WO2020057646A1 - 新型白介素2及其用途 - Google Patents

新型白介素2及其用途 Download PDF

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WO2020057646A1
WO2020057646A1 PCT/CN2019/107055 CN2019107055W WO2020057646A1 WO 2020057646 A1 WO2020057646 A1 WO 2020057646A1 CN 2019107055 W CN2019107055 W CN 2019107055W WO 2020057646 A1 WO2020057646 A1 WO 2020057646A1
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mutein
mutation
cells
mutations
receptor
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PCT/CN2019/107055
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English (en)
French (fr)
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康立山
顾春银
付凤根
周帅祥
史新震
刘军建
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信达生物制药(苏州)有限公司
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Priority to CN201980029369.6A priority Critical patent/CN112105633B/zh
Priority to US17/059,583 priority patent/US20210221863A1/en
Priority to AU2019343251A priority patent/AU2019343251B2/en
Priority to CA3098930A priority patent/CA3098930A1/en
Priority to JP2020571641A priority patent/JP7512210B2/ja
Priority to EP19862253.2A priority patent/EP3854806A4/en
Publication of WO2020057646A1 publication Critical patent/WO2020057646A1/zh

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Definitions

  • the invention relates to a novel interleukin 2 (IL-2) mutant protein and use thereof.
  • the present invention relates to an IL-2 mutein having reduced IL-2R ⁇ receptor binding ability and / or improved IL-2R ⁇ receptor binding ability compared to wild-type IL-2.
  • the invention also provides a fusion protein comprising the IL-2 mutein, an immunoconjugate, and a nucleic acid encoding the IL-2 mutein, a vector comprising the nucleic acid, and a host cell.
  • the invention further provides a method for preparing the IL-2 mutein, a pharmaceutical composition comprising the IL-2 mutein, and a therapeutic use of the mutein.
  • Interleukin 2 also known as T cell growth factor (TCGF)
  • TCGF T cell growth factor
  • TCGF T cell growth factor
  • human IL-2 (uniprot: P60568) is synthesized as a precursor peptide of 153 amino acids. After removing the 20 amino acids at the N-terminus, it produces mature secreted IL-2.
  • Interleukin 2 has four antiparallel, amphiphilic alpha helices. These four alpha helices form a quaternary structure that is essential for its function (Smith, Science 240, 1169-76 (1988); Bazan, Science 257, 410-413 (1992)). In most cases, IL-2 works through three different receptors: interleukin 2 receptor alpha (IL-2R ⁇ ; CD25), interleukin 2 receptor beta (IL-2R ⁇ ; CD122), and interleukin 2 receptor gamma ( IL-2R ⁇ ; CD132).
  • IL-2R ⁇ interleukin 2 receptor alpha
  • IL-2R ⁇ interleukin 2 receptor beta
  • IL-2R ⁇ interleukin 2 receptor gamma
  • IL-2R ⁇ and IL-2R ⁇ are essential for IL-2 signaling, while IL-2R ⁇ (CD25) is not necessary for signaling, but can confer high affinity binding of IL-2 to receptors (Krieg et al., Proc Natl Acad Sci 107, 11906-11 (2010)).
  • the trimeric receptor (IL-2 ⁇ ) formed by the combination of IL-2R ⁇ , ⁇ , and ⁇ is the IL-2 high affinity receptor (K D about 10 pM), and the dimeric receptor composed of ⁇ and ⁇ (IL -2 ⁇ ) is an intermediate affinity receptor (K D is about 1 nM), and an IL-2 receptor formed by an ⁇ subunit alone is a low affinity receptor.
  • Immune cells express dimer or trimer IL-2 receptors. Dimer receptors are expressed on cytotoxic CD8 + T cells and natural killer cells (NK), while trimer receptors are mainly on activated lymphocytes and CD4 + CD25 + FoxP3 + inhibitory regulatory T cells (Treg) Expression (Byman, O. and Sprent. J. Nat. Rev. Immunol. 12, 180-190 (2012)). Because resting effector T cells and NK cells do not have CD25 on the cell surface, they are relatively insensitive to IL-2. Treg cells consistently express the highest level of CD25 in the body. Therefore, under normal circumstances, IL-2 will preferentially stimulate Treg cell proliferation.
  • IL-2 mediates multiple roles in the immune response by binding to the IL-2 receptor on different cells.
  • IL-2 can stimulate T cell proliferation and differentiation, induce the production of cytotoxic T lymphocytes (CTL), promote B cell proliferation and differentiation and immunoglobulin synthesis, and stimulate natural killer (NK)
  • CTL cytotoxic T lymphocytes
  • NK natural killer
  • IL-2 can promote the activation and proliferation of immunosuppressive CD4 + CD25 + regulatory T cells (ie, Treg cells) (Fontenot et al., Nature Immunol 6,1142-51 (2005); D'Cruz and Klein Nature Immunol 6,1152-59 (2005); Maloy and Powrie, Nature Immunol 6,1171-72 (2005)), leading to immunosuppression.
  • CD4 + CD25 + regulatory T cells ie, Treg cells
  • high doses of IL-2 can cause vascular leak syndrome (VLS) in patients.
  • IL-2 induces pulmonary edema by directly binding to the IL-2 trimer receptor (IL-2 ⁇ ) on lung endothelial cells (Krieg et al., Proc Nat Acad Sci USA 107, 11906-11 (2010)).
  • the present invention satisfies the aforementioned needs by providing a new IL-2 mutein with improved IL-2 receptor selectivity / biasing relative to wild-type IL-2.
  • an IL-2 mutein of the invention has one or more of the following characteristics:
  • the IL-2 mutein of the invention has a characteristic of high expression compared to wild-type IL-2.
  • the present invention provides an IL-2 mutein comprising at least one mutation in amino acid residue regions 35-72 of IL-2; in other embodiments, the present invention provides amino acid residues in IL-2
  • the base region 79-92 contains at least one mutated IL-2 mutein; in still other embodiments, the present invention provides an amino acid region 35-72 and 79-92 comprising two or more and preferably three or more mutations IL-2 mutein.
  • the present invention provides a fusion protein and an immunoconjugate, a pharmaceutical composition, and a combination product comprising the IL-2 mutein; a nucleic acid encoding the IL-2 mutein, a vector and a host cell comprising the nucleic acid; Invented methods of IL-mutant proteins, fusion proteins, and immunoconjugates.
  • the present invention also provides a method for treating diseases using the IL-2 mutein of the present invention, and a method and use for stimulating the immune system of a subject.
  • Figure 1 shows the crystal structure of the IL-2 and IL-2R ⁇ complex.
  • Figure 2 shows the crystal structure of the IL-2 and IL-2R ⁇ complex.
  • Figures 3A-B show primer designs used to construct mutant libraries IBYDL029 and mutant libraries IBYDL030 and IBYDL031.
  • Figures 4A-D show some IL-2 muteins and their sequences screened from the mutant libraries IBYDL029 and IBYDL031, as well as some new muteins and their sequences generated from a combination of mutations selected from the two libraries.
  • Figures 5A-D show the CD8 + CD25 to select and construct some of the IL-2 mutant -FC - / CD25 + T-cell activation signal curves of p-STAT5.
  • Figure 6 shows the mature protein sequence (SEQ ID NO: 1) of human interleukin (IL-2) and its amino acid residue numbering, and shows sequence alignments with the mutant proteins IL-2 3X and IL-2 H9 .
  • the term “comprising” or “including” means including the recited elements, integers, or steps, but does not exclude any other elements, integers, or steps.
  • the terms “comprising” or “including” are used, unless otherwise indicated, the case of consisting of the mentioned elements, integers, or steps is also covered.
  • an IL-2 mutein that "comprises” or “includes” a certain mutation or combination of mutations, it is also intended to encompass an IL-2 mutein having only that mutation or combination of mutations.
  • the wild-type "interleukin-2” or "IL-2” refers to a parent IL-2 protein as a template for introducing a mutation or a combination of mutations of the present invention, preferably a naturally occurring IL-2 protein, for example, derived from human, Natural IL-2 proteins of mice, rats, and non-human primates, including unprocessed (e.g., signal peptide removed) and processed (e.g., signal peptide removed) forms.
  • the expression also includes naturally occurring IL-2 allelic and splice variants, isotypes, homologs, and species homologs.
  • the expression also includes variants of natural IL-2, for example, the variants can have at least 95% -99% or higher identity with natural IL-2 or have no more than 1-10 or 1-5 amino acids Mutations (especially conservative amino acid substitutions) and have substantially the same IL-2R ⁇ binding affinity and / or IL2R ⁇ binding affinity as the native IL-2 protein.
  • wild-type IL-2 may contain amino acid mutations that do not affect its binding to the IL-2 receptor compared to the native IL-2 protein, for example, natural human IL with mutation C125S introduced at position 125 -2 protein (uniprot: P60568) belongs to the wild-type IL-2 of the present invention.
  • wild-type human IL-2 protein is shown in SEQ ID NO: 1.
  • the wild-type human IL-2 sequence can have at least 85%, 90%, 95%, or even at least 96%, 97%, 98%, or 99% or more of the amino acid sequence of SEQ ID NO: 1. High amino acid sequence identity.
  • amino acid mutations can be amino acid substitutions, deletions, insertions, and additions. Any combination of substitutions, deletions, insertions and additions can be made to obtain the final mutein construct with the desired properties (eg, reduced IL-2R ⁇ binding affinity).
  • Amino acid deletions and insertions include deletions and insertions at the amino and / or carboxy terminus of a polypeptide sequence. For example, alanine residues can be deleted at full-length human IL-2 position 1.
  • a preferred amino acid mutation is an amino acid substitution.
  • non-conservative amino acid substitutions are preferred.
  • preferred non-conservative amino acid substitutions include replacement of hydrophobic amino acids with hydrophilic amino acids, or replacement with amino acids of different polarity or opposite charge.
  • amino acid sequence alignment eg using BLAST; Basic Local Alignment Search Tool available from http://blast.ncbi.nlm.nih.gov/Blast.cgi?
  • F42 when referring to “F42”, it refers to the phenylalanine residue F at position 42 of SEQ ID NO: 1, or the corresponding position on other IL-2 sequences after alignment Amino acid residues.
  • F42, R81, and S87 when referring to a combination of multiple positions, such as F42, R81, and S87, it can be expressed as F42 / R81 / S87.
  • amino acid substitutions are expressed as [original amino acid residues / positions / substituted amino acid residues].
  • substitution of isoleucine at position 92 with leucine can be expressed as I92L.
  • substitution methods such as D, E, Q
  • H79D / E / Q the substitution can be expressed as: (1) H79D, E, Q; or (2) H79D / E / Q.
  • a combination mutation at multiple given positions eg, R81, R83 and S87
  • it can be expressed as: 1) R81D / N, R83E, I92L / F / Y; or (2) R81D / S87D / I92L.
  • the "percent sequence identity" can be determined by comparing the two best aligned sequences within a comparison window.
  • sequence identity is determined over the entire length of a reference sequence (e.g., SEQ ID NO: 1).
  • Sequence alignment methods for comparison are well known in the art. Algorithms suitable for determining percent sequence identity include, for example, the BLAST and BLAST 2.0 algorithms (see Altschul et al., Nuc. Acids Res. 25: 3389-402, 1977 and Altschul et al. J. Mol. Biol. 215: 403-10, 1990. Software available for BLAST analysis available through the public at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). For the purposes of this application, identity The percentage is usually determined using the BLAST 2.0 algorithm set as the default parameter.
  • conservative substitution means an amino acid substitution that does not adversely affect or alter the biological function of the protein / polypeptide comprising the amino acid sequence.
  • conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • a typical conservative amino acid substitution refers to the replacement of one amino acid with another amino acid having similar chemical properties, such as charge or hydrophobicity.
  • Each of the following six groups contains amino acids that can be conservatively substituted with each other: 1) alanine (A), serine (S), threonine (T); 2) aspartic acid (D), glutamic acid (E ); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L) , Methionine (M), valine (V); and 6) phenylalanine (F), tyrosine (Y), and tryptophan (W).
  • the wild-type IL-2 protein may have conservative amino acid substitutions relative to SEQ ID NO: 1 or only conservative amino acid substitutions.
  • the mutant IL-2 protein of the present invention may have conservative amino acid substitutions or only conservative amino acid substitutions in addition to the characteristic mutations of the present invention relative to the wild-type IL-2 protein.
  • Binding affinity reflects the intrinsic binding capacity of interactions between members of a binding pair.
  • the affinity of molecule X for its binding partner Y can be represented by the equilibrium dissociation constant (K D ), which is the ratio of the dissociation rate constant and the association rate constant (k dis and k on, respectively ).
  • Binding affinity can be measured by common methods known in the art. A specific method for measuring avidity is the biofilm interference (BLI) technique determination herein.
  • BBI biofilm interference
  • changes in the affinity of IL-2 muteins for different receptors can also be initially assessed by flow cytometry as described herein.
  • wild-type IL-2 and mutant IL-2 proteins displayed on yeast cells can be compared with wild-type IL-2 by staining with biotinylated IL-2R ⁇ or IL-2R ⁇ receptors.
  • an antibody binding molecule is a polypeptide molecule that can specifically bind an antigen, for example, an immunoglobulin molecule, an antibody or an antibody fragment, such as a Fab fragment and a scFv fragment.
  • an antibody Fc fragment refers to the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region, and may include natural sequence Fc fragments and variant Fc fragments.
  • the human IgG heavy chain Fc fragment extends from the heavy chain Cys226 or from Pro230 to the carboxy terminus.
  • the C-terminal lysine (Lys447) of the Fc-fragment may or may not be present.
  • the Fc fragment may comprise a mutation, such as a L234A / L235A mutation.
  • the numbering of amino acid residues in Fc fragments is based on the EU numbering system, also known as the EU index, such as Kabat, EA, etc., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes Health, Bethesda, MD (1991), NIH Publication 91-3242. Aspects of the invention are further detailed in the following subsections.
  • the invention provides, in one aspect, a novel IL-2 mutein that has improved selectivity / preference for the IL-2 receptor, and more specifically, reduced or eliminated binding affinity for the IL-2R ⁇ receptor And / or enhanced binding affinity for the IL-2R ⁇ receptor.
  • the IL-2 protein interacts with the IL-2 receptor to trigger signaling and function.
  • Wild-type IL-2 shows different affinity for different IL-2 receptors.
  • IL-2 ⁇ and ⁇ receptors with lower affinity to wild-type IL-2 are expressed on resting effector cells, including CD8 + cytotoxic T cells and NK cells.
  • IL-2R ⁇ with high affinity to wild-type IL-2 is expressed on regulatory T cells (Treg) cells and activated effector cells. Due to high affinity, wild-type IL-2 preferentially binds to IL-2R ⁇ on the cell surface, then recruits IL-2R ⁇ , releases downstream p-STAT5 signals through IL-2R ⁇ , and stimulates Treg cells and activated effector cells.
  • reducing or eliminating the affinity of IL-2 for the IL-2R ⁇ receptor will reduce the bias of IL-2 preferentially activating CD25 + cells and reduce the immune downregulation of Treg cells mediated by IL-2 effect.
  • maintaining or enhancing the affinity for the IL-2 ⁇ receptor will retain or enhance the activation of IL-2 on effector cells such as CD8 + cytotoxic T cells and NK cells, thus achieving an immune stimulating effect.
  • the IL-2 mutein of the present invention passes through a region where IL-2 interacts with IL-2R ⁇ (amino acid residues 35-72) and / or a region where IL-2 interacts with IL-2R ⁇ (amino acid residue 79).
  • Position -92) introduces one or more mutations, especially three or more mutations, so that the binding of IL-2 mutein to IL-2R ⁇ is reduced or unbound, and / or the binding to IL-2R ⁇ remains unchanged or enhanced.
  • the IL-2 mutein of the present invention has improved properties, including, for example, one or more of the following:
  • the IL-2 mutein of the present invention has the properties of the above (1), preferably further has one or more, especially all properties selected from the group consisting of (3) and (5)-(8). ; More preferably still has one or more selected from (2) and (9)-(12), especially all properties.
  • the IL-2 mutein of the present invention has the properties of (2) above, preferably further has one or more, especially all properties selected from (9) to (12); more preferably It further has one or more properties selected from (1), (3) and (5)-(8), especially all properties.
  • the IL-2 muteins of the invention also have the following properties relative to wild-type IL-2: reduced in vivo toxicity mediated by the binding of IL-2 to the high affinity receptor IL-2 ⁇ .
  • the IL-2 mutein of the present invention has improved pharmaceutical properties, for example, when expressed in mammalian cells, such as H293T cells, has one or more properties selected from the group consisting of: (i) better than The amount of expression of the wild-type IL-2 protein; (ii) better homogeneity than the wild-type IL-2 protein; and (iii) easy purification to a higher protein purity.
  • the IL-2 mutein of the invention exhibits an increased expression level compared to wild IL-2.
  • increased expression occurs in a mammalian cell expression system.
  • the expression level can be determined by any suitable method that allows quantitative or semi-quantitative analysis of the amount of recombinant IL-2 protein in the cell culture supernatant, preferably the supernatant after one-step affinity chromatography purification.
  • the IL-2 mutein of the invention is increased in mammalian cells by at least 1.1-fold, or at least 1.5-fold, or at least 2-fold, 2.5-fold, 3 compared to wild-type IL-2. Times, 3.5 times, or 4 times.
  • the purity of the IL-2 mutein product of the present invention can reach 70%, or 80%, or more than 90%. In some embodiments, protein purity is detected by SEC-HPLC technology.
  • the IL-2 mutein of the present invention has at least a 5-fold reduction in affinity for the IL-2R ⁇ receptor relative to wild-type IL-2 (eg, IL-2 WT shown in SEQ ID NO: 1), At least 10 times, or at least 25 times, especially at least 30 times, 50 times, or more than 100 times.
  • the muteins of the invention do not bind the IL-2R ⁇ receptor.
  • the binding affinity can be determined by the biofilm interference (BLI) technique of the IL-2 mutein of the present invention, for example, the IL-2 mutein of the present invention fused to an Fc fragment, and the equilibrium dissociation constant of the IL-2R ⁇ receptor ( K D ) to determine.
  • the IL-2 mutein of the invention (eg, IL-2 WT shown in SEQ ID NO: 1) has at least a 5-fold increased affinity for the IL-2R ⁇ receptor relative to wild-type IL-2, At least 10 times, or at least 25 times, especially at least 30 times, 50 times or 100 times, more preferably at least 150 times, 200 times, 250 times, 300 times, 350 times, 400 times, 450 times, or 500 times Or more than 550 times.
  • the binding affinity can be determined by the biological membrane layer interference (BLI) technique of the IL-2 mutein of the present invention, for example, the IL-2 mutein of the present invention fused to an Fc fragment, and the equilibrium dissociation constant of the IL-2R ⁇ receptor K D ) to determine.
  • BLI biological membrane layer interference
  • the binding affinity K D value of the IL-2 mutein of the present invention and the receptor IL-2R ⁇ receptor is less than 10.0E-09M, for example, less than 6.0E-09M, 3.0E-09M, 2.0E-09M, 1.0E-09M, more preferably less than 9.0E-10M, such as less than 6.0E-10M, 5.0E-10M, 4.0E-10M, 3.0E-10M , 2.0E-10M, 1.0E-10M, more preferably less than 9.0E-11M, 8.0E-11M, or 7.0E-11M.
  • the IL-2 mutein of the invention results in reduced activation and proliferation of CD25 + cells mediated by IL-2 relative to wild-type IL-2.
  • the CD25 + cells are CD25 + CD8 + T cells.
  • the CD25 + cells are Treg cells.
  • activation of STAT5 phosphorylation signal to identify IL-2 muteins ability of CD25 + cells activation may STAT5 phosphorylation in cells, determining the half maximal effective concentration (EC 50) analyzed by flow cytometry.
  • the IL-2 mutein of the invention is relative to a wild-type IL-2 protein (e.g., human IL- of SEQ ID NO: 1). 2)
  • the ability to activate CD25 + cells is reduced by at least 10 times, 50 times, 100 times, 300 times, 1000 times, 3000 times or more.
  • an IL-2 mutein of the invention results in maintained or enhanced CD25 - effector cell activation and proliferation mediated by IL-2 relative to wild-type IL-2.
  • activation of STAT5 phosphorylation signal by detecting IL-2 muteins in CD25- cells EC 50 value, to identify IL-2 muteins to activate CD25 - capacity of the cells.
  • an IL-2 mutein of the invention activates CD25 + cells relative to a wild-type IL-2 protein (eg, human IL-2 of SEQ ID NO: 1), as determined in a STAT5 phosphorylation assay.
  • the ability is increased by at least 1 time, such as 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, or 10 times, 20 times, 50 times, 100 times, or 150 times.
  • the IL-2 mutein of the present invention removes or reduces the bias of preferential activation of CD-2 + on CD25 + cells by IL-2 muteins of the invention relative to wild-type IL-2.
  • the CD25 + cells are CD25 + CD8 + T cells.
  • the CD25 + cells are Treg cells.
  • STAT5 phosphorylation assay by detecting IL-2 muteins were in CD25 - and activated STAT5 phosphorylation signal cells in CD25 + cells in the EC 50 value, to identify IL-2 muteins Ability to activate CD25 - cells.
  • IL-2 muteins determining activation of CD25 + cells bias.
  • the mutant protein's bias towards CD25 + is reduced by at least 100-fold, preferably at least 1000-fold, 2000-fold, and 3000-fold relative to the wild-type protein.
  • the IL-2 protein belongs to a short-chain type I cytokine family member with four alpha helix bundles (A, B, C, D) structure.
  • IL-2 has the following amino acid sites that interact with CD25 in the amino acid residue regions 35-72: 35; 37; 38; 41; 42; 43; 45; 61; 62 68; 72.
  • IL-2 has the following sites for interaction with CD122 in the amino acid residue regions 12-23 and amino acid regions 79-92: 12; 13; 15; 16; 19; 20; 23; 79; 81; 82; 83; 84; 87; 88; 91; 92.
  • CD25 interaction site ie, position 35; 37; 38; 41; 42; 43
  • CD25 binding region amino acid residue region 35-72
  • CD122 interaction site ie, position 79; 81; 82; 83; 84;
  • CD122 binding region amino acid residue region 79-92
  • mutations in two regions can be combined to provide an IL-2 mutant having both reduced or eliminated IL-2R ⁇ binding and enhanced IL-2 ⁇ binding.
  • the present invention provides an IL-2 mutein, which comprises at least one compared to wild-type IL-2 (preferably human IL-2, more preferably IL-2 comprising the SEQ ID NO: 1 sequence) Mutations that eliminate or reduce binding affinity for the IL-2R ⁇ receptor and / or increase binding affinity for the IL-2R ⁇ receptor.
  • wild-type IL-2 preferably human IL-2, more preferably IL-2 comprising the SEQ ID NO: 1 sequence
  • the IL-2 mutein of the present invention is, relative to wild-type IL-2, at the site where IL-2 interacts with its IL-2R ⁇ receptor (CD25), preferably at SEQ ID NO: 1.
  • CD25 IL-2R ⁇ receptor
  • the mutations at these positions are amino acid substitutions.
  • the mutations at these positions are substituted residues selected from: position 35: K35D, E; position 37: T37D, E, R, K, F, Y, W; position 38: R38D, E , F, Y, W, A, V; 41 digits: T41K, R, M, F, Y, W, Q, E; 42 digits: F42K, R, A, E, Q; 43 digits: K43E, D, F, Y, W; 45 bits: Y45R, K; 61 bits: E61R, K, W, Y, L; 62 bits: E62R, K, W, Y, L; 68 bits: E68R, K, W, Y; 72 bits: L72R, K, F, Y, W.
  • substitution residues at these positions are selected from the following: K35D, E; T37E, D, K, W, Y; R38W, E, D, V, F, K; T41E, Y, R, K, Q; F42E, R, K, Q, A; K43E, Y, D, W; Y45K, R; E61K, R, L, W; E68R, Y, W, K; L72K, F.
  • the substitution residues at these positions are selected from the following: K35D, E; T37D, E; R38D, E, F; T41E; F42A, E, Q; K43E, D, Y; Y45R, K; E61R, K, W, Y, L; E62R, W; E68R, K, W, Y; L72K, F.
  • the mutein comprises one or more mutations selected from the group consisting of: K35D, E; T37E; R38F, E; T41E; F42A, E; Y45K; E68Y.
  • a mutation that reduces or eliminates binding affinity to the IL-2R ⁇ receptor includes a mutation selected from a position corresponding to the following position of SEQ ID NO: 1 :
  • mutations that reduce or eliminate binding affinity for the IL-2R ⁇ receptor include a combination of mutations selected from the group consisting of:
  • mutations that reduce or eliminate binding affinity for the IL-2R ⁇ receptor include mutations at positions selected from: K35 / R38 / F42 / T37; K35 / R38 / F42 / Y45 / E61 / E68; K35 / R38 / F42 / Y45 / E61 / E68 / T37.
  • the mutation comprises: K35E, R38E, F42A, T37E; or K35E, R38W, F42Q, Y45R / K, E61K / W / R, E68R, and optionally T37D / E.
  • the mutation is a combination of mutations selected from:
  • the IL-2 protein of the present invention including these mutation combinations has increased IL-2R ⁇ binding affinity relative to wild-type IL-2; more preferably, it also has reduced activation bias toward CD25 + cells.
  • mutations that reduce or eliminate binding affinity for the IL-2R ⁇ receptor include mutations at positions selected from: K35 / R38 / F42 / Y45 / E61 / E68 / K43; K35 / R38 / F42 / Y45 / E61 / E68 / K43 / L72; K35 / R38 / F42 / Y45 / E61 / E68 / K43 / T37; K35 / R38 / F42 / Y45 / E61 / E68 / K43 / T37 / T41.
  • the mutation comprises: K35D / E, R38W / V / E, F42E / K / R, Y45R / K, E61R / L / K, E68Y / W, K43Y / W / D, and optionally T37D / W, T41Y; One or more of L72K. More preferably, the mutation is a combination of mutations selected from:
  • the mutation is:
  • the IL-2 protein of the present invention comprising these mutation combinations has reduced or eliminated IL-2R ⁇ receptor binding affinity, and has increased IL-2R ⁇ binding affinity, relative to wild-type IL-2.
