍涛
刘建光
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Xi'an Longtengjingyun Biotechnology Co Ltd
Yunquan Biotechnology Beijing Co Ltd
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Xi'an Longtengjingyun Biotechnology Co Ltd
Yunquan Biotechnology Beijing Co Ltd
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Priority to MX2024001253A priority Critical patent/MX2024001253A/es
Priority to US18/566,499 priority patent/US20240368238A1/en
Priority to AU2022320553A priority patent/AU2022320553B2/en
Priority to CA3223427A priority patent/CA3223427A1/en
Priority to KR1020237043540A priority patent/KR20240036511A/ko
Priority to EP22848285.7A priority patent/EP4357357A4/en
Application filed by Xi'an Longtengjingyun Biotechnology Co Ltd, Yunquan Biotechnology Beijing Co Ltd filed Critical Xi'an Longtengjingyun Biotechnology Co Ltd
Priority to JP2023577980A priority patent/JP7618848B2/ja
Publication of WO2023005680A1 publication Critical patent/WO2023005680A1/zh
Anticipated expiration legal-status Critical
Priority to AU2024220168A priority patent/AU2024220168A1/en
Priority to JP2025002927A priority patent/JP2025066741A/ja
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Definitions

  • 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 have abolished or reduced affinity for high affinity receptors (IL-2R ⁇ / ⁇ / ⁇ ) and retain affinity for intermediate affinity receptors (IL-2R ⁇ / ⁇ ).
  • the present disclosure also relates to immunoconjugates, encoding polynucleotides, vectors, host cells, pharmaceutical compositions, preparation methods, and treatment methods and uses comprising the human IL-2 variants.
  • Interleukin-2 Human interleukin-2 (Interleukin-2, IL-2), also known as T cell growth factor (TCGF).
  • the IL-2 gene is located on chromosome 4 (4q27) and includes a sequence of about 7 kb in total.
  • IL-2 consists of about 133 amino acids and has a molecular weight of about 15kD.
  • Doris Morgan et al. 1976 and 1977 respectively found that activated T cell culture medium can promote T cell proliferation.
  • the stimulatory factor in the culture medium was purified and identified as IL-2.
  • T cells after activation by TCR and CD28, can secrete IL-2 and express IL-2 receptor (IL-2R) on the cell surface.
  • IL-2R IL-2 receptor
  • IL-2 is a molecule that plays a central role in T cell immune responses.
  • animals developed autoimmunity. IL-2 not only activates effector cells such as T cells and NK cells, but also activates regulatory T cells, thereby suppressing excess immunity against self.
  • IL-2 acts through IL-2R.
  • IL-2R includes three subunits: IL-2R ⁇ (ie CD25), IL-2R ⁇ (ie CD122) and IL-2R ⁇ (ie CD132).
  • IL-2R ⁇ ie CD25
  • IL-2R ⁇ ie CD122
  • IL-2R ⁇ ie CD132
  • Three subunits can form three receptor forms: the high affinity receptor contains all three subunits IL-2R ⁇ / ⁇ / ⁇ , the intermediate affinity receptor contains IL-2R ⁇ / ⁇ and the low affinity receptor IL-2R ⁇ .
  • IL-2R ⁇ and IL-2R ⁇ are required for IL-2 to activate downstream signaling pathways, and when IL-2 binds IL-2R ⁇ and IL-2R ⁇ simultaneously, the two receptor subunits form a heterodimer, phosphorylating the cell
  • the STAT5 in the cell enters the nucleus and leads to the corresponding gene transcription and expression; IL-2R ⁇ is not necessary for the signal, but it can promote the combination of IL-2 and IL-2R ⁇ and IL-2R ⁇ .
  • IL-2R ⁇ is expressed in all immune cells; IL-2R ⁇ is expressed in CD8+ T cells, NK cells, and regulatory T cells, and the expression level will increase after T cells are activated; IL-2R ⁇ is expressed in Regulatory T cells are continuously highly expressed, and there will be transient expression in activated CD8+ T cells, and then the expression level will be down-regulated.
  • IL-2 is mainly synthesized by activated T cells, especially CD4+ helper T cells. It stimulates the proliferation and differentiation of T cells, induces the generation of cytotoxic T lymphocytes (CTL) and the differentiation of peripheral blood lymphocytes into cytotoxic cells and lymphokine-activated killer (LAK) cells, and promotes the expression of cytokines and cytolysis of T cells Molecules that promote the proliferation and differentiation of B cells and immunoglobulin synthesis by B cells, and stimulate the production, proliferation and activation of natural killer (NK) cells.
  • CTL cytotoxic T lymphocytes
  • LAK lymphokine-activated killer
  • NK natural killer
  • the IL-2 variants disclosed in WO2009135615 have a mutation at position 20, 88 or 126.
  • the IL-2 variants disclosed in WO2012062228 have a mutation at at least one of positions 38, 42, 45, 62, 68 or 88.
  • the IL-2 variant disclosed in US8906356 has mutations at positions 91,126.
  • the IL-2 variants disclosed in US9732134 have mutations in at least one of positions 15, 16, 22, 84, 88 or 95.
  • the IL-2 variants disclosed in US7803361 and US8124066 comprise the R38W mutation.
  • the present disclosure relates to IL-2 variants (or derivatives thereof) with one or more amino acid mutations, and conjugates thereof; and also relates to the use and preparation method of the IL-2 variants.
  • the present disclosure provides an IL-2 variant (or derivative thereof) comprising one or more amino acid mutations compared to mature wild-type human IL-2 (SEQ ID No. 1).
  • Interleukin-2 refers to any native IL-2 from any mammal, such as primates (eg, humans), rodents (eg, mice and rats).
  • the term encompasses unprocessed IL-2 as well as IL-2 derived from any processed form in the cell.
  • the term also encompasses naturally occurring variants of IL-2 (eg, splice variants or allelic variants).
  • wild-type human IL-2 is otherwise identical to an IL-2 variant, except that the IL-2 form has a wild-type amino acid at every amino acid position of the IL-2 variant.
  • wild-type is intended to encompass one or more naturally occurring amino acid mutations that do not affect the binding ability to the IL-2 receptor.
  • “Mature wild-type human IL-2” refers to the mature form of wild-type human IL-2.
  • Raw human IL-2 additionally contains a signal peptide of about 20 amino acids at the N-terminus, which is absent in the mature IL-2 molecule.
  • Exemplary amino acid sequences of mature wild-type human IL-2 are, for example but not limited to: CAA25742.1 of database accession number INSDC, P60568.1 of Swiss-Prot, paragraphs 21-153 of NP_000577.2 in Genbank.
  • the mature wild-type human IL-2 is the amino acid sequence shown in SEQ ID NO.1. It should be noted that additional inclusion of a methionine M at the N-terminus is permitted for recombinant expression purposes. However, those skilled in the art should understand that whether there is the first M or not, it all belongs to the category of "mature wild-type human IL-2" in this application.
  • the amino acid mutations made herein result in a reduced length of the IL-2R ⁇ -binding region (at positions from about amino acid 26 to about 47) of IL-2 compared to mature wild-type human IL-2. short.
  • amino acid mutations include amino acid substitutions, deletions (or truncations), insertions (or additions), modifications, and any combination thereof.
  • one amino acid can be substituted for another (e.g., with an amino acid having different structural and/or chemical properties).
  • Amino acid substitutions include unnatural amino acids or 20 standard amino acids. Amino acid mutations can be generated using methods well known in the art (including site-directed mutagenesis, PCR, gene synthesis, chemical modification, etc.).
  • IL-2 variants (or derivatives thereof) according to the present disclosure have reduced affinity for IL-2R ⁇ and unchanged or increased affinity for IL-2R ⁇ and/or IL-2R ⁇ .
  • affinity refers to the total strength of the non-covalent interaction between the binding site of a molecule and its ligand. Unless otherwise indicated, "affinity” herein refers to intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (eg, receptor and ligand). Affinity can generally be expressed in terms of a dissociation constant (K D ), which is the ratio of the dissociation and association rate constants (K dissociation and K association , respectively). Affinity can be measured by methods routine in the art, including those described herein.
  • the IL-2 variants (or derivatives thereof) according to the present disclosure have reduced affinity for high affinity receptors (IL-2R ⁇ / ⁇ / ⁇ ), but maintain or increase affinity for intermediate affinity receptors (IL-2R ⁇ / ⁇ / ⁇ ). IL-2R ⁇ / ⁇ ) affinity.
  • high affinity IL-2 receptor refers to the heterotrimeric form of the IL-2 receptor, which consists of the receptor gamma subunit (also known as the general cytokine receptor gamma subunit, ⁇ c, or CD132 ), the receptor ⁇ subunit (also known as CD122 or p70) and the receptor ⁇ subunit (also known as CD25 or p55).
  • intermediate affinity IL-2 receptor refers to an IL-2 receptor comprising only ⁇ and ⁇ subunits, but no ⁇ subunit (see Olejniczak and Kasprzak, MedSci Monit 14, RA179-189, 2008 ).
  • IL-2 variants (or derivatives thereof) according to the present disclosure have reduced activation of regulatory T cells (Treg), and/or no activation of immune effector cells (e.g., T cells, NK cells). Affected or increased.
  • Treg regulatory T cells
  • immune effector cells e.g., T cells, NK cells
  • IL-2 variant or derivative thereof disclosed herein, wherein the mutation occurs at any one or more of the following positions (continuous or discontinuous) (from SEQ ID NO: 1 1 digit starts counting):
  • the mutation occurs at any one or more positions (continuous or discontinuous) at positions 31 to 32 and 125.
  • the mutation occurs at any one or more positions (continuous or discontinuous) at positions 35 to 41 and 125.
  • the mutation occurs at any one or more positions (continuous or discontinuous) at positions 43 to 45 and 125.
  • the mutation occurs at any one or more positions (continuous or discontinuous) at positions 31 to 45 and 125.
