Polypeptide, pharmaceutical composition containing polypeptide and use thereof

Abstract

Provided is a polypeptide, which contains a first, a second and a third domain, wherein the second domain is an interleukin-2 receptor subunit α binding domain, the first domain and the third domain are non-interleukin-2 receptor subunit α binding domains, and the third domain has a first mutation substituted with cysteine and optionally a second mutation occurring in the first, second or third domain substituted with another cysteine to form a disulfide bond. The polypeptide can preferentially bind to IL-2 receptor subunit α or βγ, and has the function of modulating T cells or effector T cells to stimulate an anti-tumor immune response.

Description

Polypeptides, pharmaceutical compositions containing polypeptides and their uses Technical Field

[0001] This invention relates to protein engineering, and particularly to the stereochemical modification and uses of interleukin-2 and its protein derivatives, with uses relating to methods for preventing, alleviating or treating diseases or disorders caused by low immune cell activity. Background Technology

[0002] Cancer is the second leading cause of death worldwide. Immunotherapy is a treatment method that uses the patient's own immune system to attack cancer cells. Interleukin-2 (IL-2) can stimulate T cell activation and proliferation, and high-dose IL-2 was the first immunotherapy approved for the treatment of metastatic renal cell carcinoma and melanoma.

[0003] However, while IL-2 can produce a lasting anti-cancer response and prolong survival in a small number of patients, it is not without side effects. For example, it can cause capillary leakage syndrome, the specific cause of which remains unknown. On the other hand, continuous triggering of the IL-2 receptor subunit α will cause T cells to favor CD8+ T cells and continuously expand the number of regulatory T cells, leading to T cell exhaustion and inhibiting the activity of functional T cells. This may induce immune escape effects and excessive secretion of inhibitory cytokines, resulting in tumor deterioration or hormonal storms, leading to serious consequences such as multiple organ failure. Therefore, the use of IL-2 in treatment regimens must be approached with extreme caution.

[0004] Furthermore, due to the poor water solubility, low thermal stability, and short half-life of IL-2, it requires multiple injections and high-dose injections during cancer treatment. However, its efficacy is limited by the aforementioned side effects.

[0005] Existing technologies for mass-producing IL-2 primarily utilize chemical protein synthesis to create biologically active IL-2 analogs. While chemical synthesis can construct well-defined IL-2 variants and produce non-natural disulfide bond analogs, their half-life remains quite short, only 7 minutes. Furthermore, even with KAHA linkage, the water solubility problem of the hydrophobic fragments of IL-2 has not been overcome, resulting in low production efficiency. Moreover, IL-2 produced by existing technologies tends to bind to the IL-2 receptor subunit α, activating regulatory T cells (Tregs), which inevitably inhibits anti-tumor immune responses. Summary of the Invention

[0006] This invention is made by introducing disulfide bonds into the structural design of IL-2 to obtain a variant with disulfide bonds, which improves thermal stability, solubility and enhances its ability to activate immune cells. It can also change the binding to the surface IL-2 receptor subunit, favoring the binding to subunit α or subunit βγ, and has the potential as a biopharmaceutical.

[0007] Accordingly, one aspect of the present invention provides a polypeptide comprising: a first domain comprising at least 80%, 85%, 90%, 95%, or 99% of amino acid sequences identical to positions 1 to 34 of the amino acid sequence shown in SEQ ID NO:1; a second domain disposed at the C-terminus of the first domain comprising at least 80%, 85%, 90%, 95%, or 99% of amino acid sequences identical to positions 35 to 72 of the amino acid sequence shown in SEQ ID NO:1; and a third domain comprising at least 80%, 85%, 90%, 95%, or 99% of amino acid sequences identical to those shown in SEQ ID NO:1. The amino acid sequence shown in NO:1 consists of amino acid sequences at positions 73 to 133, and has a first mutation that replaces a first amino acid site with a cysteine. The polypeptide has a second mutation that replaces a second amino acid site with another cysteine. The second mutation can occur selectively in the first domain, the second domain, or the third domain, whereby the cysteine ​​replaced by the first mutation forms a disulfide bond with the cysteine ​​replaced by the second mutation.

[0008] For example, the first amino acid comprises asparagine (Asn, N) at position 77, asparagine (Asp, D) at position 84, isoleucine (Ile, I) at position 92, lysine (Lys, K) at position 97, threonine (Thr, T) at position 111, phenylalanine (Phe, F) at position 117, or leucine (Leu, L) at position 132; the second amino acid comprises glutamic acid (Gln, Q) at position 11, leucine (Leu, L) at position 17, leucine (Leu, L) at position 18, tyrosine (Tyr, Y) at position 31, lysine (Lys, K) at position 43, phenylalanine (Phe, F) at position 44, leucine (Leu, L) at position 56, or leucine (Leu, L) at position 80.

[0009] For example, the first mutation involves replacing lysine (Lys,K) at position 43 with cysteine, and the second mutation involves replacing threonine (Thr,T) at position 111 with cysteine.

[0010] For example, in the C-terminal downstream region of the first amino acid, the third domain further includes a third mutation that replaces the third amino acid site with a hydrophilic amino acid or a hydrophobic amino acid, wherein the third amino acid site is cysteine.

[0011] For example, the third mutation includes any of the following:

[0012] Cysteine ​​at position 125 is replaced with serine (Ser,S);

[0013] Cysteine ​​at position 125 is replaced with alanine (Ala,A);

[0014] Cysteine ​​at position 125 is replaced with leucine (Leu, L).

[0015] For example, the first domain contains an amino acid sequence as shown in any of SEQ ID NOs:2 to 6; the second domain contains an amino acid sequence as shown in any of SEQ ID NOs:7 to 10; and the third domain contains an amino acid sequence as shown in any of SEQ ID NOs:11 to 35.

[0016] For example, the polypeptide includes an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% identical to any of the amino acid sequences shown in SEQ ID NOs:36 to 57.

[0017] Another aspect of the present invention provides a pharmaceutical composition comprising the polypeptide as described above and a pharmaceutically acceptable carrier.

[0018] Another aspect of the present invention provides the use of the pharmaceutical composition as described above for preparing a pharmaceutical or cell therapy composition for treating, preventing or alleviating diseases or disorders caused by low immune cell activity.