  • mutations that reduce or eliminate binding affinity for the IL-2R ⁇ receptor include mutations at positions selected from: K35 / R38 / T41 / K43; K35 / R38 / T41 / K43 / T37; K35 / R38 / T41 / K43 / L72; K35 / R38 / T41 / K43 / T37 / L72; K35 / R38 / T41 / K43 / E61 / L72; K35 / R38 / T41 / K43 / Y45 / L72; K35 / R38 / K43 / T37 / L72; K35 / R38 / K43 / T37 / L72 / Y45.
  • the mutation comprises: K35D / E, R38D / E, T41E, K43E / Y, optionally further including one or both selected from T37K / E, Y45K, E61K, L72F; or preferably
  • the mutations include: K35D / E, R38D, K43E / Y, T37D / E, L72F, and optionally Y45K. More preferably the mutation is a combination of mutations selected from the group consisting of:
  • K35D / R38E / T41E / K43E K35D / T37K / R38D / T41E / K43E; K35E / R38E / T41E / K43E / L72F;
  • the mutation is a combination of mutations selected from:
  • the IL-2 muteins of the invention comprising these mutant combinations have reduced or eliminated IL-2R ⁇ receptor binding affinity and have increased IL-2R ⁇ binding affinity relative to wild-type IL-2; more preferably Ground also has a reduced activation bias for CD25 + cells.
  • mutations that reduce or eliminate binding affinity for the IL-2R ⁇ receptor include mutations at positions selected from: K35 / R38 / F42 / Y45 / E61 / E68; K35 / R38 / F42 / Y45 / E61 / E68 / T37; K35 / R38 / F42 / T37; K35 / R38 / T41 / K43.
  • mutations that reduce or eliminate binding affinity for the IL-2R ⁇ receptor include a combination of mutations selected from the group consisting of: K35E / R38E / F42A / T37E; K35E / R38W / F42Q / Y45K / E61K / E68R ; K35E / R38W / F42Q / Y45K / E61W / E68R; K35E / R38W / F42Q / Y45R / E61R / E68R; K35E / R38W / F42Q / Y45K / E61K / E68R / T37D; K35E / R38W / F42Q / Y45R / E61K / E68R / T37D; K35E / R38W / F42Q / Y45R / E61K / E68R / T37D; K35E / R38W
  • IL-2 muteins of the invention can allow the IL-2 muteins of the invention to have reduced or eliminated IL-2R ⁇ receptor binding affinity relative to wild-type IL-2, and have increased IL-2R ⁇ binding affinity; and result in reduced pairing
  • the activation of CD25 + cells is biased while maintaining or enhancing the activation and / or proliferation of CD25 - effector cells.
  • the mutations that reduce or eliminate the binding affinity for the IL-2R ⁇ receptor include a mutation combination selected from the group consisting of: K35D / T37E / R38E / T41E / K43E / Y45K / L72F; K35E / R38D / T41E / K43E / L72F; K35E / R38W / F42Q / Y45K / E61L / E68R; K35D / T37K / R38E / T41K / F42Q / K43D / E68Y; K35D / T37E / R38D / K43Y / Y45K / L72F; K35E / T37Y / R38W / T38 F42E / K43E / Y45K / L72K; K35D / R38W / F42E / K43E / Y45K / L72K; K35D
  • these combinations of mutations can be combined with mutations that increase IL-2R ⁇ binding in the 79-92 region of the present invention to provide an IL-2 mutein that does not bind to IL-2R ⁇ and has enhanced binding to IL-2R ⁇ .
  • the IL-2 mutein of the present invention can be at a site where IL-2 interacts with its ⁇ receptor CD122 (ie, IL-2R ⁇ ), preferably at 79,81,82 with SEQ ID NO: 1 , 83,84,87,88,91,92 corresponding positions, containing one or more mutations, said mutations enhance the binding affinity for IL-2R ⁇ .
  • the mutations at these positions are amino acid substitutions.
  • the mutations at these positions are substitution residues selected from the following: H79R, K, Y, W, D, E, Q; R81D, E, N, Q, T, H, Y, W; P82I, T, A; R83E; D84E, N, Q, H, T, V; S87T, D, N, E, Q, K, R, Y, W; N88D, E, Q, H, Y, W; V91T, L, I, M, D, N, E, Q, H; I92V, L, M, F, Y, W, N, D, E, Q.
  • the IL-2 mutein of the invention comprises one or more mutations, preferably amino acids, at positions corresponding to positions 79, 81, 82, 83, 87, 91, 92 of SEQ ID NO: 1.
  • substitutions in particular comprising one or more substitutions selected from the group consisting of: H79D, E, Q; R81D, N; P82I, T, A; R83E; S87D, E; V91L, I; I92L, M, F, Y.
  • the mutein of the invention remains unchanged relative to the wild-type IL-2 protein at positions corresponding to positions 12, 13, 15, 16, 19, 20, 23 of SEQ ID NO: 1.
  • the mutations that enhance binding affinity for IL-2R ⁇ include mutations at positions selected from H79 / R81 / S87 / I92, H79 / R81 / S87 / I92 / P82.
  • the mutation includes H79D / E, R81D, S87D / E, I92L / F, and optionally P82T.
  • the mutation comprises a combination mutation selected from the group consisting of H79D / R81D / S87D / I92L, and H79D / R81D / P82T / S87D / I92L.
  • the combination of mutations enables the IL-2 mutein of the invention to have significantly improved IL-2R ⁇ binding affinity relative to wild-type IL-2, and more preferably results in enhanced CD25 - effector cell activation and / or proliferation .
  • the mutations that enhance binding affinity for IL-2R ⁇ include mutations at positions selected from R81 / R83 / S87; R81 / R83 / S87 / I92; R81 / P82 / R83 / S87; or R81 / P82 / R83 / S87 / I92; preferably also includes mutations at one or two positions in H79 and V91.
  • the mutation comprises a combination mutation selected from: R81D, P82T / I / A, R83E, S87E / D; R81D / N, P82T / I / A, R83E, S87E / D, I92 / L / M / F / Y; R81D, R83E, S87E / D; R81D, R83E, S87E / D, I92L, and more preferably one or two of H79D / E / Q and V91L / I. More preferably, the mutation is a mutation combination selected from the group consisting of: H79D / R81D / S87D / I92L;
  • the combination of mutations enables the IL-2 mutein of the invention to have significantly improved IL-2R ⁇ binding affinity relative to wild-type IL-2, and more preferably results in enhanced CD25 - effector cell activation and / or proliferation .
  • the mutation that enhances binding affinity for IL-2R ⁇ is a combination mutation selected from:
  • these combinations of mutations can be combined with mutations that reduce the binding affinity of IL-2R ⁇ in the 35-72 position region of the present invention to provide an IL-2 mutein that does not bind to IL-2R ⁇ and has enhanced binding to IL-2R ⁇ .
  • the invention provides an IL-2 mutein comprising a mutation in the CD25 binding region and a mutation in the CD122 binding region, preferably said mutant protein has reduced (or eliminated) IL-2R ⁇ binding and has enhanced IL-2R ⁇
  • the present invention provides an IL-2 mutein comprising a combination selected from the following mutations.
  • the present invention provides an IL-2 mutein, wherein the mutein has a reduced ability to stimulate signaling in CD25 + T cells compared to wild-type IL-2, preferably the mutein Relative to the wild-type IL-2 protein (eg, the human IL-2 protein of SEQ ID NO: 1), contains (or only has) a combination of mutations selected from:
  • the present invention provides an IL-2 mutein, wherein the mutein has a reduced ability to stimulate signaling in CD25 + T cells and has an enhanced ability to stimulate CD25 compared to wild-type IL-2.
  • the mutant protein comprises (or has only) a mutation combination selected from the following relative to a wild-type IL-2 protein (such as the human IL-2 protein of SEQ ID NO: 1):
  • the IL-2 mutein of the present invention may also have one or more mutations in other regions or positions as long as it retains the one or more beneficial properties of the IL-2 muteins of the present invention Just fine.
  • the IL-2 muteins of the invention may also contain a substitution at position 125, such as C125S.C125A, C125T, or C125V, to provide additional advantages such as improved expression or homogeneity or stability (see, for example, the United States (Patent No. 4,518,584).
  • Those skilled in the art know how to determine additional mutations that can be incorporated into the IL-2 muteins of the invention.
  • the sequence difference between the IL-2 mutein and the wild-type protein can be expressed by sequence identity, or it can be expressed by the number of different amino acids between the two.
  • the IL-2 mutein has at least 85%, 86%, 87%, 88%, 89% identity, preferably 90% or more identity, and preferably 95% identity, but preferably not more than 85% More than 97%, more preferably no more than 96% identity.
  • the other mutations may be conservative substitutions.
  • the other mutation may be a mutation that confers other improved properties to IL-2.
  • the invention also provides a fusion protein comprising the IL-2 mutein of the invention.
  • the IL-mutein of the invention is fused to another polypeptide that can confer improved pharmacokinetic properties, such as albumin, and more preferably an antibody Fc fragment.
  • the Fc fragment comprises a mutation that reduces or removes effector function, such as a L234A / L235A mutation or L234A / L235E / G237A that reduces binding to the Fc [gamma] receptor.
  • the Fc-containing fusion protein has an increased serum half-life.
  • the Fc-containing fusion protein also has reduced effector functions mediated by the Fc region, such as ADCC or ADCP or CDC.
  • the invention also provides immunoconjugates comprising an IL2 mutein of the invention and an antigen binding molecule.
  • the antigen-binding molecules are immunoglobulin molecules, especially IgG molecules, or antibodies or antibody fragments, especially Fab molecules and scFv molecules.
  • the antigen-binding molecule specifically binds an antigen presented on tumor cells or in the tumor environment, such as an antigen selected from the group consisting of fibroblast activating protein (FAP), A1 domain of tenascin C (TNC A1), A2 domain of tenascin C (TNC A2), extra domain B (EDB) of fibronectin, carcinoembryonic antigen (CEA), melanoma-related chondroitin sulfate proteoglycan (MCSP ).
  • FAP fibroblast activating protein
  • TNC A1 domain of tenascin C TCC A1
  • A2 domain of tenascin C TNC A2
  • EDB extra domain B
  • CEA carcinoembryonic antigen
  • MCSP melanoma-related chondroitin sulfate proteoglycan
  • the IL-2 mutein of the present invention may be connected to another molecule or an antigen-binding molecule directly or through a linker, and in some embodiments, a proteolysis is included between the two. Cutting site.
  • the invention provides a nucleic acid encoding any of the IL-2 muteins or fusions or conjugates above.
  • the polynucleotide sequence encoding the mutein of the present invention can be generated by methods known in the art, either by de novo solid-phase DNA synthesis or by PCR mutagenesis of an existing sequence encoding wild-type IL-2.
  • the polynucleotides and nucleic acids of the present invention may contain a segment encoding a secretory signal peptide and be operably linked to a segment encoding a mutein of the present invention, so as to guide the secreted expression of the mutein of the present invention.
  • the invention also provides a vector comprising a nucleic acid of the invention.
  • the vector is an expression vector, such as a eukaryotic expression vector.
  • Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phages, or yeast artificial chromosomes (YAC).
  • YAC yeast artificial chromosomes
  • the expression vector of the present invention is a pYDO_017 expression vector (SEQ ID NO: 13).
  • the invention also provides a host cell comprising the nucleic acid or the vector.
  • Host cells suitable for replication and supporting expression of mutant IL-2 proteins or fusions or immunoconjugates are well known in the art. Such cells can be transfected or transduced with specific expression vectors, and a large number of vector-containing cells can be grown for inoculation of large-scale fermentation tanks to obtain sufficient quantities of IL-2 mutants or fusions or immunoconjugates For clinical applications.
  • the host cell is eukaryotic.
  • the host cell is selected from yeast cells, mammalian cells (e.g., CHO cells or 293 cells).
  • polypeptides can be produced in bacteria, especially when glycosylation is not required.
  • the polypeptide can be isolated from the bacterial cell paste in a soluble fraction and can be further purified.
  • eukaryotic microorganisms such as filamentous fungi or yeast are also suitable cloning or expression hosts for vectors encoding polypeptides, including fungal and yeast strains whose glycosylation pathways have been "humanized”, resulting in the production of Or a fully human glycosylation pattern polypeptide. See Gerngross, Nat Biotech 22, 1409-1414 (2004) and Li et al., Nat Biotech 24, 210-215 (2006).
  • Examples of useful mammalian host cell lines are monkey kidney CV1 line (COS-7) transformed with SV40; human embryonic kidney line (293 or 293T cells, as described for example in Graham et al., JGen Virol 36, 59 (1977)), young Rat kidney cells (BHK), mouse sertoli cells (TM4 cells, as described, for example, in Mather, BiolReprod 23, 243-251 (1980)), monkey kidney cells (CV1), African green monkey kidney cells ( VERO-76), human cervical cancer cells (HELA), canine kidney cells (MDCK), buffalo rat liver cells (BRL3A), human lung cells (W138), human liver cells (HepG2), mouse breast tumor cells (MMT060562 ), TRI cells (as described, for example, in Mather et al., Annals N.Y.
  • the host cell is a eukaryotic cell, preferably a mammalian cell such as a Chinese hamster ovary (CHO) cell, a human embryonic kidney (HEK) cell, or a lymphocyte (eg, a Y0, NSO, Sp20 cell).
  • CHO Chinese hamster ovary
  • HEK human embryonic kidney
  • a lymphocyte eg, a Y0, NSO, Sp20 cell
  • the present invention provides a method for preparing an IL-2 mutein or fusion or conjugate of the present invention, wherein the method comprises, under conditions suitable for expression of the IL-2 mutein or fusion or conjugate, Culturing a host cell comprising a nucleic acid encoding said protein or fusion or conjugate, as provided above, and optionally recovering said protein or fusion or conjugate from said host cell (or host cell culture medium) ⁇ The compound.
  • the IL-2 muteins provided herein can be identified, screened, or characterized for their physical / chemical properties and / or biological activity by a variety of assays known in the art.
  • the IL-2 mutein of the present invention can be tested for its binding activity to the IL-2 receptor.
  • binding to human IL-2R ⁇ or ⁇ protein can be determined by methods known in the art, such as ELISA, Western blot, etc., or exemplary methods disclosed in the examples herein.
  • assays can be performed using flow cytometry in which cells, such as yeast display cells, transfected to express a mutant protein on the cell surface are reacted with a labeled (eg, biotin-labeled) IL-2R ⁇ or ⁇ protein.
  • mutant proteins bind to the receptor, including binding kinetics (e.g., K D value), using recombinant mutein -Fc fusion, in a biological interference layer (BLI) assay.
  • binding kinetics e.g., K D value
  • recombinant mutein -Fc fusion e.g., K D value
  • BLI biological interference layer
  • a BLI assay is used as described in the examples.
  • the signaling and / or immune activation effects that occur downstream of receptor binding can be determined.
  • assays are provided for identifying biologically active mutant IL-2 proteins.
  • Biological activities may include, for example, the ability to induce proliferation of T and / or NK cells with IL-2 receptors, the ability to induce IL-2 signaling in T and / or NK cells with IL-2 receptors, through NK
  • the ability of cells to produce interferon (IFN) - ⁇ as a secondary cytokine reduced ability to induce apoptosis in T cells, ability to induce tumor regression and / or improve survival, and reduced in vivo toxicity properties, such as reduced Vascular permeability.
  • the invention also provides mutant IL-2 proteins having such biological activity in vivo and / or in vitro.
  • a suitable assay for testing the ability of the IL-2 mutein of the invention to stimulate NK cells to produce IFN- ⁇ may include the steps of: culturing cultured NK cells with the mutant IL-2 protein of the invention or fusion or immunoconjugate The mixture was incubated, and then the IFN- ⁇ concentration in the medium was measured by ELISA.
  • IL-2 signaling induces several signaling pathways and is involved in JAK (Janus kinase) and STAT (signal transducers and activators of transcription) signaling molecules.
  • IL-2 The interaction of IL-2 with the receptor ⁇ and ⁇ subunits results in phosphorylation of the receptor and JAK1 and JAK3, which bind to the ⁇ and ⁇ subunits, respectively.
  • STAT5 then binds to the phosphorylated receptor and is itself phosphorylated on very important tyrosine residues. This leads to dissociation of STAT5 from the receptor, dimerization of STAT5, and translocation of STAT5 dimers to the nucleus, where they promote the transcription of target genes.
  • the ability of a mutant IL-2 polypeptide to induce signaling through the IL-2 receptor can be assessed, for example, by measuring phosphorylation of STAT5. Details of this method have been disclosed in the examples. For example, PBMCs can be treated with a mutant IL-2 polypeptide or fusion or immunoconjugate of the invention and the level of phosphorylated STAT5 can be determined by flow cytometry.
  • T cells or NK can be measured by incubating T cells or NK cells isolated from blood with the mutant IL-2 polypeptide or immunoconjugate of the present invention, and then measuring the ATP content in the lysates of the treated cells Cells respond to IL-2 proliferation.
  • T cells can be pre-stimulated with lectin (PHA-M) before treatment.
  • PHA-M lectin
  • This assay allows sensitive quantification of the number of viable cells, and a number of suitable alternative assays are also known in the art (e.g. [3H] -thymidine incorporation assay, cell titration GloATP assay, AlamarBlue assay, WST- 1 measurement method, MTT measurement method).
  • mutated IL-2 on tumor growth and survival can be evaluated in a variety of animal tumor models known in the art.
  • a xenograft of a human cancer cell line can be implanted into an immunodeficient mouse and treated with a mutant IL-2 polypeptide or fusion or immunoconjugate of the invention.
  • the mutant IL of the present invention can be determined based on mortality, lifetime observations (visible symptoms of adverse effects, such as behavior, weight, body temperature), and clinical and anatomical pathology (such as measurement of blood chemical values and / or histopathological analysis).
  • vascular permeability induced by treatment with IL-2 can be examined with a vascular leak reporter in a pre-treated vascular permeability animal model.
  • the vascular leak reporter is large enough to reveal the permeability of the wild-type form of IL-2 for pretreatment.
  • the present invention provides a method for reducing elimination or reducing the binding affinity of the IL-2 protein to the IL-2R ⁇ receptor and / or enhancing the binding affinity for the IL-2R ⁇ receptor, including 2 introduces a mutation or combination of mutations described herein, and identifies (e.g., using the aforementioned assays) a wild type IL-2 protein with altered binding affinity for ILR alpha or beta, and / or improved biological activity, For example, a mutein of one or more of the properties described above for the IL-2 mutein of the present invention.
  • the parent wild-type IL-2 protein used as a mutation template preferably has at least 80%, or at least 95% or 99% or higher identity to SEQ ID NO: 1, more preferably a human source IL-2 protein.
  • the present invention also includes a composition (including a pharmaceutical composition or a pharmaceutical preparation) comprising an IL-2 mutein or a fusion or an immunoconjugate thereof, and a polynuclear core comprising an IL-2 mutein or a fusion or an immunoconjugate thereof.
  • compositions may also optionally contain suitable pharmaceutical excipients, such as pharmaceutical carriers, pharmaceutical excipients, including buffers, as known in the art.
  • Pharmaceutically acceptable carriers suitable for the present invention can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like.
  • water is the preferred carrier.
  • Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, especially for injectable solutions.
  • Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk , Glycerin, propylene, glycol, water, ethanol, etc.
  • compositions may also contain small amounts of wetting or emulsifying agents, or pH buffering agents.
  • these compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like.
  • Oral formulations may contain standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, saccharin.
  • the IL-2 muteins, fusions or immunoconjugates of the invention having the desired purity can be obtained by combining one or more optional pharmaceutical excipients (Remington's Pharmaceutical Sciences, 16th Edition, Osol, A. Ed. (1980)) to prepare a pharmaceutical formulation comprising the present invention, preferably in the form of a lyophilized formulation or an aqueous solution.
  • a pharmaceutical formulation comprising the present invention, preferably in the form of a lyophilized formulation or an aqueous solution.
  • An exemplary lyophilized antibody formulation is described in US Patent No. 6,267,958.
  • Aqueous antibody formulations include those described in US Patent No. 6,171,586 and WO2006 / 044908, the latter formulations including histidine-acetate buffers.
  • sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the protein, which matrices are in the form of shaped articles
  • the pharmaceutical composition or formulation of the present invention may also contain one or more other active ingredients that are required for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other .
  • active ingredients such as chemotherapeutic agents, PD-1 axis binding antagonists (such as anti-PD-1 antibodies or anti-PD-L1 antibodies or anti-PD-L2 antibodies).
  • the active ingredients are suitably present in combination in an amount effective for the intended use.
  • the composition further comprises a second therapeutic agent.
  • the second therapeutic agent may be an immune checkpoint inhibitor.
  • the second therapeutic agent may be selected from, including but not limited to, for example, anti-CTLA-4 antibodies, anti-CD47 antibodies, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-CD40 antibodies, anti-OX40 (also known as CD134, TNFRSF4, ACT35 and / or TXGP1L) antibody, anti-LAG-3 antibody, anti-CD73 antibody, anti-CD137 antibody, anti-CD27 antibody, anti-CSF-1R antibody, TLR agonist or IDO or TGF ⁇ One or more small molecule antagonists.
  • the second therapeutic agent is a PD-1 antagonist, especially an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-LAG-3, an anti-CD47.
  • the second therapeutic agent may also be other radiotherapy or chemotherapy drugs.
  • the invention also provides a combination product comprising a mutein of the invention or a fusion or immunoconjugate thereof, and one or more other therapeutic agents (e.g., chemotherapeutic agents, other antibodies, cytotoxic agents, Vaccines, anti-infective agents, etc.).
  • therapeutic agents e.g., chemotherapeutic agents, other antibodies, cytotoxic agents, Vaccines, anti-infective agents, etc.
  • the combination product of the present invention can be used in the treatment method of the present invention.
  • the present invention provides a combination product, wherein the other therapeutic agent is, for example, a therapeutic agent, such as an antibody, effective to stimulate an immune response to further enhance, stimulate or up-regulate the immune response in a subject.
  • the other antibodies are, for example, anti-PD-1 antibodies or anti-PD-L1 antibodies or anti-PD-L2 antibodies or anti-LAG-3 antibodies or anti-CTLA-4 antibodies or anti-TIM-3 antibodies.
  • the combination product is used to prevent or treat a tumor.
  • the tumor is a cancer, such as a gastrointestinal cancer, such as gastric cancer, rectal cancer, colon cancer, colorectal cancer, etc .; or a skin cancer such as melanoma; or renal cell carcinoma, bladder cancer, non-small cell Lung cancer etc.
  • the combination product is used to prevent or treat infections, such as bacterial infections, viral infections, fungal infections, protozoan infections, and the like.
  • mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., human and non-human primates such as monkeys), rabbits and rodents (e.g., mice and rats mouse).
  • domesticated animals e.g., cows, sheep, cats, dogs, and horses
  • primates e.g., human and non-human primates such as monkeys
  • rabbits and rodents e.g., mice and rats mouse.
  • the subject is a human.
  • treatment refers to a clinical intervention intended to alter the natural process of a disease in an individual being treated. Desired therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of the disease, reducing symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the state of the disease, and alleviating or improving the prognosis.
  • the invention provides a method of stimulating the immune system of a subject, the method comprising administering to the subject an effective amount of a pharmaceutical combination comprising an IL-2 mutein or fusion or immunoconjugate of the invention Thing.
  • the IL-2 mutein of the present invention has high activity and selectivity for CD25 - CD122 + effector cells (cytotoxic CD8 + T cells and NK cells), and has reduced and removed stimulation effects on CD25 + Treg cells. Therefore, the IL-2 mutein of the present invention can be used at a low dose to stimulate the immune system of a subject.
  • the invention relates to a method of enhancing an immune response in a subject, the method comprising administering to the subject an effective amount of any of the IL-2 muteins described herein, or Its fusions or immunoconjugates.
  • an IL-2 mutein of the invention, or a fusion or immunoconjugate thereof is administered to a tumor-bearing subject to stimulate an anti-tumor immune response.
  • an antibody of the invention or an antigen-binding portion thereof is administered to a subject carrying an infection to stimulate an anti-infective immune response.
  • the IL-2 muteins of the invention can be used in combination with a Treg depleting antibody (eg, Fc ⁇ R-mediated Treg depletion) to further reduce the immunosuppressive effects caused by Treg.
  • a Treg depleting antibody eg, Fc ⁇ R-mediated Treg depletion
  • the IL-2 muteins of the invention can be administered in combination with an immune checkpoint inhibitor to, for example, enhance the effect of cancer immunotherapy, such as in combination with anti-PD-1 and anti-CTLA-4.
  • the invention in another aspect, relates to a method of treating a subject's diseases, such as tumors and cancers and infections, the method comprising administering to the subject an effective amount of any of the IL-2 muteins described herein, or Its fusions or immunoconjugates.
  • Cancer can be early, intermediate, or advanced or metastatic.
  • the tumor or tumor cell may be selected from colorectal tumor, ovarian tumor, pancreatic tumor, lung tumor, lung tumor, liver tumor, breast tumor, kidney tumor, prostate tumor, gastrointestinal tumor, melanoma, cervical Tumors, bladder tumors, glioblastomas, and head and neck tumors.
  • the cancer can be selected from colorectal cancer, ovarian cancer, pancreatic cancer, lung cancer, liver cancer, breast cancer, kidney cancer, prostate cancer, gastrointestinal cancer, melanoma, cervical cancer, bladder cancer, glia Cell tumor and head and neck cancer.
  • the tumor is melanoma, renal cell carcinoma, colorectal cancer, bladder cancer, non-small cell lung cancer.
  • the invention in another aspect, relates to a method of treating an infectious disease, such as a chronic infection, in a subject, the method comprising administering to the subject an effective amount of any of the IL-2 muteins or fragments thereof described herein Or an immunoconjugate, a multispecific antibody, or a pharmaceutical composition comprising the antibody or fragment.
  • the infection is a viral infection.
  • the method of the invention further comprises administering to the subject one or more therapies (e.g., treatment modality and / Or other therapeutic agents).
  • the treatment modality includes surgical treatment and / or radiation therapy.
  • the methods of the invention further comprise administering at least one other immunostimulatory antibody, such as an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-LAG-3 antibody, an anti-CD43 antibody, and / or CTLA-4 antibodies.
  • these antibodies can be, for example, fully human, chimeric, or humanized antibodies.
  • the anti-PD-1 antibody is selected from the group consisting of: IBI308 (sintilizumab, WO2017 / 025016A1), MDX-1106 (nivolumab, OPDIVO), Merck 3475 (MK-3475, pembrolizumab, KEYTRUDA), and CT-011 (Pidilizumab).
  • the anti-PD-1 antibody is MDX-1106.