  • IL-2 variant or derivative thereof disclosed herein, wherein the mutation occurs at any one or more of positions 31 to 32, 35 to 41, and 125 ( continuous or discontinuous).
  • IL-2 variant or derivative thereof disclosed herein, wherein the mutation occurs at any one or more of positions 35 to 41, 43 to 45, and 125 ( continuous or discontinuous).
  • the mutation occurs at any one or more positions (continuous or discontinuous) at positions 31 to 43 and 125
  • the amino acid mutation site is counted from the first amino acid A.
  • an IL-2 variant comprising any of the following mutations or a combination thereof:
  • the 31st-45th position is mutated into Gly-Gly-Asn-Pro-Met-His-Gly-Leu-Asp-Gly-Phe-Gly (SEQ ID No.11);
  • the 31st-32nd position is mutated to Gly-Gly;
  • the 35th-41st position is mutated to Met-His-Gly-Leu-Asp-Gly (SEQ ID No.12);
  • the 35th-43rd mutation is Met-Gly-Gly-Leu-Gly-Gly (SEQ ID No.13);
  • the 35th-41st position is mutated to Gly-Gly-Gly-Gly-Gly-Gly-Gly (SEQ ID No.14);
  • the 43rd-45th position is mutated to Gly
  • Mutation at position 125 is Ala.
  • the affinity of the IL-2 variant to IL-2R ⁇ is reduced, and the affinity to IL-2R ⁇ / ⁇ is maintained or improved:
  • YK at position 31-32 is mutated to GG; KLTRMLT at position 35-41 is mutated to MHGLDG; KFY at 43-45 is mutated to G; C at 125 is mutated to A; or
  • the mutated sequence is SEQ ID No.2 or 15.
  • the affinity of the IL-2 variant to IL-2R ⁇ is reduced, and the affinity to IL-2R ⁇ / ⁇ is maintained or improved:
  • KLTRMLT at positions 35-41 is mutated to MHGLDG, and position 125 is mutated to A; the mutated sequence is SEQ ID No.3.
  • the affinity of the IL-2 variant to IL-2R ⁇ is reduced, and the affinity to IL-2R ⁇ / ⁇ is maintained or increased:
  • YK at position 31-32 is mutated to GG, and position 125 is mutated to A; the mutated sequence is SEQ ID No.4.
  • the affinity of the IL-2 variant to IL-2R ⁇ is reduced, and the affinity to IL-2R ⁇ / ⁇ is maintained or increased:
  • KFY at positions 43-45 is mutated to G, and position 125 is mutated to A; the mutated sequence is SEQ ID No.5.
  • the IL-2 variant when the IL-2 variant comprises a mutation or combination selected from the following, the IL-2 variant has reduced affinity for IL-2R ⁇ and maintained or increased affinity for IL-2R ⁇ / ⁇ :
  • the mutated sequence is SEQ ID No.6.
  • the affinity of the IL-2 variant to IL-2R ⁇ is reduced, and the affinity to IL-2R ⁇ / ⁇ is maintained or increased:
  • KLTRMLT at positions 35-41 is mutated to MHGLDG.
  • the mutated sequence is: SEQ ID No.7.
  • the affinity of the IL-2 variant to IL-2R ⁇ is reduced, and the affinity to IL-2R ⁇ / ⁇ is maintained or increased:
  • the mutated sequence is SEQ ID No.8.
  • the affinity of the IL-2 variant to IL-2R ⁇ is reduced, and the affinity to IL-2R ⁇ / ⁇ is maintained or increased:
  • KLTRMLTFK at positions 35-43 is mutated to MGGLGG, and position 125 is mutated to A; the mutated sequence is SEQ ID No.9.
  • the affinity of the IL-2 variant to IL-2R ⁇ is reduced, and the affinity to IL-2R ⁇ / ⁇ is maintained or improved:
  • KLTRMLT at position 35-41 is mutated to GGGGGG, position 43-45 is mutated to G, and position 125 is mutated to A; the mutated sequence is SEQ ID No.10.
  • the amino acid sequence of the IL-2 variant is selected from SEQ ID No. NO: 2-10.
  • Naturally expressed human IL-2 has a total of 153 amino acids, and amino acids 1-20 are signal peptides. A total of 133 amino acids (i.e. mature human IL-2) after cutting off the signal peptide correspond to positions 1-133 of SEQ ID No. 1 of the present disclosure.
  • the artificially produced IL-2 additionally has a methionine in the first position (corresponding to the start codon AUG).
  • the IL-2 variant is in monomeric form.
  • the present disclosure provides a derivative of an IL-2 variant comprising the IL-2 variant of the present disclosure, the IL-2 variant having a modification selected from any of the following or a combination thereof : PEGylation, glycosylation, conjugation to albumin, conjugation to Fc, hydroxyethylation, de-O-glycosylation.
  • derivatives of IL-2 variants include full-length, part, or functional derivatives and functional fragments of the IL-2 variants disclosed in the present disclosure.
  • fusion proteins comprising IL-2 variants, dimers or trimers or polymers of said IL-2 variants.
  • modified forms of the IL-2 variants eg, PEGylation, glycosylation, conjugation to albumin, conjugation to Fc, hydroxyethylation, removal of O-glycosylation, etc.).
  • derivatives of IL-2 variants are PEGylated (may be denoted as PEG-IL-2).
  • the PEG-IL-2 variant comprises a methoxy-PEG-aldehyde (mPEG-ALD) linker.
  • the average molecular weight of PEG is from about 5 kD to about 50 kD; 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 to about 30 kD; or between about 15 kD to about 20 kD.
  • 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, or about 20 kDa.
  • the aldehyde group of mPEG-ALD may be acetaldehyde, propionaldehyde, butyraldehyde, or the like.
  • the derivative of the IL-2 variant has an increased serum half-life compared to mature wild-type human IL-2.
  • 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 specific embodiment, the derivative of the IL-2 variant is an IL-2 variant conjugated at its C-terminus to the Fc fragment of a human antibody (i.e., an IL-2 variant according to the application conjugated to a human antibody N-terminus of the Fc fragment).
  • the derivatives of the IL-2 variants assume the form of dimers, homodimers or heterodimers.
  • modifications were introduced in the Fc fragment to promote heterodimerization.
  • the Fc fragment comprises a knob-into-hole (KIH) structure, which involves introducing a knob at the interface of a first Fc fragment and a hole at the interface of a second Fc fragment structure (hole), and vice versa.
  • KH knob-into-hole
  • the bulge structure is constructed by replacing small amino acid side chains from the interface of the first Fc fragment with larger side chains (e.g. tyrosine or tryptophan); whereas the pore structure is constructed by substituting smaller amino acid side chains (such as alanine or threonine) are created in the interface of the second Fc fragment by replacing large amino acid side chains.
  • the present disclosure provides a conjugate comprising a first component and a second component; wherein the first component is directly conjugated to (or indirectly conjugated to) the second component through a linker; the first component is the present disclosure IL-2 variant (or derivative thereof); the second component is not IL-2, not IL-2 variant (or derivative thereof).
  • the IL-2 variant (or derivative thereof) is linked to at least one second module.
  • the IL-2 variant (or derivative thereof) forms a fusion protein via a peptide bond with the second module.
  • the IL-2 variant (or derivative thereof) is conjugated to (or indirectly conjugated to via a linker) the carboxyl terminus of the second module.
  • the IL-2 variant (or derivative thereof) is conjugated (or indirectly conjugated via a linker) to the amino terminus of the second module.
  • the second component is an antigen binding component.
  • the antigen-binding component refers to: a polypeptide molecule that specifically binds an epitope.
  • the antigen binding moiety is capable of directing a moiety to which it is attached (e.g., an IL-2 of the disclosure, a variant or derivative thereof) to a target site (e.g., to a tumor cell or a tumor stroma bearing an antigenic determinant). ).
  • the antigen binding moiety may be an antibody or an antigen binding fragment.
  • Antibodies are used herein in the broadest sense and encompass various antibody structures including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) so long as they exhibit antigen binding activity and antigen-binding fragments.
  • Antibodies may include murine antibodies, human antibodies, humanized antibodies, chimeric antibodies, camelid antibodies.
  • 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, Fab, Fv, sFv, F(ab')2, linear antibody, Single chain antibodies, scFv, sdAb, sdFv, nanobodies, peptibodies, domain antibodies and multispecific antibodies (bispecific antibodies, diabody, triabody and tetrabody, tandem di-scFv, tandem tri-scFv).
  • each antigen-binding moiety can be independently selected from antibodies and antigen-binding fragments; for example, the first antigen-binding moiety can be Fab molecules, while the second antigen binding moiety may be a scFv molecule.
  • the first antigen binding moiety can be a scFv molecule and the second antigen binding moiety is also a scFv molecule.
  • the first and second antigen binding moieties are independently directed to different antigens or directed to the same antigen.
  • the antibody or antigen-binding fragment thereof targets a tumor antigen.
  • tumor antigens such as but not limited to: MAGE family (such as 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), GAGE family (such as GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9), MART-1/Melan-A, gp75, gp100, DPPIV, ADAbp, cyclophilin, CEA, CAP-1, CAP-2, etv6, aml1, PSA (PSA-1, PSA-2 and PSA-3
  • the present disclosure provides a pharmaceutical composition, which contains the IL-2 variant (or derivative thereof) or conjugate according to the present disclosure, optionally including a pharmaceutically acceptable diluent, carrier or excipients.
  • the pharmaceutical composition can be a lyophilized preparation or an injectable solution.
  • the unit dose of the pharmaceutical composition may contain 0.01% to 99% by weight of the IL-2 variant (or derivative thereof) or conjugate; or the IL-2 contained in the unit dose of the pharmaceutical composition.
  • the amount of the variant (or derivative thereof) or conjugate is 0.1-2000 mg (eg 1-1000 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).
  • the present disclosure provides a polynucleotide encoding an IL-2 variant (or derivative thereof) of the present disclosure.