[0019] For example, the immune cells include natural killer (NK) cells, NK-T cells, T cells, B cells, macrophages (MAC), dendritic cells (DC), or oligodendrocytes.

[0020] For example, the disease is a proliferative disease including hematologic malignancies, solid tumors, or metastatic tumors.

[0021] This invention introduces disulfide bonds between the α-binding domain of the interleukin-2 receptor subunit and the α-binding domain of the non-interleukin-2 receptor subunit to generate a polypeptide that is biased towards activating the β and γ subunits of the IL-2 receptor. This polypeptide not only activates effective T cells and stimulates their proliferation, but also significantly improves their thermal stability, solubility, and yield. Therefore, the number of injections and dosages can be reduced in cancer treatment to achieve the desired therapeutic effect and reduce side effects. The polypeptide provided by this invention can also be used in cell therapy. After being added to the culture medium of immune cells, it stimulates T cell activation and proliferation, reducing the amount and frequency of addition to the culture medium, saving time and production costs. Attached Figure Description

[0022] Figure 1A is a protein structure diagram, showing the three-dimensional structure of wild-type human interleukin-2 binding to the IL-2 receptor;

[0023] Figure 1B is a protein structure diagram, showing the three-dimensional structure of the human interleukin-2 variant binding to the IL-2 receptor;

[0024] Figure 2 is a protein structure diagram, showing the three-dimensional structure of the human interleukin-2 variant;

[0025] Figure 3 is a protein structure diagram, schematically showing the relative positions of disulfide bonds introduced in the human interleukin-2 variant;

[0026] Figure 4 shows the results of differential scanning calorimetry, comparing the melting temperatures of Comparative Examples 1 and 4 with those of Example 3; and

[0027] Figure 5 shows the results of the cell toxicity experiment, comparing the toxicity of immune cells and tumor cells at different ratios between Example 2 and the standard. Detailed Implementation

[0028] I. Definition of Terms

[0029] As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably and refer to a polymeric form of an amino acid of any length, which may include coding and non-coding amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having a modified peptide backbone.

[0030] As used herein, the term "interleukin-2 receptor subunit α-binding domain," unless otherwise defined, refers to the sequence segment in the protein structure of human interleukin-2, such as SEQ ID NO:1, that can bind to the interleukin-2 receptor to initiate its downstream signaling chain; that is, it corresponds to the amino acid sequence of wild-type human interleukin-2. 35 KLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNL.

[0031] As used herein, the term "non-interleukin-2 receptor subunit α-binding domain" unless otherwise defined refers to the sequence segment in the protein structure of human interleukin-2, such as SEQ ID NO:1, that is not bound to the interleukin-2 receptor; in other words, it corresponds to the amino acid sequence of wild-type human interleukin-2. 1 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNP or 73AQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT.

[0032] The percentage of "sequence similarity" between two peptides, as referred to in this article, means that when aligning two sequences, the percentage of amino acids is the same and they are in the same relative positions. Sequence similarity can be determined in different ways, such as by using various methods or computer programs (e.g., BLAST, T-COFFEE, MUSCLE, MAFFT, etc.) obtained from websites including ncbi.nlm.nili.gov / BLAST, ebi.ac.uk / Tools / msa / tcoffee / , ebi.ac.uk / Tools / msa / muscle / , mafft.cbrc.jp / alignment / software / , or by searching the global information network.

[0033] As used herein, the term "hydrophilic amino acid" refers to an amino acid with branched chains having a hydrophilicity index less than 0, where the hydrophilicity index is determined by the Δ value of vapor-to-aqueous phase transfer. tr Gm θ And the distribution of amino acids on the surface and inside during protein formation, which meets the above definition of "hydrophilic amino acids" such as serine (Ser, S; -0.8), threonine (Thr, T; -0.7), tyrosine (Tyr, Y; -1.3), aspartic acid (Asn, N; -3.5), glutamic acid (Gln, Q; -3.5), histidine (His, H; -3.2), tryptophan (Trp, W; -0.9) or proline (Pro, P; -1.6).

[0034] As used herein, the term "hydrophobic amino acid" refers to an amino acid with a branched chain having a hydrophilicity index greater than 0. Examples of "hydrophobic amino acids" that meet the above definition include alanine (Ala, A; 1.8), valine (Val, V; 4.2), leucine (Leu, L; 3.8), isoleucine (Ile, I; 4.5), methionine (Met, M; 1.9), or phenylalanine (Phe, F; 2.8).

[0035] As used herein, the term "heterogeneous polypeptide" means, unless otherwise defined, a polypeptide sequence not found in the natural protein sequence.

[0036] As used herein, unless otherwise defined, the term "purification tag" refers to a polypeptide sequence linked to a recombinant protein via gene transfer. Based on its application, purification tags can be further categorized as follows: "affinity tag" refers to a tag that alters the affinity of a recombinant protein for a specific binding site, facilitating purification of the recombinant protein using affinity techniques. Examples include chitin-binding protein (CBP), maltose-binding protein (MBP), Strep tags, glutathione-S-transferase (GST), or polyhistidine (ploy(His)); "solubilization tag" refers to a tag that enables the recombinant protein to fold correctly and prevents inclusion body aggregation. Examples include thioredoxin (TRX) and poly(NANP); "chromatographic tag"... "Label" refers to a tag that can alter the chromatographic properties of recombinant proteins, thereby providing different resolutions in specific separation techniques. These include FLAG tags or polyglutamic acid tags. "Epitope tag" refers to a tag that can alter the immunoreactivity of recombinant proteins, enabling them to generate high-affinity antibodies in different species. These include ALFA tags, V5 tags, Myc tags, HA tags, dot tags, T7 tags, and NE tags. "Fluorescence tag" refers to a tag that can provide a visual reading of recombinant proteins under specific optical conditions. These include green fluorescent protein (GFP), red fluorescent protein (RFP), or yellow fluorescent protein (YFP).