  • the anti-PD-1 antibody is nivolumab (CAS registration number: 946414-94-4).
  • the IL-2 mutein or fragment thereof, alone or in combination with a PD-1 antagonist can also be administered in combination with one or more other therapies such as a treatment modality and / or other therapeutic agents.
  • the treatment modalities include surgery (e.g., tumor resection); radiation therapy (e.g., exoparticle beam therapy, which involves three-dimensional conformal radiation therapy in which the illuminated area is designed), local irradiation (e.g., pointing at a preselected target) Or organ irradiation) or focused irradiation.
  • a disease eg, a tumor
  • methods of treating a disease comprising administering to a subject a mutein and a CTLA-4 antagonist antibody described herein.
  • the anti-CTLA-4 antibody may be, for example, an antibody selected from: (ipilimumab or antibody 10D1, described in PCT Publication No. WO 01/14424), tremelimumab (formerly known as ticilimumab, CP-675,206), and anti-CTLA-4 antibodies described in the following publications: WO 98/42752; WO 00 / 37504; U.S. Patent No. 6,207,156; Hurwitz et al. (1998) Proc. Natl. Acad. Sci.
  • a disease eg, a tumor
  • methods of treating a disease comprising administering to a subject an anti-mutant protein and an anti-LAG-3 antagonist antibody described herein.
  • the anti-LAG3 antibody may be, for example, an antibody selected from antibodies 25F7, 26H10, 25E3, 8B7, 11F2, or 17E5 described in US Patent Application Nos. US2011 / 0150892 and WO2014 / 008218, or a CDR or variable region comprising these antibodies Antibodies; BMS-986016; IMP731 described in US 2011/007023.
  • the IL-2 muteins of the invention can be administered in combination with a chemotherapeutic or chemotherapeutic agent. In some embodiments, the IL-2 muteins of the invention can be administered in combination with radiotherapy or a radiotherapy agent. In some embodiments, the IL-2 muteins of the invention can be administered in combination with a targeted therapy or a targeted therapeutic agent. In some embodiments, the IL-2 muteins of the invention can be administered in combination with an immunotherapy or immunotherapeutic agent, such as a monoclonal antibody.
  • the muteins of the present invention can be administered by any suitable method, including parenteral administration, intrapulmonary administration Drugs and intranasal administration, and if needed for local treatment, intralesional administration.
  • Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Depending on whether the medication is short-term or long-term, it can be administered by any suitable route, such as by injection, such as intravenous or subcutaneous injection.
  • Various dosing schedules are covered herein, including, but not limited to, single administration or multiple administrations at multiple time points, bolus administration, and pulse infusion.
  • the appropriate dosage of the mutein of the invention (when used alone or in combination with one or more other therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and progress of the disease , Administration for prophylactic or therapeutic purposes, previous treatment, the patient's clinical history and response to the antibody, and the judgment of the attending physician.
  • the antibody is suitably administered to a patient in one treatment or after a series of treatments.
  • the present invention also provides the use of the IL-2 mutein, composition, immunoconjugate, and fusion of the present invention in the manufacture of a medicament for the aforementioned method (for example, for treatment).
  • the IL-2 residues listing the interaction sites are mutated according to Table 2.
  • the original amino acids at each point account for 50%, and the remaining 50% are equally divided by the "mutated amino acids" in Table 2.
  • the library is split into two small libraries.
  • the mutant library at positions 12-23 is IBYDL030, which is theoretically diverse.
  • the mutation library at 79-92 is IBYDL031, and the theoretical diversity is 8 ⁇ 9 ⁇ 4 ⁇ 2 ⁇ 7 ⁇ 10 ⁇ 7 ⁇ 10 ⁇ 11 ⁇ 3.1 ⁇ 10 7 .
  • Wild type IL-2 (uniprot: P60568, aa21-153, C125S, referred to as IL-2 WT ) was placed between two BamHI digestion sites of the yeast display plasmid pYDC011.
  • the sequence of IL-2 WT is shown in SEQ ID NO: 1 in this application, and a C125S mutation was introduced at position 125 of the sequence to avoid disulfide-bridged IL-2 dimer formation.
  • the specific steps for plasmid construction are as follows:
  • the plasmid pYDC011 was digested with BamHI (New England Biolab, article number: R3136V) and then recovered (QIAGEN Gel Extraction Kit, Cat. 28704);
  • the amplified product and the digested product were recovered by 1% agarose gel;
  • the recombinant product was transferred into E. coli Top10 competent cells (Tiangen, article number: CB104-02), spread on LB plates containing ampicillin resistance, and cultured at 37 ° C overnight;
  • IL-2 mutant IL-2 3X does not bind to IL-2R ⁇ , and its binding force to IL-2R ⁇ remains unchanged (Rodrigo Vazquez-Lombardi et al., Nature Communications, 8: 15373, DOI: 10.1038 / ncomms15373) ; IL-2 mutant IL-2 H9 enhanced binding to IL-2R ⁇ and remained unchanged with bound IL-2R ⁇ (Aron M. Levin et al., Nature, Vol 484, p529-533, DOI: 10.1038 / nature10975).
  • IL-2 3X and IL-2 H9 were displayed on the surface of yeast and used as controls. The sequences of IL-2 3X and IL-2 H9 are shown in SEQ ID NOs: 2 and 3, respectively. Both proteins are identical to IL-2 WT and also contain a C125S mutation.
  • the required primers (as shown in Figure 3) were designed and synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.
  • IBYDL029 library DNA amplification 1. Using pYDC035 as a template, primers AMP0191, AMP0200 to amplify fragment 029-F; 2. Using pYDC035 as a template, primers AMP0201, AMP0199 to amplify fragment 029-R; 3. Gel recovery fragment 029-F And 029-R were used as templates for PCR amplification, and the full-length fragment 029 was amplified with primers AMP0191 and AMP0199.
  • IBYDL030 library DNA amplification 1. Using pYDC035 as a template, primers AMP0191, AMP0224 to amplify fragment 030-F; 2. Using pYDC035 as a template, primers AMP0222, AMP0199 to amplify fragment 030-R; 3. Gel recovery fragment 030-F 030-R was used as a template for PCR amplification, and the full-length fragment 030 was amplified with primers AMP0191 and AMP0199.
  • IBYDL031 library DNA amplification 1. pYDC035 as template, primers AMP0191, AMP0225 amplified fragment 031-F; 2. pYDC035 as template, primers AMP0223, AMP0199 amplified fragment 031-R; 3. Gel recovery fragment 031-F 031-R was used as a template for PCR amplification, and primers AMP0191 and AMP0199 were used to amplify the full-length fragment 031.
  • plasmid pYDC011 100 ⁇ g of plasmid pYDC011 was digested with BamHI and digested with a PCR product recovery kit (QIAGEN PCR Purification Kit, Cat. 28104) to obtain a sufficient amount of linearized plasmid.
  • the linearized plasmid and library DNA were mixed at 4 ⁇ g: 12 ⁇ g, and in accordance with existing methods (Lorenzo Benatuil et al., An improved yeast transformation method for the generation of very large human antibody libraries. Protein Engineering, Design & Selection vol. 23no. 4pp. 155 –159, 2010) Electroporation of a mixture of each library and linearized plasmid into the EBY100 yeast strain.
  • the library was diluted and coated on a SD-Trp (TAKARA, article number: 630309) plate, and the number of colonies growing was counted.
  • the actual diversity of the library was IBYDL029: 4.2 ⁇ 10 8 , IBYDL030: 4.5 ⁇ 10 8 , IBYDL031: 3.8 ⁇ 10 8 , all larger than the theoretical diversity of the library.
  • Example 2 IL-2 WT- FC, IL-2 3X- FC, IL-2R ⁇ , IL-2R ⁇ protein preparation and biotin labeling
  • the IL-2 WT and IL-2 3X gene sequences are placed between two BamHI digestion sites of the vector pYDO_017 for expression of the fusion protein of IL-2 WT- FC and IL-2 3X- FC.
  • the Fc used in the present invention It refers to human IgG1 Fc (L234A, L235A, FcLALA for short).
  • IL-2R ⁇ (Uiprot: P01589, aa22-217) and IL-2R ⁇ (Uiprot: P14784, aa27-240) were attached at the C-terminus of the sequence with an avi tag and 6 histidine tags (the sequence is shown in SEQ ID NO: 11 and 12), which were respectively constructed on the pTT5 vector for expressing the IL-2R ⁇ and IL-2R ⁇ proteins.
  • the expression plasmid vector constructed above was transferred into HEK293-F (Invitrogen, article number: R79007) cells using a chemical transfection method.
  • the chemical transfection reagent polyethyleneimine (referred to as PEI, Polysciences, article number: 23966) was used to transiently transfect cultured HEK293-F cells according to the protocol provided by the manufacturer. Take a final volume of 1/10 (v / v) Opti-MEM medium (Gibco article number: 31985-070) as the transfection buffer, add the plasmid, mix well, and filter with a 0.22 ⁇ m filter head for use.
  • PEI polyethyleneimine
  • Molar ratio of plasmid to PEI in 293F cells is 1: 3
  • the DNA / PEI mixture was gently poured into HEK293 cells and mixed.
  • VPA Sigma, article number: P4543-100G
  • VPA Sigma, article number: P4543-100G
  • Cell culture fluid expressing IL-2 WT- FC and IL-2 3X- FC fusion protein centrifuged at 13,000 rpm for 20 min, collected the supernatant, and purified the supernatant using a pre-packed Hitrap Mabselect Sure (GE, 11-0034-95) .
  • the operation is as follows: before purification, equilibrate the packed column with 5 column volumes of equilibrium solution (0.2M Tris, 1.5M NaCl, pH7.2); pass the collected supernatant through the column, and then wash the packed column with 10 column volumes of equilibration solution. To remove non-specific binding proteins; rinse the packing with 5 column volumes of elution buffer (1M sodium citrate, pH 3.5) and collect the eluate.
  • Tris (2M Tris) 80 ⁇ L was added per 1 ml of the eluent, and it was exchanged into a PBS buffer (Gibco, 70011-044) using an ultrafiltration concentration tube (Shanghai Tukai Biotechnology Co., Ltd., MCPM02C67) and the concentration was measured. Take 100 ⁇ g of purified protein, adjust the concentration to 1 mg / mL, and use a gel filtration chromatography column (TOSOH article number: 18675) to determine the protein purity.
  • the enzyme IL-2R ⁇ and IL-2R ⁇ proteins are labeled with biotin using an enzyme-catalyzed method as follows: Take appropriate amounts of IL-2R ⁇ and IL-2R ⁇ IH protein solutions, and add 1/10 (m / m) mass of His-BirA protein (uniprot : P06709), at the same time, add a final concentration of 2mM ATP (sigma article number: A2383-10G), 5mM MgCl 2 , 0.5mM D-biotin (AVIDITY article number: K0717); incubate at 30 ° C for 1h, and pass Superdex200increase (GE, 10 / 300GL) (10245605) purification to remove excess biotin and His-BirA; the purified samples were verified by Fortebio's Streptavidin (SA) sensor (PALL, 18-5019) to confirm successful biotin labeling.
  • SA Streptavidin
  • Example 3 IL-2 mutant library screening and IL-2 mutant identification
  • Yeast-based IL-2 mutant display libraries IBYDL029, IBYDL030, and IBYDL031 were cultured and induced with 2.0 ⁇ 10 9 yeast cells. The diversity of their Chinese libraries was 2.0 ⁇ 10 8 , 1.1 ⁇ 10 7 , and 3.1 ⁇ 10 7 respectively. . Due to the large diversity of the IBYDL029 library, the first round of screening was performed using the MACS system of Miltenyi Company for magnetic bead cell sorting. First, 2 ⁇ 10 9 yeast cells were incubated in FACS washing buffer (1 ⁇ PBS, containing 1% bovine serum protein) for 30 minutes at room temperature.
  • FACS washing buffer (1 ⁇ PBS, containing 1% bovine serum protein
  • the buffer contained 500 nM biotin-labeled commercially available IL-2R ⁇ (Acro Biosystems, EZ-Link Sulfo-NHS-LC-Biotin (IL-2R ⁇ -Biotin). Wash once with 50 ml of pre-chilled FACS wash buffer, resuspend the cells with 10 ml of the same wash buffer, and add 40 ⁇ l of streptavidin microbeads (Miltenyi biotec, article number: 130-090-485) and incubate at 4 ° C for 15 minute. Centrifuge at 3000 rpm for 3 minutes, discard the supernatant, resuspend the cells with 10 ml FACS washing buffer, and add the cell solution to the Miltenyi LS column.
  • IL-2R ⁇ Acro Biosystems, EZ-Link Sulfo-NHS-LC-Biotin
  • the column was washed 3 times with 3 ml FACS washing buffer.
  • the Miltenyi LS column was removed from the magnetic area and eluted with 5 ml of growth medium.
  • the eluted yeast cells were collected and grown overnight at 30 ° C.
  • the IBYDL030 and IBYDL031 libraries have less diversity, and can be directly sorted using a flow cytometer for the first round of sorting: 1 ⁇ 10 8 and 2.5 ⁇ 10 8 yeast cells were each washed three times with FACS buffer and contained 100 nM IL -2R ⁇ -Biotin, Anti Flag antibody (Sigma Cat. No.
  • F18041 diluted 1: 1000 incubated in FACS buffer for 30 minutes at room temperature; after washing the cells twice with FACS washing buffer, the cells were incubated with SA-PE (Streptomyces Avidin-PE, Thermo Fisher Cat. No .: S21388, 1: 200 dilution), goat anti-mouse conjugated Alex Flour-647 (Thermo Fisher Cat .: A21235, 1: 200 dilution) in FACS wash buffer, mixed at 4 ° C Incubate for 15 minutes in the dark. Wash twice with pre-chilled FACS wash buffer and resuspend in 1 mL of buffer. Transfer the cells to a filter-equipped separation tube. Cells were sorted using FACS MoFlo_XDP (Beckman), and the sorted yeast cells were grown at 30 ° C overnight.
  • SA-PE Streptomyces Avidin-PE, Thermo Fisher Cat. No .: S21388, 1: 200 dilution
  • Each library cell obtained after one round of screening was shake-induced at 20 ° C for 24 hours to display the IL-2 mutant , and a second round of sorting was performed using a flow cytometer. Take 3 ⁇ 10 7 yeast cells from each library and wash them three times with FACS buffer, add FACS buffer containing different concentrations of IL-2R ⁇ -Biotin (029: 300nM, 030/031: 100nM) and Anti Flag antibody, and incubate at room temperature for 30 minutes After the cells were washed twice with FACS wash buffer, the cells were mixed with FACS wash buffer containing SA-PE, goat anti-mouse conjugated Alex Flour-647, and incubated at 4 ° C in the dark for 15 minutes; pre-cooled The FACS wash buffer was washed twice and resuspended in 1 mL of buffer, and the cells were transferred to a filter-equipped separation tube. Cells were sorted using MoFlo_XDP, and the sorted yeast cells were grown overnight at 30 ° C. The third round of
  • IBYDL029 obtained 53 mutant sequences
  • IBYDL030 did not obtain mutant sequences
  • IBYDL031 obtained 71 mutant sequences.
  • the yeast-based IL-2 mutant display libraries IBYDL029, IBYDL030, and IBYDL031 were used for the second batch of screening using the homemade IL-2R ⁇ IH-Biotin obtained in Example 2.
  • the first round of screening used the MACS system for magnetic bead cell sorting. First, 2 ⁇ 10 9 yeast cells were taken from each library and incubated at room temperature for 30 minutes in FACS wash buffer containing IL-2R ⁇ IH-Biotin (IBYDL029: 500nM, IBYDL030 / 031: 200nM) at different concentrations; Magnetic bead sorting. Yeast cells obtained after magnetic bead sorting were grown overnight at 30 ° C.
  • Flow cytometry was used for the second and third rounds of sorting.
  • concentration of IL-2R ⁇ IH-Biotin in the two rounds of screening was selected from IBYDL 029: 500nM and IBYDL 030/031: 100nM.
  • the rest of the steps were the same as the second and third rounds of the first batch .
  • IBYDL029 added 25 mutant sequences
  • IBYDL030 did not obtain mutant sequences
  • IBYDL031 added 41 mutant sequences.
  • yeast cells containing a single mutant sequence were shaken at 20 ° C for 24 hours to display the IL-2 mutant ; they were stained with their receptors IL-2R ⁇ -Biotin and IL-2R ⁇ IH-Biotin, as follows:
  • steps 3-5 analyze the binding level of IL-2 mutant to IL-2R ⁇ .
  • the 25 batches of IL-2 mutants obtained in the second batch of screening do not bind to IL-2R ⁇ ; compared to IL-2 3X , binding to IL-2R ⁇ has been improved to varying degrees, and IL-2R ⁇ has the highest binding signal.
  • the 14 mutations are listed in Table 3-1 below.
  • IBYDL031 screened two batches of 112 IL-2 mutant binds both IL-2R ⁇ , IL-2R ⁇ , higher signal 25 mutations (see FIG particular sequence 4C) and IL-2R ⁇ binding are listed in Table 3-1
  • the average fluorescence signal intensity of binding of these 25 mutations to IL-2R ⁇ was significantly stronger than that of IL-2 3X and equivalent to or slightly weaker than that of IL-2 H9 .
  • Table 3-1 IL-2 mutants with high IL-2R ⁇ binding signals identified by staining
  • a mutation derived from IBYDL029 was combined with a mutation derived from IBYDL031 (the specific sequence is shown in Figure 4D) to obtain a new mutant that does not bind to IL-2R ⁇ and enhances binding to IL-2R ⁇ , as shown in Table 3-2.
  • the IL-2 mutant sequence was placed between two BamHI digestion sites in the vector pYDO_017 to express the IL-2 mutant -FC fusion protein.
  • the yeast display plasmid containing the IL-2 mutant gene was mixed in equal proportions as a template, and the fragments were amplified using primers at both ends. After amplification, the DNA fragments were recovered using a 1% agarose gel.
  • the pYDO_017BamHI digestion vector was the same as the recovered fragments. Recombination of the source, the recombinant product was transformed into E. coli competent cells, and the obtained IL-2 mutant- FC expression plasmid was verified by sequencing.
  • Chemical transfection was used to transfer the vector containing the gene encoding the fusion protein into HEK293 cells.
  • the chemical transfection reagent PEI was used to transiently transfect cultured HEK293 cells according to the protocol provided by the manufacturer.
  • the cells were cultured, the cells were centrifuged at 13,000 rpm for 20 minutes, and the supernatant was collected, and the supernatant was purified using a prepacked column, Hitrap Mabselect Sure.
  • the operation is as follows: before purification, equilibrate the packed column with 5 column volumes of equilibrium solution (0.2M Tris, 1.5M NaCl, pH7.2); pass the collected supernatant through the column, and then wash the packed column with 10 column volumes of equilibration solution.
  • K D The equilibrium dissociation constants (K D ) of 34 IL-2 mutant- FCs and their receptors of the present invention were determined using biofilm interference (BLI) technology.
  • the BLI method affinity measurement was performed according to existing methods (Estep, P, et al., High throughput solution Based measurement of antibody-antigen affinity and epitope binning. MAbs, 2013.5 (2): p.270-8). After pre-wetting the sensor in the analysis buffer for 20 minutes, according to the established method, use Octet Red96 to measure the affinity of the candidate IL-2 mutant- FC with IL-2R ⁇ and IL-2R ⁇ , respectively: first equilibrate the baseline for 120 seconds; then -2R ⁇ -Biotin or IL-2R ⁇ IH-Biotin is cured to the SA sensor (PALL, 18-5019); the sensor of the cured IL-2R ⁇ -Biotin or IL-2R ⁇ IH-Biotin is placed in a 100nM IL-2 mutant- FC The solution was allowed to reach the plateau phase (100 seconds), after which the sensor was transferred to the analysis buffer to dissociate for at least 2 minutes, and the binding and dissociation were measured respectively. The experimental results were analyzed using
  • Y29A2, Y29B2, Y30E1, Y07, Y10, Y33A4, Y33A5, Y33A6, Y33B1, Y33B4, Y33B5, Y33C5, Y33F4, Y34F4 were selected for in vitro functional experiments.
  • Example 5 In vitro function test of IL-2 mutant -FC
  • IL-2 WT has a higher affinity for IL-2R ⁇ than IL-2R ⁇ , and IL-2R ⁇ will preferentially bind to IL-2R ⁇ on the cell surface, and then recruit IL-2R ⁇ to release downstream p-STAT5 signals through IL-2R ⁇ to stimulate T cells and NK cell proliferation. Because IL-2R ⁇ is on the surface of Treg cells, and there is no IL-2R ⁇ on the surface of effector T cells and NK cells, normally IL-2 WT will preferentially stimulate Treg cells to proliferate and down-regulate the immune response.
  • IL-2 mutant does not bind to IL-2R ⁇ , which eliminates the preference of preferentially stimulating the proliferation of Treg cells, while stimulating the proliferation of T cells and NK cells, so that the number of effector T cells and NK cells is effectively increased, and the antitumor effect is improved.
  • each mutant by detecting the activation of p-STAT5 signal of primary human CD8 + T cells by each IL-2 mutant -FC, it is verified that each mutant removes the bias of activation of CD25 + cells, and screens for the effect on the activation of CD25 - cells. Strong mutant. Specific steps are as follows:
  • PBMC cells Allcells article number: PB005F, 100M pack
  • PB005F Allcells article number: PB005F, 100M pack
  • step 2 The cells in step 2 are CD8 + CD25 - T cells, and the cells in step 3 are CD8 + CD25 + T cells.