  • nucleotide sequences encoding the same amino acid sequence can be different, and due to the degeneracy of codons and the codon preference of different hosts, different nucleotide sequences can encode the same amino acid sequence, and these sequences all belong to the present disclosure within the category.
  • the present disclosure provides an expression vector containing the polynucleotide according to the present disclosure.
  • the expression vector can be a eukaryotic expression vector, a prokaryotic expression vector or a shuttle expression vector.
  • the present disclosure provides a host cell comprising the expression vector according to the present disclosure, or expressing the IL-2 variant according to the present disclosure, or expressing the conjugate according to the present disclosure.
  • the host cell comprises (eg, has been transformed or transfected with) an expression vector comprising a polynucleotide according to the present disclosure.
  • the host cell is a prokaryotic or eukaryotic cell.
  • the host cell is bacteria, yeast or mammalian cell, specifically Pichia pastoris or Saccharomyces cerevisiae.
  • the host cell is a prokaryotic microorganism (eg, E. coli).
  • a prokaryotic microorganism eg, E. coli.
  • the host cell is a eukaryotic cell.
  • host cells expressing glycosylated polypeptides such as plant and insect cells
  • Vertebrate cells can also be used as host cells, e.g., suspension-grown mammalian cell lines, monkey kidney CV1 line (COS-7), human embryonic kidney line (293 or 293T cells), baby mouse kidney cells (BHK), Mouse sertoli (sertoli) cells (TM4 cells), monkey kidney cells (CV1), VERO-76, human cervical cancer cells (HELA), canine kidney cells (MDCK), buffalo rat liver cells (BRL3A), human Lung cells (W138), human hepatocytes (Hep G2), mouse mammary tumor cells (MMT060562), MRC5 cells, FS4 cells, CHO cells, myeloma cell lines (such as YO, NSO, P3X63, and Sp2/0).
  • the present disclosure provides pharmaceutical uses of IL-2 variants (or derivatives thereof), conjugates, and pharmaceutical compositions according to the present disclosure.
  • IL-2 variants or derivatives thereof for treating proliferative diseases, immune diseases, regulating T cell-mediated immune responses, and stimulating the immune system of individuals are provided.
  • the proliferative disease can be a tumor or cancer (eg, a metastatic tumor or cancer), and can be a solid tumor.
  • the IL-2 variants (or derivatives thereof), conjugates, and pharmaceutical compositions of the present disclosure can be used to therapeutically stimulate a host's immune system (in particular, enhance cellular immune responses).
  • enhancing cellular immune response may include, but not limited to: increased T cell function, increased B cell function, recovery of lymphocyte function, increased expression of IL-2 receptor, increased T cell responsiveness, natural killer cell Elevated active or lymphokine-activated killer (LAK) cell activity.
  • LAK lymphokine-activated killer
  • the IL-2 variants (or derivatives thereof), conjugates, pharmaceutical compositions of the present disclosure are used to treat proliferative disorders (eg, cancer).
  • cancer 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, Cancers of the rectum, stomach, prostate, blood, skin, squamous cell, bone and kidney.
  • the IL-2 variants (or derivatives thereof), conjugates, and pharmaceutical compositions of the present disclosure are used to treat neoplasms located in the following locations: abdomen, bone, breast, digestive system, liver, pancreas, Peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testis, ovary, thymus, thyroid), eye, head and neck, nervous system (central and peripheral), lymphatic system, pelvis, skin, soft tissue, spleen, thoracic, and urinary reproductive system.
  • neoplasms located in the following locations: abdomen, bone, breast, digestive system, liver, pancreas, Peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testis, ovary, thymus, thyroid), eye, head and neck, nervous system (central and peripheral), lymphatic system, pelvis, skin, soft tissue, spleen, thoracic, and urinary reproductive system.
  • the IL-2 variants (or derivatives thereof), conjugates, pharmaceutical compositions of the present disclosure are used to treat precancerous conditions or cancer metastasis.
  • the cancer is selected from the following: renal cell carcinoma, skin cancer, lung cancer, colorectal cancer, breast cancer, brain cancer, head and neck cancer.
  • the IL-2 variants (or derivatives thereof), conjugates, and pharmaceutical compositions of the present disclosure are used for the treatment of: hypergammaglobulinemia, lymphoproliferative disorders, lesion proteinemia (paraproteinemias), purpura, sarcoidosis, Sezary Syndrome, Waldenstron's macroglobulinemia, Gaucher's Disease, histiocytosis.
  • the IL-2 variants (or derivatives thereof), conjugates, and pharmaceutical compositions of the present disclosure are used to treat autoimmune diseases, transplant rejection, post-traumatic immune responses, and infectious diseases.
  • the autoimmune disease can be selected from: type I diabetes (type I diabetes mellitus), rheumatoid arthritis (rheumatoid arthritis), multiple sclerosis (multiple sclerosis), chronic gastritis (chronic gastritis), Crohn's disease, Basedow disease, Bechterew disease, psoriasis, myasthenia gravis, autoimmune hepatitis , APECED, Chrug-Strauss syndrome, ulcerative colitis, glomerulonephritis, Guillain-Barré syndrome, Hashimoto's thyroid Hashimoto thyroiditis, lichen sclerosus, systemic lupus erythematodes, PANDAS, rheumatic fever, sarcoidosis, Sjögren syndrome, Stiff-Man syndrome, scleroderma, Wegener's granulomatosis, vitiligo, autoimmune enteropathy, pulmonary hemorrhage nephritic syndrome (
  • IL-2 variants may be used in combination with immunosuppressants.
  • the immunosuppressant is selected from the group consisting of: glucocorticoid; azathioprine; cyclosporin A; mycophenolatemofetil; ); anti-CD3 antibody; anti-CD25 antibody; anti-TNF-alpha antibody; methotrexate; cyclosporin; sirolimus; everolimus; fingolimus fingolimod; and cyclophosphamide.
  • a therapeutically effective amount of: an IL-2 variant (or derivative thereof), conjugate, pharmaceutical composition of the present disclosure is administered to the subject.
  • the subject (eg, patient or individual) in need of treatment is typically a mammal, eg, a human.
  • the IL-2 variant (or derivative thereof), conjugate, pharmaceutical composition of the present disclosure is administered to the subject at least once a month, or at least once every 3 months.
  • the IL-2 variants (or derivatives thereof), conjugates, pharmaceutical compositions of the present disclosure are administered by any route.
  • parenteral injection eg, subcutaneous, intravenous.
  • Figure 1 Interaction curves of wild-type human IL-2, IL-2 variants or derivatives thereof with the IL-2R ⁇ receptor.
  • Figure 2 Interaction curves of wild-type human IL-2, IL-2 variants or derivatives thereof with IL-2R ⁇ / ⁇ receptors.
  • Example 1 Recombinant expression and preparation of wild-type human IL-2 and IL-2 variants or derivatives thereof
  • IL-2 and IL-2 variants (IL-2-1 to IL-2-9) were expressed separately, and purified and prepared by relying on the HPC4 tag on the C-terminus of the molecule .
  • IL-2-10 is the human IgG1 Fc sequence added to the C-terminus of the IL-2-1 variant, which is then purified and prepared.
  • 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 accuracy of the final expression vector was confirmed by enzyme digestion and sequencing, and finally transferred to the DH5 ⁇ clone strain.
  • the transfection-grade plasmid was extracted by a plasmid extraction kit, and the plasmid was transfected into mammalian cells HEK293 by a transfection reagent for transient expression, and each protein was purified by affinity chromatography.
  • the transformed competent cells were added to LB liquid medium, placed in a shaker at 37° C., and shaken at 200 rpm for about 30 minutes.
  • the cultured competent cells were taken out, part of the suspension was sucked and coated on a plate containing ampicillin resistance, and cultured overnight at 37°C in an incubator.
  • the water bath was adjusted to 37°C; the medium was prewarmed at 37°C.
  • the cells Place the cells in an incubator at 110rpm, 37°C with 5% CO2. The cells were cultured for 2-3 days, and when the density reached 2.0 ⁇ 10 6 /mL, the cells were subcultured by adding fresh medium. Cell density and viability assays were monitored.
  • the HEK293 cells were cultured in suspension to 1 L with a seeding density of 1 ⁇ 10 6 /mL, placed in an incubator at 110 rpm, and cultured at 37°C with 5% CO2. On the day of transfection, the cell density was controlled at 1 ⁇ 10 6 /mL to 1.5 ⁇ 10 6 /mL.
  • DNA-transfection reagent mixture Add DNA and transfection reagent to the transfection buffer, mix well, and incubate at 37°C. Add the DNA-transfection reagent mixture into the cells to be transfected, put them into an incubator at 110 rpm, and culture at 37° C. with 5% CO2. About 4-6 days after transfection, the cell culture was removed, centrifuged, and the supernatant and cells collected.
  • the cell culture fluid after 5 days of culture was taken, and after centrifugation, the supernatant was taken and filtered with a 0.22 ⁇ m filter. Dialyze against buffer 1 ⁇ PBS pH 7.4 at 4°C. After the dialysis, affinity purification was carried out with the filler coupled with HPC4 antibody, and then further purification was carried out by gel filtration chromatography superdex200 to obtain the target protein with higher purity.
  • Example 2 Determination of the affinity of wild-type human IL-2 and IL-2 variants or derivatives thereof to IL-2R ⁇
  • the binding properties of wild-type human IL-2 obtained in Example 1 and its IL-2 variants or derivatives thereof (IL-2-1 to IL-2-10) to IL-2R ⁇ were detected by the Octet platform.
  • the Octet platform is based on Bio-Layer Interferometry (BLI) for detection and analysis of biomolecular interactions.
  • Octet RED96e system (Pall Fortebio Corp, Menlo Park, CA);
  • Acetate buffer (10mM, pH4.0, 5.0, 6.0);
  • AR2G (Fortebio, catalog number: 18-5092);
  • the experiment was carried out with reference to the following experimental design, and the whole experiment was completed under the conditions of 1000 rpm, 30° C., and 220 ⁇ l/well.