[0037] As used herein, unless otherwise defined, the term "recombinant" refers to a product of various combinations of selection, restriction, polymerase chain reaction (PCR), and / or conjugation steps of a specific nucleic acid (DNA or RNA) to produce a construct having a coding or non-coding sequence distinct from endogenous nucleic acids found in natural systems. DNA sequences encoding polypeptides can be assembled from cDNA fragments or a series of synthetic oligonucleotides to provide synthetic nucleic acids that can be expressed from recombinant transcriptional units contained in cellular or cell-free transcription and translation systems.

[0038] As used herein, the terms “treatment”, “treating”, and similar terms generally mean achieving the desired pharmacological and / or physiological effect, which may be therapeutic in relation to the partial or complete cure of the disease and / or adverse effects attributable to the disease.

[0039] As used herein, the term “prevention” means preventing the development of a particular disease or symptom in subjects who are susceptible to that disease or symptom but have not yet been diagnosed with it.

[0040] As used herein, the term “relieve” means to suppress or reduce the occurrence of a particular disease or symptom in a subject who has been diagnosed with or has not yet been diagnosed with that particular disease or symptom, that is, to slow the progression of that particular disease or to cause the remission of that particular disease.

[0041] As used herein, the terms “individual” and “subject” are used interchangeably and refer to any mammalian subject who is expected to receive a diagnosis, treatment or therapy; mammals include humans, non-human primates, rodents (e.g., rats; mice), rabbits (e.g., rabbits), ungulates (e.g., cows, sheep, pigs, horses, goats and the like).

[0042] Before further elaborating on the invention, it should be understood that the invention is not limited to the specific embodiments described, and therefore variations are naturally possible. It should also be understood that the terminology used herein is for the purpose of illustrating specific embodiments only and is not intended to be restrictive, as the scope of the invention will be limited only to the scope of the appended patent applications.

[0043] If a range of values ​​is provided, it should be understood that this invention covers every intermediate value between the upper and lower limits of that range (accurate to one-tenth of the lower limit unit unless the context explicitly indicates otherwise) and any other value or intermediate value within that range. The upper and lower limits of such smaller ranges may be independently included within the smaller range and are also covered within this invention, subject to any explicit exclusions within said range. If the range includes one or both of the limits, then this invention also includes ranges that exclude any or both of the limits included therein.

[0044] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of this invention, preferred methods and materials are hereby described.

[0045] It must be noted that, unless the context clearly indicates otherwise, the singular forms “a” and “the” used herein and in the claims of the appended patent application include a plurality of indicators. Thus, for example, reference to “interleukin-2 receptor subunit α-binding domain” includes one or more of such polypeptides and includes reference to one or more interleukin-2 receptor subunit α-binding domains and their equivalents known to those skilled in the art, etc. It should be further noted that the claims of the patent application may be designed to exclude any optional elements. Therefore, statements intended to serve as a precondition for incorporating elements of the claims of the patent application use exclusive terms such as “only,” “merely,” and similar terms, or use negative limiting terms.

[0046] It should be understood that certain features of the invention set forth in the context of a single embodiment for clarity may also be provided in combination in a single embodiment. Conversely, various features of the invention set forth in the context of a single embodiment for simplicity may also be provided individually or in any suitable combination. All combinations of embodiments of the invention are specifically encompassed by the invention and disclosed herein, just as each and every combination is individually and explicitly disclosed herein. In addition, all sub-combinations of the various embodiments and their elements are also explicitly encompassed by the invention and disclosed herein, just as each and every such sub-combination is individually and explicitly disclosed herein.

[0047] II. Polypeptides

[0048] A first embodiment of the present invention relates to a polypeptide comprising:

[0049] The first domain comprises at least 80%, 85%, 90%, 95%, or 99% of the amino acid sequence at positions 1 to 34 of the amino acid sequence shown in SEQ ID NO:1, which is one of the non-interleukin-2 receptor subunit α-binding domains.

[0050] A second domain, disposed at the C-terminus of the first domain, comprises at least 80%, 85%, 90%, 95%, or 99% identical amino acid sequences at positions 35 to 72 of the amino acid sequence shown in SEQ ID NO:1, which is the α-binding domain of the interleukin-2 receptor subunit; and

[0051] The third domain comprises at least 80%, 85%, 90%, 95%, or 99% of an amino acid sequence identical to positions 73 to 133 of the amino acid sequence shown in SEQ ID NO:1, which is one of the non-interleukin-2 receptor subunit α-binding domains, wherein the introduced disulfide bond bridges the third domain to the first or second domain, or the introduced disulfide bond occurs in the third domain.

[0052] In the first embodiment, the third domain has a first mutation that replaces the first amino acid site with cysteine, wherein the polypeptide also has a second mutation that replaces the second amino acid site with another cysteine, and the second mutation may selectively occur in the first domain, the second domain, or the third domain, thereby forming a disulfide bond between the cysteine ​​replaced by the first mutation and the cysteine ​​replaced by the second mutation.

[0053] Normally, the interleukin-2 receptor is a heteromer composed of three subunits: α, β, and γ. Due to the high affinity of IL-2 for the IL-2 receptor subunit α (hereinafter referred to as IL-2Rα), it can maintain signal transduction function after cytokine depletion and form an IL-2 reservoir on the cell surface to help recycle IL-2 to the cell surface. However, because T cells compete for limited cytokines, continuous triggering of the IL-2 receptor subunit α will cause T cells to favor CD8+ T cells and continuously expand the number of regulatory T cells, leading to T cell depletion. This process inhibits the activity of functional T cells.

[0054] In one or more embodiments below, when the polypeptide provided in the first embodiment binds to the interleukin-2 receptor expressed on the surface of T cells, the disulfide bonds formed by the above-mentioned mutation cause the interleukin-2 receptor subunit α-binding domain and the non-interleukin-2 receptor subunit α-binding domain to have a stereoconfiguration in three-dimensional space that is different from that of the wild type, thereby changing the affinity between the interleukin-2 receptor subunit α-binding domain and IL-2Rα.