  • IL-2 3X (SEQ ID NO: 2) with mutations (C125S, R38D, K43E, E61R)
  • IL-2 H9 (SEQ ID NO: 3) with mutations (C125S, L80F, R81D, L85V, I86V, I92F)
  • Yeast display plasmid pYDC011 (SEQ ID NO: 4)
  • AMP0191 cccggatcggactactagcagc (SEQ ID NO: 5)
  • AMP0210 GTTATTGCTTCAGTTTTAGCAGCTCCCACCAGCAGCAGCACC (SEQ ID NO: 7)
  • AMP0225 GAAGTTCTTGCTCTGGGCTAAATTG (SEQ ID NO: 10)
  • IL-2R ⁇ receptor SEQ ID NO: 11
  • avi tag and His6 tag at the C-terminus
  • IL-2R ⁇ receptor SEQ ID NO: 12
  • avi tag and His6 tag at the C-terminus

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Abstract

本发明提供了白介素2(IL-2)突变蛋白及其用途。与野生型IL-2相比，所述IL-2突变蛋白具有降低的IL-2Rα受体结合能力和/或提高的IL-2Rβ受体结合能力。本发明还提供包含该IL-2突变蛋白的融合蛋白、免疫缀合物，以及编码该IL-2突变蛋白的核酸、包含该核酸的载体和宿主细胞。进一步提供了制备该IL-2突变蛋白的方法、包含该IL-2突变蛋白的药物组合物和该突变蛋白的治疗用途。

Description

新型白介素2及其用途 技术领域
本发明涉及新型白介素2(IL-2)突变蛋白及其用途。具体地，本发明涉及与野生型IL-2 2相比，具有降低的IL-2Rα受体结合能力和/或提高的IL-2Rβ受体结合能力的IL-2突变蛋白。本发明还提供包含该IL-2突变蛋白的融合蛋白、免疫缀合物，以及编码该IL-2突变蛋白的核酸、包含该核酸的载体和宿主细胞。本发明进一步提供制备该IL-2突变蛋白的方法、包含该IL-2突变蛋白的药物组合物和该突变蛋白的治疗用途。
背景技术
白介素2(IL-2)，也称作T细胞生长因子(TCGF)，是一种主要由活化的T细胞，尤其是CD4 +T辅助细胞产生的多能细胞因子。在真核细胞中，人IL-2(uniprot:P60568)作为153个氨基酸的前体多肽合成，在去除N端20个氨基酸后，产生成熟的分泌性IL-2。其它物种的IL-2的序列也已经公开，参见NCBI Ref Seq No.NP032392(小鼠)、NP446288(大鼠)或NP517425(黑猩猩)。
白介素2具有4个反平行的、两亲性α螺旋，此4个α螺旋形成其功能必不可少的四级结构(Smith,Science 240,1169-76(1988)；Bazan,Science257,410-413(1992))。在大多数情况下，IL-2通过三种不同受体起作用：白介素2受体α(IL-2Rα；CD25)、白介素2受体β(IL-2Rβ；CD122)和白介素2受体γ(IL-2Rγ；CD132)。IL-2Rβ和IL-2Rγ对于IL-2的信号传导至关重要，而IL-2Rα(CD25)对于信号传导不是必需的，但可以赋予IL-2对受体的高亲和力结合(Krieg等,Proc Natl Acad Sci 107,11906-11(2010))。由IL-2Rα,β,和γ联合形成的三聚体受体(IL-2αβγ)为IL-2高亲和力受体(K D约10pM)，由β和γ组成的二聚体受体(IL-2βγ)为中间亲和力受体(K D约1nM)，单独由α亚基形成的IL-2受体为低亲和力受体。
免疫细胞表达二聚体或三聚体IL-2受体。二聚体受体在细胞毒性CD8 +T细胞和天然杀伤细胞(NK)上表达，而三聚体受体主要在激活的淋巴细胞和CD4 +CD25 +FoxP3 +抑制性调节T细胞(Treg)上表达(Byman,O.和Sprent.J.Nat.Rev.Immunol.12,180-190(2012))。由于静息状态的效应T细胞和NK细胞在细胞表面上没有CD25，故对于IL-2相对不敏感。而Treg细胞在体内一贯表达最高水平的CD25，因此，正常情况下IL-2会优先刺激Treg细胞增殖。
IL-2通过与不同细胞上IL-2受体的结合，在免疫反应中介导多重作用。一方面，作为免疫系统刺激剂，IL-2可以刺激T细胞增殖和分化，诱导细胞毒性T淋巴细胞(CTL)生成，促进B细胞增殖和分化和免疫球蛋白合成，并刺激天然杀伤(NK)细胞的生产、增殖和活化，并由此已经被批准作为免疫治疗剂用于癌症和慢性病毒感染的治疗。另一方面，IL-2可以促进免疫抑制性CD4 +CD25 +调节性T细胞(即，Treg细胞)的活化与增殖(Fontenot等,Nature Immunol 6,1142-51(2005)；D’Cruz和Klein,Nature Immunol 6,1152-59(2005)；Maloy和Powrie,Nature Immunol6,1171-72(2005))，从而导致免疫抑制。此外，高剂量IL-2在患者中可引起血管渗漏综合征(VLS)。已经证实，IL-2通过直接结合肺内皮细胞上的IL-2三聚体受体(IL-2αβγ)而诱导肺水肿(Krieg等,Proc Nat Acad Sci USA107,11906-11(2010))。
为了克服与IL-2免疫治疗相关的上述问题，已经提出了通过改变IL-2对不同受体的选择 性或偏好性，来降低IL-2治疗的毒性和/或提高其功效。例如，已经提出使用IL-2单克隆抗体与IL-2的复合物，通过使IL-2靶向表达CD122而非CD25的细胞，诱导对CD122 high群体的优先扩增，增强体内IL-2治疗效果(Boyman等,Science 311,1924-1927(2006))。Oliver AST等(US2018/0142037)提出在IL-2的氨基酸残基位置42,45和72上引入三重突变F42A/Y45A/L72G来降低对IL-2Rα受体的亲合力。Aron M.Levin等(Nature,Vol 484,p529-533,DOI:10.1038/nature10975)提出一种称作“superkine”的IL-2突变体IL-2 H9，该突变体包含五重突变L80F/R81D/L85V/I86V/I92F，具有增强的IL-2Rβ结合，由此提高了对CD25 -细胞的刺激作用。Rodrigo Vazquez-Lombardi等人(Nature Communications,8:15373,DOI:10.1038/ncomms15373)提出一种三重突变人IL-2突变蛋白IL-2 3X，该蛋白在氨基酸残基位置38,43和61分别具有残基突变R38D/K43E/E61R，导致该突变蛋白对IL-2Rα不结合，以达到消除IL-2对CD25 +细胞的激活偏向性。但是，IL-2 3X对CD25 +细胞的激活偏向性依然存在，且该突变蛋白表达量较低，不利于后续作为药物大规模生产。
鉴于IL-2在免疫调节和疾病中的作用，本领域仍然存在着开发具有改善性质的新IL-2分子的需求。
发明概述
本发明通过提供相对于野生型IL-2具有改善的IL-2受体选择性/偏向性的新IL-2突变蛋白，满足了上述的需求。
因此，在一个方面，本发明提供了新的IL-2突变蛋白。在一些实施方案中，本发明的IL-2突变蛋白具有以下一个或多个特性：
(i)相对于野生型IL-2，对IL-2Rα受体的结合亲合力降低；
(ii)相对于野生型IL-2，对IL-2Rβ受体的结合亲合力增强；
(iii)有效降低IL-2对CD25 +细胞的激活偏向性；
(iv)有效激活CD25 -细胞。
在一些实施方案中，本发明的IL-2突变蛋白相比于野生型IL-2具有表达量高的特性。
在一些实施方案中，本发明提供在IL-2的氨基酸残基区域35-72中包含至少一个突变的IL-2突变蛋白；在另一些实施方案中，本发明提供在IL-2的氨基酸残基区域79-92中包含至少一个突变的IL-2突变蛋白；在再一些实施方案中，本发明提供在氨基酸区域35-72和79-92中包含两个以上且优选地三个以上突变的IL-2突变蛋白。
此外，本发明提供了包含IL-2突变蛋白的融合蛋白和免疫缀合物，药物组合物和组合产品；编码IL-2突变蛋白的核酸，包含所述核酸的载体和宿主细胞；以及产生本发明的IL-突变蛋白、融合蛋白和免疫缀合物的方法。
再有，本发明也提供了利用本发明的IL-2突变蛋白治疗疾病的方法和刺激受试者免疫系统的方法和用途。
在下面的附图和具体实施方案中进一步说明本发明。然而，这些附图和具体实施方案不应被认为限制本发明的范围，并且本领域技术人员容易想到的改变将包括在本发明的精神和 所附权利要求的保护范围内。
附图说明
图1显示了IL-2与IL-2Rα复合物的晶体结构。
图2显示了IL-2与IL-2Rβ复合物的晶体结构。
图3A-B显示了用于构建突变文库IBYDL029以及突变文库IBYDL030和IBYDL031的引物设计。
图4A-D显示了从突变文库IBYDL029和IBYDL031筛选出的一些IL-2突变蛋白及其序列，以及由两个文库中选出的突变组合产生的一些新突变蛋白及其序列。
图5A-D显示了选择并构建的一些IL-2 mutant-FC在CD8 +CD25 -/CD25 +T细胞上激活p-STAT5的信号曲线。
图6显示了人白介素(IL-2)的成熟蛋白序列(SEQ ID NO:1)及其氨基酸残基编号，并显示了与突变蛋白IL-2 3X和IL-2 H9的序列比对。
发明详述
除非另有定义，否则本文中使用的所有技术和科学术语均具有与本领域一般技术人员通常所理解的含义相同的含义。为了本发明的目的，下文定义了以下术语。
术语“约”在与数字数值联合使用时意为涵盖具有比指定数字数值小5％的下限和比指定数字数值大5％的上限的范围内的数字数值。
术语“和/或”应理解为意指可选项中的任一项或可选项中的任意两项或多项的组合。
如本文中所用，术语“包含”或“包括”意指包括所述的要素、整数或步骤，但是不排除任意其他要素、整数或步骤。在本文中，当使用术语“包含”或“包括”时，除非另有指明，否则也涵盖由所述及的要素、整数或步骤组成的情形。例如，当提及“包含”或“包括”某个突变或突变组合的IL-2突变蛋白时，也旨在涵盖仅具有所述突变或突变组合的IL-2突变蛋白。
在本文中，野生型“白介素-2”或“IL-2”是指作为引入本发明突变或突变组合的模板的亲本IL-2蛋白，优选天然存在的IL-2蛋白，例如来源于人、小鼠、大鼠、非人灵长类动物的天然IL-2蛋白，包括未经加工的(例如未去除信号肽)的形式和经加工的(例如去除了信号肽)的形式。此外，该表述也包括天然存在的IL-2等位基因变体和剪接变体、同种型、同源物、和物种同源物。该表述也包括天然IL-2的变体，例如，所述变体可以与天然IL-2具有至少95％-99％或更高同一性或具有不超过1-10个或1-5个氨基酸突变(尤其是保守氨基酸取代)，并与天然IL-2蛋白具有基本相同的IL-2Rα结合亲合力和/或IL2Rβ结合亲合力。因此，在一些实施方案中，野生型IL-2相比于天然IL-2蛋白可以包含不影响其对IL-2受体结合的氨基酸突变，例如，在125位引入了突变C125S的天然人IL-2蛋白(uniprot:P60568)属于本发明的野生型IL-2。一个野生型人IL-2蛋白的实例显示于SEQ ID NO:1中。在一些实施方案中，野生型人IL-2序列可以与SEQ ID NO:1的氨基酸序列具有至少85％,90％,95％，甚至至少96％,97％,98％,或99％或更高的氨基酸序列同一性。
在本文中，氨基酸突变可以是氨基酸取代、缺失、插入和添加。可以进行取代、缺失、 插入和添加的任意组合来获得具有期望性质(例如降低的IL-2Rα结合亲合力)的最终突变蛋白构建体。氨基酸缺失和插入包括在多肽序列的氨基和/或羧基末端的缺失和插入。例如，可以在全长人IL-2位置1缺失丙氨酸残基。优选的氨基酸突变是氨基酸取代。在一些实施方案中，当旨在通过在本发明描述的具体突变氨基酸位置上引入突变来获得具有改变的受体结合特性的IL-2突变蛋白时，优选在位置上进行非保守氨基酸取代。在一些实施方案中，优选的非保守氨基酸取代包括用亲水性氨基酸替换疏水性氨基酸，或者用不同极性或相反电荷的氨基酸进行替换。
在本发明中，当提及氨基酸位置时，通过参考SEQ ID NO:1的野生型人IL-2蛋白(也称作IL-2 WT)氨基酸序列(如图6所示)，予以确定。可以通过进行氨基酸序列比对(例如使用BLAST；可从http://blast.ncbi.nlm.nih.gov/Blast.cgi？PROGRAM＝blastp&PAGE_TYPE＝BlastSearch&LINK_LOC＝blasthome获得的Basic Local Alignment Search Tool，使用默认参数，进行比对)，鉴定在其它IL-2蛋白或多肽(包括全长序列或截短片段)上的对应氨基酸位置。因此，在本发明中，例如，当提及“F42”时，是指SEQ ID NO:1的第42位苯丙氨酸残基F，或经比对在其它IL-2序列上的对应位置的氨基酸残基。当提及多个位置的组合时，例如F42位、R81位和S87位，可以表示为F42/R81/S87。
在本文中，在提及IL-2突变蛋白时，按照以下方式描述突变。氨基酸取代表示为[原始氨基酸残基/位置/取代的氨基酸残基]。例如，位置92的异亮氨酸取代为亮氨酸，可以表示为I92L。当在一个给定位置(例如H79位)可以有多种可选氨基酸取代方式(例如D,E,Q)时，取代可以表示为：(1)H79D,E,Q；或(2)H79D/E/Q。相应地，对于在多个给定位置(例如R81，R83和S87)的组合突变，可以表示为：1)R81D/N,R83E,I92L/F/Y；或(2)R81D/S87D/I92L。
在本文中，可以通过在比较窗内比较两条最佳比对的序列来确定“序列同一性百分比”。优选地，在参考序列(例如SEQ ID NO:1)的全长上确定序列同一性。用于比较的序列比对方法是本领域内公知的。适用于确定序列同一性百分比的算法包括例如BLAST和BLAST 2.0算法(参见Altschul等，Nuc.Acids Res.25:3389-402,1977和Altschul等J.Mol.Biol.215:403-10,1990。可通过美国国家生物技术信息中心(National Center for Biotechnology Information)公众获取(http://www.ncbi.nlm.nih.gov/)用于进行BLAST分析的软件。出于本申请的目的，同一性百分比通常用设置为缺省参数的BLAST2.0算法来确定。
如本文中使用的，术语“保守取代”意指不会不利地影响或改变包含氨基酸序列的蛋白/多肽的生物学功能的氨基酸取代。例如，可通过本领域内已知的标准技术例如定点诱变和PCR介导的诱变引入保守取代。典型的保守型氨基酸取代是指将一种氨基酸取代为具有相似的化学性质(例如电荷或疏水性)的另一种氨基酸。以下六组各自包含彼此可进行典型保守取代的氨基酸：1)丙氨酸(A)、丝氨酸(S)、苏氨酸(T)；2)天冬氨酸(D)、谷氨酸(E)；3)天冬酰胺(N)、谷氨酰胺(Q)；4)精氨酸(R)、赖氨酸(K)；5)异亮氨酸(I)、亮氨酸(L)、甲硫氨酸(M)、缬氨酸(V)；和6)苯丙氨酸(F)、酪氨酸(Y)、色氨酸(W)。例如，野生型IL-2蛋白可以相对于SEQ ID NO:1具有保守氨基酸取代，或仅具有保守氨基酸取代。再例如，本发明的突变IL-2蛋白相对于野生型IL-2蛋白除了具有本发明的特征性突变外还可以具有保守氨基酸取代，或仅具有保守氨基酸取代。
“亲合力”或“结合亲合力”反映结合对子的成员之间相互作用的内在结合能力。分子X对其结合配偶体Y的亲合力可以由平衡解离常数(K D)表示，平衡解离常数是解离速率常数和结 合速率常数(分别是k dis和k on)的比值。结合亲合力可以由本领域已知的常见方法测量。用于测量亲合力的一个具体方法是本文中的生物膜层干涉(BLI)技术测定。此外，也可以通过本文中所述的流式细胞染色法来初步评估IL-2突变蛋白对不同受体的亲合力的变化。例如，可以将展示在酵母细胞上的野生型IL-2和突变IL-2蛋白，用生物素化的IL-2Rβ或IL-2Rα受体进行染色分析，来鉴定相比于野生型IL-2，与IL-2R受体IL-2Rβ或IL-2Rα的结合亲合力发生改变的IL-2突变蛋白。
在本文中，抗体结合分子是可以特异性结合抗原的多肽分子，例如，免疫球蛋白分子、抗体或抗体片段，例如Fab片段和scFv片段。
在本文中，抗体Fc片段是指含有至少一部分的恒定区的免疫球蛋白重链的C-端区域，并且可以包括天然序列Fc片段和变体Fc片段。在一个实施方案中，人IgG重链Fc片段从重链的Cys226或从Pro230延伸至羧基端。在另一实施方案中，Fc-片段的C-端赖氨酸(Lys447)可以存在或可以不存在。在另一些实施方案中，Fc片段可以包含突变，例如L234A/L235A突变。除非本文中另外指出，Fc片段中的氨基酸残基的编号根据EU编号系统，也称为EU索引，如Kabat，E.A.等，Sequences of Proteins of Immunological Interest，第5版，Public Health Service,National Institutes of Health，Bethesda，MD(1991)，NIH Publication 91-3242中所述。本发明的各方面将在下面各小节中进一步详述。
1.本发明的IL-2突变蛋白
本发明在一方面提供新IL-2突变蛋白，所述突变蛋白具有改善的IL-2受体选择性/偏好性，更为具体地，降低或消除的对IL-2Rα受体的结合亲合力和/或增强的对IL-2Rβ受体的结合亲合力。
本发明IL-2突变蛋白的有利生物学性质
IL-2蛋白通过与IL-2受体相互作用来引发信号传导和发挥功能。野生型IL-2对不同IL-2受体显示出不同的亲合力。在静息效应细胞(包括CD8 +细胞毒性T细胞和NK细胞)上表达与野生型IL-2具有较低亲合力的IL-2β和γ受体。在调节性T细胞(Treg)细胞和激活的效应细胞上表达与野生型IL-2具有高亲合力的IL-2Rα。由于高亲合力的原因，野生型IL-2会优先结合细胞表面的IL-2Rα，再招募IL-2Rβγ，通过IL-2Rβγ释放下游p-STAT5信号，刺激Treg细胞和激活的效应细胞。因此，不受理论的束缚，降低或消除IL-2对IL-2Rα受体的亲合力，将降低IL-2优先激活CD25 +细胞的偏向性，降低IL-2介导的Treg细胞的免疫下调作用。不受理论的束缚，维持或增强对IL-2β受体的亲合力将保留或增强IL-2对效应细胞如CD8 +细胞毒性T细胞和NK细胞的激活作用，因此达到免疫刺激作用。
本发明的IL-2突变蛋白通过在IL-2与IL-2Rα相互作用的区域(氨基酸残基35-72位)和/或在IL-2与IL-2Rβ相互作用的区域(氨基酸残基79-92位)引入一个或多个突变，尤其是三个以上突变，使得IL-2突变蛋白与IL-2Rα的结合降低或不结合，和/或与IL-2Rβ的结合保持不变或增强。由此，相对于野生型IL-2，本发明的IL-2突变蛋白具有改善的性质，包括例如以下的一项或多项：
(1)对IL-2Rα受体的结合亲合力降低或消除；
(2)对IL-2Rβ受体的结合亲合力增强；
(3)对高亲合力IL-2R受体(IL-2Rαβγ)的结合亲合力降低；
(4)对中等亲合力IL-2R受体(IL-2Rβγ)的结合亲合力增加；
(5)在CD25 +细胞(特别是激活的CD8 +T细胞和Treg细胞)中激活IL-2信号传导，尤其是激活STAT5磷酸化信号的能力降低；
(6)导致减少的由IL-2介导的CD25 +细胞(特别是激活的CD8 +T细胞和Treg细胞)激活和增殖；
(7)降低或消除IL-2优先刺激Treg细胞增殖的偏向性；
(8)降低Treg细胞在IL-2诱导下的免疫下调作用；
(9)保持或增强，尤其是增强，对CD25 -细胞(尤其是CD25 -T效应细胞和NK细胞)的激活作用；
(10)导致增加的由IL-2介导的效应T细胞与NK细胞激活和增殖；
(11)导致提高的免疫刺激作用；
(12)提高抗肿瘤效应。
在一些实施方案中，本发明IL-2突变蛋白具有上述(1)的性质，优选地进一步具有选自(3)和(5)-(8)的一项或多项，尤其是所有的性质；更优选地还进一步具有选自(2)和(9)-(12)的一项或多项，尤其是所有性质。在一些实施方案中，本发明IL-2突变蛋白具有上述(2)的性质，优选地进一步具有选自(9)-(12)的一项或多项，尤其是所有的性质；更优选地还进一步具有选自(1),(3)和(5)-(8)的一项或多项，尤其是所有的性质。
在一些优选地实施方案中，本发明IL-2突变蛋白相对于野生型IL-2还具有以下性质：减小的由IL-2与高亲合力受体IL-2αβγ结合介导的体内毒性。
在一些实施方案中，本发明IL-2突变蛋白具有改善的成药性质，例如，当在哺乳动物细胞例如H293T细胞中表达时，具有选自以下的一项或多项性质：(i)优于野生型IL-2蛋白的表达量；(ii)优于野生型IL-2蛋白的同质性；和(iii)易于纯化至更高的蛋白纯度。
在本发明的一些实施方案中，本发明IL-2突变蛋白与野生IL-2相比表现出表达水平的增加。在本发明的一些实施方案中，增加的表达发生在哺乳动物细胞表达系统中。表达水平可通过允许定量或半定量分析细胞培养上清液(优选一步亲和层析纯化后的上清液)中的重组IL-2蛋白量的任何合适方法来测定。在一些实施方案中，本发明IL-2突变蛋白，与野生型IL-2相比，在哺乳动物细胞中的表达量增加至少1.1倍，或至少1.5倍，或至少2倍、2.5倍、3倍、3.5倍或4倍以上。
在一些优选的实施方案中，一步蛋白A亲和层析纯化后，本发明IL-2突变蛋白产物的纯度可以达到70％，或80％，或90％以上。在一些实施方案中，蛋白纯度通过SEC-HPLC技术检测。
在一些实施方案中，相对于野生型IL-2(例如SEQ ID NO:1中所示IL-2 WT)，本发明IL-2突变蛋白对IL-2Rα受体的亲合力降低至少5倍、至少10倍、或至少25倍，尤其是至少30倍、50倍或100倍以上。在优选的实施方案中，本发明的突变蛋白不结合IL-2Rα受体。结合亲合力可以通过生物膜层干涉(BLI)技术测定本发明IL-2突变蛋白，例如与Fc片段融合的本发明IL-2突变蛋白，与受体IL-2Rα受体的平衡解离常数(K D)来确定。
在一些实施方案中，相对于野生型IL-2，本发明IL-2突变蛋白(例如SEQ ID NO:1中所 示IL-2 WT)对IL-2Rβ受体的亲合力增强至少5倍，至少10倍，或至少25倍，尤其是至少30倍、50倍或100倍，更优选地，至少150倍、200倍、250倍、300倍、350倍、400倍、450倍、或500倍或550倍以上。结合亲合力可以通过生物膜层干涉(BLI)技术测定本发明IL-2突变蛋白，例如与Fc片段融合的本发明IL-2突变蛋白，与受体IL-2Rβ受体的平衡解离常数(K D)来确定。在一个实施方案中，以IL-2-Fc融合蛋白形式，在BLI测定中，本发明IL-2突变蛋白与受体IL-2Rβ受体的结亲合力K D值小于10.0E-09M，例如小于6.0E-09M，3.0E-09M，2.0E-09M,1.0E-09M,更优选地小于9.0E-10M,例如小于6.0E-10M，5.0E-10M,4.0E-10M,3.0E-10M，2.0E-10M,1.0E-10M,更优选小于9.0E-11M,8.0E-11M,或7.0E-11M。
在一个实施方案中，相对于野生型IL-2，本发明IL-2突变蛋白导致减少的由IL-2介导的CD25 +细胞激活和增殖。在一个实施方案中，CD25 +细胞是CD25 +CD8 +T细胞。在另一实施方案中，CD25 +细胞是Treg细胞。在一个实施方案中，在STAT5磷酸化测定试验中，通过检测IL-2突变蛋白在CD25 +细胞中对STAT5磷酸化信号的激活，来鉴定IL-2突变蛋白激活CD25 +细胞的能力。例如，如本申请实施例中所述，可以通过流式细胞术分析细胞中的STAT5磷酸化，确定半最大有效浓度(EC 50)。在一个实施方案中，如在STAT5磷酸化测定试验中测定的，例如以FC融合蛋白形式，本发明IL-2突变蛋白相对于野生型IL-2蛋白(例如SEQ ID NO:1的人IL-2)，激活CD25 +细胞的能力降低了至少10倍，50倍，100倍，300倍，1000倍，3000倍或更高。
在一个实施方案中，相对于野生型IL-2，本发明IL-2突变蛋白导致保持的或增强的由IL-2介导的CD25 -效应细胞激活和增殖。在一个实施方案中，CD25 -细胞是CD8 +效应T细胞或NK细胞。在一个实施方案中，在STAT5磷酸化测定试验中，通过检测IL-2突变蛋白在CD25-细胞中激活STAT5磷酸化信号的EC 50值，来鉴定IL-2突变蛋白激活CD25 -细胞的能力。在一个实施方案中，如在STAT5磷酸化测定试验中测定的，本发明IL-2突变蛋白相对于野生型IL-2蛋白(例如SEQ ID NO:1的人IL-2)，激活CD25 +细胞的能力提高了至少1倍、例如2倍，3倍、4倍、5倍、6倍、7倍、8倍、9倍、或10倍，20倍、50倍，100倍，或150倍。
在一个实施方案中，相对于野生型IL-2，本发明IL-2突变蛋白去除或降低IL-2对CD25 +细胞优先激活的偏向性在一个实施方案中，CD25 +细胞是CD25 +CD8 +T细胞。在另一实施方案中，CD25 +细胞是Treg细胞。在一个实施方案中，在STAT5磷酸化测定试验中，通过检测IL-2突变蛋白分别在CD25 -细胞中和在CD25 +细胞中激活STAT5磷酸化信号的EC 50值，来鉴定IL-2突变蛋白激活CD25 -细胞的能力。例如，通过计算在CD25 -和CD25 +T细胞上激活STAT5磷酸化信号的EC 50值的比值，确定IL-2突变蛋白对CD25 +细胞的激活偏向性。优选地，相对于野生型蛋白，突变蛋白对CD25 +的偏向性降低了至少100倍，优选至少1000倍，2000倍，3000倍。
本发明的突变蛋白
IL-2蛋白属于具有四个α螺旋束(A,B,C,D)结构的短链I型细胞因子家族成员。根据晶体结构(PDB:1Z92)的分析，IL-2在氨基酸残基区域35-72具有如下与CD25相互作用的氨基酸位点：35；37；38；41；42；43；45；61；62；68；72。根据晶体结构(PDB:2ERJ)的分析，IL-2在氨基酸残基区域12-23和氨基酸区域79-92具有如下与CD122相互作用的位点：12；13；15；16；19；20；23；79；81；82；83；84；87；88；91；92。
本发明人发现，通过在IL-2的氨基酸残基区域35-72(以下简称“CD25结合区”)的CD25相互作用位点上(即，位点35；37；38；41；42；43；45；61；62；68；72)，引入特定突变，可以降低或消除IL-2与IL-2Rα的结合，同时保持或增强与IL-2Rβ的结合。本发明人也发现，通过在IL-2的氨基酸残基区域79-92(以下简称“CD122结合区”)的CD122相互作用位点上(即，位点79；81；82；83；84；87；88；91；92)，引入特定突变，可以增强IL-2与IL-2β的结合。此外，本发明人也发现，可以将两个区域的突变进行组合，以提供同时具有降低或消除的IL-2Rα结合和增强的IL-2β结合的IL-2突变体。
由此，本发明提供了IL-2突变蛋白，与野生型IL-2(优选人IL-2，更优选包含SEQ ID NO:1序列的IL-2)相比，所述突变蛋白包含至少一个突变，所述突变消除或降低对IL-2Rα受体的结合亲合力和/或增强对IL-2Rβ受体的结合亲合力。
CD25结合区突变