  • the protein used in the experiment is IL2-R ⁇ -his and IL-2 variants or derivatives obtained by HEK293 transient expression and affinity purification.
  • the sample to be tested is: IL-2R ⁇ , and the dilution buffer is SD buffer;
  • the concentration range of the analyte is: 500nM, 240nM, 120nM, 60nM, 30nM, 15nM (some variants increase 1000nM concentration measurement).
  • the experimental data were processed by Data Analysis software 9.0.
  • a 1:1 model was chosen for the fitted model, ie 1 wild-type human IL-2 or IL-2 variant (specifically IL-2-1 to IL-2-10) combined with 1 IL-2R ⁇ .
  • the fitting method is global; that is, the 6 concentrations are analyzed as a group. The fitting results can be seen in Figure 1.
  • the data obtained from the experiment were fitted with a 1:1 binding model to obtain the affinity Kd value of wild-type human IL-2 or IL2 variants or derivatives to the receptor IL-2R ⁇ .
  • Example 3 Determination of the affinity of wild-type human IL-2 and IL-2 variants or derivatives thereof to IL-2R ⁇ / ⁇
  • Octet RED96e system (Pall Fortebio Corp, Menlo Park, CA);
  • Acetate buffer (10mM, pH4.0, 5.0, 6.0);
  • AR2G (Fortebio, catalog number: 18-5092);
  • the IL-2R ⁇ and IL-2R ⁇ subunits were cloned and fused to Fc hole and Fc knob respectively for the preparation of IL-2R ⁇ / ⁇ -Fc heterodimer.
  • IL-2R ⁇ -Fc-hole and IL-2R ⁇ -Fc-knob were simultaneously transfected into HEK293 cells.
  • the heterodimer was purified with Protein A and molecular sieve Superdex 200.
  • the sample to be tested is IL-2R ⁇ / ⁇ heterodimer, and the dilution buffer is SD buffer.
  • the concentration range of the analyte is 750nM, 500nM, 250nM, 125nM, 62.5nM, 31.3nM.
  • the experimental data were processed by Data Analysis software 9.0.
  • a 1:1 model was chosen for the fitted model, ie 1 wild-type human IL-2 or IL-2 variant or its derivatives (IL-2-1 to IL-2-10) combined with 1 IL-2R ⁇ / ⁇ .
  • the fitting method is global, that is, the 6 concentrations are analyzed as a group.
  • the cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum and 1% double antibody (penicillin-streptomycin mixed solution) in an incubator at 37°C and 5% CO 2 .
  • the cells in the logarithmic growth phase were digested with trypsin and counted under a microscope to make a cell suspension of 1 ⁇ 10 4 to 5 ⁇ 10 4 cells/ml.
  • the absorbance at 490 nm wavelength was measured with a microplate reader. Record the value for each plate.
  • IL-2 variants or their derivatives have CTLL-2 cell proliferation activity, indicating that the mutation does not significantly affect the signal transduction function of the IL-2R ⁇ / ⁇ receptor subunit complex.
  • IL-2-1, IL-2-6, IL-2-8, IL-2-9 variants had significantly lower proliferative activity on CTLL-2 cells than positive controls because these IL-2 variants do not bind IL -2R ⁇ receptor, and the positive control can enhance the combination with IL-2R ⁇ / ⁇ after binding to IL-2R ⁇ receptor, so it has higher proliferation activity for CTLL-2 cells.
  • IL-2-10 is a recombinant protein formed after the C-terminus of IL-2-1 is linked to the N segment of the Fc fragment (this application defines a derivative of the IL-2 variant), because it has sufficient molecular weight to enhance Combined with IL-2R ⁇ / ⁇ , it can maintain the proliferative activity of CTLL-2 cells similar to that of wild-type human IL-2.
  • Table 4 The data are shown in Table 4.
  • IL-2-5 90% IL-2-6 13% IL-2-7 58% IL-2-8 8% IL-2-9 8% IL-2-10 74.9%

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Abstract

提供了一种人白细胞介素2变体及其用途。具体提供了一种IL-2变体(或其衍生物)，其对高亲和力受体IL-2Rα/β/γ具有消除或降低的亲和力，并保留对中等亲和力受体IL-2Rβ/γ的亲和力。还提供了一种重组蛋白/融合蛋白，其包含IL-2变体与人抗体片段，其提高了IL-2变体的活性，与野生人IL-2的活性接近。

Description

一种人白细胞介素2变体及其用途
本申请要求2021年7月30日提交的专利申请(申请号2021108684095)和2022年6月1日提交的专利申请(2022106214259)的优先权。
技术领域
本公开涉及具有一个或多个氨基酸突变的人白细胞介素-2(IL-2)变体或其衍生物。所述IL-2变体或衍生物具有消除或降低的对高亲和力受体(IL-2Rα/β/γ)的亲和力，并保留对中等亲和力受体(IL-2Rβ/γ)的亲和力。本公开还涉及包含所述人IL-2变体的免疫缀合物、编码多核苷酸、载体、宿主细胞、药物组合物、制备方法及治疗方法和用途。
背景技术
人白细胞介素-2(Interleukin-2，IL-2)，也称为T细胞生长因子(TCGF)。IL-2基因位于4号染色体(4q27)，包括共约7kb的序列。IL-2由约133个氨基酸组成，分子量约15kD。Doris Morgan等人1976和1977分别发现活化后的T细胞培养液可以促进T细胞增殖。培养液中的刺激因子被纯化并被鉴定为IL-2。
最初的体外细胞实验表明T细胞在通过TCR和CD28活化之后，可以分泌IL-2并在细胞表面表达IL-2受体(IL-2R)。IL-2和IL-2R的结合可以引起T细胞的增殖和使T细胞产生效应。IL-2是T细胞免疫反应中起到中心作用的分子。体内实验发现在敲除IL-2或其受体后，动物产生了自身免疫。IL-2不仅可以激活效应细胞(如T细胞和NK细胞)，还可以激活调节性T细胞，从而抑制过量针对自身的免疫。