[0055] In a particular embodiment, to improve the structural stability of the peptide and alter the affinity of the α-binding domain of the interleukin-2 receptor subunit for IL-2Ra, suitably, the first amino acid comprises asparagine (Asn, N) at position 77, aspartic acid (Asp, D) at position 84, isoleucine (Ile, I) at position 92, lysine (Lys, K) at position 97, threonine (Thr, T) at position 111, and phenylalanine (Phe, ...) at position 117. F) or leucine (Leu,L) at position 132; the second amino acid comprises: glutamic acid (Gln,Q) at position 11, leucine (Leu,L) at position 17, leucine (Leu,L) at position 18, tyrosine (Tyr,Y) at position 31, lysine (Lys,K) at position 43 replaced by cysteine, phenylalanine (Phe,F) at position 44, leucine (Leu,L) at position 56, or leucine (Leu,L) at position 80.

[0056] It should be noted that the selection of the second amino acid site is based on the fact that the amino acid branch at this site has the ability to form a non-covalent bond with the interleukin-2 binding site on IL-2Rα, or an amino acid residue adjacent to this site that has a decisive influence on the configuration of the interleukin-2 binding site; please refer to Figure 1A, taking the binding of wild-type human interleukin-2 (hereinafter referred to as hIL-2) to IL-2Rα as an example, the protein structure established by X-ray single crystal diffraction shows that, for example, the sites that form a non-covalent bond with glutamate (E) at position 29, arginine (R) at position 35, or arginine (R) at position 36 on the IL-2Rα protein sequence correspond to lysine (K) at position 43, tyrosine (Y) at position 45, or glutamate (E) at position 62 on the hIL-2 protein sequence.

[0057] Please refer to Figures 1A and 1B. After introducing the first mutation and the second mutation, taking cysteine ​​at position 43 and cysteine ​​at position 111 as an example, the stable structure of the disulfide bond formed by these two mutations disrupts the hydrogen bond between lysine at position 43 of the interleukin-2 receptor subunit α-binding domain and glutamic acid at position 29 of IL-2Rα. This reduces the affinity of the interleukin-2 receptor subunit α-binding domain for IL-2Rα, making the peptide more inclined to bind to IL-2R subunits β and γ.

[0058] In the first embodiment, the above sequence configuration was made to alter the binding affinity of the interleukin-2 receptor subunit α-binding domain to IL-2Rα and / or other biological properties.

[0059] In a specific implementation, this is used to create interleukin-2 variants with higher affinity for IL-2Rα and / or IL-2Rβγ. Such interleukin-2 variants can be created by introducing appropriate mutations (deletion, insertion, and substitution) into the nucleotide sequence encoding interleukin-2, or by peptide synthesis. Depending on the improved properties, amino acid variations other than the first and second amino acid sites can be introduced into the interleukin-2 receptor subunit α-binding domain antibody and / or non-interleukin-2 receptor subunit α-binding domain. Screening can be performed for maintaining or improving binding affinity, stability of the stereostructure, etc.

[0060] Understandably, the polypeptide sequence in the first embodiment is not limited to introducing amino acid variations into the intact interleukin-2 polypeptide chain, but can also be any of the following sequence configurations:

[0061] The C-terminus of the first structural domain is linked to the N-terminus of the second structural domain, and the C-terminus of the second structural domain is linked to the N-terminus of the third structural domain.

[0062] Heterogeneous peptides are configured at the N-terminus of the first domain or the C-terminus of the third domain;

[0063] The first structural field is further linked to the second structural field by the first connector, and the second structural field is further linked to the third structural field by the second connector;

[0064] The heteropeptide is configured by linking the N-terminus of the first domain or the C-terminus of the third domain with a third connector.

[0065] As used herein, the term "linker," unless otherwise defined, refers to an oligopeptide molecule or other portion that may be optionally linked between two polypeptide molecules to form a single continuous polypeptide molecule; some strategies involve molecularly covalently linking these polypeptide molecules, including but not limited to polypeptide bonds, disulfide bonds, or links formed by chemical cross-linking agents between the N- and C-termini of proteins or protein domains; the selection of a suitable linker in the specific case of linking two polypeptides depends on various parameters, including but not limited to the properties of the two polypeptide chains, the distance between the N- and C-termini, and / or the stability of the linker to protein hydrolysis and oxidation. Furthermore, the linker may also contain amino acid residues that provide flexibility.

[0066] The connectors mentioned above can refer to a first connector, a second connector, and / or a third connector, and the three can be the same or different; the length of the connector should be sufficient to connect the two polypeptide molecules in the correct conformational manner to maintain the desired activity, and it can have 2 to 30 amino acid residues; the amino acid residues in the connector should exhibit properties that do not significantly interfere with the activity of the polypeptide molecules; therefore, the connector should have a charge that is relatively consistent with the activity of the polypeptide molecules to avoid interfering with the folding pattern of the polypeptide molecules, or to avoid forming bonds or other interactions with one or more amino acid residues in the polypeptide molecules.

[0067] Suitablely, the linker includes the glycine-serine linker (or GS linker), which refers to polymers of glycine and serine in series, such as (Gly-Ser)n, (GSGGS)n, (GGGGS)n, and (GGGS)n, where n is an integer of at least 1; or refers to glycine-alanine polymer, alanine-serine polymer, and other flexible linkers.

[0068] In some embodiments, to improve the free properties of the peptide, increase its solubility and melting temperature (Tm), and reduce the tendency to form inclusion bodies, thereby improving the separation and purification efficiency of the peptide and increasing its production efficiency, in the downstream region of the C-terminus of the first amino acid, the third domain further includes a third mutation that replaces the third amino acid site with a hydrophilic or hydrophobic amino acid, wherein the third amino acid site is cysteine.

[0069] In a particular embodiment, the hydrophilic amino acid may be serine (Ser,S), threonine (Thr,T), tyrosine (Tyr,Y), aspartic acid (Asn,N), glutamic acid (Gln,Q), histidine (His,H), tryptophan (Trp,W), or methionine (Met,M); the hydrophobic amino acid may be alanine (Ala,A), valine (Val,V), leucine (Leu,L), isoleucine (Ile,I), proline (Pro,P), or phenylalanine (Phe,F); preferably, the third mutation is to replace cysteine ​​at position 125 with serine (Ser,S), alanine (Ala,A), or leucine (Leu,L).