在一个方面，本发明的IL-2突变蛋白，相对于野生型IL-2，在IL-2与其IL-2Rα受体(CD25)相互作用的位点，优选地在与SEQ ID NO:1的35,37,38,41,42,43,45,61,62,68和72位置相应的位置上，包含一个或多个突变(优选至少3个)，所述突变消除或降低对IL-2Rα受体的结合亲合力，且优选地导致增强的IL-2Rβ结合亲合力。优选地，在上述这些位置上的突变为氨基酸取代。更优选地，在这些位置上的突变分别为选自如下的取代残基：35位：K35D,E；37位：T37D,E,R,K,F,Y,W；38位：R38D,E,F,Y,W,A,V；41位：T41K,R,M,F,Y,W,Q,E；42位：F42K,R,A,E,Q；43位：K43E,D,F,Y,W；45位：Y45R,K；61位：E61R,K,W,Y,L；62位：E62R,K,W,Y,L；68位：E68R,K,W,Y；72位：L72R,K,F,Y,W。更优选地，这些位置上的取代残基分别选自如下：K35D,E；T37E,D,K,W,Y；R38W,E,D,V,F,K；T41E,Y,R,K,Q；F42E,R,K,Q,A；K43E,Y,D,W；Y45K,R；E61K,R,L,W；E68R,Y,W,K；L72K,F。再优选地，这些位置上的取代残基分别选自如下：K35D,E；T37D,E；R38D,E,F；T41E；F42A,E,Q；K43E,D,Y；Y45R,K；E61R,K,W,Y,L；E62R,W；E68R,K,W,Y；L72K,F。最优选所述突变蛋白包含选自以下的一个或多个突变：K35D,E；T37E；R38F,E；T41E；F42A,E；Y45K；E68Y。在进一步实施方案中，在本发明IL-2突变蛋白中，降低或消除对IL-2Rα受体的结合亲合力的突变包括在选自与SEQ ID NO:1的以下位置相应的位置上的突变：
K35/T37/R38/T41/K43；
K35/T37/R38/T41/K43/L72；
K35/T37/R38/K43/Y45/L72；
K35/R38/T41/K43；
K35/R38/T41/K43/L72；
K35/R38/T41/K43/E61/L72；
K35/R38/T41/K43/Y45/L72；
K35/R38/T41/K43/Y45/E61/L72；
K35/R38/F42/Y45；
K35/R38/F42/Y45/E61/E68；
K35/R38/F42/E68/L72；
K35/R38/F42/K43/Y45/E68；
K35/R38/F42/K43/Y45/E61/E68；
K35/R38/F42/K43/Y45/E61/E68/L72；
K35/T37/R38/F42；
K35/T37/R38/F42/Y45/E61/E68；
K35/T37/R38/F42/K43/Y45/E61/E68；
K35/T37/R38/T41/F42/K43/Y45/E61/E68；
K35/T37/R38/F42/Y45/E61/E62/E68/L72。
K35/T37/R38/F42/Y45/E62/E68；
K35/T37/R38/F42/K43/E68；
K35/T37/F42/K43；
K35/T37/R38/T41/L72；
K35/T37/R38/T41/F42；
K35/T37/R38/T41/F42/K43/E68；
K35/T37/R38/T41/F42/K43/Y45；
K35/T37/R38/T41/F42/K43/Y45/L72；
T37/R38/T41/F42/K43/Y45；
K35/T37/T41/F42/K43/Y45；
K35/T37/T41/F42/K43/E61/E68/L72；
K35/T41/F42/K43/E68；
K35/T37/R38/T41/K43/Y45；
K35/T37/R38/T41/K43/Y45/L72；
K35/T37/R38/T41/K43/Y45/E61/L72；
K35/T37/R38/K43/L72；
K35/R38/K43/L72；
K35/T37/E61/L72；
K35/Y45/E61/E68；
K35/R38/T41/F42；
K35/R38/T41/F42/Y45；
K35/R38/T41/F42/Y45/E68；
K35/R38/T41/E68；
K35/R38/Y45/E68/L72；
T37/K43/E68/L72；
T37/K43/Y45/E68/L72；
T37/K43/Y45/E61/E68/L72；
T37/F42/Y45/E61/E68/L72；
T37/T41/Y45/E61/E68/L72；
R38/T41/F42/Y45/E61/E68；
T41/K43/Y45/E61/E68/L72。
在一些优选的实施方案中，降低或消除对IL-2Rα受体的结合亲合力的突变包括选自以下的突变组合：
K35D/T37K/R38E/T41K/F42Q/K43D/E68Y；K35D/T37D/T41F/F42E/K43D/Y45K；
K35D/T37K/R38D/T41E/K43E；K35D/R38E/T41E/K43E；K35D/R38F/F42E/Y45K；
K35E/R38D/T41E/K43E/L72F；K35D/T37E/R38D/K43E/L72F；
K35E/R38D/T41E/K43E/E61K/L72F；K35E/T37D/R38W/F42Q/Y45K/E61K/E68R；
T37E/K43E/Y45K/E68K/L72K；K35E/R38E/T41M/F42E/Y45K；K35D/T37E/R38D/T41E/K43E；
K35D/T37D/R38E/K43E/L72F；K35E/T37D/R38D/K43E/L72F；
K35D/R38D/T41E/K43E/Y45K/E61W/L72F；K35D/T37E/R38D/T41E/K43E/L72F；
K35D/R38W/F42E/E68R/L72F；K35D/T37E/R38D/K43Y/Y45K/L72F；
K35E/T37D/R38W/T41E/F42A/K43F/Y45K；K35D/T37E/R38D/T41R/F42Q；
K35E/R38D/T41E/K43D/L72F；K35E/T41Q/F42R/K43D/E68Y；
K35E/T37Y/R38W/T41Y/F42E/K43E/Y45K/L72K；K35D/T37E/R38D/K43Y/Y45R/L72F；
K35E/T37E/R38D/T41E/K43Y；T37E/T41K/Y45R/E61L/E68K/L72K；
K35E/R38W/F42Q/Y45R/E61W/E68R；K35D/T37E/R38E/T41E/L72F；
K35D/T37D/R38D/T41E/L72F；K35D/R38E/T41E/K43E/L72F；
K35E/T37D/R38D/T41E/K43Y/Y45K；K35D/T37D/F42A/K43E；
K35E/R38E/T41E/K43Y/Y45K/L72F；K35D/R38W/F42E/K43Y/Y45R/E61R/E68Y；
R38K/T41R/F42Q/Y45K/E61Y/E68W；K35E/R38W/F42Q/Y45K/E61L/E68R；
K35D/T37E/R38E/T41E/K43E/Y45K/L72F；K35E/R38D/K43E/L72F；K35E/R38E/K43E/L72F；
K35D/T37E/R38D/L72F；K35E/R38E/T41E/K43E/L72F；K35E/T37E/R38E/F42A；
K35D/R38F/T41E/F42E/Y45K/E68Y；K35E/T37K/R38E/T41E/K43E/L72F；
K35D/R38D/K43E/L72F；K35D/R38A/T41Q/F42R；K35D/T37E/R38F/F42E/K43E/E68R；
K35D/T37E/R38D/T41E/K43E/Y45K/E61W/L72F；T37D/R38F/T41F/F42E/K43E/Y45R；
K35D/R38W/F42E/K43E/Y45K/E68K；K35E/T37D/R38E/T41E/K43Y；K35E/R38W/F42E/Y45K；
K35E/T37D/R38W/F42E/Y45K/E68Y/L72R；K35E/T37D/R38W/F42Q/Y45R/E62R/E68R；
K35E/T37D/T41F/F42A/K43E/E61W/E68K/L72W；T41K/K43D/Y45R/E61L/E68K/L72K；
K35E/T37E/R38W/F42Q/Y45R/E61R/E68R；K35E/T37D/R38W/F42Q/Y45R/E61K/E68R；
K35E/T37D/R38W/F42Q/Y45K/E61W/E68R；K35E/R38W/F42Q/Y45K/E61K/E68R；
K35E/R38W/F42Q/Y45K/E61W/E68R；K35E/T37D/R38W/F42Q/Y45K/E61R/E68R；
K35E/R38D/Y45K/E68R/L72K；K35E/R38W/F42Q/Y45R/E61R/E68R；T37E/K43D/E68Y/L72R；
K35E/Y45R/E61L/E68W；K35E/T37D/R38W/F42Q/Y45R/E61R/E68R；
K35E/T37D/R38V/F42E/K43W/Y45R/E61L/E68W；K35E/T37A/R38W/F42Q/Y45K/E61R/E68R；
K35E/T37W/R38E/T41Y/F42R/K43D/Y45K/E61K/E68W；
K35D/R38W/F42K/K43Y/Y45R/E61R/E68W/L72K；K35E/T37E/E61W/L72K；
K35E/R38Y/T41E/E68Y；K35E/T37E/R38W/F42Q/Y45R/E61K/E68R；
T37D/K43D/Y45R/E61Y/E68K/L72R；K35D/R38W/F42E/K43Y/Y45R/E61R/E68Y/L72K；
T37E/F42R/Y45R/E61Y/E68K/L72K；K35E/T37D/R38V/F42A/Y45K/E61R/E62W/E68Y/L72R。
在一些优选实施方案中，降低或消除对IL-2Rα受体的结合亲合力的突变包括在选自以下的位置上的突变：K35/R38/F42/T37；K35/R38/F42/Y45/E61/E68；K35/R38/F42/Y45/E61/E68/T37。优选地，所述突变包括：K35E,R38E,F42A,T37E；或K35E,R38W,F42Q,Y45R/K,E61K/W/R,E68R，和任选地T37D/E。最优选地所述突变为选自以下的突变组合：
K35E/R38E/F42A/T37E；K35E/R38W/F42Q/Y45K/E61K/E68R；
K35E/R38W/F42Q/Y45K/E61W/E68R；K35E/R38W/F42Q/Y45R/E61R/E68R；
K35E/R38W/F42Q/Y45K/E61K/E68R/T37D；K35E/R38W/F42Q/Y45R/E61K/E68R/T37D；
K35E/R38W/F42Q/Y45K/E61W/E68R/T37D；K35E/R38W/F42Q/Y45K/E61R/E68R/T37D；
K35E/R38W/F42Q/Y45R/E61R/E68R/T37D；K35E/R38W/F42Q/Y45R/E61R/E68R/T37E；
K35E/R38W/F42Q/Y45R/E61K/E68R/T37E。优选地，包括这些突变组合的本发明IL-2蛋白相对于野生型IL-2具有提高的IL-2Rβ结合亲合力；更优选地，还具有降低的对CD25 +细胞的激活偏向性。
在一些优选实施方案中，降低或消除对IL-2Rα受体的结合亲合力的突变包括在选自以下的位置上的突变：K35/R38/F42/Y45/E61/E68/K43；K35/R38/F42/Y45/E61/E68/K43/L72；K35/R38/F42/Y45/E61/E68/K43/T37；K35/R38/F42/Y45/E61/E68/K43/T37/T41。优选地，所述突变包括：K35D/E,R38W/V/E,F42E/K/R,Y45R/K,E61R/L/K,E68Y/W,K43Y/W/D,任选地还包含T37D/W,T41Y；L72K中的一个或多个。更优选，所述突变为选自以下的突变组合：
K35D/R38W/F42E/Y45R/E61R/E68Y/K43Y；K35D/R38W/F42E/Y45R/E61R/E68Y/K43Y/L72K；
K35D/R38W/F42K/Y45R/E61R/E68W/K43Y/L72K；
K35E/R38V/F42E/Y45R/E61L/E68W/T37D/K43W；
K35E/R38E/F42R/Y45K/E61K/E68W/T37W/K43D/T41Y。更优选所述突变为：
K35D/R38W/F42E/Y45R/E61R/E68Y/K43Y/L72K。优选地，包括这些突变组合的本发明IL-2蛋白相对于野生型IL-2具有降低的或消除的IL-2Rα受体结合亲合力，且具有具有提高的IL-2Rβ结合亲合力。
在一些实施方案中，降低或消除对IL-2Rα受体的结合亲合力的突变包括在选自以下的位置上的突变：K35/R38/T41/K43；K35/R38/T41/K43/T37；K35/R38/T41/K43/L72；K35/R38/T41/K43/T37/L72；K35/R38/T41/K43/E61/L72；K35/R38/T41/K43/Y45/L72；K35/R38/K43/T37/L72；K35/R38/K43/T37/L72/Y45。优选地，所述突变包括：K35D/E,R38D/E,T41E,K43E/Y,任选地还包括选自T37K/E,Y45K,E61K,L72F中的一者或两者；或者优选地所述突变包括：K35D/E,R38D,K43E/Y,T37D/E,L72F,任选地还包括Y45K。更优选地所述突变为选自以下的突变组合：
K35D/R38E/T41E/K43E；K35D/T37K/R38D/T41E/K43E；K35E/R38E/T41E/K43E/L72F；
K35D/T37E/R38D/T41E/K43E/L72F；K35E/R38D/T41E/K43E/E61K/L72F；
K35E/R38E/T41E/K43Y/Y45K/L72F；K35D/T37E/R38D/K43E/L72F；
K35E/T37D/R38D/K43E/L72F；K35D/T37E/R38D/K43Y/Y45K/L72F；
更优选所述突变为选自以下的突变组合：
K35D/T37E/R38D/T41E/K43E/L72F；K35E/R38D/T41E/K43E/E61K/L72F；
K35E/R38E/T41E/K43Y/Y45K/L72F；K35D/T37E/R38D/K43E/L72F；
K35E/T37D/R38D/K43E/L72F；K35D/T37E/R38D/K43Y/Y45K/L72F；最优选地所述突变为：
K35D/R38E/T41E/K43E。优选地，包括这些突变组合的本发明IL-2突变蛋白相对于野生型IL-2具有降低的或消除的IL-2Rα受体结合亲合力，且具有提高的IL-2Rβ结合亲合力；更优选地，还具有降低的对CD25 +细胞的激活偏向性。
在一些优选实施方案中，降低或消除对IL-2Rα受体的结合亲合力的突变包括在选自以下的位置上的突变：K35/R38/F42/Y45/E61/E68；K35/R38/F42/Y45/E61/E68/T37；K35/R38/F42/T37；K35/R38/T41/K43。
在一些优选的实施方案中，降低或消除对IL-2Rα受体的结合亲合力的突变包括选自以下的突变组合：K35E/R38E/F42A/T37E；K35E/R38W/F42Q/Y45K/E61K/E68R；K35E/R38W/F42Q/Y45K/E61W/E68R；K35E/R38W/F42Q/Y45R/E61R/E68R；K35E/R38W/F42Q/Y45K/E61K/E68R/T37D；K35E/R38W/F42Q/Y45R/E61K/E68R/T37D；K35E/R38W/F42Q/Y45K/E61W/E68R/T37D；K35E/R38W/F42Q/Y45K/E61R/E68R/T37D；K35E/R38W/F42Q/Y45R/E61R/E68R/T37D；K35E/R38W/F42Q/Y45R/E61R/E68R/T37E；K35E/R38W/F42Q/Y45R/E61K/E68R/T37E；K35D/R38E/T41E/K43E。这些突变组合可以使本发明IL-2突变蛋白相对于野生型IL-2具有降低的或消除的IL-2Rα受体结合亲合力，且具有 提高的IL-2Rβ结合亲合力；并且导致降低的对CD25 +细胞的激活偏向性，同时保持或增强CD25 -效应细胞的激活和/或增殖。
再一些优选实施方案中，降低或消除对IL-2Rα受体的结合亲合力的突变包括选自以下的突变组合：K35D/T37E/R38E/T41E/K43E/Y45K/L72F；K35E/R38D/T41E/K43E/L72F；K35E/R38W/F42Q/Y45K/E61L/E68R；K35D/T37K/R38E/T41K/F42Q/K43D/E68Y；K35D/T37E/R38D/K43Y/Y45K/L72F；K35E/T37Y/R38W/T41Y/F42E/K43E/Y45K/L72K；K35D/R38W/F42E/K43E/Y45K/E68K；K35E/T41Q/F42R/K43D/E68Y；K35D/R38W/F42E/Y45R/E61R/E68Y/K43Y；K35D/R38F/T41E/F42E/Y45K/E68Y；K35E/T37K/R38E/T41E/K43E/L72F；R38K/T41R/F42Q/Y45K/E61Y/E68W。有利地，这些突变组合可以与本发明79-92区域中增加IL-2Rβ结合的突变组合，以提供与IL-2Rα不结合同时与IL-2Rβ结合增强的IL-2突变蛋白。
CD122结合区突变
在再一方面，本发明的IL-2突变蛋白可以在IL-2与其β受体CD122(即IL-2Rβ)相互作用的位点，优选地在与SEQ ID NO:1的79,81,82,83,84,87,88,91,92位置相应的位置上，包含一个或多个突变，所述突变增强对IL-2Rβ的结合亲合力。优选地，在上述这些位置上的突变为氨基酸取代。更优选地，在这些位置上的突变分别为选自如下的取代残基：H79R,K,Y,W,D,E,Q；R81D,E,N,Q,T,H,Y,W；P82I,T,A；R83E；D84E,N,Q,H,T,V；S87T,D,N,E,Q,K,R,Y,W；N88D,E,Q,H,Y,W；V91T,L,I,M,D,N,E,Q,H；I92V,L,M,F,Y,W,N,D,E,Q。
在一个优选实施方案中，本发明IL-2突变蛋白在与SEQ ID NO:1的79,81,82,83,87,91,92位置相应的位置上包含一个或多个突变,优选地氨基酸取代，尤其是包含选自以下的一个或多个取代：H79D,E,Q；R81D,N；P82I,T,A；R83E；S87D,E；V91L,I；I92L,M,F,Y。在一个实施方案中，本发明突变蛋白，在与SEQ ID NO:1的12,13,15,16,19,20,23位置相应的位置上相对于野生型IL-2蛋白保持不变。
在一些优选实施方案中，所述增强对IL-2Rβ的结合亲合力的突变包括在选自以下的位置上的突变：H79/R81/S87/I92，H79/R81/S87/I92/P82。优选地，所述突变包括H79D/E,R81D,S87D/E,I92L/F,任选还包括P82T。更优选所述突变包括选自以下的组合突变：H79D/R81D/S87D/I92L，和H79D/R81D/P82T/S87D/I92L。优选地，所述突变组合可以使本发明IL-2突变蛋白相对于野生型IL-2具有显著提高的IL-2Rβ结合亲合力，且更优选地导致增强的CD25 -效应细胞激活和/或增殖。
在一些优选实施方案中，所述增强对IL-2Rβ的结合亲合力的突变包括在选自以下的位置上的突变：R81/R83/S87；R81/R83/S87/I92；R81/P82/R83/S87；或R81/P82/R83/S87/I92；优选还包括在H79和V91中的一个或两个位置上的突变。优选地，所述突变包括选自以下的组合突变：R81D,P82T/I/A,R83E,S87E/D；R81D/N,P82T/I/A,R83E,S87E/D,I92/L/M/F/Y；R81D,R83E,S87E/D；R81D,R83E,S87E/D,I92L，更优选还包括H79D/E/Q和V91L/I中的一个或两个。更优选地，所述突变为选自以下的突变组合：H79D/R81D/S87D/I92L；
H79D/R81D/P82T/S87D/I92L；R81D/P82T/R83E/S87E；R81D/P82T/R83E/S87E/V91L；
R81D/P82A/R83E/S87E/V91L；H79E/R81D/P82T/R83E/S87E/V91L；
H79E/R81D/P82I/R83E/S87D；H79Q/R81D/P82A/R83E/S87E；
R81N/P82T/R83E/S87E/V91L/I92F；H79D/R81N/P82T/R83E/S87E/V91L/I92F；
R81D/P82T/R83E/S87D/I92M；H79E/R81D/P82I/R83E/S87E/I92M；
H79Q/R81D/P82T/R83E/S87D/V91L/I92Y；R81D/P82I/R83E/S87D/V91I/I92Y；
R81D/P82T/R83E/S87D/I92L；R81D/P82T/R83E/S87D/V91L/I92L；
R81D/P82A/R83E/S87E/I92L；H79E/R81N/P82A/R83E/S87D/I92L；
R81D/P82T/R83E/S87E/I92M；R81D/P82A/R83E/S87E/I92M；
H79E/R81D/P82A/R83E/S87E/I92M；R81D/R83E/S87D/I92L；R81D/R83E/S87E/I92L；
H79D/R81D/R83E/S87D；H79D/R81D/R83E/S87E/V91I/I92L。优选地，所述突变组合可以使本发明IL-2突变蛋白相对于野生型IL-2具有显著提高的IL-2Rβ结合亲合力，且更优选地导致增强的CD25 -效应细胞激活和/或增殖。
在一些实施方案中，增强对IL-2Rβ的结合亲合力的突变为选自下的组合突变：
H79D/R81D/S87D/I92L；H79E/R81D/P82T/R83E/S87E/V91L；H79E/R81D/P82I/R83E/S87D；
H79Q/R81D/P82A/R83E/S87E；R81D/P82T/R83E/S87D/I92L；
R81D/P82T/R83E/S87D/V91L/I92L；R81D/P82A/R83E/S87E/I92L；
H79E/R81N/P82A/R83E/S87D/I92L；R81D/P82T/R83E/S87E/I92M；
R81D/P82A/R83E/S87E/I92M；H79E/R81D/P82A/R83E/S87E/I92M；
H79D/R81D/R83E/S87E/V91I/I92L。有利地，这些突变组合可以与本发明35-72位置区域中降低IL-2Rα结合亲合力的突变组合，以提供与IL-2Rα不结合同时与IL-2Rβ结合增强的IL-2突变蛋白。
CD25结合区突变和CD122结合区突变的组合
在再一方面，本发明提供包含CD25结合区突变和CD122结合区突变的IL-2突变蛋白，优选地所述突变蛋白具有降低的(或消除的)IL-2Rα结合且具有增强的IL-2Rα结合，更优选地还具有改善的成药性能，例如更高的表达量和产品纯度。
在一些优选方案中，本发明提供包含选自如下突变组合的IL-2突变蛋白。
Figure PCTCN2019107055-appb-000001
在一些优选实施方案中，本发明提供IL-2突变蛋白，其中所述突变蛋白与野生型IL-2相比具有降低的刺激CD25 +T细胞中的信号传导的能力，优选地所述突变蛋白相对于野生型IL-2蛋白(例如SEQ ID NO:1的人IL-2蛋白)包含(或者仅具有)选自以下的突变组合：
K35E/R38E/F42A/T37E；
K35E/R38W/F42Q/Y45K/E61K/E68R；
K35E/R38W/F42Q/Y45K/E61W/E68R；
K35E/R38W/F42Q/Y45R/E61R/E68R；
K35E/R38W/F42Q/Y45K/E61K/E68R/T37D；
K35E/R38W/F42Q/Y45R/E61K/E68R/T37D；
K35E/R38W/F42Q/Y45K/E61W/E68R/T37D；
K35E/R38W/F42Q/Y45K/E61R/E68R/T37D；
K35E/R38W/F42Q/Y45R/E61R/E68R/T37D；
K35E/R38W/F42Q/Y45R/E61R/E68R/T37E；
K35E/R38W/F42Q/Y45R/E61K/E68R/T37E。
K35D/R38E/T41E/K43E。
在一些优选实施方案中，本发明提供IL-2突变蛋白，其中所述突变蛋白与野生型IL-2相比具有降低的刺激CD25 +T细胞中的信号传导的能力，且具有增强的刺激CD25 -T细胞中信号传导的能力，优选地所述突变蛋白相对于野生型IL-2蛋白(例如SEQ ID NO:1的人IL-2蛋白)包含(或者仅具有)选自以下的突变组合：
K35D/R38W/F42E/K43E/Y45K/E68K/R81D/P82T/R83E/S87E/I92M；
K35D/R38F/T41E/F42E/Y45K/E68Y/R81D/P82T/R83E/S87D/V91L/I92L。
其它突变
除了上述区域和位置中的突变，本发明的IL-2突变蛋白还可以在其它区域或位置上具有一个或多个突变，只要其保留本发明IL-2突变蛋白的上述一个或多个有益性质即可。例如，本发明IL-2突变蛋白还可以包含在位置125的取代，例如C125S.C125A,C125T,或C125V，以提供额外的优点，例如改善的表达或同质性或稳定性(参见例如，美国专利号4,518,584)。本领域技术人员知晓如何确定可以并入本发明IL-2突变蛋白中的额外突变。
IL-2突变蛋白与野生型蛋白之间的序列差异性可以用序列同一性表述，也可以用两者之间差异氨基酸的数量来表达。在一个实施方案中，IL-2突变蛋白与野生型蛋白之间具有至少85％，86％,87％,88％,89％同一性，优选90％以上同一性，优选95％，但优选不超过97％，更优选不超过96％同一性。在另一实施方案中，除了本发明的上述CD25结合区突变或CD122结合区突变或两者的组合突变外，IL-2突变蛋白与野生型蛋白之间还可以具有不超过15个，例如1-10个，或1-5个突变。在一个实施方案中，所述其它突变可以是保守取代。在一个实施方案中，所述其它突变可以是赋予IL-2其它改善性质的突变。
2.融合蛋白和免疫缀合物
本发明还提供包含本发明IL-2突变蛋白的融合蛋白。在一个优选的实施方案中，本发明IL-突变蛋白与可以赋予改善的药代动力学性质的另一多肽融合，例如清蛋白，更优选抗体Fc片段。优选地，Fc片段包含减小或去除效应子功能的突变，例如降低与Fcγ受体结合的L234A/L235A突变或L234A/L235E/G237A。优选地，包含Fc的融合蛋白具有增加的血清半 衰期。在一个优选的实施方案中，包含Fc的融合蛋白同时还具有减少的由Fc区介导的效应子功能，例如ADCC或ADCP或CDC。
本发明还提供免疫缀合物，其包含本发明的IL2突变蛋白和抗原结合分子。优选地，抗原结合分子是免疫球蛋白分子，特别是IgG分子，或抗体或抗体片段，特别是Fab分子和scFv分子。在一些实施方案中，所述抗原结合分子特异性结合肿瘤细胞上或肿瘤环境中呈现的抗原，例如选自以下的抗原：成纤维细胞活化蛋白(FAP)、生腱蛋白C的A1域(TNC A1)、生腱蛋白C的A2域(TNC A2)、纤连蛋白的外域B(Extra Domain B,EDB)、癌胚抗原(CEA)、和黑素瘤有关的硫酸软骨素蛋白聚糖(MCSP)。由此，本发明免疫缀合物在施用于受试者体内后可以靶向肿瘤细胞或肿瘤环境，从而提供进一步的治疗益处，例如以更低的剂量进行治疗的可行性和由此带来的低副作用；增强的抗肿瘤效应等。