IL-2通过IL-2R发生作用。IL-2R包括三个亚基：IL-2Rα(即CD25)、IL-2Rβ(即CD122)和IL-2Rγ(即CD132)。三个亚基可以形成三种受体形式：高亲和力受体包含所有三个亚基IL-2Rα/β/γ，中亲和力受体包含IL-2Rβ/γ和低亲和力受体IL-2Rα。IL-2Rβ和IL-2Rγ是IL-2激活下游信号通路所必需的，当IL-2同时结合IL-2Rβ和IL-2Rγ时，两个受体亚基形成异源二聚体，磷酸化细胞内的STAT5，进入细胞核导致相应的基因转录和表达；IL-2Rα并非信号所必需，但可以促进IL-2与IL-2Rβ和IL-2Rγ的结合。
IL-2Rγ在所有免疫细胞中均有表达；IL-2Rβ在CD8+T细胞、NK细胞、调节性T细胞中均有表达，而且在T细胞被激活后还会提升表达水平；IL-2Rα在调节性T细胞持续高表达，在被激活的CD8+T细胞中会有短暂表达，随即下调表达水平。
IL-2主要由活化的T细胞，尤其是CD4+辅助T细胞合成。它刺激T细胞的增殖和分化，诱导细胞毒性T淋巴细胞(CTL)的生成以及外周血淋巴细胞分化 成细胞毒性细胞和淋巴因子激活的杀伤(LAK)细胞，促进T细胞表达细胞因子和细胞溶解分子，促进B细胞的增殖和分化以及通过B细胞的免疫球蛋白合成，并刺激天然杀伤(NK)细胞的生成、增殖和活化。IL-2在体内扩充淋巴细胞群体和提高这些细胞的效应器功能的能力使得其具有抗肿瘤效果，IL-2免疫疗法成为某些转移性癌症患者的治疗选择。目前，高剂量IL-2已被批准用于治疗转移性肾细胞癌和恶性黑素瘤。
WO2009135615公开的IL-2变体在20、88或126位具有突变。WO2012062228公开的IL-2变体在38、42、45、62、68或88中的至少一个位置上具有突变。US8906356公开的IL-2变体在91、126位具有突变。US9732134公开的IL-2变体在15、16、22、84、88或95中的至少一个位置上具有突变。US7803361和US8124066公开的IL-2变体包含R38W突变。
发明内容
本公开涉及具有一个或多个氨基酸突变的IL-2变体(或其衍生物)、及其缀合物；还涉及所述IL-2变体的用途、制备方法。
IL-2变体或其衍生物
第一方面，本公开提供一种IL-2变体(或其衍生物)，其与成熟野生型人IL-2(SEQ ID No.1)相比包含一个或多个氨基酸突变。
“白介素-2”或“IL-2”指来自任何哺乳动物(如灵长类(如人)、啮齿动物(例如小鼠和大鼠))的任何天然的IL-2。该术语涵盖未加工的IL-2以及源自细胞中任何加工形式的IL-2。该术语还涵盖天然存在的IL-2变体(例如剪接变体或等位变体)。
“野生型人IL-2”在其它方面与IL-2变体相同，只是在IL-2变体的每个氨基酸位置具有野生型氨基酸的IL-2形式。术语“野生型”意图涵盖：包含一处或多处不影响对IL-2受体结合能力的天然存在的氨基酸突变。
“成熟野生型人IL-2”是指野生型人IL-2的成熟形式。未加工的人IL-2额外包含N端约20个氨基酸的信号肽，在成熟IL-2分子中缺乏信号肽。示例性成熟野生型人IL-2的氨基酸序列例如但不限于：数据库登录号INSDC的CAA25742.1、Swiss-Prot的P60568.1、Genbank中NP_000577.2的21-153段。在一些实施方案中，成熟野生型人IL-2是SEQ ID NO.1所示的氨基酸序列。应当注意的是，出于重组表达的目的，允许在N端额外包含甲硫氨酸M。然而，技术人员应当理解，无论有无首位M，均属于本申请中“成熟野生型人IL-2”的范畴。
一些实施方案中，与成熟野生型人IL-2相比，本申请所进行的氨基酸突变使得IL-2上与IL-2Rα相结合的区域(位于氨基酸约26至约47的位置)其长度变短。
一些实施方案中，氨基酸突变包括氨基酸的取代、缺失(或截短)、插入(或添加)、修饰及其任意组合。为了改变例如IL-2的结合特性，可以用氨基酸(例 如，用具有不同结构和/或化学特性的氨基酸)取代另一种氨基酸替换。氨基酸的取代包括由非天然氨基酸或20种标准氨基酸。可以使用本领域中公知的方法生成氨基酸突变(包括定点诱变、PCR、基因合成、化学修饰等)。
一些实施方案中，根据本公开的IL-2变体(或其衍生物)与IL-2Rα的亲和力降低，与IL-2Rβ和/或IL-2Rγ的亲和力不变或提高。
“亲和力”指分子的结合位点与其配体之间的非共价相互作用总强度。除非另外指示，本文的“亲和力”指反映结合对的成员(例如受体和配体)之间1:1相互作用的内在结合亲和力。亲和力通常可以用解离常数(K D)来表述，其为解离与结合速率常数(分别为K 解离和K 结合)的比率。亲和力可以通过本领域常规的方法来测量，包括本文中所描述的方法。
一些实施方案中，根据本公开的IL-2变体(或其衍生物)与高亲和力受体(IL-2Rα/β/γ)的亲和力是降低的，但维持或提高对中等亲和力受体(IL-2Rβ/γ)的亲和力。
一些实施方案中，“高亲和力IL-2受体”指IL-2受体的异三聚体形式，其由受体γ亚基(也称为通用细胞因子受体γ亚基、γc或CD132)、受体β亚基(也称为CD122或p70)和受体α亚基(也称为CD25或p55)组成。
一些实施方案中，“中等亲和力IL-2受体”指仅包含γ亚基和β亚基的IL-2受体，而无α亚基(参见Olejniczak和Kasprzak，MedSci Monit14，RA179-189，2008)。
一些实施方案中，根据本公开的IL-2变体(或其衍生物)对调节性T细胞(Treg)的激活降低，和/或对免疫效应细胞(例如T细胞，NK细胞)的激活不受影响或增高。
一些实施方案中，根据本公开的IL-2变体(或其衍生物)，其中所述突变发生在以下任一个或多个位点(连续或不连续)(从SEQ ID NO:1的第1位开始计数)：
31位酪氨酸(Y)、32位赖氨酸(K)、33位天冬酰胺(N)、
34位脯氨酸(P)、35位赖氨酸(K)、36位亮氨酸(L)、
37位苏氨酸(T)、38位精氨酸(R)、39位甲硫氨酸(M)、
40位亮氨酸(L)、41位苏氨酸(T)、42位苯丙氨酸(F)、
43位赖氨酸(K)、44位苯丙氨酸(F)突变、45位酪氨酸(Y)、
125位半胱氨酸(C)。
一些实施方案中，根据本公开的IL-2变体(或其衍生物)，其中所述突变发生在第31至32位、125位的任一个或多个位点(连续或不连续)。
一些实施方案中，根据本公开的IL-2变体(或其衍生物)，其中所述突变发生在第35至41位、125位的任一个或多个位点(连续或不连续)。
一些实施方案中，根据本公开的IL-2变体(或其衍生物)，其中所述突变发生在第43至45位、125位的任一个或多个位点(连续或不连续)。
一些实施方案中，根据本公开的IL-2变体(或其衍生物)，其中所述突变发生在第31至45位、125位的任一个或多个位点(连续或不连续)。
一些实施方案中，根据本公开的IL-2变体(或其衍生物)，其中所述突变发生在第31至32位、第35至41位、125位的任一个或多个位点(连续或不连续)。
一些实施方案中，根据本公开的IL-2变体(或其衍生物)，其中所述突变发生在第35至41位、第43至45位、125位的任一个或多个位点(连续或不连续)。
一些实施方案中，根据本公开的IL-2变体(或其衍生物)，其中所述突变发生在第31至43位、125位的任一个或多个位点(连续或不连续)
另一些实施方案中，当以公共数据库或公知文献中记载的天然序列作为野生型成熟IL-2时，氨基酸突变位点是从第1位氨基酸A开始计数。
一些具体的实施方案中，提供一种IL-2变体，其包含以下任一突变或其组合：
第31-45位突变为Gly-Gly-Asn-Pro-Met-His-Gly-Leu-Asp-Gly-Phe-Gly(SEQ ID No.11)；
第31-32位突变为Gly-Gly；
第35-41位突变为Met-His-Gly-Leu-Asp-Gly(SEQ ID No.12)；
第35-43位突变为Met-Gly-Gly-Leu-Gly-Gly(SEQ ID No.13)；
第35-41位突变为Gly-Gly-Gly-Gly-Gly-Gly(SEQ ID No.14)；
第43-45位突变为Gly；
第125位突变为Ala。
一些具体的实施方案中，当IL-2变体包含选自以下突变或组合时，IL-2变体对IL-2Rα的亲和力降低，对IL-2Rβ/γ亲和力维持或提高：
(1)31-32位的YK突变为GG；且35-41位的KLTRMLT突变为MHGLDG；且43-45位的KFY突变为G；125位的C突变为A；或者
(2)31-45位突变为GGNPMHGLDGFG，125位突变为A；
突变后的序列为SEQ ID No.2或15。
一些具体的实施方案中，当IL-2变体包含选自以下突变或组合时，IL-2变体对IL-2Rα的亲和力降低，对IL-2Rβ/γ亲和力维持或提高：
35-41位的KLTRMLT突变为MHGLDG，125位突变为A；突变后的序列为SEQ ID No.3。
一些具体的实施方案中，当IL-2变体包含选自以下突变或组合时，IL-2变体对IL-2Rα的亲和力降低，对IL-2Rβ/γ亲和力维持或提高：
31-32位的YK突变为GG，125位突变为A；突变后的序列为SEQ ID No.4。
一些具体的实施方案中，当IL-2变体包含选自以下突变或组合时，IL-2变体对IL-2Rα的亲和力降低，对IL-2Rβ/γ亲和力维持或提高：
43-45位的KFY突变为G，125位突变为A；突变后的序列为SEQ ID No.5。
一些具体的实施方案中，当IL-2变体包含选自以下突变或组合时，IL-2变体 对IL-2Rα的亲和力降低，对IL-2Rβ/γ亲和力维持或提高：
(1)31-32位的YK突变为GG；且
(2)35-41位的KLTRMLT突变为MHGLDG；
(3)125位突变为A；
突变后的序列为SEQ ID No.6。
一些具体的实施方案中，当IL-2变体包含选自以下突变或组合时，IL-2变体对IL-2Rα的亲和力降低，对IL-2Rβ/γ亲和力维持或提高：
(1)35-41位的KLTRMLT突变为MHGLDG；且
(2)43-45位的KFY突变为G；
(3)125位突变为A；
突变后的序列为：SEQ ID No.7。
一些具体的实施方案中，当IL-2变体包含选自以下突变或组合时，IL-2变体对IL-2Rα的亲和力降低，对IL-2Rβ/γ亲和力维持或提高：