[0070] Please refer to Figure 2. Taking the third mutation, which replaces cysteine ​​at position 125 with serine (Ser,S), as an example, the complete human interleukin-2 variant (hereinafter referred to as mhIL-2) protein sequence has at least two sets of disulfide bonds: cysteine ​​at position 43-cysteine ​​at position 111 (C43-C111) and cysteine ​​at position 58-cysteine ​​at position 105 (C58-C105). Since cysteine ​​at position 125 is replaced with serine (C125S), the C-terminal fragment of mhIL-2 is given hydrophilic properties, which helps to improve its solubility.

[0071] In a particular embodiment, the first domain contains an amino acid sequence as shown in any of SEQ ID NOs:2 to 6; the second domain contains an amino acid sequence as shown in any of SEQ ID NOs:7 to 10; and the third domain contains an amino acid sequence as shown in any of SEQ ID NOs:11 to 35.

[0072] In specific cases, the first domain contains an amino acid sequence as shown in SEQ ID NO:2, the second domain contains an amino acid sequence as shown in any one of SEQ ID NOs:8 to 10, and the third domain contains an amino acid sequence as shown in any one of SEQ ID NOs:24 to 32; the first domain contains an amino acid sequence as shown in any one of SEQ ID NOs:3 to 6, the second domain contains an amino acid sequence as shown in SEQ ID NO:7, and the third domain contains an amino acid sequence as shown in any one of SEQ ID NOs:11, 15 to 17, 21 to 23, or 33 to 35; and the first domain contains an amino acid sequence as shown in SEQ ID NOs:2, the second domain contains an amino acid sequence as shown in SEQ ID NO:6, and the third domain contains an amino acid sequence as shown in any one of SEQ ID NOs:18 to 20.

[0073] Using complete mhIL-2 as an embodiment, the polypeptide provided in the first embodiment comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% identical to any of the amino acid sequences shown in SEQ ID NOs:36 to 57; more preferably, the polypeptide comprises an amino acid sequence identical to any of the amino acid sequences shown in SEQ ID NOs:36 to 57; more preferably, the polypeptide comprises an amino acid sequence identical to any of the amino acid sequences shown in SEQ ID NOs:46 to 48.

[0074] In the first embodiment, optionally, the polypeptide is a fusion protein having the function of binding to multiple cell surface interleukin receptors. (protein); suitably, the heterologous polypeptide may be replaced at least with a member of the interleukin family, such as interleukin-1, interleukin-3, interleukin-4, interleukin-5, interleukin-6, interleukin-7, interleukin-8, interleukin-9, interleukin-10, interleukin-11, interleukin-12, interleukin-13, interleukin-14, interleukin-15, interleukin-16, interleukin-17, interleukin-18, interleukin-19, interleukin-20, interleukin-21, interleukin-22, interleukin-23, interleukin-24, interleukin-25, interleukin-26, interleukin-27, interleukin-28, interleukin-29, interleukin-30, interleukin-31, interleukin-32 or interleukin-33.

[0075] Alternatively, the heterologous peptide or the PEG-covalently bonded peptide may be replaced with a serum half-life-extending peptide to increase the half-life of the peptide of the present invention in the individual circulatory system and reduce the number of administrations in subsequent embodiments. Specifically, the serum half-life-extending peptide may be the crystallizable region (Fc region) of an immunoglobulin fragment, serum albumin, albumin-binding protein or peptide, IgG, XTEN peptide, proline / alanine / serine (PAS)-rich peptide or elastin-like peptide, PEG bio-co-linking (PEGylation), and is not limited thereto.

[0076] Alternatively, the heterologous peptide can be replaced with a purification tag to facilitate purification and enrichment in the protein expression system of subsequent examples, improve the solubility of the peptide in the protein expression system, and maintain the activity of the peptide during the separation and purification process. Specifically, purification tags may include, but are not limited to, histidine tags (His-tag), glutathione S-transferase tags (GST-tag), maltose-binding protein tags (MBP-tag), transcription termination / anti-termination protein (NusA-tag), or microubiquitin-related modifier tags (SUMO-tag).

[0077] III. Pharmaceutical Compositions

[0078] The second embodiment of the present invention relates to a pharmaceutical composition comprising a polypeptide as described in the first embodiment and a pharmaceutically acceptable carrier, wherein the "pharmaceutically acceptable carrier" refers to a component of the pharmaceutical composition other than the active ingredient that is non-toxic to the subject, including but not limited to buffers, vehicles, stabilizers or preservatives.

[0079] In several embodiments, the pharmaceutical composition may be an oral administration formulation, an injection administration formulation, an inhalation administration formulation, or a topical or transdermal administration formulation.

[0080] IV. Uses of Pharmaceutical Compositions

[0081] The third embodiment of the present invention relates to the use of a pharmaceutical composition, wherein the pharmaceutical composition is as described in the second embodiment, and is a pharmaceutical or cell therapy composition for the treatment, prevention or relief of diseases or disorders caused by low immune cell activity.

[0082] In several implementations, the immune cells include natural killer (NK) cells, T cells, B cells, macrophages (MAC), dendritic cells (DC), or oligodendrocytes.

[0083] As used in this context, the term "T cell" refers to immune cells that express CD3, including T helper cells (CD4+).+ Cells), cytotoxic T cells (CD8) + (Th cells), functional Th cells, memory Th cells, T-regulatory cells (Tregs), and NK-T cells.

[0084] In a specific implementation scheme, the immune cell may be an active Th cell, a memory Th cell, a T-regulatory cell (Treg), or an NK-T cell; alternatively, the immune cell may be a cytokine-induced killer cell (CIK) composed of natural killer (NK) cells, NK-T cells, and T cells, or a γδT cell.

[0085] In several implementations, the disease is a proliferative disease including hematologic malignancies, solid tumors, or metastatic tumors; specifically, the solid tumor may be a renal solid tumor selected from the group consisting of ALK rearranged renal cell carcinoma, chromophobe renal cell carcinoma, clear cell renal cell carcinoma, clear cell sarcoma, metanephrotic adenoma, metanephrotic fibroma, myxoid tubular and spindle cell carcinoma, nephroma, Wilms' tumor, papillary adenoma, papillary renal cell carcinoma, renal eosinophilic cell carcinoma, renal cell carcinoma, succinate dehydrogenase deficient renal cell carcinoma, and collecting duct carcinoma; or a skin solid tumor selected from the group consisting of cutaneous melanoma and mucosal melanoma; or a hematologic malignancy selected from the group consisting of leukemia, lymphoma, and multiple myeloma.