在本发明的融合蛋白和免疫缀合物中，本发明IL-2突变蛋白可以直接或通过接头与另一分子或抗原结合分子连接，且在一些实施方案中，在两者之间包含蛋白水解切割位点。
3.多核苷酸、载体和宿主
本发明提供编码以上任何IL-2突变蛋白或融合物或缀合物的核酸。可以采用本领域熟知的方法，通过从头固相DNA合成或通过PCR诱变编码野生型IL-2的现有序列，产生编码本发明突变蛋白的多核苷酸序列。此外，本发明的多核苷酸和核酸可以包含编码分泌信号肽的区段，并与编码本发明突变蛋白的区段可操作连接，从而可以指导本发明突变蛋白的分泌性表达。
本发明也提供包含本发明核酸的载体。在一个实施方案中，载体是表达载体，例如真核表达载体。载体包括但不限于病毒、质粒、粘粒、λ噬菌体或酵母人工染色体(YAC)。在优选的实施方案中，本发明的表达载体是pYDO_017表达载体(SEQ ID NO:13)。
本发明也提供包含所述核酸或所述载体的宿主细胞。适用于复制并支持突变IL-2蛋白或融合物或免疫缀合物表达的宿主细胞是本领域中公知的。可以用特定的表达载体转染或转导这类细胞，并且可以生长大量的含载体细胞以用于接种大规模发酵罐，从而获得充足量的IL-2突变体或融合物或免疫缀合物用于临床应用。在一个实施方案中，宿主细胞是真核的。在另一个实施方案中，宿主细胞选自酵母细胞、哺乳动物细胞(例如CHO细胞或293细胞)。例如，可以在细菌中生成多肽，尤其在不需要糖基化时。在表达后，可以在可溶性级分中将多肽从细菌细胞糊分离并可以进一步纯化。除了原核生物外，真核微生物如丝状真菌或酵母也是适合编码多肽的载体的克隆或表达宿主，其中包括糖基化途径已被“人源化”的真菌和酵母菌株，这导致生成具有部分或完全的人糖基化模式的多肽。参见Gerngross,NatBiotech22,1409-1414(2004)和Li等,NatBiotech24,210-215(2006)。可用的哺乳动物宿主细胞系的例子是由SV40转化的猴肾CV1系(COS-7)；人胚胎肾系(293或293T细胞，如例如记载于Graham等,JGenVirol36,59(1977))、幼仑鼠肾细胞(BHK)、小鼠塞托利(sertoli)细胞(TM4细胞，如例如记载于Mather,BiolReprod23,243-251(1980))、猴肾细胞(CV1)、非洲绿猴肾细胞(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(HepG2)、小鼠乳房肿瘤细胞(MMT060562)、TRI细胞(如例如记载于Mather等,AnnalsN.Y.AcadSci383,44-68(1982))、MRC5细胞和FS4细胞。其它可用的哺乳动物宿主细胞系包括中国仓鼠卵巢(CHO)细胞，包括dhfr-CHO细胞(Urlaub 等,ProcNatlAcadSciUSA77,4216(1980))；和骨髓瘤细胞系如YO、NS0、P3X63和Sp2/0。在一个实施方案中，宿主细胞是真核生物细胞，优选为哺乳动物细胞如中国仓鼠卵巢(CHO)细胞、人胚胎肾(HEK)细胞或淋巴细胞(例如Y0、NS0、Sp20细胞)。
4.制备方法
再一方面，本发明提供制备本发明IL-2突变蛋白或融合物或缀合物的方法，其中所述方法包括，在适合IL-2突变蛋白或融合物或缀合物表达的条件下，培养包含编码所述蛋白或融合物或缀合物的核酸的宿主细胞，如上文所提供的，和任选地从所述宿主细胞(或宿主细胞培养基)回收所述蛋白或融合物或缀合物。
5.测定法
可以通过本领域中已知的多种测定法对本文中提供IL-2突变蛋白进行鉴定、筛选，或表征其物理/化学特性和/或生物学活性。
一方面，可以对本发明的IL-2突变蛋白，测试其与IL-2受体的结合活性。例如，可以通过本领域已知的方法，诸如ELISA，Western印迹等，或本文实施例公开的例示性方法，来测定与人IL-2Rα或β蛋白的结合。例如，可以使用流式细胞术进行测定，其中使经转染在细胞表面上表达突变蛋白的细胞例如酵母展示细胞，与标记的(例如生物素标记的)IL-2Rα或β蛋白进行反应。备选地，突变蛋白与受体的结合，包括结合动力学(例如K D值)，可以使用重组突变蛋白-Fc融合物，在生物膜层干涉(BLI)测定法中测定。在一些实施方案中，使用如实施例所描述的BLI测定法。
再一方面，可以通过测定在受体结合下游发生的信号传导和/或免疫激活效应。来间接测量IL-2突变蛋白结合IL-2受体的能力。
因此，在一些实施方案中，提供了用于鉴定具有生物学活性的突变IL-2蛋白的测定法。生物学活性可以包括，例如诱导具有IL-2受体的T和/或NK细胞增殖的能力、诱导具有IL-2受体的T和/或NK细胞中IL-2信号传导的能力、通过NK细胞生成干扰素(IFN)-γ作为次级细胞因子的能力、降低的诱导T细胞中细胞凋亡的能力、诱导肿瘤消退和/或改善存活的能力、和降低的体内毒性性质，例如降低的血管通透性。本发明也提供体内和/或体外具有这类生物学活性的突变IL-2蛋白。
本领域中公知多种方法可以用于测定IL-2的生物学活性。例如，用于测试本发明IL-2突变蛋白刺激NK细胞生成IFN-γ的能力的合适测定法，可以包括如下步骤：将培养的NK细胞与本发明的突变IL-2蛋白或融合或免疫缀合物温育，并随后通过ELISA测量培养基中的IFN-γ浓度。IL-2信号传导诱导数个信号传导途径，并且牵涉JAK(Janus激酶)和STAT(信号转导物和转录的激活剂)信号传导分子。
IL-2与受体β和γ亚基的相互作用导致受体以及JAK1和JAK3(分别与β和γ亚基结合)的磷酸化。然后，STAT5与磷酸化受体结合，并自身在非常重要的酪氨酸残基上磷酸化。这导致STAT5从受体解离、STAT5二聚化以及STAT5二聚体移位至细胞核，在该处它们促进靶基因的转录。由此，可以例如通过测量STAT5的磷酸化，评估突变体IL-2多肽经由IL-2受体诱导信号传导的能力。此方法的详情已经披露在实施例中。例如，可以将PBMC用本发明的突变体IL-2多肽或融合物或免疫缀合物处理，并通过流式细胞术测定磷酸化STAT5的 水平。
此外，可以通过将从血液分离的T细胞或NK细胞与本发明的突变体IL-2多肽或免疫缀合物温育来，接着测定经处理细胞的裂解物中的ATP含量，以测量T细胞或NK细胞应答IL-2的增殖。在处理前，可以用植物凝集素(PHA-M)预刺激T细胞。此测定法允许对存活细胞数目的灵敏定量，本领域中还已知大量合适的备选测定法(例如[3H]-胸苷掺入测定法、细胞滴定GloATP测定法、AlamarBlue测定法、WST-1测定法、MTT测定法)。
再有，可以在多种本领域中已知的动物肿瘤模型中评估突变的IL-2对肿瘤生长和存活的影响。例如，可以将人癌症细胞系的异种移植物植入免疫缺陷型小鼠，并用本发明的突变体IL-2多肽或融合物或免疫缀合物处理。可以基于死亡率、生命期观察(不良作用的可见症状，例如行为、体重、体温)以及临床和解剖病理学(例如测量血液化学值和/或组织病理学分析)来测定本发明的突变体IL-2多肽、融合和免疫缀合物在体内的毒性。例如，可以在预处理血管通透性动物模型中用血管渗漏报告分子检查通过用IL-2处理所诱导的血管通透性。优选地，血管渗漏报告分子足够大以揭示用于预处理的IL-2野生型形式的通透性。
6.筛选方法
再一方面，本发明提供一种降低消除或降低IL-2蛋白对IL-2Rα受体的结合亲合力和/或增强对IL-2Rβ受体的结合亲合力的方法，包括在野生型IL-2蛋白中引入本文中描述的突变或突变组合，和鉴定(例如使用前述的测定方法)相对于野生型IL-2蛋白对ILRα或β具有改变的结合亲合力、和/或改善的生物活性，例如前文中针对本发明IL-2突变蛋白描述的性质之一或多个的突变蛋白。在一些实施方案中，用作突变模板的亲本野生型IL-2蛋白优选与SEQ ID NO:1具有至少80％，或至少95％或99％或更高的同一性，更优选地为来源人的IL-2蛋白。
7.药物组合物和药物制剂
本发明还包括包含IL-2突变蛋白或其融合物或免疫缀合物的组合物(包括药物组合物或药物制剂)和包含编码IL-2突变蛋白或其融合物或免疫缀合物的多核苷酸的组合物。这些组合物还可以任选地包含合适的药用辅料，如本领域中已知的药用载体、药用赋形剂，包括缓冲剂。
适用于本发明的药用载体可以是无菌液体，如水和油，包括那些具有石油、动物、植物或合成起源的，如花生油、大豆油、矿物油、芝麻油等。当静脉内施用药物组合物时，水是优选的载体。还可以将盐水溶液和水性右旋糖以及甘油溶液用作液体载体，特别是用于可注射溶液。合适的药用赋形剂包括淀粉、葡萄糖、乳糖、蔗糖、明胶、麦芽、米、面粉、白垩、硅胶、硬脂酸钠、甘油单硬脂酸酯、滑石、氯化钠、干燥的脱脂乳、甘油、丙烯、二醇、水、乙醇等。对于赋形剂的使用及其用途，亦参见“Handbook of Pharmaceutical Excipients”,第五版,R.C.Rowe,P.J.Seskey和S.C.Owen,PharmaceuticalPress,London,Chicago。若期望的话，所述组合物还可以含有少量的润湿剂或乳化剂，或pH缓冲剂。这些组合物可以采用溶液、悬浮液、乳剂、片剂、丸剂、胶囊剂、粉末、持续释放配制剂等的形式。口服配制剂可以包含标准载体，如药用级甘露醇、乳糖、淀粉、硬脂酸镁、糖精。
可以通过将具有所需纯度的本发明的IL-2突变蛋白、融合物或免疫缀合物，与一种或多种任选的药用辅料(Remington′s Pharmaceutical Sciences，第16版，Osol，A.编(1980))混合， 来制备包含本发明的药物制剂，优选地以冻干制剂或水溶液的形式。示例性的冻干抗体制剂描述于美国专利号6,267,958。水性抗体制剂包括美国专利号6,171,586和WO2006/044908中所述的那些，后一种制剂包括组氨酸-乙酸盐缓冲剂。此外，可制备持续释放制剂。持续释放制剂的合适实例包括含有蛋白的固体疏水聚合物的半渗透基质，所述基质呈成形物品，例如薄膜或微囊形式。
本发明的药物组合物或制剂还可以包含一种或多种其它活性成分，所述活性成分是被治疗的特定适应症所需的，优选具有不会不利地影响彼此的互补活性的那些活性成分。例如，理想的是还提供其它抗癌活性成分，例如化疗剂、PD-1轴结合拮抗剂(例如抗PD-1抗体或抗PD-L1抗体或抗PD-L2抗体)。所述活性成分以对于目的用途有效的量合适地组合存在。
因此，在一个实施方案中，组合物还包含第二治疗剂。例如，第二治疗剂可以是免疫检查点抑制剂。例如第二治疗剂可以选自包括但不限于：例如抗-CTLA-4抗体、抗CD47抗体、抗-PD-1抗体、抗-PD-L1抗体、抗-CD40抗体、抗-OX40(亦称为CD134,TNFRSF4,ACT35和/或TXGP1L)抗体、抗-LAG-3抗体、抗-CD73抗体、抗-CD137抗体、抗-CD27抗体、抗-CSF-1R抗体、TLR激动剂或IDO或TGFβ的小分子拮抗剂的一种或多种。优选地，第二治疗剂是PD-1拮抗剂，尤其是抗PD-1抗体，抗PD-L1抗体、抗LAG-3，抗CD47。除了免疫治疗药物，第二治疗剂也可以是其它放疗或化疗药物。
8.组合产品
在一方面，本发明还提供了组合产品，其包含本发明的突变蛋白或其融合物或免疫缀合物，以及一种或多种其它治疗剂(例如化疗剂、其他抗体、细胞毒性剂、疫苗、抗感染活性剂等)。本发明的组合产品可用于本发明的治疗方法中。
在一些实施方案中，本发明提供组合产品，其中所述其它治疗剂为例如有效刺激免疫反应从而进一步增强、刺激或上调受试者的免疫反应的治疗剂如抗体。在一些实施方案中，其它抗体为例如抗PD-1抗体或抗PD-L1抗体或抗PD-L2抗体或抗-LAG-3抗体或抗-CTLA-4抗体或抗TIM-3抗体。
在一些实施方案中，所述组合产品用于预防或治疗肿瘤。在一些实施方案中，肿瘤为癌症，例如胃肠道癌症，例如胃癌、直肠癌、结肠癌、结肠直肠癌等；或皮肤癌，例如黑素瘤；或肾细胞癌、膀胱癌、非小细胞肺癌等。在一些实施方案中，所述组合产品用于预防或治疗感染，例如细菌感染、病毒感染、真菌感染、原生动物感染等。
9.治疗方法和用途
在本文中，术语“个体”或“受试者”可互换地使用，是指哺乳动物。哺乳动物包括但不限于驯化动物(例如，奶牛、绵羊、猫、犬和马)、灵长类(例如，人和非人灵长类如猴)、兔和啮齿类(例如，小鼠和大鼠)。特别地，受试者是人。
在本文中，术语“治疗”指意欲改变正在接受治疗的个体中疾病之天然过程的临床介入。想要的治疗效果包括但不限于防止疾病出现或复发、减轻症状、减小疾病的任何直接或间接病理学后果、防止转移、降低病情进展速率、改善或缓和疾病状态，以及缓解或改善预后。
在一方面中，本发明提供刺激受试者免疫系统的方法，所述方法包括向所述受试者施用 有效量的包含本发明IL-2突变蛋白或融合物或免疫缀合物的药物组合物。本发明IL-2突变蛋白对于CD25 -CD122 +效应细胞(细胞毒性CD8 +T细胞和NK细胞)具有高活性和选择性，并具有降低的和去除的对CD25 +Treg细胞的刺激作用。因此，可以以低剂量使用本发明IL-2突变蛋白，以刺激受试者的免疫系统。
因此，在一些实施方案中，本发明涉及在受试者中增强机体的免疫应答的方法，所述方法包括向所述受试者施用有效量的本文所述的任何IL-2突变蛋白、或其融合物或免疫缀合物。在一些实施方案，将本发明的IL-2突变蛋白或其融合物或免疫缀合物施用于携带肿瘤的受试者，刺激抗肿瘤免疫应答。在另一些实施方案中，将本发明的抗体或其抗原结合部分施用于携带感染的受试者，刺激抗感染免疫应答。在一个实施方案中本发明IL-2突变蛋白可以与Treg耗竭抗体(例如，FcγR介导的Treg耗竭)组合使用，以进一步降低由Treg引起的免疫抑制作用。在一个实施方案中，本发明IL-2突变蛋白可以与免疫检查点抑制剂组合施用，以例如提高癌症免疫治疗效果，例如与抗PD-1和抗CTLA-4组合。
在另一方面中，本发明涉及治疗受试者疾病，如肿瘤和癌症和感染的方法，所述方法包括向所述受试者施用有效量的本文所述的任何IL-2突变蛋白、或其融合物或免疫缀合物。
癌症可以处于早期、中期或晚期或是转移性癌。在一些实施方案中，肿瘤或肿瘤细胞可以选自结直肠肿瘤、卵巢肿瘤、胰腺肿瘤、肺肿瘤、肺肿瘤，肝肿瘤，乳房肿瘤，肾肿瘤，前列腺肿瘤，胃肠肿瘤，黑素瘤，宫颈肿瘤，膀胱肿瘤，成胶质细胞瘤和头颈部肿瘤。在一些实施方案中，癌症可以选自结直肠癌，卵巢癌，胰腺癌，肺癌，肝癌，乳腺癌，肾癌，前列腺癌，胃肠癌，黑素瘤，宫颈癌，膀胱癌，成胶质细胞瘤和头颈癌。在一些实施方案中，肿瘤是黑色素瘤、肾细胞癌、结直肠癌、膀胱癌、非小细胞肺癌。
在另一方面中，本发明涉及治疗受试者感染性疾病，例如慢性感染的方法，所述方法包括向所述受试者施用有效量的本文所述的任何IL-2突变蛋白或其片段，或包含所述抗体或片段的免疫缀合物、多特异性抗体，或药物组合物。在一个实施方案中，所述感染是病毒感染。
在一些实施方案中，除了本发明IL-2突变蛋白或其融合物或缀合物外，本发明的方法还包括向所述受试者联合施用一种或多种疗法(例如治疗方式和/或其它治疗剂)。在一些实施方案中，治疗方式包括手术治疗和/或放射疗法。在一些实施方案中，本发明方法还包括施用至少一种其它的免疫刺激性抗体，例如抗PD-1抗体、抗PD-L1抗体、抗-LAG-3抗体、抗CD43抗体、和/或抗CTLA-4抗体，这些抗体可以是例如全人源的、嵌合的、或人源化的抗体。
在一些实施方案中，抗PD-1抗体选自下组：IBI308(信迪利单抗，WO2017/025016A1)，MDX-1106(nivolumab，OPDIVO)，Merck 3475(MK-3475，pembrolizumab，KEYTRUDA)和CT-011(Pidilizumab)。在一些实施方案中，抗PD-1抗体是MDX-1106。在一些实施方案中，抗PD-1抗体是nivolumab(CAS注册号：946414-94-4)。在进一步的一些实施方案中，单独或与PD-1拮抗剂组合的IL-2突变蛋白或其片段还能与一种或多种其它疗法例如治疗方式和/或其它治疗剂组合施用。在一些实施方案中，治疗方式包括外科手术(例如肿瘤切除术)；放射疗法(例如，外粒子束疗法，它涉及其中设计照射区域的三维适形放射疗法)、局部照射(例如，指向预选靶或器官的照射)或聚焦照射等。
在一些实施方案中，本文提供了治疗疾病(例如，肿瘤)的方法，包括给予受试者本文所述的突变蛋白和CTLA-4拮抗剂抗体。抗-CTLA-4抗体可以是例如选自以下的抗体:
Figure PCTCN2019107055-appb-000002
(ipilimumab或抗体10D1，描述于PCT公开号WO 01/14424),tremelimumab(旧称ticilimumab,CP-675,206)，和描述于以下公开文献中的抗-CTLA-4抗体：WO 98/42752；WO 00/37504；美国专利号6,207,156；Hurwitz等(1998)Proc.Natl.Acad.Sci.USA 95(17):10067-10071；Camacho等(2004)J.Clin.Oncology 22(145):Abstract No.2505(抗体CP-675206)；和Mokyr等(1998)Cancer Res.58:5301-5304。
在一些实施方案中，本文提供了治疗疾病(例如，肿瘤)的方法，包括给予受试者本文所述的抗-突变蛋白和抗-LAG-3拮抗剂抗体。抗-LAG3抗体可以是例如选自以下的抗体：描述于美国专利申请号US2011/0150892和WO2014/008218的抗体25F7,26H10,25E3,8B7,11F2或17E5，或包含这些抗体的CDR或可变区的抗体；BMS-986016；描述于US 2011/007023中的IMP731。
在一些实施方案中，本发明的IL-2突变蛋白可以与化疗或化疗剂联合施用。在一些实施方案中，本发明的IL-2突变蛋白可以与放疗或放疗剂联合施用。在一些实施方案中，本发明的IL-2突变蛋白可以与靶向疗法或靶向治疗剂联合施用。在一些实施方案中，本发明的IL-2突变蛋白可以与免疫疗法或免疫治疗剂，例如单克隆抗体联合施用。
本发明的突变蛋白(以及包含其的药物组合物或其融合物或免疫缀合物，和任选地另外的治疗剂)可以通过任何合适的方法给药，包括肠胃外给药，肺内给药和鼻内给药，并且，如果局部治疗需要，病灶内给药。肠胃外输注包括肌内、静脉内、动脉内、腹膜内或皮下给药。在一定程度上根据用药是短期或长期性而定，可通过任何适合途径，例如通过注射，例如静脉内或皮下注射用药。本文中涵盖各种用药时程，包括，但不限于，单次给药或在多个时间点多次给药、推注给药及脉冲输注。
为了预防或治疗疾病，本发明突变蛋白的合适剂量(当单独或与一种或多种其他的治疗剂组合使用时)将取决于待治疗疾病的类型、抗体的类型、疾病的严重性和进程、以预防目的施用还是以治疗目的施用、以前的治疗、患者的临床病史和对所述抗体的应答，和主治医师的判断力。所述抗体以一次治疗或经过一系列治疗合适地施用于患者。
再一方面，本发明也提供本发明IL-2突变蛋白、组合物、免疫缀合物、融合物在制备用于前述方法(例如用于治疗)的药物中的用途。
描述以下实施例以辅助对本发明的理解。不意在且不应当以任何方式将实施例解释成限制本发明的保护范围。
实施例
实施例1：白细胞介素2突变文库设计与构建
白细胞介素2突变文库设计
根据白细胞介素2(Interleukin-2，简称IL-2)与其α受体CD25(简称IL-2Rα)复合物的晶体结构(PDB:1Z92)(如图1所示)，列出相互作用位点的IL-2残基按表1突变。各位点原始氨基酸占50％比例，其余50％比例由表1中“突变氨基酸”均分。针对IL-2与IL-2Rα的相互结合位点设计的文库理论多样性为3×8×8×9×6×6×3×6×6×5×6≈2.0×10 8，文库命名为IBYDL029(Innoventbio Yeast Display Library)。
表1.IBYDL029文库突变位点表
位点 氨基酸残基 突变氨基酸 多样性
35 Lys(K) D,E 3
37 Thr(T) D,E,R,K,F,Y,W 8
38 Arg(R) D,E,F,Y,W,A,V 8
41 Thr(T) K,R,M,F,Y,W,Q,E 9
42 Phe(F) K,R,A,E,Q 6
43 Lys(K) E,D,F,Y,W 6
45 Tyr(Y) R,K 3
61 Glu(E) R,K,W,Y,L 6
62 Glu(E) R,K,W,Y,L 6
68 Glu(E) R,K,W,Y 5
72 Leu(L) R,K,F,Y,W 6
根据IL-2与其β受体CD122(简称IL-2Rβ)复合物的晶体结构(PDB:2ERJ)(如图2所示)，列出相互作用位点的IL-2残基按表2突变。各位点原始氨基酸占50％比例，其余50％比例由表2中“突变氨基酸”均分。由于IL-2与IL-2Rβ的相互结合位点较多，文库的理论多样性太大，将文库拆分成两个小的文库，位点在12-23的突变文库为IBYDL030，理论多样性为12×8×9×12×13×7×12≈1.1×10 7；位点在79-92的突变文库为IBYDL031，理论多样性为8×9×4×2×7×10×7×10×11≈3.1×10 7。
表2.IBYDL030-IBYDL031文库突变位点表
位点 氨基酸残基 突变氨基酸 多样性
12 Leu(L) V,T,D,N,E,Q,R,K,F,Y,W 12
13 Gln(Q) D,E,V,L,F,N,T 8
15 Glu(E) D,N,Q,H,W,K,R,Y 9
16 His(H) D,N,Q,H,W,Y,F,T,I,R,K 12
19 Leu(L) V,I,M,T,D,N,E,Q,Y,H,R,K 13
20 Asp(D) E,N,Q,T,V,L 7
23 Met(M) V,L,D,N,E,Q,F,Y,W,R,K 12
79 His(H) R,K,Y,W,D,E,Q 8
81 Arg(R) D,E,N,Q,T,H,Y,W 9
82 Pro(P) I,T,A 4
83 Arg(R) E 2
84 Asp(D) E,N,Q,H,T,V 7
87 Ser(S) T,D,N,E,Q,K,R,Y,W 10
88 Asn(N) D,E,Q,H,Y,W 7
91 Val(V) T,L,I,M,D,N,E,Q,H 10
92 Ile(I) V,L,M,F,Y,W,N,D,E,Q 11
白细胞介素2突变文库构建
将野生型IL-2(uniprot:P60568,aa21-153,C125S，简称IL-2 WT)放入酵母展示质粒pYDC011的两个BamHⅠ酶切位点之间。IL-2 WT的序列在本申请中显示于SEQ ID NO:1中，在该序列的125位引入了C125S突变，以避免二硫化物桥接的IL-2二聚体形成。质粒构建的具体步骤如下：
1.用引物AMP0210与AMP0211以IL-2 WT基因为模板扩增；
2.质粒pYDC011用BamHⅠ(New England Biolab，货号：R3136V)酶切后胶回收(QIAGEN Gel Extraction Kit,Cat.28704)；
3.扩增产物与酶切产物经1％琼脂糖凝胶回收；
4.回收后，用One Step Cloning Kit(Vazyme货号：C113-02)进行体外同源重组；
5.重组后产物转入大肠杆菌Top10感受态细胞(天根，货号：CB104-02)，涂布于含氨苄抗性的LB平板，37℃培养过夜；
6.生长的单克隆菌落经测序验证后，正确的质粒命名为pYDC035。
根据已有文献，IL-2突变体IL-2 3X与IL-2Rα不结合，与IL-2Rβ结合力维持不变(Rodrigo Vazquez-Lombardi等，Nature Communications,8:15373,DOI:10.1038/ncomms15373)；IL-2突变体IL-2 H9与IL-2Rβ结合增强，与结合IL-2Rα维持不变(Aron M.Levin等,Nature,Vol 484,p529-533,DOI:10.1038/nature10975)。将IL-2 3X与IL-2 H9展示于酵母表面，作为对照使用。IL-2 3X和IL-2 H9的序列分别示于SEQ ID NO:2和3中，两个蛋白与IL-2 WT相同也包含C125S突变。
根据表1与表2的文库构建方案，设计所需引物(如图3所示)并由苏州金唯智生物科技有限公司合成。
IBYDL029文库DNA扩增：1.以pYDC035为模板，引物AMP0191,AMP0200扩增片段029-F；2.以pYDC035为模板，引物AMP0201,AMP0199扩增片段029-R；3.胶回收片段029-F、029-R作为PCR扩增模板，用引物AMP0191与AMP0199扩增全长片段029。
IBYDL030文库DNA扩增：1.以pYDC035为模板，引物AMP0191,AMP0224扩增片段030-F；2.以pYDC035为模板，引物AMP0222,AMP0199扩增片段030-R；3.胶回收片段030-F、030-R作为PCR扩增模板，用引物AMP0191与AMP0199扩增全长片段030。
IBYDL031文库DNA扩增：1.以pYDC035为模板，引物AMP0191,AMP0225扩增片段031-F；2.以pYDC035为模板，引物AMP0223,AMP0199扩增片段031-R；3.胶回收片段031-F、031-R作为PCR扩增模板，用引物AMP0191与AMP0199扩增全长片段031。
取100μg质粒pYDC011用BamHⅠ酶切，酶切后用PCR产物回收试剂盒(QIAGEN PCR  Purification Kit,Cat.28104)回收，获得足量的线性化质粒。将线性化质粒与文库DNA按4μg:12μg混合，按照现有的方法(Lorenzo Benatuil等人，An improved yeast transformation method for the generation of very large human antibody libraries.Protein Engineering,Design&Selection vol.23no.4pp.155–159,2010)将各个文库与线性化质粒的混合物电转入EBY100酵母菌株。电转后文库梯度稀释涂布于SD-Trp(TAKARA，货号：630309)的平板，统计生长的菌落数，获得文库的实际多样性为IBYDL029:4.2×10 8,IBYDL030:4.5×10 8,IBYDL031:3.8×10 8，均大于文库的理论多样性。
实施例2：IL-2 WT-FC、IL-2 3X-FC、IL-2Rα、IL-2Rβ蛋白制备与生物素标记
表达质粒的构建
将IL-2 WT,IL-2 3X基因序列放入载体pYDO_017两个BamHⅠ酶切位点之间，用于表达IL-2 WT-FC与IL-2 3X-FC融合蛋白，本发明使用的Fc是指人IgG1的Fc(L234A,L235A，简称FcLALA)。将IL-2Rα(Uiprot:P01589,aa22-217)与IL-2Rβ(Uiprot:P14784,aa27-240)在序列的C末端接上avi标签与6个组氨酸标签(序列示于SEQ ID NO:11和12)，分别构建到pTT5载体上，用于表达IL-2Rα与IL-2Rβ蛋白。
蛋白的表达及纯化
使用化学转染的方法将以上构建的表达质粒载体转入HEK293-F(Invitrogen，货号：R79007)细胞中。使用化学转染试剂聚乙烯亚胺(简称PEI,Polysciences，货号：23966)，按厂商提供的方案瞬时转染培养的HEK293-F细胞。取终体积1/10(v/v)的Opti-MEM培养基(Gibco货号：31985-070)作为转染缓冲液，加入质粒，混匀，用0.22μm的滤头过滤备用。加聚乙烯亚胺(PEI)(Polysciences，23966)到上一步的质粒中(质粒与PEI的质量比例在293F细胞中为1:3)，混匀后室温孵育10min，获得DNA/PEI混合物。将DNA/PEI混合物轻柔倒入HEK293细胞并混匀，在37℃，8％CO 2的条件下培养24h后，补加VPA(Sigma，货号：P4543-100G)使终浓度至2mM及2％(v/v)Feed(1g/L Phytone Peptone+1g/L Difco Select Phytone)，继续培养6天。