(1)31-32位的YK突变为GG；且
(2)43-45位的KFY突变为G；
(3)125位突变为A；
突变后的序列为SEQ ID No.8。
一些具体的实施方案中，当IL-2变体包含选自以下突变或组合时，IL-2变体对IL-2Rα的亲和力降低，对IL-2Rβ/γ亲和力维持或提高：
35-43位的KLTRMLTFK突变为MGGLGG，125位突变为A；突变后的序列为SEQ ID No.9。
一些具体的实施方案中，当IL-2变体包含选自以下突变或组合时，IL-2变体对IL-2Rα的亲和力降低，对IL-2Rβ/γ亲和力维持或提高：
35-41位的KLTRMLT突变为GGGGGG，43-45位突变为G，125位突变为A；突变后的序列为SEQ ID No.10。
在一些具体的实施方案中，所述的IL-2变体(或其衍生物)的氨基酸序列选自SEQ ID No.NO：2-10。
表1.人IL-2野生型及变体序列
Figure PCTCN2022105789-appb-000001
Figure PCTCN2022105789-appb-000002
Figure PCTCN2022105789-appb-000003
天然表达的人IL-2共153个氨基酸，1-20位氨基酸是信号肽。切除信号肽后共133个氨基酸(即成熟人IL-2)，对应于本公开SEQ ID No.1的1-133位。出于重组表达目的，人工生产的IL-2首位额外带有甲硫氨酸(对应起始密码子AUG)。
作为一个示例，表述“第31-32位的氨基酸突变为GG”是指：
在人IL-2成熟蛋白的序列APTSSSTKKTQLQLEHLLLDLQMILNGINN YKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT(SEQ ID No.1共计133个氨基酸)中的第31-32位的YK突变为GG。
一些实施方案中，IL-2变体是单体形式。
第二方面，本公开提供一种IL-2变体的衍生物，其包含本公开的IL-2变体，所述IL-2变体具有选自以下任一项所述的修饰或其组合：PEG化、糖基化、缀合至白蛋白、缀合至Fc、羟乙基化、去O-糖基化。
一些实施方案中，IL-2变体的衍生物包括本公开IL-2变体的全长、部分、或在本公开IL-2变体的基础上进一步获得的功能性衍生物、功能性片段、生物活性肽、融合蛋白、同种型或其盐。例如，包含IL-2变体的融合蛋白，所述IL-2变体的二聚物或三聚物或多聚物。所述IL-2变体的各种修饰形式(如PEG化、糖基化、 缀合至白蛋白、缀合至Fc、羟乙基化、去除O-糖基化等)。
一些实施方案中，IL-2变体的衍生物是PEG化的(可以表示为PEG-IL-2)。
在另一些实施方案中，PEG-IL-2变体包含甲氧基-PEG-醛(mPEG-ALD)接头。在某些实施方案中，PEG的平均分子量在约5kD至约50kD；具体地5、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、30、35、40、45、50kD；或约5kD至约40kD；或约10kD至约30kD；或约10kD至约30kD之间；或约15kD至约20kD之间。
某些具体实施方案中，mPEG-ALD接头包括具有选自下列平均分子量的PEG分子：约5kDa、约10kDa、约12kDa、或约20kDa。在某些实施方案中，mPEG-ALD的醛基可以是乙醛、丙醛或丁醛等。
在一个实施方案中，IL-2变体的衍生物，相比于成熟野生型人IL-2具有延长的血清半衰期。
一些实施方案中，IL-2变体的衍生物是缀合至抗体或其抗原结合片段的IL-2变体。在一些实施方案中，IL-2变体的衍生物是缀合至人抗体Fc片段的IL-2变体。在具体的实施方案中，IL-2变体的衍生物是在其C端缀合至人抗体Fc片段的IL-2变体(即，根据本申请的IL-2变体缀合至人抗体Fc片段的N端)。
在一些情况下，IL-2变体的衍生物呈现而二聚体的形式，同二聚体或异二聚体。作为一个示例，在Fc片段中引入修饰以促进异二聚化。在一些实施方案中，Fc片段包含杵臼(knob-into-hole，KIH)结构，该方法涉及在第一Fc片段的界面处引入凸起结构(knob)以及在第二Fc片段的界面处引入孔结构(hole)，反之亦然。使得凸起结构可以定位在孔结构中，促进异二聚体的形成，并抑制同二聚体的产生。凸起结构是通过用较大侧链(例如酪氨酸或色氨酸)取代来自第一Fc片段的界面的小氨基酸侧链而构建的；而孔结构是通过用较小的氨基酸侧链(例如丙氨酸或苏氨酸)取代大氨基酸侧链而在第二Fc片段的界面中创建的。
缀合物
第三方面，本公开提供一种缀合物，其包含第一组件和第二组件；其中第一组件直接缀合至(或通过接头间接缀合至)第二组件；第一组件是本公开的IL-2变体(或其衍生物)；第二组件不是IL-2，不是IL-2变体(或其衍生物)。
一些实施方案中，IL-2变体(或其衍生物)连接于至少一个第二组件。
一些实施方案中，IL-2变体(或其衍生物)通过肽键和第二组件形成融合蛋白。
一些具体的实施方案中，IL-2变体(或其衍生物)缀合至(或通过接头间接缀合至)第二组件的羧基端。
一些具体的实施方案中，IL-2变体(或其衍生物)缀合至(或通过接头间接缀合至)第二组件的氨基端。
一些实施方案中，第二组件为抗原结合组件。抗原结合组件指：特异性结合 表位的多肽分子。
在一些实施方案中，抗原结合组件能指导与其连接的部分(例如，本公开的IL-2、其变体或衍生物)到达靶位点(例如到达肿瘤细胞或具有抗原决定簇的肿瘤间质)。所述抗原结合组件可以是抗体或抗原结合片段。
抗体在本文中以最广义使用，并且涵盖各种抗体结构，只要它们展现出抗原结合活性，所述抗体包括但不限于单克隆抗体、多克隆抗体、多特异性抗体(例如双特异性抗体)和抗原结合片段。抗体可以包括鼠源抗体、人抗体、人源化抗体、嵌合抗体、骆驼抗体。
一些具体实施方案中，抗体或其抗原结合片段选自：包含抗体重链可变区和抗体轻链可变区的多肽复合物、Fab、Fv、sFv、F(ab’)2、线性抗体、单链抗体、scFv、sdAb、sdFv、纳米抗体、肽抗体(peptibody)、结构域抗体和多特异性抗体(双特异性抗体、diabody、triabody和tetrabody、串联二-scFv、串联三-scFv)。
一些实施方案中，IL-2变体(或其衍生物)连接于超过一个抗原结合组件时，每个抗原结合组件可以独立地选自抗体和抗原结合片段；例如，第一抗原结合组件可以是Fab分子，而第二抗原结合组件可以是scFv分子。例如，第一抗原结合组件可以是scFv分子，而第二抗原结合组件也是scFv分子。一些实施方案中，第一和第二抗原结合组件独立地针对不同的抗原或针对同一抗原。一些实施方案中，所述抗体或其抗原结合片段靶向肿瘤抗原。
一些实施方案中，肿瘤抗原例如但不限于：MAGE家族(如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)、GAGE家族(如GAGE-1、GAGE-2、GAGE-3、GAGE-4、GAGE-5、GAGE-6、GAGE-7、GAGE-8、GAGE-9)、MART-1/Melan-A、gp75、gp100、DPPIV、ADAbp、亲环素(cyclophilin)、CEA、CAP-1、CAP-2、etv6、aml1、PSA(PSA-1、PSA-2和PSA-3)、PSMA、T细胞受体、RAGE、LAGE-1、NAG、GnT-V、MUM-1、CDK4、酪氨酸酶、p53、MUC家族、HER2/neu、p21ras、RCAS1、α-胎蛋白、E-钙粘蛋白、α-连环蛋白(catenin)、β-连环蛋白、γ-连环蛋白、p120ctn、PRAME、NY-ESO-1、cdc27、SSX-1、SSX-2、SSX-1、SSX-4、SSX-5、SCP-1、CT-7。
药物组合物
第四方面，本公开提供一种药物组合物，其含有根据本公开的IL-2变体(或其衍生物)、或缀合物，任选地可包含药学上可接受的稀释剂、载体或赋形剂。所述药物组合物可以为冻干制剂或可注射溶液。
在一些具体实施方案中，药物组合物单位计量中可含有0.01重量％至99重量％的IL-2变体(或其衍生物)或缀合物；或药物组合物单位剂量中含有的IL-2 变体(或其衍生物)或缀合物的量为0.1-2000mg(例如1-1000mg；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、1000mg)。
多核苷酸
第五方面，本公开提供编码本公开的IL-2变体(或其衍生物)的多核苷酸。
技术人员理解，编码相同氨基酸序列的核苷酸序列可以不同，由于密码子的兼并性和不同宿主的密码子偏好性，不同的核苷酸序列能够编码相同的氨基酸序列，这些序列均属于本公开的范畴内。
表达载体
第六方面，本公开提供一种表达载体，其含有根据本公开的多核苷酸。表达载体可以是真核表达载体、原核表达载体或穿梭表达载体。
宿主细胞
第七方面，本公开提供一种宿主细胞，其包含根据本公开的表达载体、或表达根据本公开的IL-2变体、或表达根据本公开的缀合物。
一些实施方案中，宿主细胞包含(如已被转化或转染有)表达载体，所述表达载体包含根据本公开的多核苷酸。
一些实施方案中，所述宿主细胞是原核或真核细胞。
一些具体实施方案中，宿主细胞为细菌、酵母菌或哺乳动物细胞，具体可以为巴斯德毕赤酵母或酿酒酵母。
一些具体实施方案中，宿主细胞是原核微生物(如大肠杆菌)。
一些具体实施方案中，宿主细胞真核生物细胞。
一些实施方案中，使用表达糖基化多肽的宿主细胞，例如植物和昆虫细胞。脊椎动物细胞也可以用作宿主细胞，例如，悬浮生长的哺乳动物细胞系、猴肾CV1系(COS-7)、人胚胎肾系(293或293T细胞)、幼仑鼠肾细胞(BHK)、小鼠塞托利(sertoli)细胞(TM4细胞)、猴肾细胞(CV1)、VERO-76、人宫颈癌细胞(HELA)、犬肾细胞(MDCK)、buffalo大鼠肝细胞(BRL3A)、人肺细胞(W138)、人肝细胞(Hep G2)、小鼠乳房肿瘤细胞(MMT060562)、MRC5细胞、FS4细胞、CHO细胞、骨髓瘤细胞系(如YO、NS0、P3X63和Sp2/0)。
用途、治疗方法
第八方面，本公开提供根据本公开的IL-2变体(或其衍生物)、缀合物、药物组合物的制药用途。
一些实施方案中，提供了IL-2变体(或其衍生物)用于治疗增生性疾病、免疫性疾病、调节T细胞介导的免疫应答、刺激个体免疫系统的用途。