[0086] V. Nucleic Acid Molecules and Expression Systems

[0087] The present invention also provides nucleic acid molecules encoding a polypeptide of the first embodiment, including but not limited to RNA, DNA, or cDNA; this nucleic acid molecule may be in the form of a vector or inserted into a specific vector, the type of which is not particularly limited, such as a plastid, viscous, or YAC vector; in a particular embodiment, the vector is an expression vector for providing the polypeptide of the first embodiment in a host cell, host organism, and / or expression system; typically, the expression vector comprises the nucleic acid molecule of the present invention and one or more expression regulatory components (e.g., promoters, enhancers, terminators, etc.) linked thereto; the selection of the components and their sequences for expression in a particular host is not beyond the common understanding of those skilled in the art; examples of regulatory components and other components useful or indispensable for the expression of the polypeptide of the first embodiment include promoters, enhancers, terminators, integration components, selection markers, leader sequences, reporter genes, etc.

[0088] The aforementioned nucleic acid molecules can be prepared according to known methods (e.g., by means of automated DNA synthesis and / or DNA recombination technology) based on the amino acid sequence information of the polypeptide of the first embodiment, at least based on any one of SEQ ID NOs:1 to 52 or a reasonable combination of sequences, and / or can be isolated from suitable biological materials.

[0089] In specific cases, a host cell that exhibits or can exhibit the polypeptide of the first embodiment is provided; naturally, the host cell may contain the aforementioned nucleic acid molecule or the aforementioned expression vector; suitably, the host cell may be a bacterial cell, fungal cell, yeast cell or mammalian cell.

[0090] Appropriate bacterial cells, including Gram-negative bacterial strains (such as Escherichia coli, Proteus, and Pseudomonas) and Gram-positive bacterial strains (such as Bacillus, Streptomyces, Staphylococcus, and Lactococcus).

[0091] Suitable fungal cells, such as cells of species under the genera *Trichoderma*, *Neurospora*, and *Aspergillus*; or, for example, cells of the genera *Saccharomyces*, including but not limited to *Saccharomyces Cerevisiae*, *Schizosaccharomyces*, *Schizosaccharomyces pombe*, *Pichia*, *Pichia pastoris*, or *Pichia methanolica*; or, for example, cells of species under the genus *Hansenula*.

[0092] Suitable mammalian cells can be selected from immortalized cell lines commonly used in protein expression and purification techniques, such as HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, etc. In addition to the above cell types, amphibian cells, insect cells, plant cells, and any other cells in this technical field that can be used to express heterologous proteins can also be used in this invention.

[0093] The polypeptide of the first embodiment can be expressed in a host cell as described above, then isolated from the host cell, and further purified as desired; or it can be produced extracellularly (e.g., in a culture medium for culturing host cells) or expressed in a cell-free protein synthesis system, then isolated for purification as desired.

[0094] The transformation or transfection methods applicable to the above-mentioned expression vectors, the purification tags of peptides, the methods of their inducible expression, culture conditions, etc., are well known in the art; similarly, the separation and purification techniques suitable for producing peptides of the first embodiment are also well known to those skilled in the art; however, in addition to the above-mentioned expression systems using host cells or cell-free systems, peptides of the first embodiment can also be obtained by other protein production methods known in the art, such as solid-phase or liquid-phase chemical synthesis.

[0095] The invention is further illustrated by the following specific examples; however, the invention is not limited to these examples.

[0096] 1. Introduction of the first and second mutations (introduction of disulfide bonds)

[0097] In the following specific case, the peptide uses human interleukin-2 (hereinafter referred to as hIL-2) as a template protein to introduce amino acid mutations, thereby introducing disulfide bonds; as shown in Figure 3, hIL-2 has 4 α helices, and a ring structure is formed between each of the two adjacent α helices; the first mutation and the second mutation are introduced into the structure of the template protein to introduce another set of disulfide bonds.

[0098] As shown in Figure 3, the positions of the disulfide bonds that can be designed are located between one loop and one α helix, between two α helices, and between two loops of the template protein. The specific mutation sites are listed in Table 1.

[0099] Table 1

[0100] As shown in Table 2, the amino acid sequence of hIL-2 with introduced disulfide bonds is presented. It is obtained by substituting amino acids at the designed disulfide bond positions, using the amino acid sequence of human hIL-2 as a reference sequence. The letters underlined are the mutation sites corresponding to the disulfide bond positions in Table 1.

[0101] Table 2

[0102] 2. Introduction of the third mutation

[0103] Accordingly, IL-2-LH2, IL-2-HH1, IL-2-HH3, IL-2-LH3, IL-2-LL1, and IL-2-LL2 were further modified by introducing a third mutation at position 125 to further improve the solubility of the peptides. On the other hand, as a comparative example for the experiments below, the amino acid sequence of hIL-2 was also used as a reference sequence, and a third mutation was introduced at the corresponding position 125.

[0104] As shown in Table 3, it presents the amino acid sequences of wild-type hIL-2, mhIL-2 with the third mutation, and mhIL-2 with disulfide bonds. The letters underlined represent the amino acid sites corresponding to the first to third mutations.

[0105] Table 3

[0106] 3. Protein preparation

[0107] The hIL-2 mutant was expressed in Pichia pastoris; in short, the mutant was prepared using the pPICZαA expression vector (with Zeocin resistance) in strain X-33, where strain X-33 was cultured in YPD medium.

[0108] First, strain X-33 was cultured at 30°C in 400 mL of medium for approximately 48 hours. Then, strain X-33 was transferred to 1100 mL of medium and cultured at 30°C for approximately 60 hours. Next, cell clumps were collected by centrifugation at 3500 rpm for 15 minutes, transferred to 1000 mL of BMM induction medium, and induced to express proteins at 20°C with the addition of methanol for approximately 72 hours. The supernatant was then collected by centrifugation.