表达IL-2 WT-FC与IL-2 3X-FC融合蛋白的细胞培养液，以13000rpm离心20min，收集上清，用预装柱Hitrap Mabselect Sure(GE,11-0034-95)纯化上清液。操作如下：纯化前用5倍柱体积的平衡液(0.2M Tris,1.5M NaCl,pH7.2)平衡填料柱；将收集的上清通过柱子，再用10倍柱体积的平衡液清洗填料柱，去除非特异性结合蛋白；用5倍柱体积的洗脱缓冲液(1M sodium citrate，pH 3.5)冲洗填料，收集洗脱液。每1ml洗脱液加入80μL Tris(2M Tris)，使用超滤浓缩管(上海拓开生物科技有限公司，MCPM02C67)交换到PBS缓冲液(Gibco，70011-044)中，并测定浓度。取100μg纯化后蛋白，调整浓度至1mg/mL，使用凝胶过滤色谱柱(TOSOH货号：18675)测定蛋白纯度。
表达IL-2Rα与IL-2Rβ蛋白的细胞培养液，以13000rpm离心20min，收集上清，用0.22μm的滤器过滤；提前将镍柱(5mL Histrap excel,GE,17-3712-06)用0.1M NaOH浸泡2h，然后用10倍柱体积的超纯水冲洗，去除碱液，再用5倍柱体积的结合缓冲液(20mM Tris,300mM NaCl,pH7.4)平衡纯化柱；将细胞上清通过柱子，再用10倍柱体积的冲洗缓冲液(20mM Tris,300mM NaCl,10mM imidazole,pH7.4)清洗柱子，去除非特异性结合的杂蛋白；最后用5倍柱体积洗脱液(20mM Tris,300mM NaCl,100mM imidazole,pH7.4)将目的蛋白洗脱下来，用蛋白超滤浓缩交换到PBS，纯化测定蛋白表达量与纯度，见表3。
表3.蛋白的表达量及纯度
蛋白名称 表达量(mg/L) 纯度(SEC-HPLC)
IL-2 WT-FC 18 45％
IL-2 3X-FC 34 70％
IL-2Rα 6 65％
IL-2RβIH(in house) 6 72％
IL-2Rα与IL-2RβIH蛋白的生物素化标记
利用酶催化方法使IL-2Rα与IL-2Rβ蛋白标记生物素，方法如下：取适量的IL-2Rα与IL-2RβIH蛋白溶液，加入1/10(m/m)质量的His-BirA蛋白(uniprot:P06709),同时加入终浓度为2mM ATP(sigma货号：A2383-10G),5mM MgCl 2,0.5mM D-生物素(AVIDITY货号：K0717)；30℃孵育1h，通过Superdex200increase(GE,10/300GL,10245605)纯化，去除多余的生物素和His-BirA；纯化后的样品通过Fortebio的Streptavidin(SA)传感器(PALL,18-5019)验证，确认生物素标记成功。本实施例获得的生物素标记的IL-2Rα、IL-2RβIH蛋白分别简称IL-2Rα-Biotin和IL-2RβIH-Biotin。
实施例3：IL-2突变文库筛选获得IL-2 mutant及染色鉴定
筛选与IL-2Rβ亲和力高的IL-2 mutant
基于酵母的IL-2 mutant展示文库IBYDL029、IBYDL030、IBYDL031，均取2.0×10 9的酵母细胞进行培养与诱导，其中文库的多样性分别为2.0×10 8、1.1×10 7、3.1×10 7。由于IBYDL029文库多样性较大，第一轮筛选使用Miltenyi公司的MACS系统进行磁珠细胞分选。首先，将2×10 9酵母细胞在FACS洗涤缓冲液中(1×PBS，含有1％牛血清蛋白)室温孵育30分钟，缓冲液中含有500nM生物素标记的商购IL-2Rβ(Acro Biosystems，经EZ-Link Sulfo-NHS-LC-Biotin生物素标记，简称IL-2Rβ-Biotin)。使用50ml预冷的FACS洗涤缓冲液洗一次，再用10ml相同洗涤缓冲液重悬细胞，并加入40μl链霉亲和素微珠(Miltenyi biotec，货号：130-090-485)于4℃孵育15分钟。3000rpm离心3min弃去上清后用10ml FACS洗涤缓冲液重悬细胞，将细胞溶液加到Miltenyi LS柱中。加样完成后，用FACS洗涤缓冲液洗柱3次，每次3ml。从磁性区域取下Miltenyi LS柱，用5ml生长培养基洗脱，收集洗脱的酵母细胞并在30℃过夜生长。IBYDL030、IBYDL031文库的多样性较小，可以直接使用流式细胞仪进行第一轮的分选：各取1×10 8、2.5×10 8的酵母细胞用FACS缓冲液洗三次，于含有100nM IL-2Rβ-Biotin、Anti Flag抗体(Sigma货号：F18041，1:1000稀释)的FACS缓冲液中室温孵育30分钟；细胞用FACS洗涤缓冲液洗两次之后，将细胞与含有SA-PE(链霉亲和素-PE,Thermo Fisher货号：S21388，1:200稀释)、山羊抗小鼠偶联Alex Flour-647(Thermo Fisher货号：A21235，1:200稀释)的FACS洗涤缓冲液混合，4℃下避光孵育15分钟。用预冷的FACS洗涤缓冲液洗涤两次，并重悬于1mL缓冲液中，将细胞转移到带滤器的分离管中。使用FACS MoFlo_XDP(Beckman)分选细胞，分选后的酵母细胞在30℃过夜生长。
将经过一轮筛选获得的各文库细胞在20℃下震荡诱导24小时以展示IL-2 mutant，使用流式细胞仪进行第二轮分选。各文库取3×10 7酵母细胞用FACS缓冲液洗三次，加入含有不同浓度IL-2Rβ-Biotin(029:300nM,030/031:100nM)、Anti Flag抗体的FACS缓冲液中，室温孵育30分钟；细胞用FACS洗涤缓冲液洗两次之后，将细胞与含有SA-PE、山羊抗小鼠偶联 Alex Flour-647的FACS洗涤缓冲液混合，4℃下避光孵育15分钟；用预冷的FACS洗涤缓冲液洗涤两次，并重悬于1mL缓冲液中，将细胞转移到带滤器的分离管中。使用MoFlo_XDP分选细胞，分选后的酵母细胞并在30℃过夜生长。第三轮分选方案同第二轮，经过三轮筛选后，挑取单克隆送测序。
使用IL-2Rβ-Biotin经三轮筛选，IBYDL029获得53个突变序列，IBYDL030未获得突变序列，IBYDL031获得71个突变序列。
基于酵母的IL-2突变体展示文库IBYDL029、IBYDL030、IBYDL031，使用实施例2获得的自制IL-2RβIH-Biotin进行第二批次筛选。第一轮筛选使用MACS系统进行磁珠细胞分选。首先，各文库取2×10 9酵母细胞在含有不同浓度IL-2RβIH-Biotin(IBYDL029:500nM,IBYDL030/031:200nM)的FACS洗涤缓冲液中室温孵育30分钟；后续步骤同第一批次的磁珠分选，经磁珠分选后获得的酵母细胞在30℃过夜生长。使用流式细胞仪进行第二、三轮分选，两轮筛选IL-2RβIH-Biotin浓度均选用IBYDL 029:500nM,IBYDL 030/031:100nM，其余步骤同第一批次的二、三轮筛选。
使用IL-2RβIH-Biotin经三轮筛选，挑取单克隆送测序，IBYDL029新增25个突变序列，IBYDL030同样未获得突变序列，IBYDL031新增41个突变序列。
IL-2 mutant染色鉴定
将测序后含单一突变序列的酵母细胞，在20℃下震荡诱导24小时以展示IL-2 mutant；分别与其受体IL-2Rα-Biotin、IL-2RβIH-Biotin染色，具体步骤如下：
一、IL-2 mutant展示的酵母细胞与IL-2Rα-Biotin染色分析：
1.每个样品取1×10 6细胞，离心弃上清用FACS缓冲液洗一次后待用；
2.加入100μL含50nM IL-2Rα-Biotin与Anti Flag抗体的FACS缓冲液中室温孵育30分钟；
3.用预冷的FACS缓冲液3000rpm 4℃离心3min洗涤两次；
4.加入100μL含SA-PE、山羊抗小鼠偶联Alex Flour-647的FACS缓冲液，冰上避光孵育20min；
5.用预冷的FACS缓冲液洗涤两次后，用100μL缓冲液重悬细胞，用流式分析仪(BD,ACCURI C6)分析IL-2 mutant与IL-2Rα的结合水平。
二、IL-2 mutant展示的酵母细胞与IL-2RβIH-Biotin染色分析：
1.每个样品取1×10 6细胞，离心弃上清用FACS缓冲液洗一次后待用；
2.加入100μL含IL-2RβIH-Biotin(30nM-100nM)与Anti Flag抗体的FACS缓冲液中室温孵育30分钟；
3.同上步骤3-5，分析IL-2 mutant与IL-2Rβ的结合水平。
由流式染色的结果可知：IBYDL029第一批筛选获得的53个IL-2 mutant(具体序列见图4A)与IL-2Rα结合的平均荧光信号强度同IL-2 3X接近，均不结合；与IL-2Rβ结合的平均荧光信号强度强于IL-2 3X但弱于IL-2 H9，IL-2Rβ结合信号最高的12个突变列于下表3-1。第二批筛选获得的25个IL-2 mutant(具体序列见图4B)与IL-2Rα不结合；相对于IL-2 3X，与IL-2Rβ结合有不同程度的提高，IL-2Rβ结合信号最高的14个突变列于下表3-1。IBYDL031两批次筛选 获得的112个IL-2 mutant与IL-2Rα、IL-2Rβ均结合，与IL-2Rβ结合信号较高的25个突变(具体序列见图4C)列于下表3-1，此25个突变与IL-2Rβ结合的平均荧光信号强度显著强于IL-2 3X且相当于或稍弱于IL-2 H9。
表3-1：染色鉴定的具有高IL-2Rβ结合信号的IL-2 mutant
Figure PCTCN2019107055-appb-000003
此外，将来源于IBYDL029的突变与来源于IBYDL031的突变组合(具体序列见图4D)，以获得与IL-2Rα不结合同时与IL-2Rβ结合增强的新突变体，如表3-2。
表3-2.IBYDL029、IBYDL031来源的突变组合
克隆编号 IBYDL029 mutant IBYDL031 mutant
Y01 Y30C2 Y27C1
Y02 Y29A4 Y27C2
Y03 Y30C1 Y27C5
Y04 Y27A3 Y27D2
Y05 Y29D6 Y27D4
Y06 Y29F4 Y27E1
Y07 Y30G4 Y27F6
Y08 Y29F3 Y28A2
Y09 Y30B4 Y28A5
Y10 Y30E3 Y28F1
Y11 Y30E6 Y28F5
Y12 Y30B6 Y32D5
实施例4：IL-2 mutant-FC融合的表达及与受体的亲合力(avidity)测定
表达质粒的构建
将IL-2 mutant序列放入载体pYDO_017两个BamHⅠ酶切位点之间，用于表达IL-2 mutant-FC融合蛋白。将含IL-2 mutant的基因的酵母展示质粒等比例混合作为模板，使用两端引物扩增片段，扩增后使用1％的琼脂糖凝胶回收DNA片段；pYDO_017BamHⅠ酶切载体与回收的片段同源重组，重组后产物转化大肠杆菌感受态细胞，经测序验证获得的IL-2 mutant-FC的表达质粒。
IL-2 mutant与FC融合蛋白的表达及纯化
使用化学转染的方法将含有编码融合蛋白基因的载体转入HEK293细胞中。使用化学转染试剂PEI，按厂商提供的方案瞬时转染培养的HEK293细胞。首先在超净工作台中准备质粒DNA和转染试剂，取3mL Opti-MEM培养基(Gibco货号：31985-070)加入50ml离心管中，加入30μg对应质粒的DNA，利用0.22μm的滤头过滤含有质粒的Opti-MEM培养基，随后加入90μg PEI(1g/L)，静置20min。将DNA/PEI混合物轻柔倒入27mL HEK293细胞并混匀，在37℃，8％CO 2的条件下培养20h后，补加VPA使终浓度至2mM及2％(v/v)Feed，继续培养6天。
细胞培养后，以13000rpm离心20min，收集上清，用预装柱Hitrap Mabselect Sure纯化上清液。操作如下：纯化前用5倍柱体积的平衡液(0.2M Tris,1.5M NaCl,pH7.2)平衡填料柱；将收集的上清通过柱子，再用10倍柱体积的平衡液清洗填料柱，去除非特异性结合蛋白；用5倍柱体积的洗脱缓冲液(1M sodium citrate，pH 3.5)冲洗填料，收集洗脱液；每1ml洗脱液加入80μL Tris(2M Tris)，使用超滤浓缩管交换到PBS缓冲液中，并测定浓度。取100μg纯化后蛋白，调整浓度至1mg/mL，使用凝胶过滤色谱柱测定蛋白纯度。47个本发明IL-2 mutant的表达和纯化结果如表4所示。
表4.IL-2mutant-FC蛋白的表达水平与纯度
Figure PCTCN2019107055-appb-000004
Figure PCTCN2019107055-appb-000005
IL-2 mutant-FC与其受体的亲合力测定
采用生物膜层干涉(BLI)技术测定了本发明34个IL-2 mutant-FC与其受体的平衡解离常数(K D)。
BLI法亲和力测定按照现有的方法(Estep,P等人，High throughput solution Based measurement of antibody-antigen affinity and epitope binning.MAbs,2013.5(2):p.270-8)进行。传感器在分析缓冲液中预湿20分钟后，按照建立的方法，用Octet Red96测量候选IL-2 mutant-FC分别与IL-2Rα，IL-2Rβ的亲合力：首先平衡基线120秒；然后将IL-2Rα-Biotin或IL-2Rβ IH-Biotin固化至SA传感器(PALL,18-5019)；将已固化IL-2Rα-Biotin或IL-2Rβ IH-Biotin的传感器置于含100nM IL-2 mutant-FC的溶液中直至平台期(100秒)，之后将传感器转移至分析缓冲液解离至少2分钟，分别测定结合及解离。实验结果使用1:1结合模型进行动力学的分析。
在如以上测定法所述进行的实验中，36个由HEK293-F表达的IL-2 mutant-FC与其受体的亲合力K D值如表5所示。
表5.IL-2mutant-FC与其受体的亲合力K D值
Figure PCTCN2019107055-appb-000006
Figure PCTCN2019107055-appb-000007
由以上表结果可见：(1)经文库筛选获得的各IL-2 mutant-FC与IL-2Rβ的亲合力相比于IL-2 WT与IL-2 3X，明显增强；(2)除Y27D2、Y27F6、Y28A2、Y28A5外，各IL-2 mutant-FC与IL-2Rα的亲合力相比于IL-2 WT明显降低或不结合，与IL-2 3X接近。综合表4、5结果，选取Y29A2、Y29B2、Y30E1、Y07、Y10、Y33A4、Y33A5、Y33A6、Y33B1、Y33B4、Y33B5、Y33C5、Y33F4、Y34F4进行体外功能实验。
实施例5：IL-2 mutant-FC体外功能实验
IL-2 WT与IL-2Rα亲和力高于IL-2Rβ,IL-2Rγ，会优先结合细胞表面的IL-2Rα，再招募IL-2Rβγ，通过IL-2Rβγ释放下游p-STAT5信号，刺激T细胞与NK细胞增殖。由于Treg细胞表面有IL-2Rα，效应T细胞与NK细胞表面没有IL-2Rα，正常情况下IL-2 WT会优先刺激Treg细胞增殖，下调免疫反应。IL-2 mutant与IL-2Rα不结合，消除了优先刺激Treg细胞增殖的偏好，同时刺激T细胞与NK细胞增殖，使得效应T细胞与NK细胞的数量有效增加，提高抗肿瘤效应。
本实施例通过检测各IL-2 mutant-FC对原代人CD8 +T细胞p-STAT5信号的激活，验证各突变体对CD25 +细胞激活偏向性的去除，并筛选对CD25 -细胞激活作用较强的突变体。具体步骤如下：
1.复苏PBMC细胞：
a)从液氮取出PBMC细胞(Allcells货号：PB005F，100M装)，迅速置于37℃水浴锅中，复苏PBMC细胞；
b)将细胞加入10mL已预热的、含5％人AB血清(GemCell货号：100-512)与1‰DNA酶(STRMCELL货号：07900)的X-VIVO15(Lonza货号：04-418Q)培养基中，400G、25℃离心10分钟(后续离心均为此条件)洗涤一次；
c)加入20mL培养基重悬细胞，37℃二氧化碳培养箱静置培养过夜。
2.纯化人CD8 +T细胞：
a)吸取步骤1中悬浮细胞液，离心弃上清；
b)加入1mL Robosep缓冲液(STEMCELL货号：20104)与100μL人AB血清及100μL人CD8 +T细胞纯化试剂盒(Invitrogen货号：11348D)中负性筛选抗体混合液重悬细胞；
c)混匀后，4℃孵育20分钟，每5分钟摇晃一次；
d)孵育后，加入10mL Robosep缓冲液，离心洗涤两次；
e)同时，取1mL磁性微球(人CD8 +T细胞纯化试剂盒)，加入7mL Robosep缓冲液置于磁力架上1分钟弃上清，预洗磁性微球；
f)各加入1mL Robosep缓冲液分别重悬微球与细胞，混匀后室温旋转孵育30分钟；
g)孵育后，加入6mL Robosep缓冲液，置于磁力架上1分钟，收集上清；
h)收集液再次置于磁力架上1分钟，收集上清；
i)离心弃上清，使用预热的T培养基重悬，调整密度至1×10 6/mL；
j)取1/3细胞待后需刺激CD25表达，其余细胞置于37℃二氧化碳培养箱静置过夜培养。
3.刺激CD8 +T细胞表达CD25：
a)取1/3步骤2中纯化后的CD8 +T细胞，加入抗人CD3/CD28抗体的磁性微球(GIBCO货号：11131D)，细胞与微球的比例为3:1；
b)置于37℃二氧化碳培养箱静置三天；
c)加入10mL培养基清洗2次；
d)加入培养基调整细胞密度至1×10 6/mL，37℃二氧化碳培养箱中静置培养2天。
4.检测细胞纯度与表达水平：
a)使用抗人CD8-PE(Invitrogen货号：12-0086-42)、抗人CD25-PE(eBioscience货号：12-0259-42)、同型对照抗体(BD货号：556653)检测细胞的CD8与CD25；
b)步骤2中细胞为CD8 +CD25 -T细胞，步骤3中细胞为CD8 +CD25 +T细胞。
5.检测各IL-2 mutant-FC对CD8 +CD25 -T细胞激活p-STAT5信号的EC 50：
a)取CD8 +CD25 -T细胞，以每孔1×10 5细胞铺96孔U底培养板(Costar货号：CLS3799-50EA)；
b)加入100μL各IL-2 mutant-FC、商品化IL-2(R&D货号：202-IL-500)、IL-2 WT-FC、IL-2 3X-FC，最高浓度从266.7nM开始4倍梯度稀释共12个梯度，37℃培养箱中孵育20分钟；
c)加入取55.5μL 4.2％甲醛溶液，室温固定10分钟；
d)离心弃上清，加入200μL冰甲醇(Fisher货号：A452-4)重悬细胞，4℃冰箱孵育30分钟；
e)离心弃上清，用200μL染色缓冲液(BD货号：554657)洗涤3次；
f)加入200μL含抗p-STAT5-AlexFlour647(BD货号：562076，1:200稀释)的破膜/固定缓冲液(BD货号：51-2091KZ)，室温避光孵育3小时；
g)使用染色缓冲液洗涤三次，100μL染色缓冲液重悬细胞，进行流式细胞仪检测；
h)以抗体浓度为横坐标、AlexFlour647中间荧光度值为纵坐标，制作p-STAT5信号的EC 50值及曲线，结果如图5.A,C。
6.检测各IL-2 mutant-FC对CD8 +CD25 +T细胞激活p-STAT5信号的EC 50：
a)取CD8 +CD25 +T细胞，以每孔1×10 5细胞铺96孔U底培养板；
b)同步骤5中b)-h)，制作p-STAT5信号的EC 50值及曲线，结果如图5.B,D。
根据实验结果，比较IL-2 mutant-FC对CD8 +CD25 +T细胞的激活效应与CD8 +CD25 -T细胞的激活效应的比值(如表6.1,6.2)可知：1)本发明的各个IL-2 mutant-FC对CD8 +CD25 -T细胞的激活效应好于IL-2 3X-FC，与IL-2 WT-FC比较接近，其中Y07及Y10效果甚至强于商品化的IL-2；2)当刺激CD25表达后，本发明的各个IL-2 mutant-FC对CD8 +CD25 +T细胞的激活效应比CD25 -时有小幅提高，但相比于商品化IL-2、IL-2 WT-FC的巨大幅度有显著差别。总而言之，根据CD25 +EC 50/CD25 -EC 50倍数显而易见，本发明的各个IL-2的突变体有效消除了对CD25 +细胞激活偏向性，且Y07、Y10激活CD25 -细胞的效应好于其余突变。
表6.1 IL-2 mutant-FC对CD25 +/-T细胞激活p-STAT5信号的EC 50及其比值(donor1)
Figure PCTCN2019107055-appb-000008
表6.2 IL-2 mutant-FC对CD25 +/-T细胞激活p-STAT5信号的EC 50及其比值(donor2)
Figure PCTCN2019107055-appb-000009
序列表
IL-2 WT(SEQ ID NO:1)，具有突变(C125S)
Figure PCTCN2019107055-appb-000010
IL-2 3X(SEQ ID NO:2)，具有突变(C125S,R38D,K43E,E61R)
Figure PCTCN2019107055-appb-000011
IL-2 H9(SEQ ID NO:3)，具有突变(C125S,L80F,R81D,L85V,I86V,I92F)
Figure PCTCN2019107055-appb-000012
酵母展示质粒pYDC011(SEQ ID NO:4)
Figure PCTCN2019107055-appb-000013
Figure PCTCN2019107055-appb-000014
引物序列：
AMP0191:cccggatcggactactagcagc(SEQ ID NO:5)
AMP0199:CTCCTTGCATTGCCTTCCCGTTG(SEQ ID NO:6)
AMP0210:GTTATTGCTTCAGTTTTAGCAGCTCCCACCAGCAGCAGCACC(SEQ ID NO:7)AMP0211:CATCGTCATCCTTGTAATCgGAtCCaCCgCCtCCGGTCAGTGTGCTGATGATGC(SEQ ID NO:8)
AMP0224:CTGGGTCTTCTTGGTGCTGC(SEQ ID NO:9)
AMP0225:GAAGTTCTTGCTCTGGGCTAAATTG(SEQ ID NO:10)
IL-2受体序列：
IL-2Rα受体(SEQ ID NO:11)，C末端带avi标签和His6标签
Figure PCTCN2019107055-appb-000015
IL-2Rβ受体(SEQ ID NO:12)，C末端带avi标签和His6标签
Figure PCTCN2019107055-appb-000016
载体pYDO_017：(SEQ ID NO:13)
Figure PCTCN2019107055-appb-000017
Figure PCTCN2019107055-appb-000018

Claims (30)

  1. 一种IL-2突变蛋白，其与野生型IL-2(优选人IL-2，更优选包含SEQ ID NO:1序列的IL-2)相比包含至少一个突变，所述突变消除或降低对IL-2Rα受体的结合亲合力和/或增强对IL-2Rβ受体的结合亲合力,优选地，所述突变蛋白与SEQ ID NO:1具有至少80％，85％,90％，95％，或96％的同一性，优选地，所述突变蛋白在125位置具有氨基酸残基S。
  2. 权利要求1的IL-2突变蛋白，其包含在选自与SEQ ID NO:1的以下位置的相应的位置上的突变：
    K35/R38/T41/K43；K35/R38/F42/Y45；K35/T37/R38/F42；K35/T37/F42/K43；K35/R38/K43/L72；K35/T37/E61/L72；K35/Y45/E61/E68；K35/R38/T41/F42；K35/R38/T41/E68；T37/K43/E68/L72；T41/F42/K43/E68；F42/Y45/E61/E68；
    尤其是以下位置的突变：K35/T37/R38/F42；K35/R38/T41/K43；K35/R38/F42/Y45；K35/R38/K43/L72，
    优选地，上述位点的突变选自以下：
    位点 优选的突变 35 K35D/E 37 T37D/E/W/K/Y,尤其是T37D/E 38 R38D/E/W/V/F/K,尤其是R38D/E/W 41 T41Y/E/K/Q/R,尤其是T41E 42 F42A/Q/E/K/R,尤其是F42A/Q/E 43 K43W/D/E/Y/D，尤其是K43E/Y 45 Y45R/K 61 E61R/K/W/L/Y,尤其是E61R//K/W 68 E68K/R/Y/W，尤其是E68R/Y 72 L72K/F
    任选地，所述突变蛋白还可以包含在与SEQ ID NO:1的35,37,38,41,42,43,45,61,62,68或72位置相应的位置上的一个或多个其它突变。
  3. 权利要求1-2的IL-2突变蛋白，其中降低或消除对IL-2Rα受体的结合亲合力的突变包括在选自与SEQ ID NO:1的以下位置相应的位置上的突变：
    K35/T37/R38/T41/K43；
    K35/T37/R38/T41/K43/L72；
    K35/T37/R38/K43/Y45/L72；
    K35/R38/T41/K43；
    K35/R38/T41/K43/L72；
    K35/R38/T41/K43/E61/L72；
    K35/R38/T41/K43/Y45/L72；
    K35/R38/T41/K43/Y45/E61/L72；
    K35/R38/F42/Y45；
    K35/R38/F42/Y45/E61/E68；
    K35/R38/F42/E68/L72；
    K35/R38/F42/K43/Y45/E68；
    K35/R38/F42/K43/Y45/E61/E68；
    K35/R38/F42/K43/Y45/E61/E68/L72；
    K35/T37/R38/F42；
    K35/T37/R38/F42/Y45/E61/E68；
    K35/T37/R38/F42/K43/Y45/E61/E68；
    K35/T37/R38/T41/F42/K43/Y45/E61/E68；
    K35/T37/R38/F42/Y45/E61/E62/E68/L72；
    K35/T37/R38/F42/Y45/E62/E68；
    K35/T37/R38/F42/K43/E68；
    K35/T37/F42/K43；
    K35/T37/R38/T41/L72；
    K35/T37/R38/T41/F42；
    K35/T37/R38/T41/F42/K43/E68；
    K35/T37/R38/T41/F42/K43/Y45；
    K35/T37/R38/T41/F42/K43/Y45/L72；
    T37/R38/T41/F42/K43/Y45；
    K35/T37/T41/F42/K43/Y45；
    K35/T37/T41/F42/K43/E61/E68/L72；
    K35/T41/F42/K43/E68；
    K35/T37/R38/T41/K43/Y45；
    K35/T37/R38/T41/K43/Y45/L72；
    K35/T37/R38/T41/K43/Y45/E61/L72；
    K35/T37/R38/K43/L72；
    K35/R38/K43/L72；
    K35/T37/E61/L72；
    K35/Y45/E61/E68；
    K35/R38/T41/F42；
    K35/R38/T41/F42/Y45；
    K35/R38/T41/F42/Y45/E68；
    K35/R38/T41/E68；
    K35/R38/Y45/E68/L72；