一些实施方案中，所述增生性疾病可以是肿瘤或癌(例如转移性肿瘤或癌)，可以是实体瘤。
一些实施方案中，本公开的IL-2变体(或其衍生物)、缀合物、药物组合物可用于治疗刺激宿主的免疫系统(特别是增强细胞免疫应答)。
一些实施方案中，增强细胞免疫应答可以包括但不限于：T细胞功能升高、B细胞功能升高、淋巴细胞功能的恢复、IL-2受体表达提高、T细胞应答性提高、天然杀伤细胞活性或淋巴因子激活的杀伤(LAK)细胞活性升高。
一些实施方案中，本公开的IL-2变体(或其衍生物)、缀合物、药物组合物用于治疗增殖性病症(例如癌症)。癌症的非限制性例子包括膀胱癌、脑癌、头和颈癌、胰腺癌、肺癌、乳腺癌、卵巢癌、子宫癌、宫颈癌、子宫内膜癌、食道癌、结肠癌、结肠直肠癌、直肠癌、胃癌、前列腺癌、血液癌、皮肤癌、鳞状细胞癌、骨癌和肾癌。
一些实施方案中，本公开的IL-2变体(或其衍生物)、缀合物、药物组合物用于治疗位于以下位置的赘生物：腹部、骨、乳房、消化系统、肝、胰、腹膜、内分泌腺(肾上腺、副甲状腺、垂体、睾丸、卵巢、胸腺、甲状腺)、眼、头和颈、神经系统(中枢和外周)、淋巴系统、骨盆、皮肤、软组织、脾、胸部、和泌尿生殖系统。
一些实施方案中，本公开的IL-2变体(或其衍生物)、缀合物、药物组合物用于治疗癌前状况或癌症转移。癌症选自以下：肾细胞癌、皮肤癌、肺癌、结肠直肠癌、乳腺癌、脑癌、头和颈癌。
一些实施方案中，本公开的IL-2变体(或其衍生物)、缀合物、药物组合物用于治疗：高丙种球蛋白血症(hypergammaglobulinemia)、淋巴增生性病症、病变蛋白血症(paraproteinemias)、紫癜(purpura)、结节病、塞扎里综合征(Sezary Syndrome)、Waldenstron's巨球蛋白血症、高歇氏病(Gaucher's Disease)、组织细胞增多病(histiocytosis)。
一些实施方案中，本公开的IL-2变体(或其衍生物)、缀合物、药物组合物用于治疗自身免疫疾病、移植排斥、外伤后免疫应答和传染性疾病。
一些实施方案中，自身免疫性疾病可以是选自：I型糖尿病(type I diabetes mellitus)、类风湿性关节炎(rheumatoid arthritis)、多发性硬化症(multiple sclerosis)、慢性胃炎(chronic gastritis)、克罗恩病(Crohn’s disease)、巴塞多病(Basedow disease)、别赫捷列夫病(Bechterew disease)、银屑病(psoriasis)、重症肌无力(myasthenia gravis)、自身免疫肝炎(autoimmune hepatitis)、APECED、变应性肉芽肿血管炎(Chrug-Strauss syndrome)、溃疡性结肠炎(ulcerative colitis)、肾小球肾炎(glomerulonephritis)、吉兰-巴雷综合征(Guillain-Barrésyndrome)、桥本甲状腺炎(Hashimoto thyroiditis)、硬化萎缩性苔藓(lichen sclerosus)、系统性红斑狼疮(systemic lupus erythematodes)、PANDAS、风湿热(rheumatic fever)、结节病(sarcoidosis)、舍格伦综合征(syndrome)、僵人综合症(Stiff-Man syndrome)、硬皮病(scleroderma)、韦格纳肉芽肿病(Wegener’s granulomatosis)、白斑(vitiligo)、 自身免疫性肠病(autoimmune enteropathy)、肺出血肾炎综合征(Goodpasturesyndrome)、皮肌炎(dermatomyositis)、多发性肌炎(polymyositis)、自身免疫性过敏症(autoimmune allergy)、哮喘(asthma)。
在一些实施方案中，IL-2变体(或其衍生物)可以与免疫抑制剂联用。
一些实施方案中，免疫抑制剂选自：糖皮质激素(glucocorticoid)；硫唑嘌呤(azathioprine)；环孢素A(cyclosporin A)；吗替麦考酚酯(mycophenolatemofetil)；他克莫司(tacrolimus)；抗CD3抗体；抗CD25抗体；抗TNF-α抗体；甲氨蝶呤(methotrexate)；环孢素(cyclosporin)；西罗莫司(sirolimus)；依维莫司(everolimus)；芬戈莫德(fingolimod)；以及环磷酰胺(cyclophosphamide)。
在一些实施方案中，向受试者施用治疗有效量的：本公开的IL-2变体(或其衍生物)、缀合物、药物组合物。
一些实施方案中，需要治疗的受试者(如患者或个体)通常为哺乳动物，例如人。
一些实施方案中，每日至少2次、每日至少1次、每48小时至少1次、每72小时至少一次、每周至少一次、每2周至少一次、每个月至少一次、每2个月至少一次、或每3个月至少一次向受试者施用本公开的IL-2变体(或其衍生物)、缀合物、药物组合物。
一些实施方案中，通过任何途径施用本公开的IL-2变体(或其衍生物)、缀合物、药物组合物。例如，通过肠胃外注射(例如，皮下、静脉注射)施用。
附图说明
图1.野生型人IL-2、IL-2变体或其衍生物与IL-2Rα受体的相互作用曲线。
图2.野生型人IL-2、IL-2变体或其衍生物与IL-2Rβ/γ受体的相互作用曲线。
具体实施方式
以下结合实施例用于进一步描述本公开，但这些实施例并非限制本公开的范围。
实施例1.野生型人IL-2及IL-2变体或其衍生物的重组表达与制备
合成、表达和纯化IL-2变体的核酸序列。
1.本实施例中对野生型人IL-2和IL-2变体(IL-2-1到IL-2-9)进行分别表达，并依靠分子C端带有的HPC4标签进行纯化和制备。IL-2-10是IL-2-1变体C端添加的人IgG1 Fc序列，然后进行纯化和制备。
合成用全基因，并通过双酶切法将各蛋白的编码序列亚克隆到表达载体pcDNA3.1中；通过酶切法和测序确认最终表达载体的准确性，最终转到DH5α克隆菌株中。通过质粒提取试剂盒提取转染级质粒，将质粒通过转染试剂转染到哺 乳动物细胞HEK293中进行瞬时表达，通过亲和层析纯化各蛋白。
2.质粒转化DH5α
吸取含有编码序列的表达质粒80-100ng加入已制备的DH5α感受态细胞中。将加入质粒后的感受态细胞通过热激进行转化。
将转化后的感受态细胞加入LB液体培养基，放入摇床37℃，200rpm震荡培养约30min。将培养后的感受态细胞取出，吸取部分悬液涂在含有氨苄抗性的平板上，放入培养箱37℃过夜培养。
从新鲜的培养板中挑取单克隆于2-5mL LB培养基，37℃200rpm培养8h。按1/500比例接种于200mL LB培养基中，37℃200rpm培养16h。收集培养后的菌液离心，去掉上清。
3.HEK293细胞复苏与传代
将水浴调节至37℃；培养基在37℃预热。
从液氮罐中取出HEK293细胞，立即投入37℃水浴中，轻微摇动使其快速融化(约1min)。
用75％乙醇消毒管外壁，放入生物安全柜内；将细胞转移到含10mL培养基的15mL离心管中，离心800rpm 5min。离心后的细胞去掉上清，取少许新鲜培养基重悬细胞，再转移到培养瓶中；加入新鲜培养基轻轻摇动，使细胞分散均匀，取细胞计数及活力检测。使密度控制在3×10 5至4×10 5细胞/mL，活力>95％。
将细胞置于培养箱中110rpm，37℃5％CO2培养。细胞培养2-3天，密度达到2.0×10 6/mL时，通过补加新鲜培养基对细胞进行传代。对细胞密度及活力检测进行监测。
4.质粒转染HEK293细胞
转染前1天，将HEK293细胞悬浮培养至1L，接种密度为1×10 6/mL，置于培养箱中110rpm，37℃5％CO2培养。转染当天，使细胞密度控制在1×10 6/mL至1.5×10 6/mL。
DNA-转染试剂混合物：向转染缓冲液中加入DNA和转染试剂混匀，37℃温育。将DNA-转染试剂混合物加入待转染细胞中，放入培养箱中110rpm，37℃5％CO2培养。转染后约4-6天，取出细胞培养物，离心，收集上清和细胞。
5.蛋白纯化
取培养5天后的细胞培养液，离心后取上清用0.22μm滤器进行过滤。在4℃下对缓冲液1×PBS pH7.4进行透析。透析结束后，用偶联有HPC4抗体的填料进行亲和纯化，然后再经过凝胶过滤层析superdex200进行进一步纯化得到纯度较高的目标蛋白。
实施例2.测定野生型人IL-2及IL-2变体或其衍生物对IL-2Rα的亲和力
通过Octet平台检测实施例1中获得的野生型人IL-2及其IL-2变体或其衍生 物(IL-2-1至IL-2-10)对IL-2Rα的结合特性。Octet平台是基于生物膜干涉技术(Bio-Layer Interferometry,BLI)对生物分子相互作用进行检测分析。
1.实验仪器及试剂耗材
Figure PCTCN2022105789-appb-000004
Octet RED96e system(Pall Fortebio Corp,Menlo Park,CA)；
分析缓冲液SD缓冲液(PBS(pH 7.4)+0.01％BSA+0.02％Tween20)；
醋酸缓冲液(10mM，pH4.0、5.0、6.0)；
AR2G(Fortebio，货号：18-5092)；
96-孔板(Greiner Bio-One part no.655209)；
2.参考下面的实验设计进行实验，整个实验是在1000rpm，30℃，220μl/孔的条件下完成。实验所用蛋白为IL2-Rα-his以及IL-2变体或其衍生物是通过HEK293瞬时表达以及亲和纯化后获得的。
待测物样品为：IL-2Rα，稀释缓冲液为SD缓冲液；
待测物浓度范围为：500nM、240nM、120nM、60nM、30nM、15nM(部分变体增加1000nM浓度测量)。
3.实验数据及处理
通过Data Analysis software 9.0对实验数据进行处理。拟合模型选择1:1模型，即1个野生型人IL-2或IL-2变体(具体为IL-2-1至IL-2-10)结合1个IL-2Rα。拟合方式为global；即把6个浓度作为一组进行分析。拟合结果参见图1。