[0109] Purification was performed using a salt-tolerant cation exchange column (Capto MMC) and a reverse high-performance liquid chromatography (RP-HPLC, C18) column. In short, the supernatant was mixed with one-tenth of its volume of binding buffer (50 mM sodium acetate, pH 4.0) and the pH was adjusted to match the binding buffer. After low-temperature high-speed centrifugation (4°C, 8000 rpm), the supernatant was filtered through filter paper and loaded onto a cation exchange column pre-equilibrated with binding buffer. The target protein was extracted using a gradient of extraction buffers (50 mM ammonium bicarbonate in Buffer B, pH 9.8). The accuracy of the extracted target protein was confirmed by 15% glycine SDS-PAGE gel electrophoresis. Finally, a second purification step was performed using a C18 RP-HPLC column, with the purification method involving water (Buffer A) and acetonitrile (Buffer B). B) Gradient elution to obtain target protein with a purity of over 95% and freeze-drying to dry the target protein into powder and store it at -80°C until use.

[0110] 4. Analysis of protein characteristics

[0111] The melting temperature of the protein was determined using differential scanning calorimetry (DSC). As shown in Table 4 and Figure 4, compared with Comparative Example 1, Comparative Example 4, after replacing cysteine ​​at position 125 with leucine (hereinafter referred to as C125L), showed a good melting temperature difference ΔTm1 between mhIL-2 and wild-type hIL-2, reaching +7.52, indicating that it helps to improve the thermal stability of mhIL-2. Conversely, replacing cysteine ​​at position 125 with serine (hereinafter referred to as C125S) and alanine (hereinafter referred to as C125A) resulted in melting temperature differences ΔTm1 of -8.76 and -7.73, respectively, which led to a decrease in the melting temperature of mhIL-2 and reduced its thermal stability.

[0112] On the other hand, compared with Comparative Examples 2 to 4, the melting temperature difference ΔTm2 of other Examples 1 to 3 obtained after introducing disulfide bonds showed a significant increase, reaching +12.25, +17.08 and +17.60 respectively. In particular, the melting temperature difference ΔTm2 of Example 3 was as high as +25.12 compared with Comparative Example 1, indicating that the disulfide bonds introduced by IL-2-LL2 can further improve thermal stability.

[0113] The procedure for the solubility test of the above proteins is briefly described as follows: 10 μL of phosphate-buffered saline (pH 7.4) was dissolved in a microtube at a known weight of the mhIL-2 protein sample powder, and the solution was thoroughly and uniformly dissolved; this is the theoretical concentration value. The sample was centrifuged at 13,500 rpm at 4°C for ten minutes using a microcentrifuge. The supernatant was then transferred to another clean microtube, and the concentration A280 was measured twice and the average value was calculated. The protein solubility test results are shown in Table 4. Compared with Comparative Example 1, Comparative Examples 2 to 4, after replacing cysteine ​​at position 125 with serine (hereinafter referred to as C125S), alanine (hereinafter referred to as C125A), and leucine (hereinafter referred to as C125L), the solubility of mhIL-2 increased by 1.6 to 3.0 times compared with wild-type hIL-2. On the other hand, Examples 1 to 3, which were obtained by introducing disulfide bonds, showed a solubility increase of 3.7 to 5.6 times.

[0114] Table 4

[0115] 5. Effect on immune cell proliferation

[0116] Furthermore, the specific activity of the above-mentioned peptide was measured using the mouse cytotoxic T lymphocyte line CTLL-2 to evaluate the peptide's immune cell proliferation effect. The specific steps are briefly described below:

[0117] (1) Dilute hIL-2 standard (Comparative Examples 1 to 4) and mhIL-2 test samples (Examples 1 to 3) with RPMI 1640. The standard was serially diluted starting from a concentration of 100 ng / ml, and the test samples were also serially diluted in the same way. After dilution, the volume of each test culture well in a 96-well plate was 50 μL, and each concentration was set up in 3 replicates.

[0118] (2) Collect CTLL-2 cells in the log phase and detect cell proliferation;

[0119] (3) Adjust the cell density to 8x10 4 Add 50 μL of cells to each culture well, for a final volume of 100 μL, and culture for 48 hours.

[0120] (4) Add 10 μL of CCK-8 to each culture well, shake on a shaker for 1 minute, incubate at 37°C and 5% CO2 for 4 hours, remove the 96-well plate, and measure the absorbance values ​​of OD450-OD600.

[0121] (5) Results analysis: Plot the dilution concentrations of hIL-2 standard and mhIL-2 test sample on the horizontal axis and the OD value of the detected cells on the vertical axis. Calculate the dilution concentration of the standard at 50% of the highest OD value and find the corresponding hIL-2 dilution concentration at 50% of the highest OD value of the test sample.

[0122] The test results are shown in Table 4. The specific activity of Comparative Example 1 was 0.61 ± 0.19 × 10⁻⁶. 6 U / mg; in comparison, the specific activity of comparative examples 2 and 3 was only 0.26 ± 0.02 × 10⁻⁶ U / mg; 6 U / mg, 0.43±0.08×10 6 U / mg, on the contrary, reduced the cell viability of CTLL-2, indicating that C125S and C125A mutations not only reduced the thermostability of mhIL-2 but also reduced its ability to promote cell proliferation; the specific activity of Comparative Example 4 was 0.75±0.11×10 6 U / mg showed that the C125L mutation increased the cell proliferation activity of mhIL-2, with a promoting effect of 22.9%, and improved thermal stability.

[0123] Compared with Comparative Examples 1 to 4, Examples 1 to 3 further introduced disulfide bonds between the α-binding domain of the interleukin-2 receptor subunit and the α-binding domain of the non-interleukin-2 receptor subunit, which significantly enhanced the cell proliferation activity of mhIL-2, with specific activities reaching 1.72±0.37, 1.80±0.22 and 2.23±0.55, respectively, and their promoting effects on CTLL-2 cell proliferation reached 181.9%, 195.1% and 265.6%, respectively.

[0124] 6. Cytotoxicity assay

[0125] The cytotoxicity of the above-mentioned peptides was assessed using peripheral blood mononuclear cells (PBMCs) to evaluate the effect of the peptides on killing human hepatocellular carcinoma cells (HepG2) after activating immune cells. The specific steps are briefly described below:

[0126] (1) Use Ficoll to separate 20 mL of peripheral venous blood. After centrifugation, the blood is separated into four layers from the bottom of the tube to the surface of the liquid: red blood cell and granulocyte layer, layered liquid layer, mononuclear cell layer, and plasma layer.