    T37/K43/E68/L72；
    T37/K43/Y45/E68/L72；
    T37/K43/Y45/E61/E68/L72；
    T37/F42/Y45/E61/E68/L72；
    T37/T41/Y45/E61/E68/L72；
    R38/T41/F42/Y45/E61/E68；
    T41/K43/Y45/E61/E68/L72。
  4. 权利要求1-3的IL2突变蛋白，其中降低或消除对IL-2Rα受体的结合亲合力的突变包括选自以下的突变组合：
    K35D/T37K/R38E/T41K/F42Q/K43D/E68Y；
    K35D/T37D/T41F/F42E/K43D/Y45K；
    K35D/T37K/R38D/T41E/K43E；
    K35D/R38E/T41E/K43E；
    K35D/R38F/F42E/Y45K；
    K35E/R38D/T41E/K43E/L72F；
    K35D/T37E/R38D/K43E/L72F；
    K35E/R38D/T41E/K43E/E61K/L72F；
    K35E/T37D/R38W/F42Q/Y45K/E61K/E68R；
    T37E/K43E/Y45K/E68K/L72K；
    K35E/R38E/T41M/F42E/Y45K；
    K35D/T37E/R38D/T41E/K43E；
    K35D/T37D/R38E/K43E/L72F；
    K35E/T37D/R38D/K43E/L72F；
    K35D/R38D/T41E/K43E/Y45K/E61W/L72F；
    K35D/T37E/R38D/T41E/K43E/L72F；
    K35D/R38W/F42E/E68R/L72F；
    K35D/T37E/R38D/K43Y/Y45K/L72F；
    K35E/T37D/R38W/T41E/F42A/K43F/Y45K；
    K35D/T37E/R38D/T41R/F42Q；
    K35E/R38D/T41E/K43D/L72F；
    K35E/T41Q/F42R/K43D/E68Y；
    K35E/T37Y/R38W/T41Y/F42E/K43E/Y45K/L72K；
    K35D/T37E/R38D/K43Y/Y45R/L72F；
    K35E/T37E/R38D/T41E/K43Y；
    T37E/T41K/Y45R/E61L/E68K/L72K；
    K35E/R38W/F42Q/Y45R/E61W/E68R；
    K35D/T37E/R38E/T41E/L72F；
    K35D/T37D/R38D/T41E/L72F；
    K35D/R38E/T41E/K43E/L72F；
    K35E/T37D/R38D/T41E/K43Y/Y45K；
    K35D/T37D/F42A/K43E；
    K35E/R38E/T41E/K43Y/Y45K/L72F；
    K35D/R38W/F42E/K43Y/Y45R/E61R/E68Y；
    R38K/T41R/F42Q/Y45K/E61Y/E68W；
    K35E/R38W/F42Q/Y45K/E61L/E68R；
    K35D/T37E/R38E/T41E/K43E/Y45K/L72F；
    K35E/R38D/K43E/L72F；
    K35E/R38E/K43E/L72F；
    K35D/T37E/R38D/L72F；
    K35E/R38E/T41E/K43E/L72F；
    K35E/T37E/R38E/F42A；
    K35D/R38F/T41E/F42E/Y45K/E68Y；
    K35E/T37K/R38E/T41E/K43E/L72F；
    K35D/R38D/K43E/L72F；
    K35D/R38A/T41Q/F42R；
    K35D/T37E/R38F/F42E/K43E/E68R；
    K35D/T37E/R38D/T41E/K43E/Y45K/E61W/L72F；
    T37D/R38F/T41F/F42E/K43E/Y45R；
    K35D/R38W/F42E/K43E/Y45K/E68K；
    K35E/T37D/R38E/T41E/K43Y；
    K35E/R38W/F42E/Y45K；
    K35E/T37D/R38W/F42E/Y45K/E68Y/L72R；
    K35E/T37D/R38W/F42Q/Y45R/E62R/E68R；
    K35E/T37D/T41F/F42A/K43E/E61W/E68K/L72W；
    T41K/K43D/Y45R/E61L/E68K/L72K；
    K35E/T37E/R38W/F42Q/Y45R/E61R/E68R；
    K35E/T37D/R38W/F42Q/Y45R/E61K/E68R；
    K35E/T37D/R38W/F42Q/Y45K/E61W/E68R；
    K35E/R38W/F42Q/Y45K/E61K/E68R；
    K35E/R38W/F42Q/Y45K/E61W/E68R；
    K35E/T37D/R38W/F42Q/Y45K/E61R/E68R；
    K35E/R38D/Y45K/E68R/L72K；
    K35E/R38W/F42Q/Y45R/E61R/E68R；
    T37E/K43D/E68Y/L72R；
    K35E/Y45R/E61L/E68W；
    K35E/T37D/R38W/F42Q/Y45R/E61R/E68R；
    K35E/T37D/R38V/F42E/K43W/Y45R/E61L/E68W；
    K35E/T37W/R38E/T41Y/F42R/K43D/Y45K/E61K/E68W；
    K35D/R38W/F42K/K43Y/Y45R/E61R/E68W/L72K；
    K35E/T37E/E61W/L72K；
    K35E/R38Y/T41E/E68Y；
    K35E/T37E/R38W/F42Q/Y45R/E61K/E68R；
    T37D/K43D/Y45R/E61Y/E68K/L72R；
    K35D/R38W/F42E/K43Y/Y45R/E61R/E68Y/L72K；
    T37E/F42R/Y45R/E61Y/E68K/L72K；
    K35E/T37D/R38V/F42A/Y45K/E61R/E62W/E68Y/L72R。
  5. 权利要求1-2的IL2突变蛋白，其中降低或消除对IL-2Rα受体的结合亲合力的突变包括在选自以下的位置上的突变：
    K35/R38/F42/T37；
    K35/R38/F42/Y45/E61/E68；
    K35/R38/F42/Y45/E61/E68/T37；
    优选地所述突变包括：
    K35E,R38E,F42A,T37E；或
    K35E,R38W,F42Q,Y45R/K,E61K/W/R,E68R，和任选地T37D/E；
    最优选地所述突变为选自以下的突变组合：
    K35E/R38E/F42A/T37E；
    K35E/R38W/F42Q/Y45K/E61K/E68R；
    K35E/R38W/F42Q/Y45K/E61W/E68R；
    K35E/R38W/F42Q/Y45R/E61R/E68R；
    K35E/R38W/F42Q/Y45K/E61K/E68R/T37D；
    K35E/R38W/F42Q/Y45R/E61K/E68R/T37D；
    K35E/R38W/F42Q/Y45K/E61W/E68R/T37D；
    K35E/R38W/F42Q/Y45K/E61R/E68R/T37D；
    K35E/R38W/F42Q/Y45R/E61R/E68R/T37D；
    K35E/R38W/F42Q/Y45R/E61R/E68R/T37E；
    K35E/R38W/F42Q/Y45R/E61K/E68R/T37E。
  6. 权利要求1-2的IL2突变蛋白，其中降低或消除对IL-2Rα受体的结合亲合力的突变包括在选自以下的位置上的突变：
    K35/R38/F42/Y45/E61/E68/K43；
    K35/R38/F42/Y45/E61/E68/K43/L72；
    K35/R38/F42/Y45/E61/E68/K43/T37；
    K35/R38/F42/Y45/E61/E68/K43/T37/T41；
    优选地所述突变包括：
    K35D/E,R38W/V/E,F42E/K/R,Y45R/K,E61R/L/K,E68Y/W,K43Y/W/D,任选地还包含T37D/W,T41Y；L72K中的一个或多个；
    更优选所述突变为选自以下的突变组合：
    K35D/R38W/F42E/Y45R/E61R/E68Y/K43Y；
    K35D/R38W/F42E/Y45R/E61R/E68Y/K43Y/L72K；
    K35D/R38W/F42K/Y45R/E61R/E68W/K43Y/L72K；
    K35E/R38V/F42E/Y45R/E61L/E68W/T37D/K43W；
    K35E/R38E/F42R/Y45K/E61K/E68W/T37W/K43D/T41Y；
    更优选所述突变为：K35D/R38W/F42E/Y45R/E61R/E68Y/K43Y/L72K。
  7. 权利要求1-2的IL2突变蛋白，其中降低或消除对IL-2Rα受体的结合亲合力的突变包括在选自以下的位置上的突变：
    K35/R38/T41/K43；
    K35/R38/T41/K43/T37；
    K35/R38/T41/K43/L72；
    K35/R38/T41/K43/T37/L72；
    K35/R38/T41/K43/E61/L72；
    K35/R38/T41/K43/Y45/L72；
    K35/R38/K43/T37/L72；
    K35/R38/K43/T37/L72/Y45；
    优选地所述突变包括：K35D/E,R38D/E,T41E,K43E/Y,任选地还包括选自T37K/E,Y45K,E61K,L72F中的一者或两者；或者
    优选地所述突变包括：K35D/E,R38D,K43E/Y,T37D/E,L72F,任选地还包括Y45K；
    更优选地所述突变为选自以下的突变组合：
    K35D/R38E/T41E/K43E；
    K35D/T37K/R38D/T41E/K43E；
    K35E/R38E/T41E/K43E/L72F；
    K35D/T37E/R38D/T41E/K43E/L72F；
    K35E/R38D/T41E/K43E/E61K/L72F；
    K35E/R38E/T41E/K43Y/Y45K/L72F；
    K35D/T37E/R38D/K43E/L72F；
    K35E/T37D/R38D/K43E/L72F；
    K35D/T37E/R38D/K43Y/Y45K/L72F；
    更优选所述突变为选自以下的突变组合：
    K35D/T37E/R38D/T41E/K43E/L72F；
    K35E/R38D/T41E/K43E/E61K/L72F；
    K35E/R38E/T41E/K43Y/Y45K/L72F；
    K35D/T37E/R38D/K43E/L72F；
    K35E/T37D/R38D/K43E/L72F；
    K35D/T37E/R38D/K43Y/Y45K/L72F；
    最优选地所述突变为：
    K35D/R38E/T41E/K43E。
  8. 权利要求1-2的IL2突变蛋白，其中降低或消除对IL-2Rα受体的结合亲合力的突变包括在选自以下的位置上的突变：
    K35/R38/F42/Y45/E61/E68；
    K35/R38/F42/Y45/E61/E68/T37；
    K35/R38/F42/T37；
    K35/R38/T41/K43。
  9. 权利要求1-2的IL2突变蛋白，其中降低或消除对IL-2Rα受体的结合亲合力的突变包括选自以下的突变组合：
    K35E/R38E/F42A/T37E；
    K35E/R38W/F42Q/Y45K/E61K/E68R；
    K35E/R38W/F42Q/Y45K/E61W/E68R；
    K35E/R38W/F42Q/Y45R/E61R/E68R；
    K35E/R38W/F42Q/Y45K/E61K/E68R/T37D；
    K35E/R38W/F42Q/Y45R/E61K/E68R/T37D；
    K35E/R38W/F42Q/Y45K/E61W/E68R/T37D；
    K35E/R38W/F42Q/Y45K/E61R/E68R/T37D；
    K35E/R38W/F42Q/Y45R/E61R/E68R/T37D；
    K35E/R38W/F42Q/Y45R/E61R/E68R/T37E；
    K35E/R38W/F42Q/Y45R/E61K/E68R/T37E；
    K35D/R38E/T41E/K43E。
  10. 权利要求1-2的IL2突变蛋白，其中降低或消除对IL-2Rα受体的结合亲合力的突变包括选自以下的突变组合：
    K35D/T37E/R38E/T41E/K43E/Y45K/L72F；
    K35E/R38D/T41E/K43E/L72F；
    K35E/R38W/F42Q/Y45K/E61L/E68R；
    K35D/T37K/R38E/T41K/F42Q/K43D/E68Y；
    K35D/T37E/R38D/K43Y/Y45K/L72F；
    K35E/T37Y/R38W/T41Y/F42E/K43E/Y45K/L72K；
    K35D/R38W/F42E/K43E/Y45K/E68K；
    K35E/T41Q/F42R/K43D/E68Y；
    K35D/R38W/F42E/Y45R/E61R/E68Y/K43Y；
    K35D/R38F/T41E/F42E/Y45K/E68Y；
    K35E/T37K/R38E/T41E/K43E/L72F；
    R38K/T41R/F42Q/Y45K/E61Y/E68W。
  11. 权利要求1-10的IL-2突变蛋白，其中所述突变蛋白包含在选自与SEQ ID NO:1的以下位置的相应的位置上的突变：R81和S87；且优选地以下位置上的突变：H79/R81/S87/I92，R81/R83/S87；R81/R83/S87/I92；R81/P82/R83/S87；或R81/P82/R83/S87/I92；
    优选地，上述位点的突变选自以下：
    位点 优选的突变 79 H79D/E/Q 81 R81D/N 82 P82T/A/I 83 R83E 87 S87D/E 91 V91L/I 92 I92L/F/M/Y
    任选地，所述突变蛋白还可以包含在与SEQ ID NO:1的79,81,82,83,84,87,88,91,92位置相应的位置上的一个或多个突变。
  12. 权利要求11的突变蛋白，其中所述增强对IL-2Rβ的结合亲合力的突变包括在选自以下的位置上的突变：
    H79/R81/S87/I92，
    H79/R81/S87/I92/P82；
    优选地，所述突变包括H79D/E,R81D,S87D/E,I92L/F,任选还包括P82T；
    更优选所述突变包括选自以下的组合突变：
    H79D/R81D/S87D/I92L；
    H79D/R81D/P82T/S87D/I92L。
  13. 权利要求11的突变蛋白，所述增强对IL-2Rβ的结合亲合力的突变包括在选自以下的位置上的突变：
    R81/R83/S87；
    R81/R83/S87/I92；
    R81/P82/R83/S87；或
    R81/P82/R83/S87/I92；
    优选还包括在H79和V91中的一个或两个位置上的突变；
    优选地，所述突变包括选自以下的突变组合：
    R81D,P82T/I/A,R83E,S87E/D；
    R81D/N,P82T/I/A,R83E,S87E/D,I92/L/M/F/Y,
    R81D,R83E,S87E/D；
    R81D,R83E,S87E/D,I92L；
    更优选还包括H79D/E/Q和V91L/I中的一个或两个；
    更优选地，所述突变为选自以下的突变组合：
    H79D/R81D/S87D/I92L；
    H79D/R81D/P82T/S87D/I92L；
    R81D/P82T/R83E/S87E；
    R81D/P82T/R83E/S87E/V91L；
    R81D/P82A/R83E/S87E/V91L；
    H79E/R81D/P82T/R83E/S87E/V91L；
    H79E/R81D/P82I/R83E/S87D；
    H79Q/R81D/P82A/R83E/S87E；
    R81N/P82T/R83E/S87E/V91L/I92F；
    H79D/R81N/P82T/R83E/S87E/V91L/I92F；
    R81D/P82T/R83E/S87D/I92M；
    H79E/R81D/P82I/R83E/S87E/I92M；
    H79Q/R81D/P82T/R83E/S87D/V91L/I92Y；
    R81D/P82I/R83E/S87D/V91I/I92Y；
    R81D/P82T/R83E/S87D/I92L；
    R81D/P82T/R83E/S87D/V91L/I92L；
    R81D/P82A/R83E/S87E/I92L；
    H79E/R81N/P82A/R83E/S87D/I92L；
    R81D/P82T/R83E/S87E/I92M；
    R81D/P82A/R83E/S87E/I92M；
    H79E/R81D/P82A/R83E/S87E/I92M；
    R81D/R83E/S87D/I92L；
    R81D/R83E/S87E/I92L；
    H79D/R81D/R83E/S87D；
    H79D/R81D/R83E/S87E/V91I/I92L。
  14. 权利要求11的突变蛋白，其中增强对IL-2Rβ的结合亲合力的突变为选自下的突变组合：
    H79D/R81D/S87D/I92L；
    H79E/R81D/P82T/R83E/S87E/V91L；
    H79E/R81D/P82I/R83E/S87D；
    H79Q/R81D/P82A/R83E/S87E；
    R81D/P82T/R83E/S87D/I92L；
    R81D/P82T/R83E/S87D/V91L/I92L；
    R81D/P82A/R83E/S87E/I92L；
    H79E/R81N/P82A/R83E/S87D/I92L；
    R81D/P82T/R83E/S87E/I92M；
    R81D/P82A/R83E/S87E/I92M；
    H79E/R81D/P82A/R83E/S87E/I92M；
    H79D/R81D/R83E/S87E/V91I/I92L。
  15. 权利要求1的突变蛋白，其中所述突变蛋白包含选自如下组合的突变：
    组合 降低或消除对IL-2Rα受体的结合亲合力的突变 增强对IL-2Rβ的结合亲合力的突变 1 K35D/T37E/R38E/T41E/K43E/Y45K/L72F H79E/R81D/P82I/R83E/S87D/ 2 K35E/R38D/T41E/K43E/L72F R81D/P82A/R83E/S87E/I92M/ 3 K35E/R38W/F42Q/Y45K/E61L/E68R R81D/P82A/R83E/S87E/I92L/ 4 K35D/T37K/R38E/T41K/F42Q/K43D/E68Y H79E/R81D/P82T/R83E/S87E/V91L/ 5 K35D/T37E/R38D/K43Y/Y45K/L72F R81D/P82T/R83E/S87D/I92L/ 6 K35E/T37Y/R38W/T41Y/F42E/K43E/Y45K/L72K H79Q/R81D/P82A/R83E/S87E/ 7 K35D/R38W/F42E/K43E/Y45K/E68K R81D/P82T/R83E/S87E/I92M/ 8 K35E/T41Q/F42R/K43D/E68Y H79D/R81D/S87D/I92L/ 9 K35D/R38W/F42E/Y45R/E61R/E68Y/K43Y H79E/R81N/P82A/R83E/S87D/I92L/ 10 K35D/R38F/T41E/F42E/Y45K/E68Y R81D/P82T/R83E/S87D/V91L/I92L/ 11 K35E/T37K/R38E/T41E/K43E/L72F H79E/R81D/P82A/R83E/S87E/I92M/ 12 R38K/T41R/F42Q/Y45K/E61Y/E68W H79D/R81D/R83E/S87E/V91I/I92L/
  16. 根据权利要求1-15中任一项所述的IL-2突变蛋白，其中所述突变蛋白与野生型IL-2(尤其是野生型人IL-2)相比，具有以下特性之一或多项：
    -具有消除或降低的对IL-2Rα受体的结合亲合力，且具有增强的对IL-2Rβ受体的结合亲合力；
    -具有降低的对高亲合力IL-2R受体(IL-2Rαβγ)的结合亲合力；
    -具有增加的对中等亲合力IL-2R受体(IL-2Rβγ)的结合亲合力；
    -降低对CD25 +细胞(尤其是CD8 +T细胞，特别是Treg细胞)的激活；
    -降低对CD25 +细胞(尤其是CD8 +T细胞)中IL-2介导的信号传导的刺激，尤其是对STAT5磷酸化信号的刺激作用；
    -去除或降低IL-2对CD25 +细胞优先激活的偏向性，特别是降低IL-2优先刺激Treg细胞增殖的偏向性；
    -降低由IL-2诱导的Treg细胞引起的免疫反应下调作用；
    -保持或增强对CD25 -细胞(尤其是T细胞和NK细胞，特别是CD8 +T细胞)的激活作用；
    -刺激效应细胞T细胞和NK细胞的增殖和激活；
    -提高抗肿瘤效应。
  17. 根据权利要求16所述的IL-2突变蛋白，其中所述突变蛋白在哺乳动物细胞如HEK293细胞中表达时，还具有以下一个或多个特征:
    -优于野生型IL-2蛋白的表达量；
    -优于野生型IL-2蛋白的同质性；
    -易于纯化至更高的蛋白纯度，
    优选地，所述突变蛋白具有优于野生型IL-2蛋白的表达量，更优选地，所述突变蛋白包含选自以下的突变组合：
    -K35E/R38W/F42Q/Y45K/E61K/E68R/T37D；
    -K35E/R38E/F42A/T37E；
    -K35E/R38W/F42Q/Y45R/E61K/E68R/T37E；
    -K35E/R38W/F42Q/Y45K/E61L/E68R/R81D/P82A/R83E/S87E/I92L；
    -K35D/R38W/F42E/K43E/Y45K/E68K/R81D/P82T/R83E/S87E/I92M；
    -K35D/R38F/T41E/F42E/Y45K/E68Y/R81D/P82T/R83E/S87D/V91L/I92L。
  18. 根据权利要求1-17中任一项所述的IL-2突变蛋白，其中所述突变蛋白与野生型IL-2相比具有降低的刺激CD25+T细胞中的信号传导的能力，优选地所述突变蛋白包含选自以下的突变组合：
    K35E/R38E/F42A/T37E
    K35E/R38W/F42Q/Y45K/E61K/E68R/
    K35E/R38W/F42Q/Y45K/E61W/E68R/
    K35E/R38W/F42Q/Y45R/E61R/E68R/
    K35E/R38W/F42Q/Y45K/E61K/E68R/T37D/
    K35E/R38W/F42Q/Y45R/E61K/E68R/T37D/
    K35E/R38W/F42Q/Y45K/E61W/E68R/T37D/
    K35E/R38W/F42Q/Y45K/E61R/E68R/T37D/
    K35E/R38W/F42Q/Y45R/E61R/E68R/T37D/
    K35E/R38W/F42Q/Y45R/E61R/E68R/T37E/
    K35E/R38W/F42Q/Y45R/E61K/E68R/T37E。
    K35D/R38E/T41E/K43E。
  19. 根据权利要求1-17中任一项所述的IL-2突变蛋白，其中所述突变蛋白与野生型IL-2相比具有降低的刺激CD25 +T细胞中的信号传导的能力，且具有增强的刺激CD25 -T细胞中信号传导的能力，优选地所述突变蛋白包含选自以下的突变组合：
    K35D/R38W/F42E/K43E/Y45K/E68K/R81D/P82T/R83E/S87E/I92M；
    K35D/R38F/T41E/F42E/Y45K/E68Y/R81D/P82T/R83E/S87D/V91L/I92L。
  20. 一种融合蛋白，其包含根据权利要求1-19的IL2突变蛋白，优选地与Fc抗体片段融合。
  21. 一种免疫缀合物，其包含权利要求1-19的IL2突变蛋白和抗原结合分子，优选地，抗原结合分子是免疫球蛋白分子，特别是IgG分子，或抗体或抗体片段，特别是Fab分子和scFv分子。
  22. 权要求21的免疫缀合物，其中所述抗原结合分子特异性结合肿瘤细胞上或肿瘤环境中呈现的抗原，例如选自以下的抗原：成纤维细胞活化蛋白(FAP)、生腱蛋白C的A1域(TNC A1)、生腱蛋白C的A2域(TNC A2)、纤连蛋白的外域B(Extra Domain B,EDB)、癌胚抗原(CEA)、和黑素瘤有关的硫酸软骨素蛋白聚糖(MCSP)。
  23. 一种分离的多核苷酸，其编码权利要求1-19的IL-2突变蛋白或权利要求20的融合物或权利要求21-22的免疫缀合物。
  24. 一种表达载体，其包含权利要求23的多核苷酸。
  25. 一种宿主细胞，其包含权利要求23的多核苷酸或权利要求24的载体，优选所述宿主细胞是哺乳动物细胞，特别是HEK293细胞，和酵母。
  26. 一种生产IL-2突变蛋白或其融合物或免疫缀合物的方法，包括在适于表达所述IL-2突变蛋白或融合物或缀合物的条件下培养权利要求25的宿主细胞。
  27. 一种药物组合物，其包含权利要求1-19的IL-2突变蛋白或权利要求19的融合物或权利要求21-22的免疫缀合物和药学可接受载体。
  28. 一种治疗受试者疾病的方法，所述方法包括向所述受试者施用权利要求1-19的IL-2突变蛋白或权利要求20的融合物或权利要求21-22的免疫缀合物或权利要求27的药物组合物，优选地，所述疾病是癌症。
  29. 一种刺激受试者免疫系统的方法，所述方法包括向所述受试者施用有效量的包含权利要求1-19的IL-2突变蛋白或权利要求20的融合物或权利要求21-22的免疫缀合物的药物组合物。
  30. 一种降低消除或降低IL-2蛋白对IL-2Rα受体的结合亲合力和/或增强对IL-2Rβ受体的结合亲合力的方法，包括在IL-2蛋白中引入权利要求2-19中所描述的突变或突变组合，其中所述IL-2蛋白优选与SEQ ID NO:1具有至少80％-99％或更高的同一性，更优选地为来源于人的IL-2蛋白。
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