实验得到的数据以1:1binding模型进行拟合，得出野生型人IL-2或IL2变体或衍生物对受体IL-2Rα的亲和力Kd数值。
野生型人IL-2和IL-2变体或其衍生物与IL-2Rα的结合数据见图1和表2。
结果显示IL-2-1、IL-2-6、IL-2-8、IL-2-9和IL-2-10同IL-2Rα间没有结合，IL-2-2、IL-2-3、IL-2-4、IL-2-5、IL-2-7同IL-2Rα间相互作用有一定程度降低或维持。
这表明：
1)31-32位突变成GG没有显著影响IL-2与IL-2Rα的结合；
2)43-45突变为G会对IL-2与IL-2Rα的结合产生一定的影响；
3)第35-41位的KLTRMLT突变为MHGLDG没有显著影响IL-2与IL-2Rα的结合；
4)将前述三种突变进行组合，使蛋白质结构产生较大改变，则极大降低了IL-2与IL-2Rα的结合，在实验条件下观测不到二者的结合。
表2.IL-2野生型及其变体或衍生物与IL-2受体α的亲和力
Figure PCTCN2022105789-appb-000005
Figure PCTCN2022105789-appb-000006
实施例3.测定野生型人IL-2及IL-2变体或其衍生物对IL-2Rβ/γ的亲和力
通过实验来检测实施例1中野生型人IL-2及其变体或其衍生物对IL-2Rβ/γ的亲和力。
1.实验仪器及试剂耗材
Figure PCTCN2022105789-appb-000007
Octet RED96e system(Pall Fortebio Corp,Menlo Park,CA)；
分析缓冲液SD缓冲液(PBS(pH 7.4)+0.01％BSA+0.02％Tween20)；
醋酸缓冲液(10mM，pH4.0、5.0、6.0)；
AR2G(Fortebio，货号：18-5092)；
96-孔板(Greiner Bio-One part no.655209)。
对IL-2Rβ和IL-2Rγ亚基分别进行克隆、融合到Fc hole和Fc knob上，用于制备IL-2Rβ/γ-Fc异源二聚体。IL-2Rβ-Fc-hole和IL-2Rγ-Fc-knob同时转染到HEK293细胞中。异源二聚体用Protein A和分子筛Superdex 200纯化。
2.整个实验是在1000rpm，30℃，220μl/孔条件下完成。
待测物样品为IL-2Rβ/γ异源二聚体，稀释缓冲液为SD缓冲液。待测物浓度范围为750nM、500nM、250nM、125nM、62.5nM、31.3nM。
3.实验数据及处理
通过Data Analysis software 9.0对实验数据进行处理。拟合模型选择1:1模型，即1个野生型人IL-2或IL-2变体或其衍生物(IL-2-1至IL-2-10)结合1个IL-2Rβ/γ。拟合方式global，即把6个浓度作为一组进行分析。
实验得到的数据以1:1binding模型进行拟合，得出实施例1中野生型人IL-2或IL2变体或其衍生物与IL-2Rβ/γ的亲和力Kd数值，结合数据见图2和表3。
结果显示，IL-2变体或其衍生物(IL-2-1至IL-2-10)与IL-2Rβ/γ之间相互作用维持不变。
表3.IL-2野生型及其变体或其衍生物与IL-2Rβ/γ的亲和力
名称 Kd(nM)
IL-2WT 4.9
IL-2-1 17.9
IL-2-2 4.87
IL-2-3 3.74
IL-2-4 4.11
IL-2-5 5.24
IL-2-6 19.56
IL-2-7 6.48
IL-2-8 21.34
IL-2-9 27.2
IL-2-10 9.83
实施例4.野生型IL-2、IL-2变体或其衍生物的CTLL-2细胞增殖活性测定
依据在不同IL-2浓度下细胞依赖株CTLL-2的增殖速率，检测IL-2的生物学活性。
细胞采用含10％胎牛血清，1％双抗(青链霉素混合液)的RPMI 1640培养液，置于37℃，5％CO 2的培养箱中进行培养。将处于对数生长期的细胞经胰蛋白酶消化，显微镜下计数后制成1×10 4至5×10 4个细胞/ml的细胞悬液。分别取100μl至96孔培养板，每种细胞每块板接种3个同样的孔作为复孔，1×10 3至5×10 3个细胞/孔，以100μl培养液做空白对照，37℃培养过夜。
设置以下不同浓度的组：空白组、0.001nM、0.01nM、0.1nM、1nM、10nM。
分别在0h、24h时，按1：10体积比混合Cell Counting Kit-8(CCK-8)和无血清必需基本培养基，每孔100μL加入待测孔中，在37℃，5％CO2培养箱中孵育1h。
用酶标仪测定490nm波长吸光度。记录每块板的数值。
结果如表4所示，IL-2变体或其衍生物都具有CTLL-2细胞增殖活性，说明突变没有显著影响IL-2Rβ/γ受体亚基复合物的信号传导功能。IL-2-1、IL-2-6、IL-2-8、IL-2-9变体对CTLL-2细胞的增殖活性明显低于阳性对照，是因为这些IL-2变体不结合IL-2Rα受体，而阳性对照结合IL-2Rα受体后，可以增强与IL-2Rβ/γ的结合，所以对于CTLL-2细胞增殖活性更高。
IL-2-10是IL-2-1变体在其C端连接Fc片段N段后形成的重组蛋白(本申请限定的是IL-2变体的衍生物)，因为具有足够的分子量可以增强与IL-2Rβ/γ的结合，所以可以对于CTLL-2细胞维持与野生型人IL-2相近的增殖活性。数据见表4。
表4
名称 相对活性
IL-2WT 100％
IL-2-1 13％
IL-2-2 64％
IL-2-3 100％
IL-2-4 100％
IL-2-5 90％
IL-2-6 13％
IL-2-7 58％
IL-2-8 8％
IL-2-9 8％
IL-2-10 74.9％

Claims (10)

  1. 一种人白细胞介素2(IL-2)变体，相对于成熟野生型人IL-2，所述IL-2变体包含氨基酸突变，所述突变是取代、缺失、添加中的至少一种；优选地，所述成熟野生型人IL-2是SEQ ID No.1所示；
    其中，所述突变是选自以下的任一项或其组合：
    成熟野生型人IL-2第31至45位氨基酸残基突变为SEQ ID No.11所示序列；
    成熟野生型人IL-2第35至41位氨基酸残基突变为SEQ ID No.12、或14所示序列；
    成熟野生型人IL-2第35至43位氨基酸残基突变为SEQ ID No.13所示序列；
    成熟野生型人IL-2第31至32位氨基酸残基突变为GG；
    成熟野生型人IL-2第43至45位氨基酸残基突变为G；
    成熟野生型人IL-2第125位氨基酸残基突变为A；
    任选地，成熟野生型人IL-2的N端添加M。
  2. 根据权利要求1所述的IL-2变体，其中所述IL-2变体是选自以下任一序列所示：
    SEQ ID No.2至10、SEQ ID No.15；
    N端添加M的SEQ ID No.2至10、N端添加M的SEQ ID No.15。
  3. 根据权利要求1至2中任一项所述的IL-2变体，其中：
    所述IL-2变体与IL-2Rα的亲和力降低；和/或
    所述IL-2变体与IL-2Rβ的亲和力不变或提高；和/或
    所述IL-2变体与IL-2Rγ的亲和力不变或提高；
    优选地，所述IL-2变体与IL-2Rα/β/γ的亲和力降低，并且维持或提高所述IL-2变体对IL-2Rβ/γ的亲和力。
  4. 一种IL-2变体的衍生物，其包含权利要求1至3中任一项所述的IL-2变体，所述IL-2变体具有选自以下任一项的修饰或其组合：
    PEG化、糖基化、缀合至白蛋白、缀合至Fc、羟乙基化、去O-糖基化；
    优选地，所述缀合至Fc是指在权利要求1至3中任一项所述IL-2变体在其C端缀合人IgG1 Fc；
    所述PEG化是指PEG连接至所述IL-2变体的N端；
    优选地，PEG的分子量为5kD至80kD；
    更优选地，PEG的分子量为10kD至20kD。
  5. 一种缀合物，其包含：
    第一组件，和
    第二组件；
    其中：所述第一组件直接缀合至、或通过接头间接缀合至所述第二组件；
    所述第一组件是权利要求1至3中任一项所述的IL-2变体或权利要求4所述的IL-2变体的衍生物；
    优选地，所述第二组件是抗体或其抗原结合片段；
    更优选地，所述抗体或其抗原结合片段靶向肿瘤抗原。
  6. 一种药物组合物，其含有：
    -权利要求1至3中任一项所述的IL-2变体、或权利要求4所述的IL-2变体的衍生物、或权利要求5所述的缀合物；
    -药学上可接受的稀释剂、载体或赋形剂。
  7. 一种核酸分子，其编码权利要求1至3中任一项所述的IL-2变体、权利要求4所述的IL-2变体的衍生物、或权利要求5所述的缀合物。
  8. 一种表达载体，其包含权利要求7所述的核酸分子。
  9. 一种宿主细胞，其包含或表达选自以下的任一项：
    包含权利要求8所述的表达载体、
    表达权利要求1至3中任一项所述的IL-2变体、
    表达权利要求4所述的IL-2变体的衍生物、
    表达权利要求5所述的缀合物；
    优选地，所述宿主细胞为原核或真核细胞；
    更优选地，所述宿主细胞选自以下任一项：细菌细胞、酵母细胞、哺乳动物细胞；
    最优选地，所述宿主细胞为酿酒酵母细胞或大肠杆菌细胞。
  10. 选自以下任一项在制备药物中的用途：
    权利要求1至3中任一项所述的IL-2变体、权利要求4所述的IL-2变体的衍生物、权利要求5所述的缀合物；
    所述药物用于预防或治疗选自以下任一项：增生性疾病、增生性疾病的转移、免疫性疾病；
    优选地，所述增生性疾病是肿瘤或癌症；
    优选地，所述免疫性疾病选自：糖尿病、类风湿性关节炎、多发性硬化、系统性红斑狼疮、湿疹、哮喘、自身免疫疾病、器官移植后的自身免疫反应；
    更优选地，所述肿瘤或癌症选自：上皮细胞癌、内皮细胞癌、鳞状细胞癌、 乳头状瘤病毒引起的癌、腺癌、黑素瘤、肉瘤、畸胎癌、肺癌、转移性肺癌、淋巴瘤、转移性肾细胞癌。
PCT/CN2022/105789 2021-07-30 2022-07-14 一种人白细胞介素2变体及其用途 Ceased WO2023005680A1 (zh)

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