[0127] (2) Take a mononuclear cell layer to achieve a final cell density of 2×10⁻⁶. 6 / mL, add 1000U / mL γ-interferon (IFN-γ) on the same day, incubate at 37℃ and 5% CO2 for 24 hours, then add 100ng / mL anti-CD3 monoclonal antibody and 300IU / mL mhIL-2 test sample (Example 2) and hIL-2 standard (Comparative Example 1), incubate at 37℃ and 5% CO2 for 48 hours;

[0128] (3) After that, add 300 IU / mL mhIL-2 test sample and hIL-2 standard every four days for one week. In the second and third weeks, adjust to add 1000 IU / mL mhIL-2 test sample and hIL-2 standard every four days.

[0129] (4) One day before co-culturing peripheral blood mononuclear cells with human hepatocellular carcinoma cell lines after three weeks of culture, human hepatocellular carcinoma cells carrying the green fluorescent protein gene were attached to 96-well plates at a cell density of 8 × 10⁻⁶ cells / well. 4 / mL, volume 100mL;

[0130] (5) On the day of co-culture, the activated immune cells and tumor cells were set up in 3 replicates at a ratio of 80:1, 40:1, 20:1, 10:1 and 5:1. After culturing at 37°C and 5% CO2 for 24 hours, the 96-well plate was removed and the absorbance values ​​of OD535-OD485 were measured.

[0131] (6) Results analysis: The cytotoxicity of mhIL-2 test samples and hIL-2 standard products on tumor cells was analyzed by plotting the culture concentration of mhIL-2 test samples and hIL-2 standard products on the horizontal axis and the percentage of cell cytotoxicity on the vertical axis.

[0132] The test results are shown in Figure 5. Four days after the addition of stimulated and activated immune cells, the cytotoxicity was 71% under the condition of an 80:1 ratio of activated immune cells to tumor cells in Comparative Example 1; in comparison, the cytotoxicity of Example 2 reached 95%; under the condition of a ratio of 40:1, the cytotoxicity of Comparative Example 1 was 27%, and the cytotoxicity of Example 2 was 61%; under the condition of a ratio of 20:1, the cytotoxicity of Comparative Example 1 was 13%, and the cytotoxicity of Example 2 was 26%. The cytotoxicity test showed that by introducing disulfide bonds between the α-binding domain of the interleukin-2 receptor subunit and the α-binding domain of the non-interleukin-2 receptor subunit, with an addition cycle of 4 days, Example 2 had high stability and could continuously activate immune cells to maintain the activity of killing cancer cells.

Claims

1. A polypeptide, characterized in that, Include: The first structural domain includes at least 80%, 85%, 90%, 95%, or 99% of the amino acid sequence at positions 1 to 34 of the amino acid sequence shown in SEQ ID NO:1; A second domain, disposed at the C-terminus of the first domain, comprises at least 80%, 85%, 90%, 95%, or 99% of the amino acid sequence at positions 35 to 72 of the amino acid sequence shown in SEQ ID NO:1; and The third domain includes at least 80%, 85%, 90%, 95%, or 99% of the amino acid sequence at positions 73 to 133 of the amino acid sequence shown in SEQ ID NO:1, and has a first mutation that replaces a first amino acid site with a cysteine, wherein the polypeptide has a second mutation that replaces a second amino acid site with another cysteine, and the second mutation may selectively occur in the first domain, the second domain, or the third domain, whereby the cysteine ​​replaced by the first mutation forms a disulfide bond with the cysteine ​​replaced by the second mutation.

2. The polypeptide according to claim 1, characterized in that, The first amino acid includes asparagine (Asn, N) at position 77, asparagine (Asp, D) at position 84, isoleucine (Ile, I) at position 92, lysine (Lys, K) at position 97, threonine (Thr, T) at position 111, phenylalanine (Phe, F) at position 117, or leucine (Leu, L) at position 132. The second amino acid comprises: glutamic acid (Gln, Q) at position 11, leucine (Leu, L) at position 17, leucine (Leu, L) at position 18, tyrosine (Tyr, Y) at position 31, lysine (Lys, K) at position 43 replaced by cysteine, phenylalanine (Phe, F) at position 44, leucine (Leu, L) at position 56, or leucine (Leu, L) at position 80.

3. The polypeptide according to claim 1, characterized in that, In the C-terminal downstream region of the first amino acid, the third domain further includes a third mutation that replaces the third amino acid site with a hydrophilic or hydrophobic amino acid, wherein the third amino acid site is cysteine.

4. The polypeptide according to claim 3, characterized in that, The third mutation includes any of the following: Cysteine ​​at position 125 is replaced with serine (Ser,S); Cysteine ​​at position 125 is replaced with alanine (Ala,A); Cysteine ​​at position 125 is replaced with leucine (Leu, L).

5. The polypeptide according to claim 1, characterized in that, The first domain contains any of the amino acid sequences shown in SEQ ID NOs:2 to 6; the second domain contains any of the amino acid sequences shown in SEQ ID NOs:7 to 10; and the third domain contains any of the amino acid sequences shown in SEQ ID NOs:11 to 35.

6. The polypeptide according to claim 1, characterized in that, Includes amino acid sequences that are at least 80%, 85%, 90%, 95%, or 99% identical to any of the amino acid sequences shown in SEQ ID NOs:36 to 57.

7. A pharmaceutical composition, characterized in that, Includes the polypeptide and pharmaceutically acceptable carrier as described in any one of claims 1 to 6.

8. The use of the pharmaceutical composition according to claim 7, characterized in that, It is a pharmaceutical or cell therapy composition used to prepare for the treatment, prevention or relief of diseases or disorders caused by low immune cell activity.

9. The use according to claim 8, characterized in that, These immune cells include natural killer (NK) cells, NK-T cells, T cells, B cells, macrophages (MAC), dendritic cells (DC), oligodendrocytes, cytokine-induced killer cells (CIK), or γδT cells.

10. The use according to claim 8, characterized in that, The disease is a proliferative disorder, including hematologic malignancies, solid tumors, or metastatic tumors.

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